Renin Angiotensin System and Cardiovascular Disease (Contemporary Cardiology)

Contemporary Cardiology C HRISTOPHER P. C ANNON , SERIES EDITOR

For other titles published in this series, go to http://www.springer.com/7677

MD

Walmor C. DeMello · Edward D. Frohlich Editors

Renin Angiotensin System and Cardiovascular Disease

Editors Walmor C. DeMello Department of Pharmacology University of Puerto Rico P.O. Box 5067 San Juan PR 00936 Medical Sciences Campus USA [email protected]

Edward D. Frohlich Ochsner Clinic Foundation 1514 Jefferson Highway New Orleans LA 70121 USA [email protected]

ISBN 978-1-60761-185-1 e-ISBN 978-1-60761-186-8 DOI 10.1007/978-1-60761-186-8 Library of Congress Control Number: 2009933642 © Humana Press, a part of Springer Science+Business Media, LLC 2009 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper springer.com

Preface

Experimental and clinical evidence supports the view that the activation of the renin angiotensin aldosterone system is involved in cardiovascular pathology including hypertension, heart failure, myocardial ischemia, and atherosclerosis. The present volume describes the intricacies involved in these processes, including the influence of prorenin/renin, angiotensin II, angiotensin (1-7), and aldosterone on cardiac and vascular functions as well as their involvement in the generation of cardiovascular diseases. Fundamental aspects like intracellular signaling, regulation of cell volume in the failing heart, and the presence of an intracrine renin angiotensin system are discussed. Moreover, the role of the mineralocorticoid receptor as an important component of the intracrine renin angiotensin system and as a regulator of extracellular action of angiotensin II is described, reinforcing the view that aldosterone inhibitors are helpful in the treatment of heart failure and hypertension. Let us hope the important topics included here motivate basic and clinical investigators and contribute to the development of new therapeutic approaches for cardiovascular diseases. We want to thank the distinguished authors and Humana Press for the opportunity to publish this important book. Walmor C. DeMello Edward Frohlich

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Contents

1 Systemic Versus Local Renin Angiotensin Systems. An Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Walmor C. DeMello and Richard N. Re

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2 Clinical Import of the Local Renin Angiotensin Aldosterone Systems . . . . . . . . . . . . . . . . . . . . . . . . . Edward D. Frohlich

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3 Renin, Prorenin, and the (Pro)Renin Receptor . . . . . . . . . . . Genevieve Nguyen and Aurelie Contrepas

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4 Local Renin Angiotensin Systems in the Cardiovascular System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Richard N. Re

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5 Renin-Angiotensin-Aldosterone System and Pathobiology of Hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pierre Paradis and Ernesto L. Schiffrin

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6 AT1 Receptors, Angiotensin Receptor Blockade, and Clinical Hypertensive Disease . . . . . . . . . . . . . . . . . . Robert M. Carey

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7 Structural and Electrophysiological Remodeling of the Failing Heart . . . . . . . . . . . . . . . . . . . . . . . . . . Walmor C. DeMello

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8 Inhibiting the Renin Angiotensin Aldosterone System in Patients with Heart Failure and Myocardial Infarction . . . . . . Marc A. Pfeffer

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9 Left Ventricular Hypertrophy and Treatment with Renin Angiotensin System Inhibition . . . . . . . . . . . . . . . . . . . . Edward D. Frohlich and Javier Díez

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10 Angiotensin-(1-7), Angiotensin-Converting Enzyme 2, and New Components of the Renin Angiotensin System . . . . . . Aaron J. Trask, Jasmina Varagic, Sarfaraz Ahmad, and Carlos M. Ferrario

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11 Kinin Receptors and ACE Inhibitors: An Interrelationship . . . . Ervin G. Erdös, Fulong Tan, and Randal A. Skidgel

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12 Kinins and Cardiovascular Disease . . . . . . . . . . . . . . . . . Oscar A. Carretero, Xiao-Ping Yang, and Nour-Eddine Rhaleb

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13 CMS and Type 2 Diabetes Mellitus: Bound Together by the Renin Angiotensin Aldosterone System . . . . . . . . . . . . . Deepashree Gupta, Guido Lastra, Camila Manrique, and James R. Sowers 14 Renin Angiotensin Aldosterone System and Cardiovascular Disease . . . . . . . . . . . . . . . . . . . . . Swynghedauw Bernard, Milliez Paul, Messaoudi Smail, Benard Ludovic, Samuel Jane-Lise, and Delcayre Claude

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15 Renin Angiotensin System and Atherosclerosis . . . . . . . . . . . Changping Hu and Jawahar L. Mehta

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16 Renin Angiotensin System and Aging . . . . . . . . . . . . . . . . León F. Ferder

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Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors

Sarfaraz Ahmad, MD, PhD Hypertension and Vascular Research Center, Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, NC Robert M. Carey, MD, MACP Division of Endocrinology and Metabolism, Department of Medicine, University of Virginia Health System, Charlottesville, VA Oscar A. Carretero, MD Hypertension and Vascular Research Division, Department of Medicine and Heart and Vascular Institute, Henry Ford Hospital, Detroit, MI Aurelie Contrepas, BS Institut de la Santé et de la Recherche Médicale and Collège de France, Experimental Medecine Unit, Paris, France Claude Delcayre, PhD Centre de Recherches Cardiovasculaires INSERM Lariboisière, PARIS, France Walmor C. DeMello, MD, PhD Department of Pharmacology, School of Medicine, Medical Sciences Campus, University of Puerto Rico, San Juan, PR Javier Díez, MD, PhD Área de Ciencias Cardiovasculares, Edificio CIMA, Pamplona, Spain Ervin G. Erdös, MD Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL León F. Ferder, MD Departments of Physiology, Pharmacology and Medicine, Ponce School of Medicine, Ponce, PR Carlos M. Ferrario, MD Hypertension and Vascular Research Center, Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, NC Edward D. Frohlich, MD Ochsner Clinic Foundation, Louisiana State University School of Medicine, New Orleans, LA Deepashree Gupta, MD Diabetes and Cardiovascular Center, University of Missouri School of Medicine, and VA Medical Center, Columbia, MO

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Changping Hu, MD, PhD Division of Cardiovascular Medicine, University of Arkansas for Medical Sciences and the Central Arkansas Veterans Healthcare System, Little Rock, AR Guido Lastra, MD Diabetes and Cardiovascular Center, University of Missouri School of Medicine, and VA Medical Center, Columbia, MO Benard Ludovic, BS Centre de Recherches Cardiovasculaires INSERM Lariboisière, Paris, France Camila Manrique, MD Diabetes and Cardiovascular Center, University of Missouri School of Medicine, and VA Medical Center, Columbia, MO Jawahar L. Mehta, MD, PhD Division of Cardiovascular Medicine, University of Arkansas for Medical Sciences and the Central Arkansas Veterans Healthcare System, Little Rock, AR Genevieve Nguyen, MD, PhD Institut de la Santé et de la Recherche Médicale, (INSERM) and Collège de France, Experimental Medecine Unit Marcelin Berthelot Paris, France Pierre Paradis, MD Hypertension and Vascular Research Unit, Lady Davis Institute for Medical Research, McGill University, Montreal. Canada Milliez Paul, MD Centre de Recherches Cardiovasculaires INSERM Lariboisière, Paris, France Marc A. Pfeffer, MD, PhD Department of Medicine, Division of Cardiology, Brigham and Women’s Hospital, Dzau Professor of Medicine, Harvard Medical School, Brigham and Women’s Hospital, Boston, MA Richard N. Re, MD Ochsner Clinic Foundation, New Orleans, LA Nour-Eddine Rhaleb, PhD, FAHA Hypertension and Vascular Research Division, Department of Medicine and Heart and Vascular Institute, Henry Ford Hospital, Detroit, MI Jane-Lise Samuel, MD, PhD Centre de Recherches Cardiovasculaires INSERM Lariboisière, Paris, France Ernesto L. Schiffrin, MD Department of Medicine, Sir Mortimer B. Davis-Jewish General Hospital, Hypertension and Vascular Research Unit, Lady Davis Institute for Medical Research, McGill University, Montreal, Canada Randhal Skidgel, PhD Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL Messamoudi Smail, PhD Centre de Recherches Cardiovasculaires INSERM Lariboisière, Paris, France James R. Sowers, MD Diabetes and Cardiovascular Center, University of Missouri School of Medicine, and VA Medical Center, Columbia, MO

Contributors

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Bernard Swyngedauw, MD, PhD Centre de Recherches Cardiovasculaires INSERM Lariboisière, Paris, France Fulong Tan, PhD Department of Pharmacology, University of Illinois, College of Medicine, Chicago, IL Aaron J. Trask, BS Hypertension and Vascular Research Center, Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, North Carolina Winston-Salem, NC Jasmina Varagic, MD, PhD Hypertension and Vascular Research Center, Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, NC Xiao-Ping Yang, MD Hypertension and Vascular Research Division, Department of Medicine and Heart and Vascular Institute, Henry Ford Hospital, Detroit, MI

Chapter 1

Systemic Versus Local Renin Angiotensin Systems. An Overview Walmor C. DeMello and Richard N. Re

Abstract The concept of local renin angiotensin systems in the cardiovascular system is discussed, and evidence is presented that these systems work independently of the systemic one. Particular attention was given to the presence of an intracrine renin angiotensin aldosterone system in the heart and the novel role of the mineralocorticoid receptor. Furthermore, the influence of the renin angiotensin system on cell volume regulation is briefly discussed. This chapter includes an overview of these important biological concepts and provides an introduction to the topics that are discussed in detail by different authors throughout the book. The renin angiotensin system (RAS) is an enzymatic cascade in which renin derived from the juxtaglomerular cells (JG) of the kidney acts on an hepatically synthesized substrate, angiotensinogen, to generate the decapeptide angiotensin I. This peptide is cleaved by angiotensin-converting enzyme (ACE), primarily in the pulmonary circulation, to the vasoconstrictor and aldosterone secretagogue, angiotensin II. The blood pressure-elevating action of angiotensin II, together with its direct suppressive action on JG cells and the volume expansion produced by enhanced aldosteronedriven sodium retention, leads to the suppression of JG renin secretion, thereby forming a negative feedback loop. Volume depletion or lowered blood pressure stimulates renin release, leading to pressure elevation and volume retention. Elevated blood pressure or hypervolemia suppresses renin release and tends to lower blood pressure and intravascular volume. However, as powerful as this construct is, accumulating evidence indicates that it is incomplete in that it focuses solely on angiotensin synthesis in the circulation. For example, the blood pressure response to ACE inhibitors, which block ACE-driven angiotensin I generation, is not predicted by circulating renin activity, suggesting that RAS activity in tissues may be relevant [1]. Indeed, early on it was shown that most angiotensin II generation takes place in the arterial wall where angiotensin II is generated from RAS components taken W.C. DeMello (B) School of Medicine, Medical Sciences Campus, UPR, San Juan, PR USA e-mail: [email protected] W.C. DeMello, E.D. Frohlich (eds.), Renin Angiotensin System and Cardiovascular Disease, Contemporary Cardiology, DOI 10.1007/978-1-60761-186-8_1,  C Humana Press, a part of Springer Science+Business Media, LLC 2009

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up from the circulation [2]. In the follow-up to these observations, it was then noted that components of RAS were taken up by many tissues [3], leading to the possibility that angiotensin II synthesis could be locally influenced by the relative uptake of components at those sites [4] and the synthesis of the various components can be detected in various tissues under various conditions [3]. Together, these observations made the concept of local RASs in tissues compelling. It must be recalled that renin in the circulation is not strictly rate-limiting for angiotensin I production. That is, angiotensinogen circulates at a concentration close to the K m for the generation of AI by renin. Therefore, an increase in angiotensinogen will lead to an increased production of angiotensin I [3, 5]. Thus, if a tissue were to augment tissue concentration of angiotensinogen by local production, more angiotensin I and likely angiotensin II would be generated in that tissue as compared to a tissue that did not augment the concentration of angiotensinogen with local synthesis. Also, it must be noted that the alteration of JG renin secretion cannot possibly equalize the angiotensin concentrations in the two tissues, which clearly indicates local regulation of local angiotensin II [5]. This, in turn, is particularly important because, of all the components of the RAS, the synthesis of renin in tissues (with a few exceptions) is the most contentious. Indeed, in many tissues the reported renin secretion is very low, suggesting that this renin could influence angiotensin production in only a small area [4]. Nonetheless, renin upregulation has been reported in the adrenal gland following nephrectomy where it helps maintain aldosterone secretion, as well as in the left ventricle and other tissues such as the heart in specific circumstances [6]. But it is clear from the arguments presented above that even in the absence of local renin synthesis, local regulation of angiotensin production can occur through local synthesis of other RAS cascade components [3]. Differential uptake of renin into tissues provides another mechanism for local RAS regulation. Although in normal heart cardiac renin seems to be related to its uptake from plasma [5], evidence is available that renin expression is increased after myocardial infarction [7] and after stretch of the cardiomyocytes [8]. On the other hand, a renin transcript that does not encode a secretory signal [9] and remains inside the cell is overexpressed during myocardial infarction – suggesting that intracellular renin has functional properties. Indeed, previous studies showed that intracellular renin and Ang II administration impairs cell coupling in the heart [10, 11] and intracellular Ang II reduces the inward calcium current in the failing heart [12], supporting the view that there is a functional intracrine renin angiotensin system [13–17]. This intracrine angiotensin II must properly also be considered an aspect of the tissue RASs, and it may well play an important role in such pathological processes as left ventricular hypertrophy, cardiac arrhythmias and cardiac myocyte apoptosis [13, 17] (see also Chapters 4 and 7). Other studies have demonstrated upregulation of angiotensinogen and angiotensin-converting enzyme (ACE) in tissues under normal or pathological conditions. The enzyme chymase, which can substitute for ACE in the conversion of angiotensin I to angiotesnin II, is also expressed in multiple tissues and upregulated in some circumstances [17]. Even more telling is the recent demonstration of a (pro)renin receptor in mesangial and other cells, which signals using classical

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second messengers following the binding of prorenin or renin [18, 19]. This reveals the hormonal nature of (pro)renin. At the same time, binding of prorenin to the receptor activates its binding site so that the prohormone becomes enzymatically active, generating angiotensin I in the vicinity of cell surface receptors [19]. Similarly, renin bound to the receptor becomes more enzymatically active [4, 20]. These observations make clear that the biological activity of the RAS in a tissue can be powerfully influenced by the level of expression of the (pro)renin receptor in the tissue – a variable totally hidden from any analysis of the concentrations of circulating RAS components. The potential importance of this finding is suggested by the fact that prorenin levels are elevated in diabetic patients, and high concentrations of circulating prorenin are a predictor of retinopathy – a finding made all the more compelling by the observation that prorenin can be synthesized locally in the Mueller cells of the retina [19]. In addition, it now appears that there exist countervailing systems which while not influencing angiotensin II action at the receptor nonetheless offset some of its effect. For example, an ACE homologue, ACE2, has recently been described and studied [21]. ACE2, unlike ACE, does not convert angiotensin I to angiotensin II, but rather its principal action seems to be the conversion of angiotensin II to the hepatapeptide angiotenin (1–7), which operating through its own receptor offsets many of the vasoconstrictive and growth-promoting actions of angiotensin II [22, 23], improving impulse propagation during ischemia reperfusion through activation of the sodium pump, reducing the incidence of slow conduction and the generation of cardiac arrhythmias [24] (see also Chapter 10). Recently, it was found that chronic administration of eplerenone, a mineralocorticoid receptor blocker, reduces the expression of AT1 receptors at surface cell membrane as well as intracellularly inhibiting the intracrine and extracellular actions of Ang II on the inward calcium current in the failing heart [25]. These findings indicate that the mineralocorticoid receptor is involved in the regulation of intracellular and extracellular actions of Ang II and lead to the concept that there is an intracrine renin angiotensin aldosterone system (see also Chapter 7). It is possible to conclude that the beneficial effects of eplerenone in patients with heart failure are in part explained by the suppression of fibrosis, hypertrophy and electrophysiological abnormalities elicited by Ang II [26]. It is well known that regulation of cell volume is essential for normal cellular function. Recent evidence is available that the renin angiotensin system is involved in the regulation of heart cell volume [27] because extracellular Ang II increases cell volume through inhibition of the sodium pump and activation of the Na-K-2Cl cotransporter, while intracellular Ang II reduces the cell volume by activating the Na-K pump [27]. These findings are relevant particularly to myocardial ischemia which by itself causes cell swelling. According to these observations, the activation of the circulating renin angiotensin system is particularly harmful during myocardial ischemia while the activation of the intracrine renin angiotensin system might be beneficial by decreasing the cell volume (see Chapter 7). In conclusion, evidence is available that there are local renin angiotensin systems in the cardiovascular system, and that a functional intracrine renin angiotensin aldosterone system contributes to cardiovascular pathology [13, 25, 28, 29].

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References 1. Mazzolai, L., Nussberger, J., and Aubert, J.F. et al. (1998) Blood-pressure independent cardiac hypertrophy induced by local activated renin-angiotensin system. Hypertension 31, 1324–30 2. Muller, D.N., and Luft, F.C. (1998) The renin angiotensin system in vessel wall. Basic Res Cardiol 93(Supl 2), 7–14 3. Kurdi, M., DeMello, W.C., and Booz, G.W. (2005) Working outside the system: an update on unconventional behavior of the renin angiotensin system components. Intern J Biochem Cell Biol 37, 1357–67 4. Nguyen, G., Delarue, F., and Bu, C. et al. (2002) Pivotal role of the renin/prorenin receptor in angiotensin II production and cellular responses to renin. J Clin Invest 109, 1417–27. 5. Danser, A.H.J., van Katz, J.P., and Admiraal, P.J.J. et al. (1994) Cardiac renin and angiotensins; uptake from plasma versus in situ synthesis. Hypertension 24, 37–48 6. Peters, J., Obermuller, N., Woyth, A., Peters, B., Maser-Gluth, C., Kranzlin, B., and Gretz N (1999) Losatan and angiotensin II inhibit aldosterone production in anephric rats via different actions on the intraadrenal renin-angiotensin system. Endocrinology 140, 675–82. 7. Passier, R.C.J.J., Smits, J.F.M., Verluyten, M.J.A., and Daemen, M.J.A.P (1996) Expression and localization of renin and angiotensinogen in rat heart after myocardial infarction. Am J Physiol 271, H1040–8 8. Malhotra, R., Sadoshima, J., Broscius, F.C., and Izumo, S. (1999) Mechanical stretch and angiotensin II differentially upregulated the renin angiotensin system in cardiac myocytes in vitro. Circ Res 85, 137–46 9. Clausmeyer, S., Reinecke, A., and Farrenkopf, R. et al. (2000) Tissue-specific expression of a rat renin transcript lacking the coding sequence for the prefragment and its stimulation by myocardial infarction. Endocrinology 141, 2963–70 10. DeMello, W.C. (1994) Is an intracellular renin angiotensin system involved in the control of cell communication in the heart? J Cardiovasc Pharmacol 23, 640–6 11. DeMello, W.C. (1995) Influence of intracellular renin on heart cell communication. Hypertension 25, 1172–7 12. DeMello, W.C. (1998) Intracellular angiotensin II regulates the inward calcium current in cardiac myocytes. Hypertension 32, 976–82 13. DeMello, W.C., and Danser, A.J.H. (2000) Angiotensin II and the heart: on the intracrine renin angiotrensin system. Hypertension 35, 1183–8 14. Re, R.N (2000) On the biological actions of intracellular angiotensin. Hypertension 35, 1189–90 15. Cook, J.L., Zhang, Z., and Re, R.N. (2001) In vitro evidence for an intracellular site of angiotensin action. Circ Res 89, 1138–46 16. Singh, V.P., Le, B., Bhat, V.B., Baker, K.M., and Kumar, R. (2007) High-glucose-induced regulation of intracellular ANG II synthesis and nuclear redistribution in cardiac myocytes. Am J Physiol Heart Circ Physiol 293(2), H939–48. 17. Paul, M., Poyan, M.A., and Kreutz, R. (2006) Physiology of local renin-angiotensin systems. Physiol Rev 86(3), 747–803. 18. Nguyen, G., Burckle, C.A., and Sraer, J.D. (2004) Renin/prorenin receptor biochemistry and functional significance. Curr Hypertens Rep 6, 129–32 19. Nguyen, G., Delarue, F., Berrou, J., Rondeau, E., and Sraer, J.D. (1996) Specific receptor binding of renin on human mesangial cells in culture increases plasminogen activator inhibitor-1 antigen. Kidney Int 50, 1897–903. 20. Nguyen, G., and Danser, A.H. (2008) Prorenin and (pro)renin receptor: a review of available data from in vitro studies and experimental models in rodents. Exp Physiol 93(5), 557–63 21. Donoghue, M., Hsieh, F., and Baronas, E. et al. (2000) A novel angiotensin converting enzyme-related carboxypeptidase(ACE2) converts angiotensin I to angiotensin (1–9). Circ Res 87, E1–E9.

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22. Ferrario, C., Chappell, M., and Tallant, E.K. et al. (1997) Counterregulatory actions of angiotensin (1-7). Hypertension 30, 535–41 23. Crackower, M.A., Sarao, R., and Oudit, G.Y. et al. (2002) Angiotensin-converting enzyme 2 is an essential regulator of heart function. Nature 417, 799–802. 24. DeMello, W.C. (2004) Angiotensin (1-7) re-establishes impulse conduction in cardiac muscle during ischaemia-reperfusion. The role of the sodium pump. J Renin Angiotensin Aldosterone Syst Dec 5(4), 203–8. 25. DeMello, W.C., and Gerena, Y. (2008) Eplerenone inhibits the intracrine and extracellular actions of angiotensin II on the inward calcium current in the failing heart. On the presence of an intracrine renin angiotensin aldosterone system. Regul Pept 151, 54–60. 26. De Mello, W.C. (2006) Beneficial effect of eplerenone on cardiac remodelling and electrical properties of the failing heart. J Renin Angiotensin Aldosterone Syst 7(1), 40–6. 27. DeMello, W.C. (2008) Intracellular and extracellular renin have opposite effects on the regulation of heart cell volume. Implications for myocardial ischaemia. J Renin Angiotensin Aldosterone Syst. 9(2), 112–8. 28. Re, R.N., and Cook, J.L. (2008) The basis of intracrine physiology. J Clin Pharmacol 48, 344–50. 29. Re, R.N., and Cook, J.L.M. (2007) Mechanisms of disease: intracrine physiology in the cardiovascular system. Nat Clin Pract Cardiovasc Med Oct 4(10), 549–57.

Chapter 2

Clinical Import of the Local Renin Angiotensin Aldosterone Systems Edward D. Frohlich

Abstract The concept of local renin angiotensin (and possibly aldosterone) systems has been a relatively recent interjection to the investigative milieu. Much interest and important studies have resulted, and reference to applicability to disease and disease mechanisms is still of innovative and imaginative clinical and experimental studies. To this end, there are several areas of pertinence which have evolved including the underlying causations, mechanisms, and treatment of a number of diseases. Among those fascinating and provocative study areas is the need for additional motivated investigation related to ventricular and vascular hypertrophy, remodeling, and cardiac and renal failure and new thinking related to lifestyle modifications (including those related to salt excess, obesity, and responses to various drugs, clinically useful or otherwise). We have much confidence that these and other areas for study will be productive and useful and will lead to important clinical approaches and contributions on the issue of existing local RAAS. Much of the present-day clinical and investigative considerations of the renin angiotensin aldosterone system (RAAS) as well as this monograph concern the classically accepted endocrine RAAS system. The overall concepts involved have been extremely important in understanding the biology, physiology, and clinical relevance of this system as it pertains to cardiovascular and renal diseases, and they have led to the synthesis of new classes of therapeutic agents which have changed dramatically approaches to disease. Consequently, these changes have resulted in remarkable reductions in the morbidity and mortality of cardiovascular, renal, brain, and other diseases.

2.1 The Classical System The framework of this classically understood system embodies the synthesis of the enzyme renin in the kidney, the variety of mechanisms that promote and stimulate its release by the renal juxtaglomerular apparatus, and its action on the complex E.D. Frohlich (B) Ochsner Clinic Foundation, New Orleans, LA e-mail: [email protected] W.C. DeMello, E.D. Frohlich (eds.), Renin Angiotensin System and Cardiovascular Disease, Contemporary Cardiology, DOI 10.1007/978-1-60761-186-8_2,  C Humana Press, a part of Springer Science+Business Media, LLC 2009

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protein angiotensinogen which is synthesized in the liver. The consequence of this action is the production of the decapeptide angiotensin I which, as it passes through the pulmonary circulation, loses its terminal two peptides by virtue of the proteolytic action of the angiotensin-converting enzyme. The resultant octapeptide angiotensin II is the potent vasopressor agent which is responsible for vasoconstriction; release of aldosterone by the adrenal cortex and consequent retention of sodium and water by the kidney; stimulation of specific brain centers responsible for increased cardiovascular adrenergic outflow and thirst; and local endothelial actions that promote mitogenesis, hypertrophy, collagen synthesis and tissue fibrosis, apoptosis, inflammation, and, no doubt, other intracellular signaling and other biological and pathophysiological effects [1]. Already, many of these latter actions have been incorporated in our consideration of the clinical diagnosis used clinically with respect to “endothelial dysfunction” [2, 3]. Although relatively recently described, there have been several additional components to the RAAS which have intriguing biological actions that have the potential for developing new physiological and pathological implications [4].

2.2 The Local Systems Although several of the foregoing actions of angiotensin II are relatively new, they have already been inculcated into a new dimension of the classical RAAS. This then relates to the overall concept of this monograph. It therefore concerns the concept of local RAAS (hereafter to be considered in plurality) that affect the structure and function of specific target organs of disease, including heart, blood vessels, kidney, and, no doubt, other organs [5]. To this end, although to some extent considered by some to be controversial, each of the components of these local RAAS has been identified within these foregoing organs although certain specific components (e.g., the putative synthesis of the enzyme renin within the heart) of the system. Indeed, these local systems already have important clinical and even therapeutic considerations and implications in health and disease [6]. In this respect, we also have deliberately included the hormone aldosterone in this local system since this hormone has already been identified to be present in some of these systems as for vital consideration of the existence of local RAAS [7]. Thus, although perhaps still in the realm of speculation, consideration of these local systems and newer components and metabolites of the system is neither premature, irrelevant, nor speculative for present-day consideration in this monograph. This monograph has been conceived and organized to stimulate further fundamental and clinical investigations dealing with the impact of the RAAS in disease. Thus, the participants of this workshop are of the unanimous opinion that these local RAAS are no longer a subject of debate; indeed, this is an important area of fundamental and clinical study, which is the intellectual commitment of this entire volume. To this end, the existence of these local systems in certain organs and the information derived from recent and current investigations provide the substance of

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tentative (but appealing) and exciting information which relates to specific clinical problems. Thus, this rather selective and speculative discussion of local RAAS in disease is included to tantalize the interested reader, student, and investigator in certain specific clinical situations including the pathogenesis and pathophysiology of ventricular hypertrophy in hypertension; ventricular and vascular remodeling in hypertensive and ischemic cardiovascular diseases; secondary (e.g., renal) therapeutic responses to disease; structural and functional responses of organs and in toxemias of pregnancy, to certain lifestyle and other interventions (e.g., salt excess) in hypertension. Ever since the Framingham Heart Study demonstrated that left ventricular hypertrophy (LVH) was a major risk factor predisposing the hypertensive patient to increased morbidity and mortality associated with coronary heart disease [8], we have been intrigued about the fundamental pathophysiological mechanisms of LVH that account for this risk. Thus, soon after this landmark epidemiological study, we initiated our earliest clinical and pathophysiological studies of this problem in which we elucidated the clinical correlates associated with the development of LVH [9]. We perceived the well-recognized concept that arterial pressure increased as an adaptive response of the left ventricle to the progressive increase in afterload in response to the increasing total peripheral resistance imposed by arteriolar constriction imposed. Our subsequent studies introduced the feasibility of the new noninvasive technology of M-mode echocardiography in order to identify the pathophysiological sequence in the clinical development of LVH [10]. We confirmed that coincident with the developing increased left ventricular (LV) mass and wall thicknesses, the earlier events associated with electrocardiographic evidence of left atrial abnormality were also identified with increased left ventricular mass and LVH. Moreover, these structural changes were associated with functional changes of LV functional impairment early in LVH [10]. These early findings suggested to us our ongoing concern that the development of LVH in hypertension were not solely the consequence of “adaptive hypertrophy”. We soon focused our attention on the functional events associated with antihypertensive therapy and whether it reversed the increased LV mass [11–14]. These studies indicated that certain agents decreased LV mass and impaired the ventricular functional responses. However, other agents decreased LV mass and were associated with normal ventricular function following reversal. We also showed some of those therapeutic agents that reduced LV mass also maintained normal function when the ventricular afterload was abruptly increased to pretreatment levels; other agents did not maintain that normal function [15–23]. These findings suggested to us that associated with treatment were intrinsic biological and physiological alterations which were related to the “reversal of hypertrophy” and were also responsible for these disparate functional changes. Our ensuing hypothesis was supported by our subsequent reports that the reduction of LA mass was achieved within only 3 weeks of therapy at a time when arterial pressure had not been reduced. In some studies, this was achieved with doses of some of these agents that had not even reduced arterial pressure [18, 20]. We therefore restated our concept to the development and reversal of the increased LV mass in hypertension, which were associated with nonhemodyanamic as well

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as hemodynamic factors [24, 25]. These provocative findings permitted a further assessment of the issue concerning whether there were additional comorbid pathophysiological alterations associated with LVH. This concept was soon supported by our studies in untreated naturally developing spontaneously hypertensive rats (SHR) and their normotensive (control) Wistar-Kyoto (WKY) rats, matched for gender and age. In these studies we learned that they developed progressive ventricular ischemia not only in the hypertrophied LV but also in the nonhypertrophied right ventricles and in the LV of the WKY rats. Furthermore, this progressive ischemia with aging was closely related to increased hydroxyproline deposition and histological evidence of fibrosis in the extracellular matrix of the ventricle as well as surrounding the intramural arterioles in the chamber [26]. These findings were supported further by additional reports demonstrating pathological changes of apoptosis [27] and inflammatory changes [25]. Hence, we concluded that the underlying mechanisms of risk associated with LVH in hypertension related to ischemia, fibrosis, apoptosis, and inflammatory changes [23, 25]. More recently, we added yet another factor that complicates risk associated with LV – certain environment factors including excessive dietary salt-loading (vide infra) (28–30]. In this chapter, I shall not discuss the important experimental and clinical evidence that provides abundant clinical and experimental data demonstrating that angiotensin II contributes importantly to the development of LVH as well as the remodeling of the LV and the arterioles in clinical and experimental hypertension. This is the subject of separate chapters in this monograph [31, 32]. The evidence is abundant with reference to the numerous well-designed placebo-controlled multicenter pharmacological clinical trials involving administration of either angiotensinconverting enzyme agents or angiotensin II type 1 receptor blocking agents to patients following myocardial infarction. These trials demonstrated the efficacy of these drugs in reducing not only arterial pressure but cardiovascular morbidity and mortality, cardiac failure, and even a second myocardial infarction [6].

2.3 Structural and Function Response of Organs to Salt-Loading Abundant clinical and experimental evidence has accumulated in recent years to the response of various organs (i.e., heart, vessels, kidney) to excessive salt-loading [28–30, 33). Until relatively recently, much evidence of risk with salt-loading has been ascribed to increase in arterial pressure; however, more recent reports have demonstrated clearly that salt-loading (experimentally as well as clinically) was associated with increased cardiovascular morbidity and mortality as well as structural and functional alterations of heart, aorta, and kidney [34–36). Recent data have shown that co-treatment with angiotensin II receptor antagonists or angiotensinconverting enzyme inhibitors along with the salt-loading will prevent the structural and functional end-organ damage [30, 33, 35]. The reader is referred to those specific references that provide abundant data and references to support the foregoing statements. Moreover, sodium-restricted diets in prehypertension patients will significantly reduce cardiovascular morbidity and mortality as compared with

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control group patients whose daily sodium diet was not reduced [36]. These findings provide important data that relates the data derived from chronic salt-loading diets in the earlier epidemiological studies that demonstrated a close relationship between salt-loading and the prevalence of hypertension in large population groups [37–39].

2.4 Secondary Organ Responses of Therapy to Certain Treatment Over the past five or more decades of antihypertensive therapy and the welldocumented evidence of associated reduction in cardiovascular morbidity and mortality, a disturbing conundrum has complicated this therapeutic effort [33]. Thus, each national and international report has attested to the remarkable reduction in morbidity and mortality of such disease endpoints in hypertensive emergencies, stroke, and coronary heart disease [40, 41]. However, over the years, the successive publications of these very same reports have continued to provide an ever-increasing prevalence, morbidity and mortality resulting from end-stage renal disease and of cardiac failure [40, 41]. How can we reconcile these startling data? In response to this shocking and as yet unresolved conundrum, we have suggested that this may be the result of long-term stimulation or ineffective inhibition of the local cardiac and renal renin angiotensin systems. Indeed, there are abundant experimental data which have demonstrated that prolonged diuretic treatment promotes structural and functional renal abnormalities which can be prevented by co-existent treatment with an angiotensin-converting enzyme agent or an angiotensin II type 1 angiotensin receptor blocker [42, 43]. This led to our suggestion resulting from long-term diuretic therapy, there is a secondary increase of renin generation in the kidney that promotes the local synthesis of angiotensin II and its attendant pathophysiological alterations from secondary renal renin generation [34]. These latter studies have demonstrated that in addition to promoting renin release from the juxtaglomerular apparatus of the kidney, a second source of renin production occurs in renal tubular cells [44, 45]. In addition, salt-loading without adequate treatment with either an ACE inhibitor or an angiotensin II type 1 receptor blocker may not protect or prevent stimulation of the local cardiac RAAS. These salt/pharmacological stimuli or inhibition of local renal and cardiac RAAS may be analogous to the multiplicity of Yin/Yang biological systems in the body. Therefore, unless the consequent events stimulating the increased renin synthesis and angiotensin II generation are prevented, the adverse structural and functional cardiac and renal biological events may result.

2.5 Toxemias of Pregnancy Finally, a word or two may be in order concerning yet another clinical expression of pathological stimulation of a local RAAS in the uterus or other female genital organs. Several recent reports have suggested that the utero-placental unit may be the source of stimulated synthesis of components of the RAAS [46, 47]. In part, this

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may be related to inadequate perfusion of the utero-placental unit and/or a consequent relative hypoxemia stimulation, endothelial dysfunction of that unit, upregulation of specific genes, generation of autoantibodies, and generation of certain humoral or hormonal factors, inflammatory changes and production of an increased arterial pressure and proteinuria that are characteristic of pre-eclampsia or eclampsia [48, 49]. Each of these possible pathophysiological changes may be responsible for the establishment of toxemia alone or in association with preexisting or otherwise predisposed underlying mechanisms of hypertensive disease. Although these provocative findings are of great significance, what is most important is that this much neglected area for study has now captured much needed interest and work.

References 1. Fyhrquist, F., and Saijonmaa, O. (2008) Renin-angiotensin system revisited. Intern Med 264, 224–236. 2. Harrison, D.G., and Cai, H. (2003) Endothelial control of vasomotion and nitric oxide production. Cardio Clin 21, 289–302. 3. Besler, C., Doerries,C., Giannotti,G., Luscher,T.F., and Landmesse, U. (2008) Pharmacological approaches to improve endothelial repair mechanisms. Expert Rev Cardiovasc Ther 6, 1071–1082. 4. Varagic, J., Trask, A.J., Jessup, J.A., Chappell, M.C., and Ferrario, C.M. (2008) New angiotensins. J Mol Med 86, 663–671. 5. Paul, M., Mehr, A.P., and Kreutz R (2006) Physiology of local renin angiotensin systems. Physiol Rev 86, 747–803. 6. Pfeffer, M.A., and Frohlich, E.D. (2006) Improvements in clinical outcomes with the use of angiotensin converting enzyme inhibitors: cross-fertilization between clinical and basic investigation. Am J Physiol Heart Circ 291, H2021–H2025. 7. Frohlich, E.D., and Re, R.N. (eds) (2006) The Local Cardiac Renin Angiotensin-Aldosterone System. Springer, New York. 8. Kannel, W.B., Dawber, T.R., Kagan, A., Revorskie, N., and Sacks, J. (1961) Factors of risk in the development of coronary heart disease: six year follow up experience: the Framingham Study. Ann Intern Med 55, 33–56. 9. Frohlich, E.D., Tarazi, R.C., and Dustan, H.P. (1971) Clinical-physiological correlations in the development of hypertensive heart disease. Circulation 44, 446–455. 10. Dunn, F.G., Chandraratna, P., de Carvalho, J.G.R., Basta, L.L., and Frohlich. E.D. (1977) Pathophysiologic assessment of hypertensive heart disease with echocardiography. Am J Cardiol 39, 789–795. 11. Frohlich, E.D., and Tarazi, R.C. (1979) Is arterial pressure the sole factor responsible for hypertensive cardiac hypertrophy? Am J Cardiol 44, 959–963. 12. Frohlich, E.D. (1983) Hemodynamics and other determinants in development of left ventricular hypertrophy: conflicting factors in its regression. Fed Proceed 42, 2709–2715. 13. Tarazi, R.C., and Frohlich, E.D. (1987) Is reversal of cardiac hypertrophy a desirable goal of antihypertensive therapy? Circulation 75, 113–117. 14. Frohlich, E.D. (1988) State of the Art. The heart in hypertension: unresolved conceptual challenges. Hypertension 11, 19–24. 15. Sasaki, O., Kardon, M.B., Pegram, B.L., and Frohlich, E.D. (1989) Aortic distensibility and left ventricular pumping ability after methyldopa in Wistar-Kyoto and spontaneously hypertensive rats. J Vascular Med Biol 1, 59–66.

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16. Natsume, T., Kardon, M.B., Pegram, B.L., and Frohlich, E.D. (1989) Ventricular performance in spontaneously hypertensive rats with reduced cardiac mass. Cardiovasc Drug Ther 3, 433–439. 17. Frohlich, E.D. (1989) Overview of hemodynamic and non-hemodynamic factors associated with LVH. J Mol Cell Cardio 21(Suppl V), 3–10. 18. Frohlich, E.D., and Sasaki, O. (1990) Dissociation of changes in cardiovascular mass and performance with angiotensin converting enzyme inhibitors in Wistar-Kyoto and spontaneously hypertensive rats. J Am Coll Cardiol 16, 1492–1499. 19. Frohlich, E.D., and Horinaka, S.(1991) Cardiac and aortic effects of angiotensin converting enzyme inhibitors. Hypertension 18, 2–7. 20. Ando, K., Frohlich, E.D., Chien, Y., and Pegram, B.L. (1991) Effects of quinapril on systemic and regional hemodynamics and cardiac mass in spontaneously hypertensive and WistarKyoto rats. J Vascular Med Biol 3, 117–123. 21. Frohlich, E.D., Sasaki, O., Chien, Y., and Arita, M. (1992) Changes in cardiovascular mass, left ventricular pumping ability, and aortic distensibility after calcium antagonist in Wistar-Kyoto and spontaneously hypertensive rats. J Hypertens 10, 1369–1378. 22. Soria, F., Frohlich, E.D., Aristizabal, D., Kaneko, K., Kardon, M.B., Hunter, J., and Pegram, B.L. (1994) Preserved cardiac performance with reduced left ventricular mass in conscious exercising spontaneously hypertensive rats. J Hypertens 12, 585–589. 23. Frohlich, E.D (1994) Okamoto International Award Lecture: The spontaneously hypertensive rat. Jpn Heart J 35, 487–491. 24. Susic, D., Nunez, E., Hosoya, H., and Frohlich, E.D. (1998) Coronary hemodynamics in aging spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. J Hypertens 16, 231–237 25. Frohlich, E.D. (1999) Risk mechanisms in hypertensive heart disease. Hypertension 34, 782–789. 26. Frohlich, E.D. (2001) Fibrosis and Ischemia: The real risks in hypertensive heart disease. Am J Hypertension 14, 194S–199S. 27. Fortuño, M.A., González, A., Ravassa, S., López, B., and Díez, J. (2003) Clinical implications of apoptosis in hypertensive heart disease. Am J Physiol Heart Circ Physio 284, H1495– H1506. 28. Ahn, J., Varagic, J., Slama, M., Susic, D., and Frohlich, E.D. (2004) Cardiac structural and functional responses to salt loading in SHR. Am J Physiol (Heart Circ Physiol) 287, H767–H772. 29. Varagic, J., Frohlich, E.D., Diez, J., Susic, D., Ahn, J., Gonzalez, A., and Lopez, B. (2006) Myocardial fibrosis, impaired coronary hemodynamics, and biventricular dysfunction in saltloaded SHR. Am J Physiol (Heart Circ Physiol) 290, H1503–H1509. 30. Matavelli, L.C., Zhou, X., Varagic, J., Susic, D., and Frohlich, E.D. (2007) Saltloading produces severe renal hemodynamic dysfunction independent of arterial pressure in spontaneously hypertensive rats. Am J Physiol (Heart Circ Physiol) 292, H814–H819. 31. Pfeffer, M.A. (2009) Inhibiting the renin angiotensin aldosterone system in patients with heart failure and myocardial infarction. In: DeMello, W.C., Frohlich, E.D., (eds.) Renin Angiotensin Aldosterone System and Cardiovascular Disease. Humana Press, Totowa, NJ, Chapter 8 of this book. 32. Diez, J., and Frohlich, E.D. (2009) Left ventricular hypertrophy and treatment with renin angiotensin system inhibition. In: DeMello, W.C., Frohlich, E.D., (eds.) Renin Angiotensin Aldosterone System and Cardiovascular Disease. Humana Press, Totowa, NJ, Chapter 9 of this book. 33. Frohlich, E.D. (2007) The salt conundrum: a hypothesis. Hypertension 50, 161–166. 34. Frohlich, E.D. (2008) The role of salt in hypertension: the complexity seems to become clearer. Nat Clin Pract Cardiovasc Med 5, 2–3.

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35. Varagic, J., Frohlich, E.D., Susic, D., Ahn, J., Matavelli, L., Lopez, B., and Diez, J. (2008) AT1 receptor antagonism attenuates target organ effects of salt excess in SHRs without affecting pressure. Am J Physiol Heart Circ 294, H853–H353. 36. Cook, N.R., Cutler, J.A., Obarzanek, E., Buring, J.E., Rexrode, K.M., Kumanyika, S, K., Appel, L.J., and Whelton, P. K. (2007) Long term effects of dietary sodium reduction on cardiovascular disease outcomes: observational follow-up of the trials of hypertension prevention (TOHP), BMJ 334, 885–894. 37. Kurlansky, M. (2003) Salt: A World History. Penguin Books, New York 38. Dahl, L.K., and Love, R.A. (1954) Evidence for a relationship between sodium (chloride) intake and human essential hypertension. Arch Intern Med 94, 525–531. 39. Stamler, J. (1997) The INTERSALT study: background, methods, findings, and implications. Am J Clin Natr 65, 626–642. 40. The Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC-7) (2003). JAMA, 289, 2560–2572. 41. International Society of Hypertension Writing Group. International Society of Hypertension (ISH): Statement on blood pressure lowering and stroke prevention (2003). J Hypertens 21, 651–663. 42. Ono, Y., Ono, H., and Frohlich, E.D. (1996) Hydrochlorothiazide exacerbates nitric oxideblockade nephrosclerosis with glomerular hypertension in spontaneously hypertensive rats. J Hypertens 14, 823–828. 43. Zhou, X., Matavelli, L.C., Ono, H., and Frohlich, E.D. (2005) Superiority of combination of thiazide with angiotensin-converting enzyme inhibitor or AT1 – receptor blocker over thiazide alone on renoprotection in L-NAME/SHR. Am J Physiol Renal 289, F871–F879. 44. Schunkert, H., Ingelfinger, J.R., Jacob, H., Jackson, B., Bouyounes, B., and Dzau, V.J. (1992) Reciprocal feedback regulation of kidney angiotensinogen and renin RNA expressions by angiotensin II. Am J Physiol E863–E869. 45. Navar, L.G., Prieto-Carrasquero, M.C., and Kobori, H. (2005) Regulation of renin in JGA and tubules in hypertension. In: Frohlich, E.D., Re, R.N., (eds.) The Local Cardiac Renin Angiotensin-Aldosterone System. Springer Science –Business Media, Inc, New York, 22–29. 46. Herse, F., Dechend, R., Harsem, N.K., Wallukat, G., Jurgen, J., Fatimunnisa, Q., Hering, L., Muller, D.N., Lucct, F.C., and Staff, A.C. (2007) Dysregulation of the circulating and tissuebased renin-angiotensin system in preeclampsia. Hypertension 49(2), 604–611. 47. LaMarca, B.D., Gilbert, J., and Granger, J.P. (2008) Recent progress toward the understanding of the pathophysiolsogy of hypertensions during preeclampsia. Hypertension 51, 982–988. 48. Robert, J.M., Pearson, G., Cutler, J., and Lindheimer, M. (2003) Summary of the NHLBI working group on research on hypertension during pregnancy. Hypertension 41, 437–445. 49. Robert, J.M., and Von Versen-Hoeynck, F. (2007) Maternal fetal/placental interactions and abnormal pregnancy outcomes. Hypertension 49, 15–16.

Chapter 3

Renin, Prorenin, and the (Pro)Renin Receptor Genevieve Nguyen and Aurelie Contrepas

Abstract The discovery of a receptor for renin and for its inactive precursor prorenin, and the introduction of renin inhibitors in therapeutic, has renewed the interest for the physiology of the renin angiotensin system (RAS) and has brought prorenin back in the spotlight. The receptor known as renin for (Pro)Renin Receptor binds both renin and prorenin, and binding triggers intracellular signaling involving the MAP kinases ERK1/2 and p38. The MAP kinases activation in turn upregulates the expression of profibrotic genes, potentially leading to fibrosis, growth, and remodeling. Simultaneously, binding of renin to (P)RR increases its angiotensin I-generating activity, whereas binding of prorenin induces the inactive prorenin to become enzymatically active. These biochemical characteristics of (pro)renin binding to (P)RR allow to distinguish two aspects for the new (pro)renin/(P)RR system, an angiotensin-independent function related to the intracellular signaling and its downstream effects and an angiotensin-dependent aspect related to the increased generation of angiotensin I on the cell surface. Ongoing experimental studies should now determine which of the two aspects is the most important in pathological situations.

List of Abbreviations (pro)renin: AOG: Ang I and Ang II: ACE: HRP: (P)RRB:

designate renin and prorenin angiotensinogen angiotensin I and angiotensin II angiotensin-converting enzyme handle region peptide (pro)renin receptor blocker

G. Nguyen (B) Institut de la Santé et de la Recherche Médicale, París, France e-mail: [email protected]

W.C. DeMello, E.D. Frohlich (eds.), Renin Angiotensin System and Cardiovascular Disease, Contemporary Cardiology, DOI 10.1007/978-1-60761-186-8_3,  C Humana Press, a part of Springer Science+Business Media, LLC 2009

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3.1 Introduction The discovery of a specific receptor for renin and for its precursor, prorenin, has modified our conception of renin being just an enzyme responsible for the cleavage of angiotensinogen and of prorenin being just an “inactive” proenzyme. The receptor named (P)RR binds with similar affinity to renin and prorenin. Binding to the receptor allows these enzymes to display increased enzymatic activity on the cell surface and trigger intracellular signaling that in turn modifies gene expression. This implies that renin may also be considered as a hormone and that a function was finally found for prorenin. Information on the role of the (P)RR in organ damage was obtained only recently, and experimental models suggest that (P)RR may play a role in the development of high blood pressure and of glomerulosclerosis, in cardiac fibrosis, in diabetic nephropathy and retinopathy by “non-proteolytically” activating prorenin. Importantly, blocking prorenin/(P)RR interaction with a putative (P)RR blocker called “handle region peptide” (HRP) was claimed to not only prevent diabetic nephropathy but also reverse the glomerulosclerosis of diabetic nephropathy. If this is true, then it would make (P)RR a major therapeutic goal.

3.2 Renin and Prorenin The term “renin” is used to cover two entities: – renin, the mature enzyme which is catalytically active in solution and – prorenin, the proenzyme form of renin which is virtually inactive in solution. Prorenin is synthesized in many organs, the kidney of course, and also the eye, the brain, the adrenal gland, the submandibular gland, the glands of the reproductive system and the adipose tissue. All these tissues are able to secrete inactive prorenin in the surrounding milieu and in plasma, but the only tissue able to mature and secrete active renin is the kidney. Indeed, prorenin, but not renin, is still detectable in blood after bilateral nephrectomy, although prorenin levels are lower than in normal subjects indicating that, although kidney is the main if not the only source of renin in the body, other tissues are able to release prorenin in the circulation [1, 2].

3.2.1 Renin Renin is an aspartyl protease with a typical structure made of two lobes. The cleft in between the lobes contains the active site characterized by two catalytic aspartic residues. Renin is a highly specific enzyme and has only one known substrate, angiotensinogen (AOG). Renin cleaves AOG to generate angiotensin (Ang) I that is converted into Ang II by the angiotensin-converting enzyme. In addition to its substrate specificity, renin catalytic activity is species-specific and renin can only

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cleave AOG of the same species. Renin is synthesized by the renin-producing cells of the juxtaglomerular apparatus (JGA) and is stored as active enzyme in secretory granules from which it is released upon acute stimulation of the JGA. Renin has also been called “active” renin in opposition to the enzymatically “inactive” form of renin, prorenin [3].

3.2.2 Prorenin Being the precursor of renin, prorenin was assumed to have no function of its own, and yet it represents 70–90% of total renin in human plasma. The absence of enzymatic activity of prorenin is due to the fact that a 43-amino acid N-terminal prosegment covers the cleft of the active site. Unlike for the proenzymes of trypsin and of cathepsin D, prorenin does not undergo auto-activation in the plasma ant its activation takes place under two circumstances: a proteolytic activation by a proconvertase which identity is still not established and that removes the prosegment, an irreversible process that occurs in physiology in the renin- producing cells of the juxtaglomerular apparatus exclusively; and non-proteolytic activation in a test tube by exposure to low pH (pH < 3.0) or cold (4◦ C) and which can be imagined as a reversible unfolding of the prosegment. In plasma and in physiological conditions, however, approximately 2% of prorenin is in the open, active form and can display enzymatic activity, whereas 98% is in closed and inactive form [3]. In contrast to renin, prorenin is released constitutively and renin and prorenin levels are usually well correlated. However, under some physiopathological circumstances such as pregnancy and diabetes, prorenin levels exceed by far those of renin. In diabetes mellitus complicated by retinopathy and nephropathy, prorenin is increased out of proportion to renin and this increase starts before the occurrence of microalbuminuria, so that prorenin level was suggested to be a marker of microvascular complications in diabetic patients [4, 5]. Pregnant women also have high plasma prorenin levels, likely derived from the ovaries. The reason for the elevated prorenin levels in diabetes and pregnancy is unknown.

3.3 The (Pro)Renin Receptor Interestingly, the renal vasodilator response to captopril in diabetic subjects correlated better with plasma prorenin than plasma renin [6]. Possibly therefore, prorenin rather than renin is responsible for tissue angiotensin generation despite the absence of prorenin–renin conversion that cannot occur elsewhere than in the JGA cells [7]. In support of this concept, transgenic rodents with inducible prorenin expression in the liver display increased cardiac Ang I levels, cardiac hypertrophy, hypertension, and/or vascular damage without evidence for increased circulating renin or angiotensin [8–10]. Even more surprising, increased tissue Ang I formation occurred even when expressing a non-activatable prorenin variant mutated in the

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site of cleavage of the prosegment [11]. Therefore, it seems logical to assume that prorenin accumulates in tissues, e.g., via a receptor-dependent mechanism, where it can be activated in a non-proteolytic manner. Several proteins able to bind renin and prorenin have been described, an intracellular renin-binding protein (RnBP) [12] and the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) [13–15]. The intracellular RnBP was found to be an inhibitor of renin activity and its deletion affected neither blood pressure nor plasma renin [16], and it is now believed that the M6P/IGF2R is a clearance receptor for renin/prorenin [17]. This leaves the (Pro)Renin Receptor [(P)RR] as the most promising candidate for the tissue uptake of circulating renin/prorenin.

3.3.1 Biochemistry of the (P)RR The (pro)renin receptor is a 350-amino acid receptor with a single transmembrane domain, like receptors for growth factors [18]. There is no homology with any known protein based on the nucleotide and the amino acid sequence of (P)RR. Homologies in the tertiary structure have not yet been determined due to the lack of knowledge on the crystal structure of (P)RR. The receptor binds both renin and prorenin, with affinities in the nanomolar range, and the encoding gene, called ATP6AP2 (see below), is located on the X chromosome in locus p11.4. The initial characteristics of the (P)RR were: 1. Renin and prorenin bound to the receptor are not internalized or degraded but remain on the cell surface. 2. Renin bound to the receptor displays increased catalytic activity as compared to renin in solution. 3. Receptor-bound prorenin displays Ang I-generating activity in the absence of cleavage of the prosegment, most likely due a conformational change induced by binding and non-proteolytic activation of prorenin. 4. (Pro)renin binding triggers intracellular signalization involving the mitogenactivated protein (MAP) kinase ERK1/2 and p38. Further studies confirmed ERK1/2 phosphorylation and showed that it was due to MEK phosphorylation and provoked Elk phosphorylation [19–22]. Moreover, ERK 1/2 activation resulted in the upregulation of transforming growth factor ß1 gene expression, the subsequent upregulation of genes coding for profibrotic molecules such as plasminogen-activator inhibitor-1, fibronectin, and collagens, and the induction of mesangial cell proliferation [19, 20, 23]. The ERK1/2 pathway is not the only signaling pathway linked to the (P)RR as the receptor also appears to activate the MAP kinase p38-heat shock protein 27 cascade [24] and the PI3K-p85 pathway [25]. Importantly, the latter results in the nuclear translocation of the promyelocytic zinc finger transcription factor, which downregulates the expression of the (P)RR

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itself [25, 26]. In other words, high (pro)renin levels will suppress (P)RR expression, thereby preventing excessive receptor activation. Prorenin binding (Fig. 3.1) and its subsequent non-proteolytic activation was confirmed both in primary cells [27] and in cells with transient overexpression of (P)RR [28]. Data in rat aortic vascular smooth muscle cells overexpressing the human (P)RR suggested that prorenin binds with higher affinity to the receptor than renin, so that in vivo prorenin might be the endogenous agonist of the receptor [27]. The fact that both prorenin and renin are capable of binding to the (P)RR implies that the domains involved in the interaction between (P)RR and the (pro)renin molecule are different from the active site and are not restricted to the prosegment of prorenin. Unfortunately, due to the difficulties in generating purified recombinant (P)RR, no structure–function studies are currently available, which would allow the identification of the domains of the (P)RR and (pro)renin involved in binding. In the absence of such structure–function studies or of an X-ray crystallographic structure of the (P)RR, it is difficult to design antagonists for the (P)RR. Nevertheless, Suzuki et al. [29] made the interesting observation that, when bound to prorenin, an antibody against the sequence I11P FLKR15P of the prosegment was able to open the pro-fragment, yielding a “non-proteolytically” activated prorenin in a manner similar to the putative mechanism of (P)RR binding-induced prorenin activation. They named this region of the prosegment the “handle” region. Based on this observation, Ichihara et al. [30] tested a 10 amino acid peptide encompassing the handle region and called HRP for handle region peptide as a blocker of prorenin-(P)RR binding. In diabetic rodents, they reasoned that diabetes would increase prorenin synthesis, thus

Fig. 3.1 Schematic representation of the angiotensin II-dependent and independent consequences of (pro)renin binding to (P)RR and of (P)RR activation. Adapted from [47]

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creating optimal conditions to test the efficacy of HRP in vivo. Indeed, HRP could totally prevent or even reverse diabetic nephropathy [30, 31] and blocked ischemiainduced retinal neovascularization and ocular inflammation in endotoxin-induced uveitis [32]. Moreover, it diminished cardiac fibrosis in stroke-prone spontaneously hypertensive rats [31]. Taken together, these data strongly suggest that the prorenin(P)RR axis plays an essential role in end-organ damage in diabetic and inflammatory pathologies. HRP was subsequently renamed a (P)RR “blocker.” However, in vitro and in vivo studies by others did not reproduce the protective effect of HRP on organ damages well as they did not support the inhibition of prorenin binding to its receptor by HRP [22, 27, 33]. Even more surprising, an FITC-labeled HRP also bound to cells devoid of the (P)RR on the plasma membrane [22]. If there is no demonstration that HRP can really block (pro)renin binding to the (P)RR, thus one may wonder why it is so successful if not blocking renin– (P)RR interaction. At this moment, it cannot be ruled out that HRP also exerts other non-(P)RR related effects, particularly in diabetic animals. Clearly, more work is needed to unravel its mechanism of action before HRP can truly be called a (P)RR blocker.

3.3.2 (P)RR in Experimental Models of Cardiovascular and Renal Diseases The high blood pressure occurring in a transgenic rat model targeting human (P)RR expression to vascular smooth muscle cells suggests a pathological role of the (P)RR in raising blood pressure [34]. Ubiquitous over-expression of the human (P)RR resulted in proteinuria and glomerulosclerosis [35] and in cyclooxygenase-2 upregulation [36]. Both targeted and ubiquitous (P)RR expression left the plasma levels of renin and angiotensin unaltered, but did cause a rise in plasma aldosterone. Finally, in a Goldblatt model of hypertension, a parallel increases in (P)RR and renin was suggested to be profibrotic in the clipped kidney [37] and an increase of (P)RR expression was described in diabetic rats [38]. Although the claimed beneficial effects of HRP in diabetic rodents and stroke-prone spontaneously hypertensive rats are suggestive for a role of the (P)RR in fibrosis and glomerulosclerosis, no increased (P)RR expression was described in these models [30, 31, 39]. In addition, it should be noted that glomerulosclerosis did not occur in transgenic ren-2 rats with inducible prorenin expression [10], despite the fact that such rats, following induction, displayed 200-fold higher prorenin levels, with no change in renin. This argues against the concept that prorenin, through a direct interaction with its receptor, induces glomerulosclerosis. Of the two means classically used to establish the role of a receptor in pathology, the antagonist, HRP, is still speculative and the total knock-out of the (P)RR is, surprisingly for a component of the rennin angiotensin system (RAS), not possible [40]. Therefore, the generation of (P)RR conditional knock-out mice is becoming mandatory and such animals will allow to further establish the role of (P)RR in disease.

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3.3.3 Unexpected Properties and Ontogeny of the (P)RR There is only one gene called ATP6ap2 coding for the full-length protein known as (P)RR. All other truncated forms of (P)RR derive from intracellular processing of the full-length form. The reason why the (P)RR gene is called ATP6ap2 was because a truncated form of the (P)RR, composed of the transmembrane and cytoplasmic domains of (P)RR, had been co-purified with a vacuolar H+ -ATPase (V-ATPase) [41]. This V-ATPase is a complex, 13-subunit protein, essential to maintain an acidic pH in intracellular vesicles and to regulate cellular pH homeostasis [42], but (P)RR is not a subunit of this V-ATPase. The necessity of an intact (P)RR/ATP6ap2 gene in early development is stressed by the observations that in zebra fish, the mutation of (P)RR/ATP6ap2 gene provoked the death of the fish before the end of the embryogenesis [43] and that in rodents (P)RR/ATP6ap2 gene expression is ubiquitous and early in development [44]. Whereas renin expression can be detected in large intrarenal arteries only at 15.5 days of gestation, (P)RR mRNA is already present on day 12 in the ureteric bud and at later stages in vesicles and S-shaped bodies (Fig. 3.2). In newborn mice (P)RR expression is high in epithelial cells of distal, proximal, and collecting tubules and low in glomeruli and arteries [44]. These observations in zebra fish and in the developing kidney suggest that the (P)RR has functions essential for cell survival and proliferation that are unrelated to the RAS.

Fig. 3.2 In situ hybridization with riboprobes

35 S-labelled

mouse (P)RR (left) and mouse renin (right)

Analysis of the sequence of (P)RR-coding cDNA shows that sequence coding for the transmembrane and the intracellular domain putatively associated with the V-ATPase is remarkably conserved between invertebrates and vertebrates, whereas the cDNA sequence coding for the extracellular domain responsible for renin and prorenin binding is conserved in vertebrates only [45]. This leads to the postulate that the (P)RR/ATP6ap2 gene may result from the fusion of two genes, an ancient gene (corresponding with the C-terminus) coding for a protein essential for cell survival and a more recent gene in vertebrates (corresponding with the N-terminus)

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which binds renin and prorenin [40]. However, to date, we have no arguments to confirm or to infirm that the (P)RR role in cell survival is related to V-ATPase activity.

3.4 Conclusion The discovery of the (P)RR has confirmed the hypothesis of Tigerstedt and Bergman more than a century ago that renin is a hormone [46]. Now, the (P)RR also endows prorenin with a function that was suspected over 25 years ago by Luetscher and Wilson in diabetic patients (1985). Experimental studies suggest that the (P)RR might be a major target in cardiovascular disease and in diabetes-induced organ damage, and tissue-specific knock-out of (P)RR should soon establish whether the (P)RR plays a role in cardiovascular pathologies and in diabetes and to what degree HRP exerts (P)RR-dependent effects.

References 1. Danser, A.H.J., Derkx, F.H.M., Schalekamp, M.A.D.H., et al. (1998). Determinants of interindividual variation of renin and prorenin concentrations: evidence for a sexual dimorphism of (pro)renin levels in humans. J Hypertens 16, 853–862. 2. Krop, M., and Danser, A.H.J. (2008). Circulating versus tissue renin-angiotensin system: on the origin of (pro)renin. Curr Hyp Rep 10, 112–118. 3. Danser, A.H.J., and Deinum, J. (2005). Renin, prorenin and the putative (pro)renin receptor. Hypertension 46, 1069–1076. 4. Luetscher, J.A., Kraemer, F.B., Wilson, D.M., et al. (1985). Increased plasma inactive renin in diabetes mellitus. A marker of microvascular complications. N Engl J Med 312, 1412–1417. 5. Wilson, D.M., and Luetscher, J.A. (1990) Plasma prorenin activity and complications in children with insulin-dependent diabetes mellitus. N Engl J Med 323, 1101–1106. 6. Stankovic, A.R., Fisher, N.D.L., and Hollenberg, N.K. (2006). Prorenin and angiotensindependent renal vasoconstriction in type 1 and type 2 diabetes. J Am Soc Nephrol 17, 3293–3299. 7. Lenz, T., Sealey, J.E., Maack, T., et al. (1991). Half-life, hemodynamic, renal, and hormonal effects of prorenin in cynomolgus monkeys. Am J Physiol 260, R804–R810. 8. Véniant, M., Ménard, J., Bruneval, P., et al. (1996). Vascular damage without hypertension in transgenic rats expressing prorenin exclusively in the liver. J Clin Invest 98, 1966–1970. 9. Prescott, G., Silversides, D.W., and Reudelhuber, T.L. (2002). Tissue activity of circulating prorenin. Am J Hypertens 15, 280–285. 10. Peters, B., Grisk, O., Becher, B., et al. (2008). Dose-dependent titration of prorenin and blood pressure in Cyp1a1ren-2 transgenic rats: absence of prorenin-induced glomerulosclerosis. J Hypertens 26, 102–109. 11. Methot, D., Silversides, D.W., and Reudelhuber, T.L. (1999). In vivo enzymatic assay reveals catalytic activity of the human renin precursor in tissues. Circ Res 84, 1067–1072. 12. Maru, I., Ohta, Y., Murata, K., et al. (1996). Molecular cloning and identification of N-acylD-glucosamine 2-epimerase from porcine kidney as a renin-binding protein. J Biol Chem 271, 16294–16299. 13. van Kesteren, C.A.M., Danser, A.H.J., Derkx, F.H.M., et al. (1997). Mannose 6-phosphate receptor-mediated internalization and activation of prorenin by cardiac cells. Hypertension 30, 1389–1396.

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14. Saris, J.J., Derkx, F.H.M., de Bruin, R.J.A., et al. (2001a). High-affinity prorenin binding to cardiac man-6-P/IGF-II receptors precedes proteolytic activation to renin. Am J Physiol 280, H1706–H1715. 15. van den Eijnden, M.M.E.D., Saris, J.J., et al. (2001). Prorenin accumulation and activation in human endothelial cells. Importance of mannose 6-phosphate receptors. Arterioscler Thromb Vasc Biol 21, 911–916. 16. Schmitz, C., Gotthardt, M., Hinderlich, S., et al. (2000). Normal blood pressure and plasma renin activity in mice lacking the renin-binding protein, a cellular renin inhibitor. J Biol Chem 275, 15357–15362. 17. Saris, J.J., van den Eijnden, M.M.E.D., Lamers, J.M.J., et al. (2002). Prorenin-induced myocyte proliferation: no role for intracellular angiotensin II. Hypertension 39, 573–577. 18. Nguyen, G., Delarue, F., et al. (2002). Pivotal role of the renin/prorenin receptor in angiotensin II production and cellular responses to renin. J Clin Invest 109, 1417–1427. 19. Huang, Y., Wongamorntham, S., Kasting, J., et al. (2006). Renin increases mesangial cell transforming growth factor-beta1 and matrix proteins through receptor-mediated, angiotensin II-independent mechanisms. Kidney Int 69, 105–113. 20. Huang, Y., Noble, N.A., Zhang, J., Xu, C., et al. (2007b). Renin-stimulated TGF-beta1 expression is regulated by a mitogen-activated protein kinase in mesangial cells. Kidney Int 72, 45–52. 21. Sakoda, M., Ichihara, A., Kaneshiro, Y., et al. (2007). (Pro)renin receptor-mediated activation of mitogen-activated protein kinases in human vascular smooth muscle cells. Hypertens Res 30, 1139–1146. 22. Feldt, S., Batenburg, W.W., Mazak, I., et al. (2008a). Prorenin and renin-induced extracellular signal-regulated kinase 1/2 activation in monocytes is not blocked by aliskiren or the handleregion peptide. Hypertension 51, 682–688. 23. Huang, Y., Border, W.A., and Noble, N.A. (2007a). Functional renin receptors in renal mesangial cells. Curr Hypertens Rep 9, 133–139. 24. Saris, J.J., ’t Hoen, P.A.C., Garrelds, I.M., et al. (2006). Prorenin induces intracellular signalling in cardiomyocytes independently of angiotensin II. Hypertension 48, 564–571. 25. Schefe, J.H., Menk, M., Reinemund, J., et al. (2006). A novel signal transduction cascade involving direct physical interaction of the renin/prorenin receptor with the transcription factor promyelocytic zinc finger protein. Circ Res 99, 1355–1366. 26. Schefe, J.H., Neumann, C., Goebel, M., et al. (2008) Prorenin engages the (pro)renin receptor like renin and both ligand activities are unopposed by aliskiren. J Hypertens 26, 1787–1794. 27. Batenburg, W.W., Krop, M., and Garrelds, I.M., et al. (2007). Prorenin is the endogenous agonist of the (pro)renin receptor. Binding kinetics of renin and prorenin in rat vascular smooth muscle cells overexpressing the human (pro)renin receptor. J Hypertens 25, 2441–2453. 28. Nabi, A.H., Kageshina, A., Uddin, M.N., Nakagawa, T. ,et al. (2006) Binding properties of rat prorein and renin to recombinant rat renin prorein receptor prepared by a baculovir expression system. Int. I Mol Med 18, 483–488 29. Suzuki, F., Hayakawa, M., Nakagawa, T., et al. (2003). Human prorenin has “gate and handle” regions for its non-proteolytic activation. J Biol Chem 278, 22217–22222. 30. Ichihara, A., Hayashi, M., Kaneshiro, Y., et al. (2004). Inhibition of diabetic nephropathy by a decoy peptide corresponding to the “handle” region for nonproteolytic activation of prorenin. J Clin Invest 114, 1128–1135. 31. Ichihara, A., Kaneshiro, Y., Takemitsu, T., et al. (2006a). Nonproteolytic activation of prorenin contributes to development of cardiac fibrosis in genetic hypertension. Hypertension 47, 894–900. 32. Satofuka S, Ichihara A, Nagai N, et al. (2006). Suppression of ocular inflammation in endotoxin-induced uveitis by inhibiting nonproteolytic activation of prorenin. Invest Ophthalmol Vis Sci 47, 2686–2692.

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33. Müller, D.N., Klanke, B., Feldt, S., et al. (2008). (Pro)renin receptor peptide inhibitor “handleregion” peptide does not affect hypertensive nephrosclerosis in Goldblatt rats. Hypertension 51, 676–681. 34. Burcklé, C.A., Danser, A.H.J., Müller, D.N., et al. (2006). Elevated blood pressure and heart rate in human renin receptor transgenic rats. Hypertension 47, 552–556. 35. Kaneshiro, Y., Ichihara, A., Sakoda, M., et al. (2007). Slowly progressive, angiotensin II-independent glomerulosclerosis in human (pro)renin receptor-transgenic rats. J Am Soc Nephrol 18, 1789–1795. 36. Kaneshiro, Y., Ichihara, A., Takemitsu, T., et al. (2006). Increased expression of cyclooxygenase-2 in the renal cortex of human prorenin receptor gene-transgenic rats. Kidney Int 70, 641–646. 37. Krebs, C., Hamming, I., and Sadaghiani, S., et al. (2007). Antihypertensive therapy upregulates renin and (pro)renin receptor in the clipped kidney of Goldblatt hypertensive rats. Kidney Int 72, 725–730 38. Siragy, H.M., and Huang, J. (2008) Renal (pro)renin receptor upregulation in diabetic rats through enhanced angiotensin AT1 receptor and NADPH oxidase activity. Exp Physiol 93(5), 709–714 39. Ichihara, A., Suzuki, F., and Nakagawa, T., et al. (2006b). Prorenin receptor blockade inhibits development of glomerulosclerosis in diabetic angiotensin II type 1a receptor-deficient mice. J Am Soc Nephrol 17, 1950–1961. 40. Burcklé, C., and Bader, M. (2006). Prorenin and its ancient receptor. Hypertension 48, 549–551. 41. Ludwig, J., Kerscher, S., Brandt, U., et al. (1998). Identification and characterization of a novel 9.2-kDa membrane sector-associated protein of vacuolar proton-ATPase from chromaffin granules. J Biol Chem 273, 10939–10947. 42. Nishi, T., and Forgac, M. (2002). The vacuolar (H+)-ATPases–nature’s most versatile proton pumps. Nat Rev Mol Cell Biol 3, 94–103. 43. Amsterdam, A., Nissen, R.M., Sun, Z., et al. (2004). Identification of 315 genes essential for early zebrafish development. Proc Natl Acad Sci USA 101, 12792–12797. 44. Contrepas, A., Praizovic, N., Duong Van Huyen, J.P., et al. (2007). Expression of (pro)renin receptor in mouse embryonic and newborn kidney and proliferative effect of soluble (P)RR on mesangial cells. Hypertension 50, e145 (Abstract). 45. L’Huillier, N., Sharp, M.G.F., Dunbar, D.R., et al. (2006). On the relationship between the renin receptor and the vacuolar proton ATPase membrane sector associated protein (M8-9). In: E.D. Frolich and R.N. Re. (eds.) The Local Cardiac Renin Angiotensin-Aldosterone System. Chapter 3. Springer, 233 spring street, new york NY10013, USA, pp. 17–34. 46. Tigerstedt, R., and Bergman, P.G. (1898). Niere und Kreislauf. Scand Arch Physiol 8, 223–271. 47. Nguyen, G., and Danser, A.H. (2008) Prorenin and (pro)renin receptor: a review of available data from in vitro studies and experimental models in rodents. Exp Physiol 93, 557–563.

Chapter 4

Local Renin Angiotensin Systems in the Cardiovascular System Richard N. Re

Abstract The renin angiotensin system (RAS) is an established regulator of intravascular volume and arterial pressure. It is now clear that complete and partial RASs exist in multiple tissues, including the cardiovascular system, with the result that local regulation of angiotensin can occur. In addition, newly identified factors such as ACE 2 and the (pro)renin receptor expand the potential physiological actions of these tissue RASs. Here evidence for the existence and functional relevance of local RASs in cardiovascular tissues is reviewed.

4.1 Introduction There is abundant evidence to indicate that components of the renin angiotensin system (RAS) are found in, and act in, cardiovascular tissues. In some cases evidence strongly points to local synthesis of these moieties, while in others uptake from the circulation predominates [1–5]. However, in either case, there exists the opportunity for local regulation of angiotensin production in various tissues. Moreover, tissue-level regulation also occurs via the actions of newly discovered angiotensinconverting enzyme homologue (ACE-2), which cleaves angiotensin II to produce angiotensin [1–7], a peptide with actions that in many instances counteract those of angiotensin II itself [6, 7]. Receptor interactions and modulation can affect the net action of RAS peptides in tissues. For example, heterodimerization of the AT1 angiotensin II receptor with the bradykinin receptor leads to enhanced activity of the AT-1 receptor, and this may play a role in the angiotensin II sensitivity seen in patients with pre-eclampsia [8, 9]. Also the AT-1 receptor can transactivate the epidermal growth factor (EGF) receptor at the cell surface and thereby enhance cell growth [10]. A reciprocal interaction between the AT-1 receptor and the lectin-like oxidized low-density lipoprotein (LDL) receptor results in upregulated R.N. Re (B) Ochsner Clinic Foundation, New Orleans, LA e-mail: [email protected]

W.C. DeMello, E.D. Frohlich (eds.), Renin Angiotensin System and Cardiovascular Disease, Contemporary Cardiology, DOI 10.1007/978-1-60761-186-8_4,  C Humana Press, a part of Springer Science+Business Media, LLC 2009

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angiotensin signalling in the environment of high-circulating oxidized LDL [11]. In addition, two receptors for (pro)renin have recently been described: one an apparent clearance receptor and the other having the capacity to enhance angiotensinogen cleavage at the cell membrane, leading to enhanced generation of angiotensin II in the near vicinity of the AT-1 receptor [12–14]. Moreover, the binding of prorenin to this receptor results in the activation of prorenin such that angiotensinogen can be cleaved and angiotensin II subsequently produced at the target cells. This latter receptor is widely expressed and in particular is found on coronary artery smooth muscle cells and on mesangial cells [12–14]. The physiological relevance of these findings is suggested by the recognition that elevated circulating prorenin levels are found in diabetics, and although this circulating prorenin was heretofore thought to be inert, circulating prorenin concentrations correlate with the extent of microvascular complications in these patients [15]. The marked expression of prorenin in the diabetic eye also suggests a role for prorenin activation at its receptor in human disease [16]. Thus, the actions of local RAS systems in cardiovascular tissues are complex and in some cases remain controversial. Early on, there was considerable debate over whether or not renin was synthesized in cardiovascular tissues [17]. Although the great majority of renin in these tissues is taken up from the circulation, there are persistent reports that renin can be synthesized in cardiovascular tissues of experimental animals and in other tissues [2, 18]. But irrespective of the degree of local synthesis in the cardiovascular tissues of man or experimental models, it must be recognized that the availability of angiotensinogen and ACE in tissues can nonetheless lead to dramatic differences in tissue angiotensin II production in different tissues – that is, tissue angiotensin II production can be locally regulated [3]. Differential uptake of these components could produce this kind of local regulation. In addition, there is evidence for the synthesis of ACE and angiotensinogen in various tissues (for example, the left ventricle in the case of angiotensinogen and the vessel wall in the case of ACE) under specific physiological conditions. These findings are not as controversial as is the issue of tissue renin synthesis; local synthesis of ACE and angiotensinogen therefore provides yet another means of local tissue RAS regulation in the cardiovascular system [3, 18, 19]. Moreover, there is good evidence for the synthesis of chymase, a second enzyme capable of converting angiotensin I to angiotensin II in heart and other tissues depending on species [20]. It must also be noted that renin gene expression resulting in prorenin synthesis occurs in multiple sites [1–5, 21–27]. Thus, even in the absence of conversion to active renin, tissuederived prorenin could generate angiotensin II independent of circulating renal renin after prorenin binding to its receptor in tissue or it could function by direct receptor stimulation [26, 28]. In order to approach this complex subject, the evidence for local cardiovascular RAS regulation in man will first be discussed. Thereafter, the more extensive literature on local cardiovascular systems in selected experimental models will be reviewed both to point the way towards new lines of experimentation and to suggest novel physiologic roles of locals RASs in human physiology.

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4.2 Studies in Man The most direct studies of the cardiovascular RAS in man have been conducted by Sernieri and colleagues. Initially, they studied normal volunteers on both lowand high-salt diets and determined the angiotensin II gradient across the heart [29]. Depending on conditions, the hearts of these volunteers either extracted or released angiotensin while transiting the heart. Surprisingly, cardiac synthesis of angiotensin I and angiotensin II was lowest when the patients were on a low-sodium diet (that is, when circulating renin and angiotensin II were high) and greatest on the highsodium diet (when circulating renin and angiotensin were low). Thus, this study provided clear evidence of locally regulated angiotensin II production. Later, this group took advantage of the fact that some patients suffering from severe heart failure undergo cardiac transplantation, meaning that both diseased and normal myocardia can easily be biopsied for study [30]. Hearts removed from patients suffering from cardiomyopathy reveal increased tissue expression of angiotensinogen, ACE and chymase by PCR and in situ hybridization as compared to normal donor hearts. No renin signal was detected in the tissues, however, and presumably the local upregulation of angiotensinogen and ACE allowed renin to be taken up from the circulation to drive local synthesis of angiotensin II. Also, the gradients of angiotensin I and II across the heart were elevated in patients with congestive heart failure and were correlated with wall stress to a greater degree than to circulating renin activity. Once again, this study points to a role for locally generated angiotensin in the heart and, taken with studies in diabetic patients, suggests that increased tissue concentrations of angiotensin II are associated with apoptosis, a role in cardiac pathobiology [2]. An area of some controversy is the possibility that cardiac tissue can synthesize aldosterone from circulating steroids or even directly from cholesterol. Catheterization studies revealed a gradient of aldosterone, suggesting cardiac production in patients with congestive heart failure or hypertension [31–33]. These studies, however, do not necessarily imply aldosterone synthesis in the heart but could alternatively reflect differential uptake and release. This, coupled with ongoing controversy regarding the possibility that cardiac tissue can synthesize aldosterone, has led to uncertainty regarding the physiologic significance of these results. But the issue of local synthesis aside – and therefore the related issue of whether the local cardiac RAS regulates aldosterone levels so as to form a local renin angiotensin aldosterone system – the variability of aldosterone release from the heart and its association with disease suggests that local regulation of cardiac aldosterone concentration by one mechanism or another does in fact occur. Additional support for local regulation of angiotensin in cardiovascular tissues comes from studies of diabetic patients. In this group plasma renin activity is low; it had been conjectured that the diabetic kidney was not capable of activating prorenin adequately. However, when renovascular resistance was determined in these patients before and after ACE inhibition, AT-1 blockade, or renin inhibition, it was found that renovascular resistance responded briskly to all three agents [34–36]. Also, treatment with ACE inhibitors resulted in marked increases in circulating plasma rennin activity [34]. Collectively, these results suggest the likelihood that renal tissue

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production of angiotensin II results in increased renovascular resistance and renin suppression. The fact that all three agents reverse the effect on resistance indicates that any tissue source of angiotensin II is renin driven. In fact, the response to the renin inhibitor aliskiren is greater than that to the other agents, raising the possibility that prorenin activation at the (pro)renin receptor – a site in close proximity to the AT-1 receptor – could account for this differential sensitivity to aliskiren, which could block the action of prorenin directly and therefore effectively blunt the RAS cascade [36]. These findings must be seen in the context of animal studies demonstrating synthesis of renin in the renal tubules with subsequent generation of angiotensin II from locally produced and filtered angiotensinogen. It is noteworthy that angiotensin II infusion actually upregulates the local production of renin and angiotensinogen, indicating a local renal tubular RAS [37, 38]. To the extent that angiotensin II so produced can migrate to the juxtaglomerular cells to suppress renin release and can traffic to the renal vasculature to cause vasoconstriction, this system could explain the findings described above in diabetic subjects.

4.3 Studies in Animal Models Compared to the relative paucity of direct data related to the cardiovascular RAS in man, there is a large body of work studying this system in animals. Interpretation of this work is made complex by virtue of known and unknown species differences in the RAS. For example, the mouse has a second renin gene (REN 2) expressed primarily in salivary glands and producing a nonglycosylated renin moiety [39]. The rat expresses two related but distinct AT-1 receptors [40]. This divergence in the RAS among species carries over to the local systems and their functioning; this demands caution in extrapolating animal studies to man. An additional caveat derives from the fact that many cell culture studies of the local RAS involve neonatal cells, which are more easily cultured than adult cardiomyocytes. Here too, caution must be exercised in extrapolating results to adult animals. That said, there is good evidence for the regulated synthesis of various RAS components in the cardiovascular and other tissues of experimental animals. Here local RAS activity in the heart, adrenal gland and kidney will be discussed.

4.3.1 Cardiac RAS More than 25 years ago, renin gene expression was first reported in the rodent heart, and this observation has been repeated in various species over the ensuing decades [2, 18, 41]. To be sure, expression is low and in some systems is only seen in pathological states. For example, rapid cardiac pacing leads to the upregulation of renin gene expression in the dog heart [42]. In rats myocardial infarction is associated with the upregulation of renin expression in monocytes and myofibroblasts in the infarction zone [18]. At the same time, cardiac tissue angiotensin I and II levels fall to

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very low levels following nephrectomy, indicating the primacy of circulating renin in the generation of angiotensin in the cardiac interstitium. This has led some to the dogmatic view that all cardiac renin is derived from the circulation [17]. This view assumes that the entire physiological relevance of tissue renin lies in the generation of interstitial angiotensin. This need not be the case. Recently, a second renin transcript, encoding a renin lacking the signal sequence for secretion, has been described by three groups. In the rodent adrenal gland both genes are expressed, while in the juxtaglomerular cells only the secreted form is found. However, in the heart only the nonsecreted form is expressed, and this expression is markedly upregulated by myocardial infarction [21–23]. This intracellular renin may be pathogenic (see Intracellular RASs). Irrespective of the role of locally produced renin in the heart, there is clear evidence for renin uptake from the circulation along with local synthesis of angiotensinogen, leading to locally regulated angiotensin I production followed by its conversion to angiotensin II by ACE or chymase. The upregulation of angiotensin production in the ventricles of transgenic animals has been shown to lead to left ventricular hypertrophy, and so this local system is likely relevant to myocardial stress responses and pathology [43]. The synthesis of aldosterone in the hearts of experimental animals has been reported, but this remains controversial.

4.3.2 Adrenal RAS There is a consistent body of evidence to indicate the synthesis of renin by the rodent adrenal gland. Indeed, following nephrectomy adrenal renin synthesis increases in these animals to the extent that circulating renin levels remain detectable as the result of spillover into the circulation [44]. Moreover, this adrenal renin appears to be biologically important in so far as it maintains adrenal aldosterone synthesis in the absence of renal renin [22, 24, 25, 45, 46]. It is also of note that following nephrectomy renin accumulates in crystalline structures in adrenal mitochondria, the sites of aldosterone synthesis. Thus, it can be conjectured that a local intracellular RAS exists in these cells. Indeed, several groups have reported the existence of a renin variant termed renin exon1A, which lacks the signal sequence for secretion and is predicted to encode a nonsecreted but active renin (as opposed to prorenin), which could act within the cell without the necessity of proteolytic activation [21– 23]. Studies indicate that the renin that accumulates in the mitochondria of anephric animals is this nonsecreted form of renin and is active in aldosterone regulation [22, 24, 25].

4.3.3 Renal RAS As noted earlier, there is good evidence for an autonomous tubular renin angiotensin system that is upregulated in high angiotensin II states, including renal artery

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stenosis. This local system appears to play a role in renal autoregulation and may spill over to influence juxtaglomerular renin secretion and renovascular resistance [37, 38]. At the same time there is evidence to suggest that direct (pro)renin effects at mesangial and possibly other kidney cells are physiologically important. It has been reported that a peptide designed to mimic the handle region of prorenin, and therefore to block the binding of prorenin to the prorenin receptor, can blunt the glomerular pathology in a rodent model of diabetes. It did this in an angiotensin-independent manner as evidenced by a beneficial effect in animals lacking the AT-1 receptor [47]. These results suggest a novel approach to the prevention of glomerulosclerosis. However, there exists controversy about these findings because the observations have not yet been successfully repeated. Also, it is unclear why a peptide directed against prorenin binding would be so effective given the fact it is expected to have no effect on renin binding, and signalling, at the receptor. Therefore, while these findings are exciting, much work remains before the role of the prorenin receptor in glomerulosclerosis will be understood. An additional point, however, is that recent in vitro studies indicate that while the clinically available renin inhibitor aliskiren will likely block angiotensin I formation by receptor-bound renin or prorenin, it does not appear to be capable of blocking direct signalling at the receptor by these factors [48].

4.3.4 Intracellular RASs In 1978, it was reported that tritiated angiotensin II injected into the circulation of a rat localized to myocardial cell nuclei and mitochondria [49]. Subsequent studies revealed that hepatic cell nuclei possessed angiotensin II receptors, some associated with the nuclear membrane and some with euchromatin and nucleosomal protein/DNA particles; binding of angiotensin II to these receptors was associated augmented RNA synthesis. Subsequently, these binding sites were found to be AT1-like in character, and binding of angiotensin II to hepatic nuclear sites was associated with the upregulation of renin and angiotensinogen transcription [50, 51]. Over the ensuing years, considerable evidence accumulated to support the existence of an intracellular site of angiotensin action: (i) Electron-microscopy immunohistology revealed angiotensin II associated with nuclear euchromatin, consistent with the previously reported chromatin receptors, in unmanipulated animals [52]. (ii) The introduction of angiotensin I or II into cardiac myocytes produced definite changes in calcium currents; the angiotensin II effects were blocked by an angiotensin II receptor blocker, the angiotensinogen effects by an ACE inhibitor. These results also suggest the presence of active renin in the cells [53–56]. (iii) Cells engineered to synthesize intracellular angiotensin II in the absence of secreted angiotensin (either by introducing an angiotensinogen construct lacking the signal sequence for secretion or by introducing a construct encoding octapeptide angiotensin II as an EGF fusion protein) showed marked proliferation in the absence of extracellular angiotensin II. This effect was not inhibited by the angiotensin II receptor blocker candesartan but was blocked by

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renin antisense, again indicating the existence of an intracellular RAS [57–59]. (iv) A second renin transcript encoding a nonsecreted but active renin was identified by several groups. This renin is found in the adrenal gland and supports aldosterone synthesis after nephrectomy. It is also upregulated in the rodent left ventricle following myocardial infarction [21–25]. (v) It was shown that nonglycosylated renin, which does in fact circulate to some extent in man, can be internalized by cardiac myocytes via a heretofore uncharacterized (pro)renin receptor and generate intracellular angiotensin II, thereby producing cardiac pathology [28]. (vi) Angiotensinogen was shown to be synthesized in some circumstances by glial cells and to reside in the nucleus. This retention of angiotensinogen by the cells appeared to result from altered posttranslational phosphorylation [60]. (vii) Immunohistochemical studies revealed renin, angiotensin [1–7] and N-terminal ACE immunoreactivity in mesangial cells [61]. (viii) The transfection of cardiac myocytes with a construct encoding a nonsecreted angiotensin II construct led to marked hypertrophy within 96 h; the same construct injected into mice produced overt myocardial hypertrophy in the same time period [62, 63]. (ix) Elevated glucose concentrations were demonstrated to upregulate renin and angiotensinogen synthesis in cultured neonatal cardiac myocytes, an effect blocked by the renin inhibitor aliskiren [62, 63]. Collectively, these and other results strongly point to the existence of intracellular RASs in the cardiovascular system. As these results were being developed, a theory of intracellular peptide action (i.e. intracrine action) was developed to encompass intracrine factors in the RAS as well as the large and growing number of other intracrine peptides/proteins. This, in turn, led to the development of an intracrine physiology and pharmacology, which remain under active investigation [50, 63–65].

4.4 Conclusion Considerable evidence has accumulated over recent decades to indicate that locally regulated angiotensin II synthesis occurs in many tissues and that this synthesis has potentially important physiological and therapeutic implications. In addition, physiologically relevant actions of tissue renin, prorenin, angiotensin [1–7] and other RAS components have been described, as has the operation of intracellular systems. Collectively, these findings demonstrate that the renin angiotensin system is more than a circulating enzymatic cascade regulating blood pressure and volume: It is an important regulator of tissue biology and structure in the cardiovascular system and elsewhere.

References 1. Re, R. N. (1987) The renin angiotensin systems. Med Clin North Am 71, 877–895. 2. Re, R. N. (2004) Tissue renin angiotensin systems. Med Clin North Am 88, 19–38. 3. Dzau, V. J., and Re, R. N. (1994) Tissue angiotensin system in cardiovascular medicine. A paradigm shift? Circulation 89, 493–498.

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24. Clausmeyer, S., Stürzebecher, R., and Peters, J. (1999) An alternative transcript of the rat renin gene can result in a truncated prorenin that is transported into adrenal mitochondria. Circ Res 84, 337–344. 25. Peters, J., Obermüller, N., Woyth, A., et al. (1999) Losartan and angiotensin II inhibit aldosterone production in anephric rats via different actions on the intraadrenal renin-angiotensin system. Endocrinology 140, 675–682. 26. Morgan, T., Craven, C., and Ward, K. (1998) Human spiral artery renin-angiotensin system. Hypertension 32, 683–687. 27. Li, C., Ansari, R., Yu, Z., and Shah, D. (2000) Definitive molecular evidence of reninangiotensin system in human uterine decidual cells. Hypertension 36, 159–164. 28. Peters, J., Farrenkopf, R., Clausmeyer, S., et al. (2002) Functional significance of prorenin internalization in the rat heart. Circ Res 90, 1135–1141. 29. Neri Serneri, G. G., Boddi, M., Coppo, M., et al. (1996) Evidence for the existence of a functional cardiac renin-angiotensin system in humans. Circulation 94, 1886–1893. 30. Serneri, G. G., Boddi, M., Cecioni, I., et al. (2001) Cardiac angiotensin II formation in the clinical course of heart failure and its relationship with left ventricular function. Circ Res 88, 961–968. 31. Delcayre, C., Silvestre, J. S., Garnier, A., et al. (2000) Cardiac aldosterone production and ventricular remodeling. Kidney Int 57, 1346–1351. 32. Mizuno, Y., Yoshimura, M., Yasue, H., et al. (2001) Aldosterone production is activated in failing ventricle in humans. Circulation 103, 72–77. 33. Yamamoto, N., Yasue, H., Mizuno, Y., et al. (2002) Aldosterone is produced from ventricles in patients with essential hypertension. Hypertension 39, 958–962. 34. Price, D. A., Porter, L. E., Gordon, M., et al. (1999) The paradox of the low-renin state in diabetic nephropathy. J Am Soc Nephrol 10, 2382–2391. 35. Lansang, M. C., Osei, S. Y., Price, D. A., Fisher, N. D., and Hollenberg, N. K. (2000) Renal hemodynamic and hormonal responses to the angiotensin II antagonist candesartan. Hypertension 36, 834–838. 36. Fisher, N. D., Jan Danser, A. H., Nussberger, J., Dole, W. P., and Hollenberg, N. K. (2008) Renal and hormonal responses to direct renin inhibition with aliskiren in healthy humans. Circulation 117, 3199–3205. 37. Prieto-Carrasquero, M. C., Botros, F. T., Pagan, J., et al. (2008) Collecting duct renin is upregulated in both kidneys of 2-kidney, 1-clip goldblatt hypertensive rats. Hypertension 51, 1590–1596. 38. Gonzalez-Villalobos, R. A., Seth, D. M., Satou, R., et al. (2008) Intrarenal angiotensin II and angiotensinogen augmentation in chronic angiotensin II-infused mice. Am J Physiol Renal Physiol 295, F772–F779. 39. Field, L. J., McGowan, R. A., Dickinson, D. P., and Gross, K. W. (1984) Tissue and gene specificity of mouse renin expression. Hypertension 6, 597–603. 40. Gembardt, F., Heringer-Walther, S., van Esch, J. H., et al. (2008) Cardiovascular phenotype of mice lacking all three subtypes of angiotensin II receptors. FASEB J 22, 3068–3077. 41. Dzau, V. J., and Re, R. N. (1987) Evidence for the existence of renin in the heart. Circulation 75, I134–I136. 42. Barlucchi, L., Leri, A., Dostal, D. E., et al. (2001) Canine ventricular myocytes possess a renin-angiotensin system that is upregulated with heart failure. Circ Res 88, 298–304. 43. Mazzolai, L., Nussberger, J., Aubert, J. F., et al. (1998) Blood pressure-independent cardiac hypertrophy induced by locally activated renin-angiotensin system. Hypertension 31, 1324–1330. 44. Volpe, M., Gigante, B., Enea, I., et al. (1997) Role of tissue renin in the regulation of aldosterone biosynthesis in the adrenal cortex of nephrectomized rats. Circ Res 81, 857–864. 45. Peters J. (2008) Secretory and cytosolic (pro)renin in kidney, heart, and adrenal gland. J Mol Med 86, 711–714.

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46. Wanka, H., Keßler, N., Ellmer, J., et al. (2008) Cytosolic renin is targeted to mitochondria and induces apoptosis in H9c2 rat cardiomyoblasts. J Cell Mol Med, Jul 30. [Epub ahead of print] 47. Inagami, T., Nakagawa, T., Ichihara, A., Suzuki, F., and Itoh, H. (2008) Renin/prorenin receptor, (P)RR, in end-organ damage: current issues in 2007. J Am Soc Hypertens 2, 205–209. 48. Feldt, S., Batenburg, W. W., Mazak, I., et al. (2008) Prorenin and renin-induced extracellular signal-regulated kinase 1/2 activation in monocytes is not blocked by aliskiren or the handleregion peptide. Hypertension 51, 682–688. 49. Robertson, A. L. Jr., and Khairallah, P. A. (1971) Angiotensin II: rapid localization in nuclei of smooth and cardiac muscle. Science 172, 1138–1139. 50. Re, R. N. (2003) The intracrine hypothesis and intracellular peptide hormone action. Bioessays 25, 401–409. 51. Erdmann, B., Fuxe, K., and Ganten, D. (1996) Subcellular localization of angiotensin II immunoreactivity in the rat cerebellar cortex. Hypertension 28, 818–824. 52. De Mello, W. C. (1995) Influence of intracellular renin on heart cell communication. Hypertension 25, 1172–1177. 53. De Mello, W. C. (2001) Cardiac arrhythmias: the possible role of the renin-angiotensin system. J Mol Med 79, 103–108. 54. Eto, K., Ohya, Y., Nakamura, Y., Abe, I., and Iida, M. (2002) Intracellular angiotensin II stimulates voltage-operated Ca(2+) channels in arterial myocytes. Hypertension 39, 474–478. 55. Haller, H., Lindschau, C., Quass, P., and Luft, F. C. (1999) Intracellular actions of angiotensin II in vascular smooth muscle cells. J Am Soc Nephrol Suppl 11, S75–S83. 56. Cook, J. L., Zhang, Z., and Re, R. N. (2001) In vitro evidence for an intracellular site of angiotensin action. Circ Res 89, 1138–1146. 57. Cook, J. L., Mills, S. J., Naquin, R., Alam, J., and Re, R. N. (2006) Nuclear accumulation of the AT1 receptor in a rat vascular smooth muscle cell line: effects upon signal transduction and cellular proliferation. J Mol Cell Cardiol 40, 696–707. 58. Cook, J. L., Giardina, J. F., Zhang, Z., and Re, R. N. (2002) Intracellular angiotensin II increases the long isoform of PDGF mRNA in rat hepatoma cells. J Mol Cell Cardiol 34, 1525–1537. 59. Sherrod, M., Liu, X., Zhang, X., and Sigmund, C. D. (2005) Nuclear localization of angiotensinogen in astrocytes. Am J Physiol Regul Integr Comp Physiol 288, R539–R546. 60. Camargo de Andrade, M. C., Di Marco, G. S., de Paulo Castro Teixeira, V., et al. (2006). Expression and localization of N-domain ANG I-converting enzymes in mesangial cells in culture from spontaneously hypertensive rats. Am J Physiol Renal Physiol 290, F364–F375. Erratum in: Am J Physiol Renal Physiol 291, F921. 61. Kumar, R., Singh, V. P., and Baker, K. M. (2008) The intracellular renin-angiotensin system: implications in cardiovascular remodeling. Curr Opin Nephrol Hypertens 17, 168–173. 62. Singh, V. P., Le, B., Bhat, V. B., Baker, K. M., and Kumar, R. (2007) High-glucose-induced regulation of intracellular ANG II synthesis and nuclear redistribution in cardiac myocytes. Am J Physiol Heart Circ Physiol 293, H939–H948. 63. Re R. N. (2003) The implications of intracrine hormone action for physiology and medicine. Am J Physiol Heart Circ Physiol 284, H751–H757. 64. Re R. N, and Cook J. L. (2006) The intracrine hypothesis: an update. Regul Pept 133, 1–9. 65. Re R. N, and Cook J. L. (2008) The basis of an intracrine pharmacology. J Clin Pharm 48, 344–350.

Chapter 5

Renin-Angiotensin-Aldosterone System and Pathobiology of Hypertension Pierre Paradis and Ernesto L. Schiffrin

Abstract The renin-angiotensin-aldosterone system (RAAS) plays a critical role in the pathophysiology of elevated blood pressure, both in experimental models and in humans, in essential hypertension and some forms of secondary hypertension, such as renovascular hypertension and primary hyperaldosteronism. This is supported by studies measuring its components, such as renin activity or concentration, angiotensin II or aldosterone in plasma and tissues, the receptors for these mediators and their signaling in cells, as well as by inhibition of the different steps in the RAAS cascade with renin inhibitors, angiotensin I–converting enzyme (ACE) inhibitors, or angiotensin receptor blockers (ARBs). The effects of the RAAS on blood pressure are exerted on blood vessels to induce vasoconstriction, inflammation, growth, and remodeling and accelerate the progression of both atherosclerosis and arteriosclerosis in large vessels and remodeling of resistance arteries, on the kidney to retain salt and water, a critical effect to induce long-term blood pressure elevation, on the heart to induce left ventricular hypertrophy and coronary artery disease, and on the brain to stimulate vasopressin secretion and sympathetic nervous system activity. These different aspects of the role of the RAAS in hypertension will be reviewed in this chapter. Classically, the renin-angiotensin-aldosterone system (RAAS) was described as a peripheral system regulating blood pressure and water and salt balance (Chapter 1). Renin secreted from juxtaglomerular (JG) cells in the afferent arterioles of the kidney cleaves angiotensinogen, an α-2-globulin produced and released into the circulation by the liver, to generate the decapeptide angiotensin (Ang) I. Ang I is then cleaved by the dipeptidyl carboxypeptidase AngI-converting enzyme (ACE) in the lungs to generate the vasoactive octapeptide Ang II. Ang II, classically considered the final mediator of the RAAS, binds to Ang type 1 (AT1 ) receptors. Ang II increases blood pressure by causing vasoconstriction of blood vessels or, indirectly, by increasing blood volume through enhanced renal sodium and water reabsorption. E.L. Schiffrin (B) Hypertension and Vascular Research Unit, Lady Davis Institute for Medical Research/Sir Mortimer B. Davis-Jewish General Hospital, McGill University, Montreal, QC, Canada e-mail: [email protected] W.C. DeMello, E.D. Frohlich (eds.), Renin Angiotensin System and Cardiovascular Disease, Contemporary Cardiology, DOI 10.1007/978-1-60761-186-8_5,  C Humana Press, a part of Springer Science+Business Media, LLC 2009

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In addition, by causing release of aldosterone from the adrenal glands which also causes increased renal reabsorption of salt and water, Ang II contributes to increased blood pressure. Ang II mediates most of its effects through AT1 receptors, which are expressed ubiquitously. In rodents there are two AT1 receptor subtypes: AT1a which is the predominant AT1 receptor subtype in most organs and AT1b which is highly expressed in the adrenal cortex and the pituitary gland [1]. Studies with AT1a receptor null (agtr1a) mice have revealed that blood pressure and vascular tone are regulated by AT1a receptors [2, 3]. Agtr1a mice had a reduced resting blood pressure and no pressor response to Ang II infusion. Ang II also binds to AT2 receptors, which are highly expressed in fetal tissues, but whose expression decreases dramatically after birth. The density of AT2 receptors is low in adult tissues. AT2 receptors are expressed in tissues involved in blood pressure regulation, such as the heart, kidneys, adrenal glands, brain, and vascular smooth muscle (VSMC) and endothelial cells (EC) in the adult [4]. Although there is some evidence that AT2 receptors may counteract the action of AT1 receptors, their role in blood pressure regulation remains unclear. In addition to the peripheral RAAS of renal origin, there are local tissue RAASs (Chapter 2). More recently, our understanding of the complexity of the RAAS has been significantly enhanced with the finding that pro-renin is more than a precursor protein but also an active molecule, and the discovery of renin receptors (Chapter 3), ACE2 (Chapter 11), and new vasoactive peptides such as Ang IV, Ang 1-7, and Ang 1-12 (Chapter 11), and new receptors like Ang 4R/IRAP and Ang 1-7R/Mas, as well as the rediscovery that Ang III may play important roles in the brain and in the kidney that had not been detected in the past. From the initial finding of the different components of the RAAS, it has been assumed that this system is involved in the development and maintenance of hypertension. This was first recognized in acquired disorders, particularly in renovascular hypertension for renin and Ang II, and primary aldosteronism in the case of aldosterone, and also in essential hypertension (reviewed in reference 2). Both Ang II, the main peptide effector of RAAS, and aldosterone exert effects that participate in the mechanisms leading to the development of hypertension by acting on the vasculature, the kidneys, and the central and peripheral nervous system. Ang II and aldosterone also act on the heart beyond their effects on the coronary vessels. These effects on the myocardium are part of the target organ damage associated with hypertension.

5.1 Blood Vessels Essential hypertension is characterized by increased peripheral vascular resistance to blood flow [5], occurring mostly through energy dissipation in small resistance arteries with a reduced lumen diameter. It is important to note that according to the law of Poiseuille, flow resistance is inversely related to the fourth power of the vessel radius, and therefore, small decreases in the diameter of the lumen significantly increase resistance. Small resistance arteries with a lumen diameter of 100–300 μm play an important role in the development of hypertension [6], and contribute to

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its complications [7], to myocardial ischemia [8–10], stroke [11], and renal failure [12]. In essential hypertension, resistance arteries undergo eutrophic remodeling characterized by reduced outer diameter and lumen and increased media-to-lumen ratio, but with unaltered media cross-sectional area [13–20]. Eutrophic remodeling has been most often observed in animal models of hypertension with an activated RAAS [4, 21, 22]. This remodeling is characterized in humans by the absence of VSMC hypertrophy or hyperplasia [23] but with rearrangement of VSMCs around the smaller lumen [24–26]. The VSMC rearrangement may result from increased constriction [27, 28] due to activation of the RAAS and/or the sympathetic nervous system or secretion of growth factors such as endothelin-1 (ET-1), which becomes embedded in an increased extracellular matrix, also resulting from the action of RAAS components, such as Ang II or aldosterone, or other agents. Hypertrophic remodeling, which is characterized by increased media-to-lumen ratio and media cross-sectional area, has been observed in small arteries of patients with renovascular hypertension [29], hypertensive diabetic subjects [30, 31], and acromegalic patients [32]. This remodeling is associated with VSMC hypertrophy without evidence of hyperplasia. It is noteworthy that VSMC hyperplasia and hypertrophy have been shown to contribute to vascular remodeling in animal models of hypertension, such as spontaneous hypertensive rats (SHRs), stroke-prone SHR (SHR-SP), and Ang II-induced hypertension in rodents [33–37]. We demonstrated that Ang II induced proliferation (hyperplasia) and growth (hypertrophy) of cultured vascular VSMCs isolated from resistance arteries from subcutaneous gluteal biopsies from human healthy subjects [38] through AT1 receptors via the ERK-dependent signaling pathway and increased generation of reactive oxygen species (ROS) [39]. Furthermore, Ang II induced growth and proliferation of VSMCs through a crosstalk between AT1 receptors and epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and insulin-like growth factor (IGF) receptors [28]. Aortic media thickening in animal models of hypertension is accompanied by re-expression of fetal genes in VSMCs associated with a shift of VSMCs from a contractile to a synthetic phenotype and/or expansion of preexisting immature VSMC population [40–42], and accordingly, in TGRen2 transgenic rats overexpressing the mouse Ren2 gene, Ang II induced re-expression of fetal muscle genes (SM-MyHC and MyHC-α2) and EIIIA-fibronectin (FN) in aortic VSMCs, which may play a role in the changes observed in VSMC phenotype [40] and vascular stiffness (see below). Apoptosis in the periphery of small arteries triggered by Ang II, combined with growth of VSMCs toward the lumen, may contribute to the VSMC rearrangement in eutrophic remodeling [28, 43]. However, Rizzoni et al. observed apoptosis in small mesenteric arteries of older SHRs while no apoptosis was present in younger rats in which eutrophic remodeling was already present [44]. Interestingly, whereas Ang II infusion induced apoptosis in aortic VSMCs of Wistar rats, AT1 or AT2 receptor blockers did not prevent but actually enhanced apoptosis [45], suggesting a role for both receptors in this process. Although the role of AT2 receptors is still unclear, they may counteract and fine-tune AT1 pro-proliferative actions [46]. Fibrosis of the media is observed in hypertension in blood vessels in both eutrophic and hypertrophic remodeling [31], and may contribute to increased resis-

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tance by augmenting vasculature stiffness [47]. Vascular fibrosis is characterized by the accumulation of extracellular matrix components such as collagen, FN, fibrillin, and proteoglycans in the media and perivascularly [48]. Ang II regulates the synthesis and degradation of collagen in the vascular media. Results from in vivo studies have indicated that Ang II through AT1 receptors induced collagen deposition in the media [40, 49]. In vitro studies further demonstrated that Ang II increased expression of collagen in VSMCs and fibroblasts through AT1 receptors [50, 51]. In addition, Ang II decreased the expression of enzymes that degrade collagen, the matrix metalloproteinases (MMPs), and stimulated production of the tissue inhibitors of MMPs (TIMPs) [52, 53]. Ang II–AT1 receptor-dependent signaling pathways upregulated the expression of a fetal extracellular matrix gene, EIIIA-FN [48]. Increased levels of Ang II in TGRen2 transgenic rats increased EIIIA-FN in aortic SMCs, thus contributing further to vascular stiffness [40]. The increase in FN preceded the rise in blood pressure in Ang II-dependent hypertensive models [48]. The profibrotic effects of Ang II via AT1 receptors were mediated by transforming growth factor (TGF) β [54] or connective tissue growth factor (CTGF) [55, 56]. Increased vasoconstriction may contribute to development of hypertension. Indeed, we have observed exaggerated vasoconstriction response to Ang II in resistance arteries of patients with essential hypertension [16]. Increased contractile sensitivity has also been found in rodent models of hypertension [57]. This enhanced contractile response may be mediated by aldosterone-induced increase in AT1 receptor expression [58, 59] and/or through enhanced signaling responses by the RhoA/Rho kinase-dependent pathways in VSMCs [57]. Endothelial dysfunction may also play a role in the increases in peripheral resistance found in both experimental and human hypertension. We and others have observed endothelial dysfunction in humans [60, 61] and in SHRs [60, 62]. This altered endothelial function is mainly dependent on reduced bioavailability of nitric oxide (NO), as reported in human subjects with essential hypertension [60, 63, 64] and in animal models such as SHR-SP and Ang II-induced hypertension in rabbits [65, 66]. Decrease in NO bioavailability could result from a reduction in synthesis or an increase in degradation of NO. Inhibition of NO synthesis could result from increases in asymmetric dimethylarginine (ADMA), which is a potent endogenous inhibitor of NO synthase (NOS) [67]. ADMA is produced by methylation of arginine by protein arginine N-methylytansferase (PRMT) and release by proteolysis. Normally, most of ADMA is degraded by dimethylamine dimethylaminohydrolase (DDAH) and the remaining is excreted in the urine. Increased levels of ADMA have been shown in hypertensive subjects [64, 68, 69] and in SHR [63]. Interestingly, the levels of ADMA and NO were, respectively, decreased and increased by blockade of the RAAS independently of blood pressure [63, 69]. Ang II-induced oxidative stress may contribute to decrease in biovailability of NO [70, 71]. Ang II induces the production of superoxide (• O2 – ) mainly through activation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which is expressed in all vascular layers including ECs, VSMCs, and adventitial fibroblasts [70], although other enzymes such as xanthine oxidase, the respiratory chain, etc., probably also

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contribute to different degrees. Increase in ROS was documented in humans with essential, renovascular, and malignant hypertension and in women with preeclampsia [63, 72–74]. In experimental models of hypertension, increases in oxidative stress in different tissues including the vasculature have been demonstrated in deoxycorticosterone acetate (DOCA)-salt mice, SHRs, and SHR-SPs [75–78]. We observed that basal and Ang II-induced NADPH oxidase-driven • O2 – production increased in VSMCs from SHRs with the level of blood pressure elevation [75]. Furthermore, we and others found that blockade of the RAAS, for example, with the AT1 receptor blocker (ARB) valsartan, decreased the production of ROS in SHRSP and in Dahl salt-sensitive hypertensive rats [77–79]. Decreases in NO bioavailability could also be caused by oxidation of NO to peroxynitrate (• ONOO– ) by • O – . In addition, Ang II-induced generation of • O – and • ONOO– may oxidize 2 2 tetrahydrobiopterin (BH4 ), an essential co-factor of endothelial NOS (eNOS), to 7,8-dihydrobiopterin (BH2 ). This will cause uncoupling of eNOS and change its state from one in which it produces NO to one in which it produces • O2 – . BH4 levels and the rate-limiting enzyme for the de novo synthesis of BH4 , guanyltriphosphate cyclohydrolase (GTPCH) I, as well as endothelium-dependent acetylcholineinduced vascular relaxation, were decreased in DOCA-salt hypertensive mice [76]. Interestingly, endothelium-specific overexpression of GTPCH I restored endothelial function in DOCA-salt mice. Several other studies have demonstrated that treatment with BH4 decreased blood pressure and improved endothelial function in Dahl saltsensitive hypertensive rats and in Ang II-induced hypertension in rats [79–81]. Peripheral resistance is modulated by up to slightly under 20% by the density of capillaries. Factors affecting angiogenesis such as hypoxia and inflammation modify capillary density and therefore blood pressure. Most forms of human and experimental hypertension are associated with decreased density of microvessels (rarefaction), which can increase blood pressure further and exacerbate hypertension-induced end-organ damage [28, 82]. Apoptosis of ECs may also be involved in microvascular rarefaction, contributing thus to hypertension [83, 84]. This could be mediated in part by Ang II, since inhibition of the RAAS with an ACE inhibitor has been shown to increase microvascular density [82]. Vascular remodeling and endothelial dysfunction are accompanied by local inflammation, which may contribute to the development of hypertension and its complications [28]. Inflammation is actually a normal process involved in recovery of tissue integrity, but if repair is not well regulated, this may lead to persistent changes and tissue damage. ROS have been implicated in all the stages of inflammation. Growing evidence indicates that low-grade inflammation plays a significant role in the pathophysiology of RAAS-induced high blood pressure and its complications. Ang II-induced • O2 – by activation of NADPH oxidase in the vasculature may be an early step in the initiation of local inflammation. Furthermore, Ang II has been implicated in all the subsequent steps of inflammation characterized by an increase in vascular permeability, leukocyte recruitment, and activation of tissue repair [28]. Ang II increased vascular permeability by inducing the synthesis of prostaglandins (PGs) and vascular endothelial growth factor (VEGF) in VSMCs and ECs through

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AT1 receptors [85]. Ang II-induced leukocyte recruitment and activation via AT1 receptors, by inducing the expression of selectins (P and E) [86, 87], integrins (β2 and α4) [86], intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 [87], cytokines such as monocyte chemotactic protein (MCP)1, interleukin (IL)-6, IL-8, IL-18, osteopontin (OPN), tumor necrosis factor (TNF)α [88–92], and chemokines such as cytokine-inducible neutrophil chemoattractant (CINC), keratinocyte-derived chemokine (KC), macrophage inflammatory protein (MIP)-2, and CC chemokine ligand 5 (CCL5) [89, 90, 93]. Furthermore, Ang II induces low-grade inflammatory effects independently of its blood pressureraising actions. Inhibition of VEGF by soluble VEGF receptor 1 (sFlt-1) gene transfer attenuated Ang II infusion-induced inflammation and vascular remodeling in mice without normalization of blood pressure [94]. A sub-pressor dose of Ang II induced leukocyte adhesion in mesenteric arteries via pressor-independent mechanisms [89]. Ang II may also induce inflammation by acting directly on lymphocytes. Nataraj et al. observed that T and B cells and macrophages isolated from the spleen express AT1a receptors and that in vitro Ang II regulated the proliferation of wildtype but not agt1ar–/– splenic lymphocytes [95]. More recently, it was demonstrated that T lymphocytes are required for Ang II to induce vascular remodeling [96]. Ang II-induced vascular remodeling was impaired in rag1–/– mice, which are deficient in both T and B lymphocytes, although expression levels of aortic AT1 and AT2 receptors were unaltered. The vascular effects of Ang II were restored by transfer of T but not B cells. Finally, the last step of inflammation, tissue repair, which is a normal process that restores tissue integrity, may be impaired by the action of Ang II. Indeed, Ang II causes VSMC growth, proliferation and apoptosis, and vascular fibrosis, leading to vascular remodeling and hypertension (see above). Furthermore, Ang II-induced inflammation is a major contributor to the progression of atherosclerosis. The ACE inhibitor (ACEi), quinapril, blunted the increased expression of the CC chemokine MCP-1 and macrophage infiltration in the neointima of injured femoral artery in a rabbit model of accelerated atherosclerosis [97]. The ARB ibesartan reduced both the progression of the lesions and the expression of CC chemokines such as MCP-1 and macrophage inflammatory protein (MIP)-1α and CXC chemokines (MIP-2 and KC) in artherosclerotic lesions of apoE–/– mice [98]. The ARB losartan decreased both intima proliferation and the expression of P-selectin and macrophage infiltration in aorta of hypercholesterolemic rabbits [99]. The ARB irbesartan reduced the serum level of • O2 – ,VCAM-1, and TNF-α in normotensive patients with coronary artery disease (CAD) who had undergone coronary artery bypass graft, percutaneous transluminal coronary angioplasty, or both prior to the study. The ARB candesartan reduced the serum levels of two proinflammatory molecules in hypertensive patients, the acute-phase reactant C-reactive protein (CRP) produced by the liver and adipocytes and CD40 ligand expressed by activated T lymphocytes, independently of blood pressure reduction [100]. In addition, Ang II-induced inflammation may further exacerbate artherosclerotic coronary artery disease (CAD) or stroke by causing atherothrombosis, which is characterized by an unpredictable, sudden rupture or erosion/fissure of an atherosclerotic plaque,

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which leads to platelet activation and thrombus formation. Plasminogen activator inhibitor (PAI)-1, the major inhibitor of fibrinolysis, is increased in atherosclerotic plaques [101, 102], and Ang II has been demonstrated in vitro to induce expression of PAI-1 in endothelial cells [103, 104] and in vivo to cause a rapid increase in circulating PAI-1 in normotensive and hypertensive patients [105]. Inhibition of the RAAS with the ACE inhibitors ramipril or fosinopril decreased, respectively, the plasma levels of PAI-1 in patients with acute CAD [106] and in type 2 diabetic hypertensive patients [107]. Administration of the ARB valsartan to diabetic hypertensive subjects corrected structural remodeling of small arteries [108] and also reduced proinflammatory cytokines [109]. Interestingly, in the diabetic hypertensive subjects, the ARB treatment was associated with upregulation of AT2 receptors, which could contribute to the beneficial effects of valsartan on remodeling and inflammation [110]. Renin receptors have been described in different tissues including blood vessels [111], but their significance with respect to blood pressure elevation remains unclear. This subject is treated extensively in Chapter 3. So far, this chapter has concentrated on effects attributable to actions of Ang II. However, aldosterone, whose secretion by the adrenal glomerulosa is stimulated by Ang II, has important vascular effects (for more complete review see reference 112). These affect not only the endothelial layer [113] and the media but also the adventitia. Aldosterone binds to the mineralocorticoid receptor (MR) and has proliferative, proinflammatory, and profibrotic actions that are mediated in part via its genomic effects and in part via its nongenomic actions. These effects in part appear to mediate vascular actions usually attributed to Ang II [114–116]. Aldosterone appears to contribute to vascular actions of angiotensin II in part via upregulation of Ang receptors [58, 117] and other components of the RAS, not only in the vasculature but also in the brain [118]. It may also act in concert with Ang II to contribute to enhance its actions, area which is under active research. Aldosterone exerts its nongenomic effects by stimulating oxidative stress mainly through activation of NADPH oxidase and stimulation of MAPK (ERK 1/2) and other kinases [119]. Through genomic effects, aldosterone and other mineralocorticoids stimulate the expression of different proteins and peptides, including ET-1, which contributes to the oxidative stress, proliferation, inflammation, and fibrosis [120]. Increased oxidative stress participates in the endothelial dysfunction associated with effects of aldosterone [121], as NO is scavenged by ROS. The contribution of aldosterone to the hypertensive process is highlighted by the fact that it participates not only when aldosterone is produced in excess by an adrenal adenoma, which will not be discussed here [122], but also in essential hypertension, where there may be inappropriate secretion of aldosterone, particularly in resistant hypertension [123] and among obese hypertensive subjects [124]. Treatment of hypertensive subjects with blockers of MR, such as eplerenone, resulted in lowering of blood pressure (demonstrating the participation of aldosterone in blood pressure elevation) but as well decreased stiffness of large and small arteries of hypertensive patients [125]. Typically, collagen and fibronectin deposition in the media was reduced by eplerenone, whereas elastin was enhanced and vessels became more distensible.

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5.2 The Kidney The effects of Ang II and aldosterone that impact on the kidney to contribute to the pathophysiology of hypertension will be dealt with only briefly. True renovascular hypertension in humans, in which increased renin secretion occurs in response to renal artery stenosis, or renin-secreting tumors, or those forms of experimental hypertension in which renin secretion by the juxtaglomerular cells is increased, such as Goldblatt hypertension in rodents or dogs, will not be addressed. Rather, it is the effects that the RAAS has on the kidney, which may lead to hypertension, and more specifically that of Ang II, which will be described. The specific effects of aldosterone on salt and water balance are dealt with in Chapter 15. The role of the kidney in long-term control of blood pressure and in the development and maintenance of hypertension was underlined by the computer modeling carried out by Arthur Guyton [126], and its relation to Ang II clarified in large measure by studies of John Hall [127] and others. Infusion of Ang II impairs pressure natriuresis, and blood pressure becomes highly sensitive to sodium intake, resulting in blood pressure elevation in the presence of small increments in sodium in the diet, and this is corrected by inhibition of Ang II generation by an ACE inhibitor (Fig. 5.1) [128]. This is particularly important in view of the fact that the kidney has large amounts of Ang II generated locally [129], which may be increased in hypertension as suggested by studies with RAAS blockade selectively administered

Fig. 5.1 Chronic relationships between blood pressure and sodium intake and excretion in dogs with a normal RAS, after blockade of Ang II formation with an ACE inhibitor and after continuous infusion of a low dose of Ang II (5 ng/kg per minute) to prevent suppression of circulating Ang II upon increased sodium intake. Inability to modulate Ang II levels decreases the slope of pressure/natriuresis relationship, causing marked salt sensitivity of blood pressure. Reproduced from [127] with permission

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to the kidney, which improve natriuresis [127]. Ang II, through its hemodynamic and tubular effects on the kidney (for review see reference 130), and aldosterone, through its sodium-retaining action, will contribute thus to blood pressure elevation. The similarity of what happens with Ang II infusion, which can be reversed by ACE inhibitors as shown in Fig. 5.1 [128], and the displacement to the right of the blood pressure/urinary volume output relationships in both salt-sensitive and nonsalt-sensitive essential hypertension compared to normal as shown in Fig. 5.2 [131] suggests that indeed Ang II plays this role in renal hemodynamics and natriuresis, leading to blood pressure elevation. Interestingly, studies by T. Coffman and his group have demonstrated the critical importance of the AT1a receptor in the kidney in blood pressure elevation in response to Ang II infusion in mice using gene deletion and cross-transplantation studies (Fig. 5.3) [132]. From these studies, it would appear that in mice, kidney AT1a receptors that seem to be tubular rather than vascular play a more important role in blood pressure elevation induced by Ang II than systemic AT1a receptors. Whether this also applies to other species remains to be determined.

Fig. 5.2 Blood pressure/volume output relationships in normal conditions to the left or in saltsensitive and non-salt-sensitive essential hypertension to the right. Reproduced from [131] with permission

A word should be said about renin receptors and the kidney [111]. These have been identified in the kidney, but little is known about their importance in relation to blood pressure control by the RAAS. However, it has been suggested that renin may

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Fig. 5.3 (a) Kidney cross-transplantation groups used in the study by Coffman’s group. Wild-type (+/+) or AT1a receptor-deficient (–/–) mice were transplanted with kidneys from either AT1a (+/+) or AT1a (–/–) mice. Systemic knockout (KO) mice express AT1a receptors only in the kidney since they have received a kidney transplant from AT1a (+/+) mice. Kidney KO animals express AT1a receptors in all tissues except the kidney, since they have been transplanted with kidneys from AT1a (–/–) mice. Total KO animals lack AT1a receptors completely. (b) Daily blood pressures in cross-transplanted mice during 21-day Ang II infusion. By day 12 of Ang II infusion, the severity of blood pressure elevation in systemic KO reaches that of the wild-type mice. Absence of renal AT1a receptors in kidney KO mice blunts the development of Ang II-induced blood pressure elevation. Total KO blood pressure shows minimal response to Ang II infusion (∗ P≤0.03 vs wild type; §P<0.008 vs systemic KO; †P<0.006 to 0.0001 vs wild type). Reproduced from [130] with permission

act directly via these receptors to induce fibrosis in the kidney [133], which could contribute to nephroangiosclerosis and blood pressure elevation. Although some evidence was produced regarding a potential role in diabetic nephropathy [134], recent data suggest that in contrast, in Goldblatt hypertension in rats, renin receptors do not seem to play a role, or at least the so-called handle-region peptide renin receptor inhibitor has no effect on hypertensive nephrosclerosis [135].

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5.3 The Heart Left ventricular hypertrophy (LVH) is one of the prototypical manifestations of target organ damage found in hypertension and is associated with increased risk of cardiovascular events [136]. Whether LVH is only a consequence of elevated blood pressure or also results in part from the effects of Ang II has been a matter of controversy for some time. In vitro, Ang II causes cardiac myocyte hypertrophy [137], and treatment of hypertension with blockers of the RAAS results in regression of LVH [138, 139]. High-level (200- to 400-fold) overexpression of AT1 receptors in the heart under control of the alpha-myosin heavy chain (αMHC) promoter resulted in ventricular hypertrophy only in mice older than 1.5 months and rapidly progressed to heart failure associated with myocyte apoptosis and fibrosis and death [140]. Accordingly, it has been concluded that Ang II may indeed participate in the induction of LVH through its action on cardiomyocytes and fibroblasts, stimulating the hypertrophy of the former and fibrosis by its action on the latter. However, some studies have provided quite definitive evidence that this may not be the case at least in mice. Coffman’s group in their studies of gene deletion of AT1a receptors and cross-transplantation showed that mice with renal AT1a receptors and no systemic (therefore cardiac) AT1a receptors, when infused with Ang II, develop high blood pressure and LVH [141]. In contrast, mice with no renal but systemic and cardiac AT1a receptors infused Ang II develop neither hypertension nor LVH. This suggests that Ang II acting on AT1a receptors in the heart does not induce LVH, whereas elevation of blood pressure even in absence of AT1a receptors in the heart will induce LVH. Production of Ang II in the heart using the αMHC promoter to target the cardiac expression of an engineered fusion protein that directly releases Ang II produced transgenic mice with 20- to 50-fold elevation of cardiac levels of Ang II with no detectable increase in circulating Ang II [142]. These mice developed slight interstitial fibrosis but no cardiac hypertrophy at 3 months of age. An engineered fusion protein was also used that released a form of Ang II that could not be degraded, which resulted in extremely high levels of cardiac Ang II and in spillover into the circulation. Cardiac hypertrophy did not occur despite the very high levels of cardiac Ang II until blood pressure rose in response to the increases in circulating Ang II. Finally, some recent studies have demonstrated that signaling pathways for pressure-mediated and direct (Ang II-mediated?) effects through AT1 receptors may be different. Mice in which the gene for Gαq /α11 has been deleted specifically in cardiomyocytes did not develop myocardial hypertrophy after pressure overload [143], whereas mice in which an AT1 receptor devoid of the Gαq -coupling intracellular loop (i2m) has been overexpressed in the heart had more ventricular hypertrophy than those in which the nonmutated receptor was overexpressed [144]. Thus, most of the evidence does not support a direct role of the RAAS in LVH. LVH can, however, be improved by RAAS inhibition even if the RAAS activation may not be the primary cause of cardiac hypertrophy [145]. However, direct activation of cardiac AT1 receptors may contribute to the development of LVH through alteration of cardiac repolarization, which is a major risk factor for ventricular arrhythmias and sudden death. Detailed analysis of ventricular repolarization parameters in mice with

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specific cardiomyocyte AT1 receptor overexpression revealed an increased incidence of cardiac arrhythmia associated with delayed repolarization in 50-dayold mice, before the development of cardiac hypertrophy [146]. Increased incidence of cardiac arrhythmias has also been observed in mice with cardiac-restricted ACE and in AT1 i2m mutant receptor transgenic mice [144, 147]. Aldosterone as well as Ang II will have effects on the heart, which contribute to the pathophysiology of hypertension, namely via its effects on cardiac fibrosis and inflammation [50, 148]. The effects of aldosterone on the heart have been particularly underlined by the clinical studies with mineralocorticoid receptor blockers such as spironolactone in heart failure in the RALES trial [149] and eplerenone postmyocardial infarction in the EPHESUS trial [150]. Interestingly, it appears that effects of aldosterone on the heart require a functional AT1 receptor [151, 152]. It has been suggested that gene inactivation of the mineralocorticoid receptor in cardiomyocytes was associated with cardiac fibrosis and heart failure in mice, which could be reversible if treated with spironolactone, although this work remains controversial and unexplained [153]. With respect to the coronary circulation, what was already reviewed in the section on the blood vessels is applicable as well to coronary vessels. Randomized clinical trials have shown that RAAS blockade with ACE inhibitors or ARBs protects patients with and without hypertension from cardiovascular events [154, 155]. The effects of Ang II on progression of atherosclerosis, on PAI-1, on thrombosis and inflammation, on MMPs and plaque rupture that have already been referred may be underlying the benefits derived from RAAS inhibition at the level of the coronary circulation, which will not be reviewed here in detail (for review see reference 156).

5.4 The Brain and the Sympathetic Nervous System The brain and the sympathetic nervous system have been implicated in the pathophysiology of hypertension by well-founded evidence for many years [157]. Brain renin, as well as all other components of the RAS, including ACE, AT1 and AT2 receptors, angiotensinogen, Ang II, Ang III, and Ang IV have been demonstrated in the brain [158]. Ang II induces pressor and dipsogenic responses and vasopressin secretion after injection into the brain [159, 160]. Moreover, there are Ang II receptors in the circumventricular organs (which are devoid of a blood–brain barrier and therefore accessible to circulating Ang II and include the subfornical organ or SFO, organum vasculosum lamina terminalis or OVLT, area postrema on the floor of the fourth ventricle), and are in the vicinity and connected to cardiovascular control nuclei of the brain [161], as well as in other regions of the brain including the cortex and the cerebellum and the choroid plexus. These findings have provided the anatomic and physiological basis involvement of the RAAS in the role that the central nervous system (CNS) plays in blood pressure control and elevation [162]. In part, these central effects of Ang II are mediated via AT1 receptors and increased activity of the sympathetic nervous system (SNS) [163]. AT2 receptors may counteract some of the actions mediated by AT1 receptors [164].

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In addition to the brain, Ang II induces increased release of norepinephrine (NE) from nerve endings of the SNS in the periphery, and epinephrine from the adrenal medulla, and as well affects baroreflex function, an important contributor to blood pressure regulation [165]. These effects may play a role in blood pressure elevation in both experimental animals and humans. Increased oxidative stress occurs in the circumventricular organs in the brain in response to Ang II administered both centrally and peripherally [166, 167]. Blockade of generation of ROS in these circumventricular organs inhibits both central and peripheral Ang II-induced blood pressure elevation [168, 169]. Mineralocorticoid receptors are also present in the hypothalamus and elsewhere in the brain, and aldosterone induces effects on the hypothalamus, which have been implicated in heart failure through interactions with other components of the RAS [170]. Indeed, aldosterone exerts effects on the brain that worsen manifestations of heart failure in rodents in part through upregulation of AT1 receptors [118], as already demonstrated many years ago in peripheral blood vessels [58]. The complex mechanisms whereby the CNS, the SNS, and the adrenal medulla may play a role in hypertension in response to the effects of Ang II and aldosterone are beyond the scope of this short review. It should be noted, however, that Ang II-like immune reactivity has been found in many areas of the brain and brain stem such as the anterior and middle hypothalamus, basal ganglia, locus coeruleus, nuleus tractus solitarius, and reticular formation of SHR [171], and that increased AT1 receptors have been found in circumventricular organs and cultured brain neurons of SHR [172, 173]. Moreover, intracerebroventricular administration of captopril, an ACE inhibitor, or saralasin, an Ang receptor blocker, or an antisense to angiotensinogen or to AT1 receptors in intact hypertensive rats, or peripherally in anephric rats, results in blood pressure lowering [174–178]. Thus, in experimental hypertensive models, Ang II stimulation of brain centers appears to play an important role. To what degree this occurs in humans has not been established.

5.5 Conclusion The RAAS is involved in the pathophysiology of hypertension at the level of vessels, the heart, the kidney, and the central and peripheral nervous system and participates in the complications of hypertension. Both Ang II and aldosterone have been proven to play roles in these different tissues and organ systems. Thus, it is not surprising that treatment of patients with hypertension with blockers of the RAAS, be it renin inhibitors, ACE inhibitors, ARBs, or mineralocorticoid receptor blockers, results in improved blood pressure control and better outcomes in humans with essential hypertension, beyond the secondary forms of hypertension such as reninsecreting tumors, true renovascular hypertension (with increased renin secretion due to a renal artery stenosis), or primary aldosteronism. However, there are many components of the RAAS whose exact contribution to pathophysiology remains unclear, including renin receptors, ACE2, Ang 1-12, Ang 1-7, Ang III, and Ang IV. The

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participation of these, and the development of treatments thereof, should help not only our understanding but also the benefits that patients with hypertension may derive from interference with the RAAS. Acknowledgments The work of the authors was supported by grants 37917 and 82790 from the Canadian Institutes of Health Research (CIHR) and by the Canada Fund for Innovation and the Canada Research Chairs program of the Government of Canada, all to ELS.

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Chapter 6

AT1 Receptors, Angiotensin Receptor Blockade, and Clinical Hypertensive Disease Robert M. Carey

Abstract The renin angiotensin system (RAS) is a coordinated hormonal cascade, the major effector peptide of which is angiotensin II (Ang II). Ang II binds to one of two principle receptors, AT1 (AT1 R) and AT2 (AT2 R). AT1 Rs mediate the vast majority of biological actions of the RAS, almost all of which are potentially detrimental. This chapter focuses on new developments in our knowledge of AT1 Rs and the actions of angiotensin receptor blockers (ARBs) in the treatment of hypertension. Several novel mechanisms have been described whereby AT1 Rs mediate detrimental actions in cardiovascular and renal function. These include [1] Ang IIindependent activation of AT1 Rs, which respond only to inverse agonist administration [2]; AT1 R-activating autoantibodies, potentially important in the pathogenesis of pre-eclampsia and other conditions associated with inflammation [3]; G protein receptor-interacting proteins which may modulate the functional activity of AT1 Rs [4]; and receptor cross-talk/receptor dimerization. New considerations of ARBs for the treatment of hypertension include [1] renal tissue RAS function independently of the systemic circulation [2], AT2 R activation in response to AT1 R blockade [3], pleiotropic actions of ARBs, and new data on the use of combination RAS blockade. The results of several major new clinical trials are discussed and placed in context with existing antihypertensive therapy.

6.1 Introduction The renin angiotensin system (RAS) is a coordinated hormonal cascade of major critical importance in the regulation of blood pressure. The principal effect or peptide of the RAS is angiotensin II (Ang II), which acts by binding to one of two major angiotensin receptors, type-1 (AT1 R) and type-2 (AT2 R) (Fig. 6.1) [1]. AT1 Rs mediate the vast majority of the biological actions of Ang II, including vasoconstriction, R.M. Carey (B) Division of Endocrinology and Metabolism, Department of Medicine, University of Virginia Health System, Charlottesville, VA, USA e-mail: [email protected]

W.C. DeMello, E.D. Frohlich (eds.), Renin Angiotensin System and Cardiovascular Disease, Contemporary Cardiology, DOI 10.1007/978-1-60761-186-8_6,  C Humana Press, a part of Springer Science+Business Media, LLC 2009

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Agt Renin Inactive fragments

Ang I ACE Inhibits

Ang II

Bradykinin Substance P

AT1R

AT2R

Fig. 6.1 Schematic diagram of the renin angiotensin system. Agt – angiotensinogen; Ang I – angiotensin I; Ang II – angiotensin II; ACE – angiotensin-converting enzyme; AT1 R – angiotensin type-1 receptor; AT2 R – angiotensin type-2 receptor

antinatriuresis, sympathetic nervous system activation, aldosterone, vasopressin and endothelin secretion, and vascular smooth muscle hypertrophy, migration, proliferation, and growth, all of which act in concert to raise blood pressure (BP). In addition to a rise in BP, AT1 R activation induces a number of detrimental effects in cardiovascular and renal tissues (Fig. 6.2), including cytokine production by monocytes and macrophages, leading to inflammation; plasminogen activator inhibitor-1 (PAI1) biosynthesis, platelet activation, aggregation and adhesion, leading to thrombosis; collagen biosynthesis leading to fibrosis; and low-density lipoprotein transport leading to atherosclerosis. These tissue actions of Ang II via AT1 Rs have been attributed in large part to a common mechanism: the production of reactive oxygen species, especially superoxide anion via stimulation of NAD(P)H oxidase with accompanying nitric oxide destruction [2]. Other actions of Ang II through AT1 Rs include increased cardiac contractility, cardiac and vascular remodeling, and reduction in vascular compliance. These detrimental actions of Ang II can be offset to some extent by AT1 R-mediated short-loop negative feedback suppression of renin biosynthesis and secretion at renal juxtaglomerular cells. In contrast to the pressor and tissue destructive mechanisms of Ang II via AT1 Rs, Ang II activation induces opposite effects via AT2 Rs, including vasodilation of both resistance and capacitance vessels, natriuresis and inhibition of cellular proliferation and growth [3, 4]. However, the relative balance between AT1 R and AT2 R functions may be influenced by receptor expression patterns in tissues. Whereas AT1 Rs are highly expressed in the cardiovascular, renal, endocrine, and nervous systems in adults, AT2 R expression is quantitatively less and its tissue distribution more limited than that of AT1 Rs. The extent to which AT2 Rs are functionally counterregulatory to AT1 Rs and play a role in the pathophysiology of hypertension continues to be a subject of intensive study.

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AT1 Receptors, Angiotensin Receptor Blockade, and Clinical Hypertensive Disease

Abnormal vasoconstriction

Renal Na+ retention

Cytokines/ inflammation ↑PAI-1/ thrombosis

Activate SNS ↑Aldosterone

Platelet aggregation/ adhesion

Angiotensin II ↑Vasopressin

Superoxide production/ NO destruction

↑Endothelin

Cardiomyocyte growth

61

Vascular smooth muscle growth

↑Collagen/ fibrosis

Cardiac and vascular remodeling Fig. 6.2 Deleterious actions of angiotensin II. SNS – sympathetic nervous system; NO – nitric oxide; PAI-1 – plasminogen activator inhibitor – 1

Hypertension is recognized as one of the leading risk factors for human morbidity and mortality and, on a worldwide basis, is ranked third as a cause of disabilityadjusted life years (DALYS) [5]. The enormity of the problem of hypertension is underscored by the fact that one-quarter of the world’s adult population, totaling nearly one billion individuals, had hypertension in the year 2000, and this number is predicted to increase to 29% by the year 2025 [6]. Despite the huge burden of disease and the availability of several different classes of antihypertensive pharmacological agents, relatively few patients achieve their target BP level. In the United States during 2003–2004, only 33% of hypertensive patients had controlled BP and only 64% of patients treated for hypertension achieved BP control [7]. Even more strikingly, in five European countries approximately 70% of hypertensive patients do not meet their BP targets [8]. As a major risk factor for myocardial infarction, congestive heart failure, stroke, and end-stage renal disease, all of which convey risk of significant morbidity and mortality, hypertension is an enormous public problem [9]. The difficulty in controlling hypertension is related, at least in part, to the complex pathogenesis of hypertension and related cardiovascular disease. Multiple signaling pathways and redundant feedback mechanisms, both positive and negative, contribute to the hypertensive disease process, which is even more confounded by the interrelationship of hypertension with associated diseases such as diabetes and renal dysfunction. The RAS plays an important role not only in the control of BP but in the pathogenesis of diabetes and kidney disease [10–12]. While it has been difficult to demonstrate in vivo activation of the RAS in early or established hypertension in humans, there is no question that inhibition of the RAS is effective in lowering

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BP in patients with primary hypertension [10]. The results of multiple clinical trials demonstrate that blocking the RAS with angiotensin-converting enzyme (ACE) inhibitors or angiotensin (AT1 ) receptor blockers (ARBs) not only lowers BP and BP variability but also reduces cardiovascular events and total morbidity and mortality [10]. This chapter will focus on new developments in our knowledge of AT1 Rs and the actions of ARBs in the treatment of hypertension. I will discuss novel mechanisms regulating AT1 Rs and their actions, review newly discovered RAS pathways with which ARBs may interact, and interpret recent clinical efficacy studies of ARBs alone and in combination with other blockers of the RAS.

6.2 Emerging Concepts of AT1 R Regulation and Action 6.2.1 Ang II-Independent Activation of AT1 Rs Ligand-independent receptor activation has been described for several G proteincoupled receptors (GPCRs) [13]. Receptors, including AT1 Rs, may exist in two states, inactive (uncoupled) and active (coupled). Docking of an agonist to the receptor causes more of the receptors to exist in the active state. In the case of the AT1 R, when the agonist Ang II couples with the receptor, the receptor activates resulting in vasoconstriction, aldosterone secretion, sodium and water retention, sympathetic nervous system activation, and increased BP. Even when Ang II is unavailable, AT1 Rs can still be in the active state. When this occurs, the receptor is said to exhibit “constitutive activity.” For example, the native AT1 R can be activated by mechanical stretching of cardiomyocytes in the absence of Ang II [14]. An inverse agonist is a compound which upon docking to the receptor causes an increase in the number of receptors to exist in the inactive state (Fig. 6.3). Inverse agonists may stabilize the inactive conformation of the receptor and drive the equilibrium away from active conformation. Thus, docking of an inverse agonist reduces the constitutive (basal) activity of the receptor. The property of inverse agonism is important because it may help explain the ability of some ARBs to lower BP even when the levels of Ang II are low, as they are in 25% of patients with primary hypertension [10]. Spontaneous receptor mutations leading to increased constitutive activity (in the absence of the agonist) have been implicated in human disease, but such mutations have not yet been described for the AT1 R [15]. Constitutive GPRC activation should lead to basal G protein and downstream effector activity. Although it is unknown whether the wild-type AT1 R has constitutive activity in native tissues, the receptor has been shown to have this activity for inositol phosphate (IP) production in recombinant systems in which basal expression levels are high [16]. A number of ARBs display inverse agonist activity, including olmesartan, EXP3174, a metabolite of losartan (but not losartan itself), valsartan, and candesartan [16]. An extensive evaluation of the inverse agonist properties of olmesartan and its

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AT1 Receptors, Angiotensin Receptor Blockade, and Clinical Hypertensive Disease

A

63

B Ligand-independent AT1R activation

Autacrine Mechanism Stretch

Mechanoreceptor

Ang II

Stretch

AT1R

AT1R

Gq

Gq

Hypertrophic response

Hypertrophic response Modified from Yasuda et al., Naunyn-Schmeideberg’s Arch Pharmacol, 2007.

Fig. 6.3 Ligand-dependent and ligand-independent activation of AT1 receptors (AT1 R) in cardiomyocytes leading to hypertrophy. Modified from Yasuda et al., Naunyn-Schmeideberg’s Arch Pharmacol, 2007

molecular mechanisms was published in 2006 [15]. Mutant AT1 Rs with constitutive activity were expressed in COS-1 cells. Olmesartan suppressed basal IP production in mutant receptor-transfected cells demonstrating inverse agonist activity. These studies suggested that the coexistence of carboxyl and hydroxyl groups in the imidazole moiety of olmesartan is essential for potent inverse agonist activity [15]. Site-directed mutagenesis of the transfected AT1 Rs demonstrated that the inverse agonist activity of olmesartan requires interactions of Lys199 and His256 in receptor transmembrane VI with the carboxyl group and Tyr113 in transmembrane IV with the hydroxyl group of the ARB [15]. The possibility of constitutive activation of AT1 Rs in the pathophysiology of human disease is intriguing. However, at this time more information is necessary before we can judge the importance of inverse agonists in the RAS and in the pharmacological therapy of hypertension and related tissue damage.

6.2.2 AT1 R-Activating Autoantibodies Pre-eclampsia is a condition characterized by hypertension, proteinuria, hypercoagulability, edema, and placental abnormalities. Pre-eclampsia affects approximately 7% of first pregnancies and is one of the leading causes of maternal and fetal mortality and morbidity worldwide. In addition to the circulating RAS, a separate

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uteroplacental RAS exists in both the mother (decidua) and the fetus (placenta), and the RAS in the decidua has been found to be activated in pre-eclampsia [17, 18]. Recently, dysregulation of the RAS in the chorionic villi, the tissue responsible for maternal–fetal blood flow, has been described in pre-eclampsia, setting up the possibility that increased Ang II generated locally may contribute to vasoconstriction with reduction in maternal–fetal exchange of nutrients [19]. In 1999, autoantibodies that bind to and activate AT1 Rs, termed AT1 agonistic antibodies (AT1 -AAs), were first described in the circulation of pre-eclamptic women [20]. AT1 -AAs have been detected only rarely in serum from normal women [20, 21]. Since the initial discovery of AT1 -AAs, multiple groups have confirmed their presence in the circulation of preeclamptic women and have substantiated that many of the clinical features of pre-eclampsia may be related to the ability of these autoantibodies to activate AT1 Rs on a variety of cells [20–26]. In addition to pre-eclampsia, AT1 -AAs are present in renal transplant recipients during an episode of rejection [26, 27]. From studies in experimental models of pre-eclampsia such as the reduced uterine perfusion pressure (RUPP) and double renin/angiotensinogen transgenic rat models, it is thought that an initiating pathogenic mechanism is decreased blood flow to the uteroplacental unit. Decreased blood flow results in hypoxia and placental ischemia, which leads to endovascular injury. The resulting vascular inflammatory response mediated by tumor necrosis factor-alpha (TNFα) probably induces the production of AT1 -AAs which activate AT1 Rs leading to hypertension and proteinuria. AT1 -AAs increase the spontaneous beating rate of cultured neonatal cardiomyocytes in an AT1 R-specific manner, and this observation has served as the basis for AT1 -AA bioassay [20]. AT1 -AAs bind to a seven-amino acid sequence, AFHYESQ, on the second extracellular loop of the AT1 R (Fig. 6.4). AT1 -AAs 7-aa peptide (AFHYESQ)

EXTRACELLULAR

PLASMA MEMBRANE

INTRACELLULAR

Fig. 6.4 Amino acid structure of the rat type-1 angiotensin receptor depicting its seventransmembrane domains and extracellular and intracellular termini. AT1 receptor-activating autoantibodies bind to a seven-amino acid sequence on the second extracellular loop of the receptor

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induce extracellular signal-related kinase (ERK) phosphorylation and stimulate reactive oxygen species generation via NAD(P)H oxidase in similar fashion to Ang II. Multiple intermediate pathophysiological steps have been stimulated by AT1 AA, including increased PAI-1 in trophoblasts and renal mesangial cells, increased interleukin-6 (IL-6) in mesangial cells, increased Ca++ in AT1 R-transfected Chinese hamster ovary (CHO) cells, and increased tissue factor in monocytes and vascular smooth muscle cells [28]. These changes are likely to contribute to the vasoconstrictor, inflammatory, and hypercoagulable state in pre-eclampsia. Currently, there is no specific and effective therapy for pre-eclampsia, which can lead to premature delivery. ARBs are contraindicated in pregnancy due to the role of the RAS in fetal tissue development. If maternal circulating AT1 -AAs contribute to the pathogenesis of pre-eclampsia, as many studies suggest, then blocking the action of these autoantibodies with a seven-amino acid epitope blocking antibody may offer the potential for effective treatment.

6.2.3 G Protein Receptor-Interacting Proteins and AT1 Rs As with most G protein-coupled receptors, AT1 Rs exhibit an endosomal internalization/trafficking and intracellular recycling process that governs its activity. Liganddependent AT1 R activation depends upon cell membrane expression, and receptor internalization is critical for desensitization and inhibition of signal transduction. Following agonist binding, AT1 Rs undergo rapid endocytosis and downregulation through β-arrestin and dynamin-dependent mechanisms in clatharin-coated vesicles, by receptor phosphorylation, and through interaction with caveolae [29]. The carboxy-terminal cytoplasmic tail of the AT1 R is involved in the regulation of receptor internalization independently of G protein coupling. Recently, AT1 R internalization has been demonstrated also to be mediated by AT1 R-interacting proteins. AT1 R-associated protein (ATRAP) is a 17.8-kDa protein with three transmembrane domains that bind to a 20 amino acid sequence on the intracellular C-terminal domain of the AT1 R [30] (Fig. 6.5). ATRAP is expressed in many tissues, such as aorta, heart, liver, and especially kidney where it associates with AT1 Rs in the renal tubules [31]. ATRAP exerts an inhibitory action on AT1 R signaling by promoting constitutive internalization of the receptor, thus reducing cell surface receptor number [32, 33]. Mice with transgenic overexpression of ATRAP exhibit reduced neointimal formation, NAD(P)H oxidase activity, and inflammation in response to injury compared to their wild-type controls [34]. Furthermore, ATRAP transgenic mice have smaller cardiac hypertrophic responses to aortic banding than their wild-type controls, indicating a role for ATRAP in inhibiting cardiovascular remodeling [35]. Recent studies using immunoprecipitation and BRET (bioluminescence resonance energy transfer) microscopy have demonstrated that ATRAP closely associates with the C-terminal domain of AT1 Rs, promotes receptor internalization into cytoplasmic endosomes, and attenuates the Ang II-mediated c-fos/transforming growth factor-β pathway and proliferative responses in vascular smooth muscle cells [36]. ATRAP is able to interact with AT1 Rs even in the absence of Ang II, but Ang

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AT1-AA NH2

NH2

Extracellular

Intracellular COOH

COOH

DAG

IP3

PKC

Ca++

ERK 1/2

NF- B

Calcineurin NAD(P)H Oxidase

NFAT

PAI-1

Fig. 6.5 Schematic illustration of the interaction of AT1 receptor activating autoantibodies (AT1 AA) with AT1 receptors and downstream cell signaling mechanisms. DAG – diacylglycerol; PKC – protein kinase C; ERK – extracellular signal-related kinase; NF-?B – nuclear factor kappa B; IP3; inositol tris-phosphate; Ca++ – calcium ion; NFAT – Nuclear factor activating T cell; PAI-1 – plasminogen activator inhibitor -1

II stimulation significantly facilitates the interaction of ATRAP with AT1 Rs. These studies have shown that while ATRAP can bind to AT1 Rs under basal conditions, the major interaction occurs with AT1 Rs that have been internalized into endocytic vesicles upon Ang II stimulation [36]. Therefore, it is thought that ATRAP may help keep AT1 Rs internalized even after removal of Ang II. Interestingly, knockdown of ATRAP increased both basal constitutive and Ang II-mediated AT1 R activity in these studies [36]. In spontaneously hypertensive rats (SHR), hypertension was accompanied by a reduction of the ATRAP:AT1 R ratio and cardiac hypertrophy [37]. ARB olmesartan recovered the suppressed cardiac ATRAP:AT1R ratio, decreased AT1 R cell surface density, inhibited p38 mitogen-activated protein kinase, and reversed the cardiac hypertrophy [37]. One remaining question is whether the inhibitory mechanism of ATRAP is different from that of ARBs. However, taken altogether, the results of these studies imply that increased expression/action of ATRAP, for example, via an activating ligand, could be helpful in reducing detrimental AT1 R actions. Another AT1 R interacting molecule is AT1 R-associated protein (ARAP), a 57.2kDa protein which also interacts with the C-terminal tail of the receptor [38]. In contrast to ATRAP, ARAP promotes recycling of AT1 Rs to the plasma membrane, suggesting a role in receptor signaling recovery (Fig. 6.6) [39]. ARAP is expressed mainly in lung, liver, and kidney, but not in the vascular system [40]. Overexpression of ARAP increases receptor number in the plasma membrane after Ang II stimulation, whereas overexpression of ATRAP does not affect Ang II-mediated AT1 R

6

AT1 Receptors, Angiotensin Receptor Blockade, and Clinical Hypertensive Disease NH2

Extracellular

NH2

ARAP

Intracellular

67

ATRAP

COOH

COOH

NH2

Receptor Internalization

Receptor recycling to the plasma membrane

COOH

Growth promotion signaling

Degradation

Hypertension Cardiovascular remodeling

Fig. 6.6 Schematic diagram of the actions of angiotensin receptor-interacting proteins ATRAP (angiotensin receptor-associated protein) and ARAP (AT1 receptor-associated protein 1) on receptor internalization, sequestration, and recycling to the plasma membrane

internalization. The exact role of ARAP in receptor cycling and its function await further study.

6.2.4 Receptor Cross-Talk and Dimerization AT1 Rs have been described to form tightly associated complexes with themselves and/or other receptors (Fig. 6.7), including AT2 Rs, mas oncogene receptors, bradykinin B2 receptors, dopamine receptors, endothelin type B receptors, and epidermal growth factor receptors [41–45]. AT2 Rs functionally heterodimerize cross-talk

AT1R

ETB Mas D1/D2/D3 EGF-R

Extracellular AT1R

B2 R

AT1R AT1R

AT1R AT2R

Intracellular Increased AT1R activation and signaling

Inhibition of AT1R signaling

Cell Proliferation and Growth

Fig. 6.7 Schematic representation of cross-talk of AT1 receptors (AT1 R) with other receptors and the functional actions of homo- and heterodimerization of the AT1 receptor (AT1 R). AT2 R – angiotensin type-2 receptor; B2 R – bradykinin B2 receptor; ETB – endothelin-B receptor; Mas – mas oncogene receptor; D1 /D2 /D3 – dopamine receptors; EGFR – epidermal growth factor receptor

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with bradykinin B2 receptors to generate nitric oxide and cyclic GMP [46]. The functional consequences of AT1 R homodimerization and heterodimerization have not been fully elucidated. AT2 Rs antagonize the activation of AT1 Rs by direct physical association [43]. AT1 Rs heterodimerize with bradykinin B2 receptors in preeclampsia, and the receptor complex contributes to Ang II hypersensitivity [44, 45]. The precise role of dimmer formation in the functional activity of AT1 Rs awaits further study.

6.3 Considerations in the Use of ARBs for Hypertension 6.3.1 Importance of Renal AT1 Rs in the Control of Blood Pressure The classical RAS has been considered predominantly as a circulating hormonal system with the enzyme renin, the production of which is supplied exclusively by renal juxtaglomerular cells, performing catalytic conversion of angiotensinogen to Ang I, the rate-limiting step in Ang II formation. However, strong evidence now exists that the RAS also functions as a series of local tissue systems that operate independently of the systemic circulation [10]. The definition of a tissue hormonal system requires that all components are present locally and that the final product (Ang II) is generated locally and engenders a biological action without being transported outside of the local environment. Local tissue hormonal systems are termed paracrine (cell-to-different cell), autacrine (cell-to-same cell), or intracrine (intracellular), depending on their relationship to the cell of origin. In 1977, the intrarenal RAS was first identified as a local cell-to-cell system [47]. Since that time, incontrovertible evidence has demonstrated that an independent tissue RAS exists within the kidney, which is important in the control of fluid and electrolyte balance, kidney function, and blood pressure [10]. Independent tissue RASs have now been demonstrated for many other organs including brain, vasculature, heart, adrenal glands, and the uteroplacental unit, but the evidence for a functionally significant intrarenal RAS remains the strongest among these (10, 48). The existence of a functionally significant intrarenal RAS raises important questions concerning the tissue penetration and distribution of ARBs. Selective activation of the intrarenal RAS in the renal proximal tubule, independent of the circulating RAS, has been demonstrated to induce hypertension in renin and angiotensinogen transgenic animal models [49, 50]. Recently, elegant renal crosstransplantation studies by Coffman and colleagues [51, 52] have demonstrated that selective Ang II activation of AT1 Rs in the kidney, particularly in the renal proximal tubules, is required to sustain a hypertensive process. In light of the critical importance of the intrarenal RAS and renal AT1 R in initiating and sustaining hypertension, the renal distribution of ARBs may be a critical characteristic for effectively blocking the intrarenal RAS and lowering BP. However, there is very little information on the renal partitioning of ARBs; this would seem an important area for future investigation.

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6.3.2 AT2 R Activation in Response to AT1 R Blockade As discussed above, AT1 R blockade increases renin biosynthesis and secretion by renal juxtaglomerular cells, leading to increased Ang I and Ang II formation in vivo. Consequently, Ang II is available to activate unblocked AT2 Rs, which have vasodilator, natriuretic, anti-inflammatory, and antigrowth properties [3]. Indeed, increasing evidence suggests that AT2 Rs are counter-regulatory to the detrimental actions of Ang II via AT1 Rs. Recently, Savoia et al. [53] reported that AT2 R expression is upregulated in the resistance arteries of hypertensive, diabetic patients treated for 1 year with ARB valsartan, but not by β-adrenergic receptor blocker atenolol. Resistance arteries from valsartan-treated patients responded to Ang II with vasodilation, while those from atenolol-treated patients did not [53]. These observations suggest that chronic AT1 R blockade may improve vasoconstriction and vascular remodeling by upregulation and activation of AT2 Rs and introduce the possibility that nonpeptide AT2 R agonists may be appropriate therapeutic agents for hypertension in the future. Other studies have shown that, while AT1 Rs promote sodium retention, AT2 Rs mediate natriuresis and that des-aspartyl1 -Ang II (Ang III) may be the preferred endogenous AT2 R agonist for this response [54, 55]. Ang III is a metabolic degradation product of Ang II via aminopeptidase A (APA) and is itself metabolized to Ang IV via aminopeptidase N (APN). Inhibiting APN in experimental animals markedly augments the natriuretic response to exogenous Ang III, indicating that APN inhibition may be an additional therapeutic target in hypertension in the future.

6.3.3 Clinical Efficacy Trials of ARBs in Hypertension Significant antihypertensive actions and positive clinical outcomes have been described during treatment with ARBs (losartan, valsartan, eprosartan, irbesartan, candesartan, telmisartan, and olmesartan). Clinical trials have included patients with varying degrees of hypertension alone or in combination with other cardiovascular risk factors, including elderly patients and patients with left ventricular hypertrophy, congestive heart failure, post-myocardial infarction, diabetes mellitus, hyperlipidemia, resistant hypertension, multiple risk factor combinations, and target organ damage including renal dysfunction (Table 6.1) [56–67]. Characteristics of ARBs that significantly contribute to their high degree of success in the treatment of hypertension are (1) placebo-equivalent tolerability, (2) rapid BP reduction and (3) general 24-hour BP control. Freedom from serious side effects with ARB therapy renders long-term compliance feasible in the vast majority of patients. Because BP surges during the early morning hours before awakening, duration of antihypertensive action of ARBs is paramount. While ARBs are generally safe and effective, in certain populations caution is required. Hypotension is sometimes observed in elderly patients and those with sodium or volume depletion due to activation of the endogenous RAS. ARBs

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R.M. Carey Table 6.1 Major angiotensin receptor blocker (ARB) monotherapy clinical efficacy trials

Clinical trial

Study drugs

Study population

VALUE

Valsartan vs amlodipine

SCOPE

N

Time (y)

HT, high CV Risk

15,245

4.2

Candesartan vs placebo

HT, elderly

4,964

3.7

LIFE

Losartan vs atenolol

HT, LVH

9,193

4.8

VALIANT

Valsartan vs captopril vs comb. Losartan vs captopril

MI, HF, LVD

4,703

2.0

AMI, HF, LVD

5,477

2.7

OPTIMAL

CHARM

Candesartan vs placebo

HF

7,601

3.0

VaL-HeFT

Valsartan vs placebo Losartan vs captopril

HF

5,010

2.3

HF

3,152

1.5

ELITE II

INDT

Irbesartan vs amlodipine vs placebo

HT, T2DM nephropathy

1,715

2.6

RENAAL

Losartan vs placebo

T2DM, nephropathy

1,513

3.4

IRMA-2

Irbesartan vs placebo

HT, T2DM, microalbuminuria

590

2.0

Primary end point Morbidity/ mortality equivalent CV death, nonfatal stroke, nonfatal MI equivalent Death, MI, stroke reduced All-cause mortality equivalent All-cause mortality equivalent All-cause mortality equivalent Mortality equivalent All-cause mortality equivalent Combined serum creatinine, ESRD, death reduced Combined serum creatinine, ESRD, death reduced Time to onset nephropathy reduced

may aggravate hyperkalemia due to their ability to reduce aldosterone secretion. In patients with bilateral renal artery stenosis, glomerular filtration rate (GFR) is dependent on Ang II-dependent renal efferent arteriolar constriction. ARB therapy in this setting can reduce efferent arteriolar resistance, lower GFR, and induce renal failure [9]. For reasons discussed above, ARBs are contraindicated in pregnancy.

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6.3.4 Pleiotropic Actions of ARBs Given the myriad of detrimental tissue effects of Ang II summarized in Fig. 6.2, cardiovascular and renal effects independent of BP have been sought in experimental animal models. Indeed, multiple pleiotropic actions of ARBs as well as angiotensin converting enzyme (ACE) inhibitors have been described, including (among many others) (1) blockade of advanced glycation end product-induced angiogenesis, (2) protection from renal injury in the subtotal nephrectomy model, (3) protection from renal injury in the metabolic syndrome and diabetes mellitus, (4) renal protection in uninephrectomized aldosterone/salt-treated rats, (5) protection from hypertension-induced renal injury, (6) reduction in oxidative stress, (7) attenuation of cardiopulmonary remodeling and inflammatory signaling in hypoxic pulmonary hypertension, (8) attenuation of post-myocardial infarction left ventricular remodelling, (9) improvement in cardiac diastolic dysfunction, (10) reversal of remodeling in atrial fibrillation-induced structural changes in the heart, and (11) blockade of the decrease in bone mass induced by estrogen deprivation (summarized in 68, 69). In these animal studies, the beneficial effects were often independent of the degree of BP control. Pleiotropic effects of RAS inhibition have been much more difficult to demonstrate in humans. Whereas relatively small studies with different surrogate endpoints have suggested the likelihood of benefits exceeding those expected from BP control [69], large clinical trials generally have not confirmed these actions [68]. In addition, large meta-analyses generally have not lent support to pleiotropic effects of ARBs. However, there are some notable exceptions: (1) A large metaanalysis from multiple clinical trials has suggested that RAS inhibition with either an ACE inhibitor or ARB can reduce the incidence of new-onset diabetes mellitus by approximately 25 % [69]. (2) In a relatively small but well-controlled study employing ARB losartan for 1 year in subjects with hypertension demonstrated amelioration, but not complete reversal, of structural changes due to vascular remodeling in small resistance arteries as compared to the absence of such improvement with β-adrenergic blocker atenolol, despite a similar degree of BP reduction [70, 71]. In this study, resistance artery media:lumen ratios were 5.91 in normotensive subjects, 8.34 in hypertensive subjects prior to treatment, 8.32 in hypertensive subjects treated with atenolol, and significantly reduced to 6.86 in hypertensives treated with losartan [70]. Endothelium-dependent vasodilator responses to acetylcholine also were improved with losartan but not by atenolol, and endothelium-independent vasodilator responses to sodium nitroprusside were indistinguishable between losartan and atenolol treatment. These studies suggest a pleiotropic action of ARB treatment beyond BP control [70, 71]. Larger carefully controlled studies will help clarify whether there are benefits of ARB treatment independently of BP. While left ventricular hypertrophy, heart failure, post-myocardial infarction cardiac remodeling, and renal disease all clearly benefit from RAS blockade, it is difficult to separate these positive tissue effects from those due to BP reduction. For a pleiotropic effect of an ARB to be regarded as valid, the effect must be assessed in the context of time-dependent precision measurements of BP differences. At this

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time, techniques for demonstrating these differences are not sensitive enough to clearly identify blood pressure-independent effects in large clinical trials.

6.4 Combination Therapy Combination therapy has been the hallmark of antihypertensive management for two decades since it has been realized that introduction of medications with different mechanisms of action is more effective than single agent dose-titration [72]. Combinations have included diuretics, RAS inhibitors, calcium channel blockers, β-adrenergic blockers, direct vasodilators, central sympatholytic drugs, and mineralocorticoid receptor antagonists. Indeed, JNC VII (2003) provides recommendations for administration of medications with different mechanisms of action as the fundamental principle in treatment of hypertension (9). Recently, it has been hypothesized that combination therapy with ACE inhibitor and ARB might provide more complete RAS blockade than use of either class of agent alone. Rationale for combination ACE inhibitor/ARB included possible increased kinin production, decreased aldosterone secretion, improvement in insulin sensitivity by different mechanisms, additive effects in heart failure and diabetic nephropathy, and increased AT2 R activation due to Ang II escape in response to ACE inhibitors alone via the chymase, tissue plasminogen activator (tPA), and cathepsin D pathways. Meta-analysis of multiple relatively small studies has suggested advantages of combination ACE inhibitor and ARB therapy over either agent alone [73]. Combination therapy reduced BP by 4/3 mm Hg when compared with monotherapy without significant escalation of untoward effects [73]. However, it could not be determined whether this additive effect on BP resulted from synergistic interactions of the combination due to differences in individual study design. Meta-analysis of 35 clinical trials also has suggested that the combination of ACE inhibitor and ARB is more effective than either alone in the reduction of proteinuria [74]. The ONTARGET trial program, a large (25,620 patients), randomized clinical trial quantifying cardiovascular events and total morbidity and mortality published in 2008, radically changed the approach to ACE inhibitor/ARB combination therapy [75]. The objective of this study was to compare the efficacy of ARB telmisartan with that of ACE inhibitor ramipril in preventing cardiovascular morbidity and mortality and to determine whether there is any additional benefit if combining telmisartan with ramipril compared with ramipril individual therapy. Subjects included in the ONTARGET trial were ≥55 years of age and had a high risk of developing a cardiovascular event with coronary artery disease, peripheral arterial occlusive disease, cerebrovascular disease, and diabetes mellitus with target organ damage. The trial was designed to demonstrate non-inferiority of telmisartan compared with ramipril. BP was significantly lower in the telmisartan group (0.9/0.6 mm Hg greater reduction) and the combination group (2.4/1.4 mm Hg greater reduction) than in the ramipril group. In this study, telmisartan and ramipril were equally effective in the prevention of cardiovascular morbidity and mortality, and combination therapy did

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not reduce the primary outcome to a greater extent compared with ramipril alone. In addition, combination therapy was associated with a higher adverse event rate, including a significant reduction in glomerular filtration rate, than ACE inhibitor monotherapy. This study demonstrates that combination therapy with ACE inhibitor and ARB should not be recommended in high-risk patients. Another method of combination blockade of the RAS is ACE inhibitor or ARB + direct renin inhibitor (DRI) therapy. Currently, aliskiren is the only available direct renin inhibitor available for treatment of humans. Aliskiren blocks the catalytic conversion of angiotensinogen to Ang I, reducing plasma renin activity and Ang II formation. Because both ACE inhibition and ARB therapy increase plasma renin activity, combination with aliskiren seems particularly attractive. Indeed, DRI therapy has been shown to be additive to ACE inhibitor or ARB therapy in lowering BP in hypertensive patients [76, 77]. Recently, results from the Aliskiren in the Evaluation of Proteinuria in Diabetes (AVOID) trial have been reported [78]. Patients (599) with hypertension and type-2 diabetes mellitus with nephropathy receiving ARB losartan were studied for their BP and proteinuric responses to combination with aliskiren. The primary outcome was urinary microalbumin:creatinine ratio at 6 months of combination therapy. Aliskiren + losartan induced a small nonsignificant (2/1 mm Hg) further BP reduction compared with that due to losartan alone. However, aliskiren + losartan induced a highly significant 20% reduction in urinary albumin:creatinine ratio compared to losartan alone, suggesting that DRI may have renoprotective effects independently of BP in this group of patients [78]. Recent studies also show beneficial effects of aliskiren added to ACE inhibitors or ARBs in heart failure [79]. Taken altogether, the evidence for combination of ACE inhibitor + DRI or ARB + DRI is incomplete and requires further study.

6.5 Conclusions Knowledge of the regulation and actions of AT1 Rs has increased dramatically during the past 5 years. We now know that AT1 Rs can be activated independently of their natural ligand Ang II, but the role of this mechanism in human disease processes and of ARBs as inverse agonists thereof remains to be determined. An interesting new mechanism for the pathogenesis of pre-eclampsia is the formation of activating AT1 R autoantibodies, which probably contribute to hypertension and proteinuria. This discovery opens the door for novel therapeutic approaches, including the potential use of blocking antibodies. Two major G protein receptor-interacting proteins have recently been discovered. One of these, ATRAP, appears to play an important role in AT1 R internalization and desensitization; this suggests the possibility that an ATRAP-activating ligand could be useful in forcing the AT1 R to remain in the internalized inactive state. In addition, receptor dimerization to both itself and to other receptors may play a role in AT1 R activation and downstream signaling events, but more work needs to be done on the functional consequences of heter- and homodimerization.

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These and other recent developments in the RAS have introduced several new considerations in angiotensin receptor blockade for the treatment of hypertension. We now know that the kidney, and specifically the proximal renal tubule, is critical for the initiation and maintenance of hypertension. When AT1 Rs are blocked, renin secretion and Ang II formation are enhanced. Consequently, Ang II is free to activate unblocked AT2 Rs with potential beneficial effects. One of the major beneficial sites of AT2 R action is the kidney, where Ang III activation induces natriuresis. Many clinical trials have demonstrated the efficacy and safety of ARB therapy in hypertension. In spite of a myriad of studies in experimental animals documenting the beneficial effects of ARB administration on tissue damage in hypertension and cardiovascular disease, whether pleiotropic actions of ARBs independently of BP exist in humans is currently a matter of controversy. While there is no question that RAS blockade reduces BP and conveys benefit in terms of cardiovascular morbidity and mortality, more complete RAS blockade using a combination of ACE inhibitor and ARB has not proven beneficial, particularly in a high-risk population. However, combination of ACE inhibitor or ARB with a DRI shows promise on both BP and microalbuminuria. New information from fundamental laboratory studies has successfully informed the pathogenesis and treatment of hypertension and its cardiovascular and renal consequences during the past 5 years. Continuing basic and clinical investigation should lead to improved understanding, which will enable better therapeutic approaches in the future.

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Chapter 7

Structural and Electrophysiological Remodeling of the Failing Heart Walmor C. DeMello

Abstract The influence of the renin angiotensin aldosterone system on the structural and electrophysiological remodeling of the failing heart is discussed including changes in cell communication, impulse propagation, and the generation of cardiac arrhythmias. Particular attention was given to the harmful effects of angiotensin II and the beneficial influence of ACE inhibitors, AT1 receptor blockers, and angiotensin [1–7] on cardiac function and cardiac remodeling. Finally, the role of the renin angiotensin aldosterone system on the regulation of cell volume in the failing heart was discussed and evidence was provided that the intracrine RAS plays an important role in the regulation of cell volume. Pathophysiological implications of the change in cell volume are discussed. Keywords Failing heart · Renin angiotensin system · Structural · Electrophysiological remodeling · Cell volume · Intracrine renin angiotensin system

7.1 Morphologic and Functional Abnormalities in the Failing Heart Heart failure is a complex pathological process characterized by a decline in contractility involving a cascade of events such as a defect in calcium-handling proteins [1, 2], enhanced oxidative stress [3], apoptosis, and changes in the release of inflammatory cytokines, endothelin, angiotensin II, and aldosterone [1]. Moreover, a decreased sarcoplasmic reticulum ATPase protein levels, myofilament dysfunction [4], a change in orientation of the T tubules toward the longitudinal axis of the fibers [5], and enhanced microtubule density, which imposes a viscous load on active filaments during contraction [6]. Abnormalities of ion pumps and alteration of hormone W.C. DeMello (B) School of Medicine, Medical Sciences Campus, UPR, San Juan, PR, USA e-mail: [email protected] W.C. DeMello, E.D. Frohlich (eds.), Renin Angiotensin System and Cardiovascular Disease, Contemporary Cardiology, DOI 10.1007/978-1-60761-186-8_7,  C Humana Press, a part of Springer Science+Business Media, LLC 2009

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receptors, fibroblast hyperplasia, recruitment of inflammatory cells are also part of the complex set of events which culminate in abnormalities of impulse conduction and arrhythmias [7, 8]. All these events contribute to the reprogramming of the heart muscle at the molecular and cellular levels. The impairment of electrical synchronization due to an appreciable reduction of gap junction conductance [9], to a decline of connexin43, which is the main gap junction protein in the heart, and to an abnormal distribution of this connexin [10, 11] leads to cardiac arrhythmias and to decreased mechanical efficiency [12]. The mechanism involved in the change of expression and distribution of Cx43 is not totally clear. However, some factors like angiotensin II [13] and vascular endothelial growth factor [14] have been suggested. Increasing evidence has been provided that connexin43 interacts with zona-occludens-1(ZO-1), that ZO1 promotes the formation and growth of gap junction plaques, and that in patients with heart failure downregulation of ZO-1 plays an important role in gap junction formation and stability [11]. Although the measurements of total Cx43 content, per se, do not provide information on the quantity of open gap junction channels [15], in the failing heart there is an agreement between immunofluorescence and electrophysiological studies. The velocity and amplitude of calcium transients are regulated by phosphorylation of calcium-cycling controllers. The protein kinase A and the Ca2+ /calmodulin-dependent protein kinases are involved in this process as well as phosphatases. Increased SR-associated protein phospatase 1 seems involved in the decline of SR Ca2+ pump activity in the failing heart [16].

7.2 On the Role of the Renin Angiotensin System The heart is a complex electrochemical and mechanical machine in which the electrical impulse is essential for the generation of contraction. The action potential which is generated in the sinoatrial node propagates from cell-to-cell through gap junctions which permit the electrical synchronization of the electrical and mechanical properties of the heart. Therefore, an increase in resistance of the gap junction reduces the electrical coupling and slows the conduction velocity what facilitates the generation of re-entrant rhythms. On the other hand, the impulse propagation is discontinuous due to changes in morphology and variations in gap junctional conductance. This situation is particularly evident under pathological conditions like heart failure due to the development of interstitial fibrosis, necrosis, and calcifications. It is known that the activation of the plasma renin angiotensin system during the process of heart failure is largely responsible for the impairment of heart function and remodeling of the ventricle [17, 18] [36].

7.3 On the Harmful Effects of Angiotensin II It is well known that RAS is activated during the process of heart failure and that it is responsible for many cardiac abnormalities including impairment of cell communication and cardiac arrhythmias [12]. The involvement of Ang II on the generation of

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cardiac arrhythmias is also supported by the finding that studies performed in type 1 knockout mice showed AT1 receptors are involved in the generation of cardiac arrhythmias [19]. The activation of angiotensin II (Ang II) AT1 receptors is involved in the decline of gap junction conductance and conduction velocity [20]. Ang II as well as aldosterone promote inflammation and apoptosis [1] enhance collagen deposition [12] with consequent generation of interstitial fibrosis which impairs impulse propagation throughout the ventricle [21]. The presence of a cardiac renin angiotensin system is substantiated by the following findings: (a) the ACE mRNA level is increased in the failing heart [22]; (b) randomized studies have shown that ACE inhibitors improve symptoms and survival of patients with heart failure [8] – an effect not necessarily related to the fall in arterial pressure; (c) enalapril increases cell coupling and conduction velocity in the failing heart [23], supporting the view that the RAS is involved in the generation of re-entrant rhythms including atrial fibrillation [24]. Ang II enhances the dispersion of repolarization of the action potential of the failing heart [24] – a phenomenon probably related to different expression of AT1 receptors throughout the ventricle [24]. Because evidence is available that ACE inhibitors improve sarcoplasmic reticulum (SR) functions and calcium handling in the failing heart [25], it is possible that abnormalities of the SR functions which are present in the failing heart including SR Ca leak induced by oxidative stress are, in part, related to the harmful effects of Ang II (see [26]).

7.4 On the Beneficial Effects of Chronic Blockade of Ang II AT1 Receptors Measurements of gap junctional conductance performed on cell pairs isolated from the failing ventricle of cardiomyopathic hamsters showed a large number of cells weakly coupled. Since Ang II reduces the gap junction conductance in the failing heart [20], the question whether chronic blockade of AT1-receptors reverses the decline in cell communication merits serious consideration. Recently, studies have been performed in cardiomyopathic hamsters treated chronically with losartan for a period of 3 months, and the results showed that the number of cells with very low values of junctional conductance (2-8 nS) was significantly reduced by losartan while the group with larger values (18-45 nS) was significantly increased [21]. Moreover, electrophysiological studies performed in the isolated ventricle of animals treated with losartan indicated a significant decline in the number of fibers showing nonpropagated action potentials and a higher conduction velocity in longitudinal and transversal directions. Confocal microscopy performed on the heart of cardiomyopathic hamsters treated with losartan for 4 months showed a decrease in percentage area of interstitial fibrosis [21]. It is known that Ang II AT1 receptor transgene in the mouse myocardium causes myocyte hyperplasia and heart block [27]. The increased gap junction conductance and impulse propagation found in animals treated chronically with losartan explains the decline in the incidence of re-entrant rhythms found in these animals [21]. The improvement of impulse propagation was in part due to the reduction of fibrosis, which is known to be produced

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by Ang II – an effect mediated by AT1-receptor activation and TGF-B1 synthesis [28]. Since interstitial fibrosis causes rupture of cell contacts, it is possible to assume that the disruption of the interstitial space impairs the flow of current through gap junctions. The mechanism by which losartan improves cell communication is probably multifactorial, but mainly related to the prevention of PKC and tyrosine kinase activation caused by Ang II [29]. Evidence is also available that AT1 blockers as well as ACE inhibitors reduce the incidence of atrial fibrillation [30] and other re-entrant arrhythmias. It is also known that AT1 antagonists restores cardiac rhyanodine receptor function in the failing heart [26], improving mechanical performance.

7.5 On the Influence of Extracellular and Intracellular Renin on Cardiac Function In nephrectomized animals the level of renin in the heart is extremely low [31], suggesting that cardiac renin is dependent on its uptake from plasma [32]. Two types of renin receptors have been described: (a) a mannose-6-phosphate receptor, which is a clearance receptor and binds exclusively to glycosylated form of renin followed by internalization and degradation without generation of angiotensins [33]; (b) a specific receptor which is highly expressed in the heart, brain, placenta, and eye [35]. Experiments performed on myocytes isolated from the rat ventricle indicated that the nonglycosylated form of renin not only binds to the cell membrane receptor but is internalized with consequent formation of Ang I and Ang II inside the cell [34]. These findings open the possibility that intracellular renin has a functional significance. Experiments performed in isolated cells from rat ventricle demonstrated for the first time that intracellular renin reduces cell coupling – an effect potentiated by simultaneous intracellular dialysis of angiotensinogen [37]. More recently, it was found that intracellular administration of renin plus angiotensinogen into cardiac myocytes isolated from the failing heart enhanced the inward calcium current – an effect abolished by intracellular but no extracellular losartan [38]. These findings lead to the conclusion that renin internalization increases the inward calcium current through Ang II formation inside the cell of the failing heart. Interestingly, an intracellular renin receptor has been recently described [38], which when activated by renin promotes the transport of a transcription factor (PLZF) to the nucleus with consequent activation of regulatory genes [38]. Moreover, a transcript of renin, which remains inside the cells, is overexpressed during myocardial infarction [39], indicating that pathological conditions lead to activation of the intracrine RAS.

7.6 Oxidative Stress, Angiotensin II, and Heart Failure It is known that Ang II stimulates superoxide production via AT1 receptor and activation of NADPH oxidase, which is located in many tissues [40]. Enhanced Ang II levels found during pathological conditions like heart failure and hypertension causes increased superoxide production, simultaneously with a decrease of NO

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activity [41–43]. On the other hand, it is known the beneficial effects of AT1 blockers as well as ACE inhibitors in diabetic cardiomyopathy are, in part, related to the increased availability of NO [44]. Indeed, olmesartan reduced oxidative stress and hypoxia-induced LV remodeling in part through inhibition of nuclear factor-KappaB(NF-KappaB) and matrix methaloprotein (MMP-9 activities [45]. In patients with essential hypertension valsartan decreased QTc-dispersion – an effect probably related to its antioxidative stress effect [46].

7.7 Ang (1-7) Counteracts the Effects of Ang II. But is the Overexpression of ACE2 Arrhythmogenic? Previous studies indicated that ACE2 hydrolyze Ang II to Ang (1-7) [47] and that Ang (1-7) is present in the failing heart [48]. On the other hand, Ang (1-7) improves the heart function after myocardial infarction in rats [49, 50], leading to the idea that Ang (1-7) counteracts the harmful effects of Ang II in different system including cardiac muscle. Supporting this view is the finding that Ang (1-7) has antiarrhythmic properties [51]. During myocardial ischemia/reperfusion, Ang (1-7) reduces the incidence of cardiac arrhythmias due to the activation of the electrogenic sodium pump and consequent membrane hyperpolarization [52]. Similar effects have been described in the failing heart [53] in which Ang (1-7) incremented the pump current – an effect suppressed by ouabain. Not only the increase of membrane potential but also the increment of refractoriness reduced the incidence of cardiac arrhythmias. Since higher doses of Ang (1-7) incremented the action potential duration appreciably and generated early-after depolarizations [53], the question remains if an overexpression of ACE2 with consequent formation of greater amounts of Ang (1-7) can be arrhythmogenic. This idea is supported by previous observations indicating that in mice overexpressing ACE2, severe cardiac arrhythmias were described [54]. Considering the high incidence of cardiac arrhythmias patients with heart failure, it is reasonable to ask which is the meaning of the increased expression in the failing heart [48]. It is quite possible that other abnormalities like apoptosis, the release of inflammatory cytokines, as well as the genetic reprogramming of the failing heart prevent the amelioration of the pathologic process.

7.8 Renin Angiotensin Aldosterone System and Regulation of Cell Volume. Intracrine Versus Extracellular Renin. An Integrated Hypothesis Cell volume activates stretch-sensitive ion channels and is an important contributor to metabolism, gene expression, and protein synthesis [55, 56]. It is known that hypotonic stress induced by ischemia, for instance, leads to accumulation of metabolites intracellularly, with consequent cell swelling due to water entering the cells. Recent observations [57, 58] indicated that the renin angiotensin aldosterone system is involved in the regulation of cell volume in normal as well as in the failing heart. In cells isolated from the failing ventricle and exposed to renin (128 pmol

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Ang I/ml) plus angiotensinogen (110 pmol Ang I generated by renin by exhaustion), an increase of cell volume was seen concurrently with the inhibition of the sodium pump while intracellular administration of renin plus angiotensinogen reduced cell volume through an activation of the sodium pump [58]. The effect of renin is related to the formation of Ang II because (a) intracellular Ang II administration activates the sodium pump – an effect abolished by intracellular losartan; (b) ouabain inhibited the effect of both renin and Ang II. The increase of cell volume elicited by extracellular renin plus Ao is related to the activation of the Na-K-2Cl cotransporter because bumetanide abolished this effect [58]. On the other hand, since intracellular Ang II reversed the cell swelling caused by hypotonic solution, it is reasonable to think that the activation of the intracrine RAS might play a protective role during myocardial ischemia by reducing cell volume [58]. These observations seem to indicate that an important function of the renin angiotensin system is the regulation of cell volume, with extracellular renin and Ang II increasing the cell volume and the intracrine RAS reducing it. The tantalizing importance of these findings to heart cell biology is related to the finding that alterations of cell volume induce the release of ATP, hormones, and neurotransmitters as well as elicit the activation of plasma membrane receptors and integrins which also participate in the regulation of cell volume (Fig. 7.1). Cell volume regulation following cell swelling involves the efflux of ions through activation of K+ channels and or anion channels and parallel activation of K+ /H+ exchange and Cl/HCO3 exchange, while cell shrinkage involves accumulation of ions through different mechanisms including activation of the Na-K-2Cl cotransporter and Na+ /H+

Fig. 7.1 Diagram showing the influence of extracellular Ang II on heart cell volume and the consequences of cell swelling including the enhanced gene expression and the activation of anionic channels

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exchange [59]. Alteration of cell volume regulation contributes to several diseases such as diabetic ketoacidosis, liver insufficiency, sickle cell anemia, and infection [59]. Cell swelling-activated Cl current (ICl swell), which is broadly distributed throughout the heart, shortens the action potential, depolarizes the cell membrane, is arrhythmogenic [60], and is also activated by agents that alter membrane tension and cause mechanical stretch [60].

7.9 Aldosterone and Heart Failure. On the Beneficial Effects of Eplerenone Aldosterone receptors are members of a large family of nuclear transcription factors, which includes thyroxine, glucocorticoids, androgen, estrogen, and progesterone receptors. Recent studies indicated the presence of aldosterone receptors in the heart of humans and animals [61]. These receptors are involved in tissue inflammation, fibrosis, and cardiac hypertrophy [2]. It is known that aldosterone contributes to cardiac remodeling including fibrosis and LV hypertrophy, congestive heart failure, and sudden deaths due to ventricular arrhythmias [62]. The Randomised Aldactone Evaluation Study (RALES), which was conducted with spironolactone in patients at advanced stages of heart failure, indicated a significant beneficial effect of the drug on both mortality and morbidity [63, 7]. Other studies indicated that eplerenone is also of benefit to patients with myocardial infarction and left ventricular dysfunction [64]. More recent studies indicated that eplerenone reduces cardiac remodeling, particularly interstitial fibrosis with consequent improvement of impulse propagation and consequent decline in the incidence of cardiac arrhythmias [65]. The decrease in the incidence of re-entrant rhythms was related to improvement of impulse propagation in part due to the decrease of fibrosis but also due to a decrease of the dispersion of QT interval [65].

7.10 Eplerenone Inhibits the Intracrine Action of Angiotensin II in the Failing Heart Recent studies performed on isolated cells from the failing heart showed that chronic administration of eplerenone, an aldosterone receptor inhibitor, suppressed the intracrine action of Ang II on peak ICa density while reduced significantly the effect of extracellular administration of the peptide on inward calcium current [66]. This is a relevant finding because it might indicate that the mineralocorticoid receptor (MR) is an essential component and a modulator of the intracrine renin angiotensin system. MR is an intracellular nuclear receptor that belongs to a large family including estrogen, thyroxine, progesterone, and glucocorticoid receptors. The finding that aldosterone reversed the inhibitory effect of eplerenone on the intracrine action of Ang II supports the view that aldosterone modulates the intracellular action of Ang II through the mineralocorticoid receptor [66]. Although the effect of eplerenone on the intracellular action of Ang II might be multifactorial, evidence was found

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that the decline in the action of Ang II was related to a decline in the expression of membrane-bound and intracellular AT1 receptors [66]. Major conclusions of these findings are as follows: (1) eplerenone inhibits the intracrine action of Ang II; (2) aldosterone reverses the effect of eplerenone; the mineralocorticoid receptor is a component of the intracrine RAS; (3) the beneficial effects of eplerenone on cardiac remodeling are related to the inhibition of the harmful effects of extracellular and intracellular Ang II.

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57. De Mello, W.C. (2007) Interamerican Soc. of Hypertension Meeting 58. De Mello, W.C. (2008) Intracellular and extracellular renin have opposite effects on the regulation of heart cell volume. Implications to myocardial ischemia. J Renin Angiotensin Aldosterone Syst 9, 112–118 59. Lang, F. (2007) Mechanisms and significance of cell volume regulation. J Am Coll Nutr 26(5), 613S–623S 60. Baumgarten, C.M., and Clemo, H.F. (2003) Swelling-activated chloride channels in cardiac physiology and pathophysiology. Prog Biophys Mol Biol 122, 689–702 61. Delcayre, C., and Swynghedauw, B. (2002) Molecular mechanisms of myocardial remodeling. J Mol Cell Cardiol 34, 1577–1584 62. Ramires, F.J.A., Mansur, A., and Coelho, O., et al. (2000) Effect of spironolactone on ventricular arrhythmias in congestive heart failure secondary to idiopathic dilated or to ischemic cardiomyopathy. Am J Cardiol 85, 207–211 63. Pitt, B., Zannad, F., and Remme, W.J., et al. for the Randomized Aldactone Evaluation Study Investigators (1999) The effect of spironolactone on morbidity and mortality in patients with severe heart failure. New Engl J Med 341, 709–717 64. Pitt, B., Remme, W., and Zannad, F., et al. (2003) Eplerenone, a selective aldosterone blocker, in patients with left ventricular dysfunction after myocardial infarction. New Engl J Med 348, 1309–1321 65. De Mello, W.C. (2006) Beneficial effect of eplerenone on cardiac remodeling and electrical properties of the failing heart. J Renin Angiotensin Aldosterone Syst 7, 40–46 66. De Mello, W.C., and Gerena, Y. (2008) Eplererenone inhibits the intracrine and extracellular actions of angiotensin II on the inward calcium current in the failing heart. On the presence of an intracrine renin angiotensin aldosterone system. Reg. Pept 151, 54–60

Chapter 8

Inhibiting the Renin Angiotensin Aldosterone System in Patients with Heart Failure and Myocardial Infarction Marc A. Pfeffer

Abstract Pharmacologic inhibitors of the renin angiotensin aldosterone system (RAAS) have played an important role in treating and preventing a variety of cardiovascular diseases. Although mainly developed as antihypertensive agents, the lifesaving benefits of inhibiting the RAAS at several steps along its neurohumoral cascade in patients with chronic heart failure and high-risk myocardial infarction have earned these compounds a central role in the pharmacologic armamentarium. This chapter will highlight proven accomplishments of various inhibitors of the RAAS and discuss optimal clinical use of these compounds individually and in combinations in both of these conditions. Keywords Heart failure · Angiotensin-converting enzyme (ACE) · Beta-blockers

8.1 Heart Failure Angiotensin-converting enzyme (ACE) inhibitors earned their well-deserved cornerstone role in the treatment of heart failure from early studies that demonstrated clear survival benefits. In a small study of patients with severe heart failure and a life expectancy of less than 1 year, the CONSENSUS trial first highlighted the importance of inhibiting the renin angiotensin system to prolong survival [1]. This was followed by the much larger SOLVD program of both symptomatic (treatment) and asymptomatic (prevention) studies of patients with left ventricular dysfunction (LVEF less than or equal to 35%) [2, 3]. Randomization to the ACE inhibitor resulted in a reduction in the risk of death and hospitalization for heart failure among those who were designated as symptomatic [2]. Those classified as asymptomatic had a better prognosis and randomization to the ACE inhibitor reduced the rates of Marc A. Pfeffer (B) Department of Medicine, Division of Cardiology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA e-mail: [email protected] W.C. DeMello, E.D. Frohlich (eds.), Renin Angiotensin System and Cardiovascular Disease, Contemporary Cardiology, DOI 10.1007/978-1-60761-186-8_8,  C Humana Press, a part of Springer Science+Business Media, LLC 2009

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hospitalization for heart failure but did not have a significant impact on mortality [3]. However, with longer follow-up it was possible to demonstrate that this early benefit eventually became translated into improved survival [4]. The importance of inhibiting the renin angiotensin system in symptomatic heart failure became further solidified by the V-HeFT II, which directly compared two active therapy strategies – the combination of hydralazine and isosorbide dinitrate versus the ACE inhibitor enalapril [5]. Although enalapril had been shown previously to be superior to placebo, this survival benefit even when compared to another effective strategy underscored the central role of ACE inhibitors in the treatment of patients with heart failure and reduced left ventricular ejection fraction [5].

8.1.1 Angiotensin Receptor Blockers With the development of angiotensin receptor blockers (ARB) and the concept of more complete inhibition by blocking at the angiotensin type I receptor rather than the ACE inhibitor reduction in the generation of the angiotensin II sparked the hope that the newer pharmacologic agent would produce even better clinical outcomes. In the ELITE trial, the first major head-to-head test, the ARB losartan (50 mg daily) was disappointingly found not even to be as effective as the previously proven dose of captopril (50 mg three times daily) in patients with symptomatic congestive heart failure [6]. However, when tested against placebo in heart failure patients that were previously considered intolerant of an ACE inhibitor, the CHARM Alternative trial demonstrated the effectiveness of the ARB candesartan in reducing cardiovascular death and hospitalization for heart failure [7]. The magnitude of the observed risk reductions was well in the range of what would be anticipated to be achieved with an ACE inhibitor. Similarly, the small subgroup of patients in Val-HeFT that were not being treated with an ACE inhibitor appeared to show a large reduction in morbidity and mortality with the use of the ARB valsartan [8]. Collectively, these findings support that at proper dosages, an ARB can offer heart failure patients the clinical benefits achieved with ACE inhibitors.

8.1.2 Aldosterone Antagonist Since angiotensin II is also an important stimulus for aldosterone release, the complete neurohormonal profile of RAAS inhibitors would include mineralocorticoid blockers. Spironolactone is an effective diuretic and has been used in the treatment of hypertension for approximately 40 years. Concerns about producing clinically important hyperkalemia with combined use of an ACE inhibitor and a mineralocorticoid blocker initially limited concomitant use. However, RALES showed that in the context of a carefully monitored clinical trial, an incremental survival benefit could be achieved in patients with severe heart failure already being treated with an ACE inhibitor with the addition of spironolactone [9]. Subsequently, an increase in admissions for hyperkalemia and deaths with non-protocol use of

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spironolactone in the general population underscored that patients participating in RALES were carefully selected and monitored under a protocol. Diligence must be exercised when trying to extrapolate the findings from RALES to an even older population with more severe renal dysfunction than those represented in the clinical trials [10, 11].

8.1.3 Preserved Left Ventricular Systolic Function Heart Failure Although approximately one-third to one-half of patients with symptomatic heart failure are not found to have markedly reduced left ventricular ejection fractions, these patients have generally not been included in most of the now landmark heart failure trials. Under names like diastolic heart failure, preserved systolic function heart failure, heart failure with normal ejection fraction, this poorly characterized large subset of heart failure patients has only recently been the subject of major randomized controlled clinical trials. As a consequence, the temporal improvements in survival achieved in the better studied patients with depressed left ventricular ejection fraction have not yet occurred in this population [12]. In CHARM Preserved, use of the ARB candesartan did not result in a significant reduction in the primary end point of cardiovascular death or hospitalization for heart failure [13]. However, in a secondary analysis, rates of hospitalization for heart failure were reduced when candesartan was added to conventional therapy. The lack of benefit of the ARB irbesartan on top of concomitant therapies in an even larger population of patients with heart failure with preserved systolic function recently reported in I-PRESERVED provided a very clear message concerning the hazards of extrapolating results from one population to another [14]. Similarly, a sustained benefit of an ACE inhibitor was not observed when perindopril was tested in the preserved left ventricular systolic function heart failure patients in PEP-CHF [15]. Although both ACE inhibitors and ARBs have not been shown to be effective in this population, TOPCAT, the largest ongoing trial in these patients, is currently testing whether the use of the aldosterone blocker spironolactone can reduce morbidity and mortality in patients with symptomatic heart failure and a left ventricular ejection fraction over 45% [16].

8.1.4 Combining RAAS Inhibitors in the Treatment of Heart Failure There was a good deal of enthusiasm for combining the newer ARBs with an ACE inhibitor with the concept of preserving all the clinical benefits of the proven ACE inhibitor and adding more by producing more complete inhibition of the adverse effects of angiotensin II. This concept was particularly attractive since it was known that angiotensin II can be generated by chymase, which is not under the influence of converting enzyme, and that chronic ACE inhibitor therapy leads to “ACE escape”

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where angiotensin II levels creep back up despite continued ACE inhibitor administration. Val-HeFT was the first major test of this concept in a trial large enough to assess clinical outcomes. Although no improvement was seen in mortality, the addition of an ARB to conventional therapy (which for over 90% included an ACE inhibitor) did result in reductions in hospitalizations for the management of heart failure [8]. The lack of a survival benefit and the directionally adverse findings in the large subgroup of patients on beta-blockers dampened the initial enthusiasm for the clinical use of this combination. Another major trial in this area, CHARM Added, directly focused on this combination with 100% of its patients on an ACE inhibitor (by design) and 55% treated with a beta-blocker at baseline [17]. The addition of the ARB candesartan was shown to reduce cardiovascular mortality and hospital admissions for heart failure. Both of these components of the primary end point were reduced and these benefits were consistently observed across a wide range of subgroups including different background doses of ACE inhibitors and importantly in the presence or absence of beta-blocker use.

8.1.5 Beta-Blockers This question of whether or not there is an interaction with beta-blockers is particularly important since the evidence for the use of beta-blockers in patients with reduced left ventricular heart failure to reduce mortality and hospital admissions for heart failure was particularly robust. Indeed, three independent major randomized controlled clinical trials (CIBIS II, COPERNICUS, MERIT-HF) each demonstrated an approximately 30% reduction in risk of death when a beta-blocker (bisoprolol, carvedilol and metoprolol, respectively) was added to conventional therapy [18–20]. Fortunately, at the time the studies were conducted, ACE inhibitors were already established as a key therapy for patients with heart failure, and as a consequence the clear benefits of a beta-blocker were proven on top of an ACE inhibitor. Therefore, by preserving previously proven advances attributed to the ACE inhibitor and demonstrating incremental value, the ACE inhibitor – beta-blocker combination is generally considered as the foundation of treatments that should be used together. This combination is uniformly endorsed by all major heart failure guideline writing groups [21, 22].

8.1.6 Triple RAAS Inhibitor Combinations Once an ACE inhibitor and beta-blocker are being used in effective doses, the information for the third agent for patients with heart failure and reduced left ventricular ejection fraction is less robust and more opinion than evidence based. The case for adding mineralocorticoid blockers such as spironolactone is based on RALES where a clear survival benefit of spironolactone was seen on top of an ACE inhibitor. However, at the time that study was conducted, the effectiveness of betablockers had not been established and only 11% of these patients were on an ACE

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inhibitor – beta-blocker regimen at baseline [9]. The case for adding the ARB is predominantly driven by the CHARM Added experience, which had 55% of its patients on an ACE inhibitor – beta-blocker combination at baseline [17]. Using subgroups to make clinical decisions is not ideal and this is an area where further studies are needed. It must be underscored that if a third inhibitor of the RAAS is employed to attempt to continue to lower cardiovascular risk, then careful monitoring for hyperkalemia, exacerbation of renal dysfunction and symptomatic hypotension must be conducted to balance risks and benefits [23].

8.2 Myocardial Infarction The most extensive clinical testing of RAAS inhibitors has been in the management of patients with myocardial infarction. The rationale for this use stems from animal studies that demonstrated that ACE inhibitors could attenuate the adverse structural alterations in left ventricular cavity size and shape produced by an experimental myocardial infarction, resulting in improved left ventricular function and survival [24, 25]. Proof-of-concept clinical studies showing attenuation of left ventricular enlargement with ACE inhibitors led the way for a series of major clinical trials of patients with recent myocardial infarctions [26, 27]. Survivors of myocardial infarction are at heightened risk for all major adverse cardiovascular events such as reinfarction, sudden death, developing chronic heart failure, and stroke. This risk is not uniformly distributed and is related to both comorbidities as well as the extent of the myocardial infarction and residual left ventricular dysfunction. Major randomized placebo-controlled clinical trials such as SAVE, AIRE, TRACE, and SMILE each demonstrated important improvements in clinical outcomes including survival with the addition of an ACE inhibitor to patients with a myocardial infarction complicated by either left ventricular dysfunction and signs of acute pulmonary congestion [28–32]. Although survival benefits were also achieved with ACE inhibitors in a broader acute myocardial infarction population in GISSI-3, ISIS-4, and the Chinese Cardiac Study Collaborative Group, the absolute and relative increases in survival in the broader populations were less than those observed in the higher-risk patients [33–35]. The consistency of the improvements in prognosis produced by ACE inhibitors led to the highest-grade recommendation for their use in patients with myocardial infarction by international guidelines [36].

8.2.1 Angiotensin Receptor Blockers (ARBs) With this success of ACE inhibitors, there was a hope, almost an assumption that ARBs, generally considered to be better tolerated and theoretically viewed as offering more complete inhibition of the adverse effects of angiotensin II, would result in even greater improvements in clinical outcomes. As in the heart failure field, the first major clinical trial of patients experiencing a high-risk myocardial infarction, OPTIMAAL, directly compared the ARB losartan (50 mg) to the

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previously proven dose of captopril from the SAVE trial (target 50 mg three times daily) [37]. As in ELITE 2, the best outcomes were achieved with the older more established ACE inhibitor regimen. Fortunately, this concept of ACE inhibitor versus ARB was further tested in the VALIANT trial evaluating a different ARB (valsartan, target 160 mg twice daily) also compared to captopril [38]. In this study, survival and other major cardiovascular outcomes including development of heart failure, recurrent myocardial infarction, and cardiovascular death were all comparable in the ACE inhibitor- and ARB-treated groups. Using a rigorous prespecified noninferiority analysis, this study was able to demonstrate that this dose of valsartan preserved the clinical benefits achieved with the previously proven ACE inhibitor. However, the prestudy hypothesis of the ARB being superior to the ACE inhibitor had to be rejected.

8.2.2 Combining RAAS Inhibitors in Myocardial Infarction Treatment: ACE Inhibitor Plus ARB In addition to directly comparing outcomes between ACE inhibitor- and ARBtreated patients, VALIANT was also designed to test the combination of the ACE inhibitor plus the ARB versus the ACE inhibitor alone. Unique to VALIANT was that it was the first study to compare this combination where the use of the ACE inhibitor was at the proven dose of a tested agent. Thus, this was the most scientifically rigorous test of the concept of adding an ARB to an ACE inhibitor to examine whether more complete inhibition of the RAAS would lead to improved prognosis. Unfortunately, there was no further reduction in risk of cardiovascular events, and the combination of therapies was less well tolerated than either monotherapy [38].

8.2.3 RAAS Inhibitor and Beta-Blocker The early studies demonstrating improvement of outcomes of acute myocardial infarction patients treated with beta-blockers for the most part excluded the higherrisk patients with left ventricular dysfunction and/or pulmonary congestion – the group of patients that had the greatest absolute and relative benefits with use of an ACE inhibitor. Although some patients in the ACE inhibitor trials were being treated with a beta-blocker and they appeared to experience the same improvements in outcomes as those not on beta-blockers, this ACE inhibitor – beta-blocker combination was not directly examined until the CAPRICORN trial. By adding either the beta-blocker carvedilol or placebo to patients with acute myocardial infarction and reduced cardiac function predominantly treated with an ACE inhibitor, the survival benefit observed in the CAPRICORN trial solidified the importance of the combination of an ACE inhibitor and beta-blocker in this patient population just as was established by multiple studies in chronic heart failure [39].

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8.2.4 ACE Inhibitor, Beta-Blockers, and Mineralocorticoid Blocker EPHESUS tested whether the mineralocorticoid inhibitor eplerenone could reduce the rates of death in a high-risk group of patients with recent myocardial infarction (those with reduced ejection fraction and at least transient pulmonary congestion) when added to conventional therapy [40]. The 15% improvement in survival, as well as important reductions in nonfatal cardiovascular events with the use of eplerenone on top of a well-managed contemporary regimen with 85% of patients at baseline being treated with both an ACE inhibitor and a beta-blocker, does indicate that this triple combination in this cohort of patients with high-risk acute myocardial infarction offers an advance in medical therapy.

8.2.5 Direct Renin Inhibitor The development of a direct renin inhibitor, which interrupts the RAAS at the origin of the angiotensin II-generating cascade, once again offers a promise that will require rigorous testing to determine its place in clinical care. The initial surrogate outcome study in patients with heart failure did show that plasma BNP was incrementally lowered with the addition of the renin inhibitor aliskiren when added to background therapy with other RAAS inhibitors [41]. Similarly, early testing as combination therapy on top of the already proven RAAS inhibitors has commenced in a high-risk myocardial infarction population. Whether these preliminary encouraging findings will lead to a safe and incrementally effective therapy for these patients remains an open question to be resolved by major morbidity mortality trials.

8.2.6 Safety Although each unique pharmacologic molecule has a distinctive (usually doserelated) risk-benefit profile, all inhibitors of RAAS can produce symptomatic hypotension and exacerbate renal problems. The ACE inhibitors have a higher incidence of angioedema and cough. Fortunately, these patients may better tolerate an ARB and with the proper dose still derive clinical benefits [7]. Small increases in creatinine can be anticipated with RAAS inhibition; however, in patients with marginal renal blood flow supported by the actions of angiotensin II, initiation of one of these neurohumoral inhibitors can result in a more profound acute reduction in renal clearances. In these instances, the RAAS inhibitors should be discontinued and an evaluation for renal vascular disease undertaken. At doses associated with survival benefits, all inhibitors of RAAS impart a risk of producing life-threatening hyperkalemia, which must be avoided by monitoring potassium levels. This risk is not uniform and those with advanced age, chronic kidney disease (particularly eGFR less than 30 ml per minute), diabetes mellitus, and being treated by combination RAAS inhibitors are more in jeopardy [42].

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The practice and art of medicine requires judicious use of the best available, though always incomplete, evidence to make clinical decisions for the individual patient. Inhibitors of the RAAS are amongst the best studied compounds in cardiovascular medicine and offer great deal of data about risks as well as benefits for skilled practitioners to use to improve the prognosis of their patients.

References 1. The CONSENSUS Trial Study Group. Effects of enalapril on mortality in severe congestive heart failure. Results of the Cooperative North Scandinavian Enalapril Survival Study (CONSENSUS). (1987) N Engl J Med 316, 1429–1435. 2. The SOLVD Investigators. Effect of enalapril on survival in patients with reduced left ventricular ejection fractions and congestive heart failure. (1991) N Engl J Med 325, 293–302. 3. The SOLVD Investigators. Effect of enalapril on mortality and the development of heart failure in asymptomatic patients with reduced left ventricular ejection fractions (1992) N Engl J Med, 327, 685–691. 4. Jong, P., Yusuf, S., Rousseau, M.F., Ahn, S.A., and Bangdiwala, S.I. (2003) Effect of enalapril on 12-year survival and life expectancy in patients with left ventricular systolic dysfunction: a follow-up study. Lancet 361, 1843–1848. 5. Cohn, J.N., Johnson, G., Ziesche, S., et al. (1991) A comparison of enalapril with hydralazine– isosorbide dinitrate in the treatment of chronic congestive heart failure. NEJM 325, 303–310. 6. Pitt, B., Poole-Wilson, P.A., Segal, R., et al. (2000) Effect of losartan compared with captopril on mortality in patients with symptomatic heart failure: randomised trial–the Losartan Heart Failure Survival Study ELITE II. Lancet 355, 1582–1587. 7. Granger, C.B., McMurray, J.J., Yusuf, S., et al. (2003) Effects of candesartan in patients with chronic heart failure and reduced left-ventricular systolic function intolerant to angiotensinconverting-enzyme inhibitors: the CHARM-Alternative trial. Lancet 362, 772–776. 8. Cohn, J.N., and Tognoni, G. (2001) A randomized trial of the angiotensin-receptor blocker valsartan in chronic heart failure. N Engl J Med 345, 1667–1675. 9. Pitt, B., Zannad, F., Remme, W.J., et al. (1999) The effect of spironolactone on morbidity and mortality in patients with severe heart failure. Randomized Aldactone Evaluation Study Investigators. N Engl J Med 341, 709–717. 10. Juurlink, D.N., Mamdani, M.M., Lee, D.S., et al. (2004) Rates of hyperkalemia after publication of the Randomized Aldactone Evaluation Study. N Engl J Med 351, 543–551. 11. McMurray, J.J., and O‘Meara, E. (2004) Treatment of heart failure with spironolactone–trial and tribulations. N Engl J Med; 351, 526–528. 12. Owan, T.E., Hodge, D.O., Herges, R.M., Jacobsen, S.J., Roger, V.L., and Redfield M.M. (2006) Trends in prevalence and outcome of heart failure with preserved ejection fraction. N Engl J Med 355, 251–259. 13. Yusuf, S., Pfeffer, M.A., Swedberg, K., et al. (2003) Effects of candesartan in patients with chronic heart failure and preserved left-ventricular ejection fraction: the CHARM-Preserved Trial. Lancet 362, 777–781. 14. Massie, B.M., Carson, P.E., McMurray, J.J., et al. (2008) Irbesartan in patients with heart failure and preserved ejection fraction. N Engl J Med 359, 2456–2467. 15. Cleland, J.G., Tendera, M., Adamus, J., Freemantle, N., Polonski, L., and Taylor, J. (2006) The perindopril in elderly people with chronic heart failure (PEP-CHF) study. Eur Heart J 27, 2338–2345. 16. http://clinicaltrial.gov/ct2/show/NCT00094302?term=TOPCAT&rank=1 17. McMurray, J.J., Östergren, J., Swedberg, K., et al. (2003) Effects of candesartan in patients with chronic heart failure and reduced left-ventricular systolic function taking angiotensinconverting-enzyme inhibitors: the CHARM-Added trial. Lancet 362, 767–771.

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18. CIBIS-II Investigators. The Cardiac Insufficiency Bisoprolol Study II (CIBIS-II): a randomised trial. (1999) Lancet 353, 9–13. 19. Packer, M., Fowler, M.B., Roecker, E.B., et al. (2002) Effect of carvedilol on the morbidity of patients with severe chronic heart failure: results of the carvedilol prospective randomized cumulative survival (COPERNICUS) study. Circulation 106, 2194–2199. 20. MERIT-HF (1999) Effect of metoprolol CR/XL in chronic heart failure: Metoprolol CR/XL Randomised Intervention Trial in Congestive Heart Failure (MERIT-HF). Lancet 353, 2001–2007. 21. Dickstein, K., Cohen-Solal, A., Filippatos, G., et al. (2008) ESC guidelines for the diagnosis and treatment of acute and chronic heart failure 2008: the Task Force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology. Developed in collaboration with the Heart Failure Association of the ESC (HFA) and endorsed by the European Society of Intensive Care Medicine (ESICM). Eur J Heart Fail 10, 933–989. 22. Hunt, S.A., Abraham, W.T., Chin, M.H., et al. (2005) ACC/AHA 2005 guideline update for the diagnosis and management of chronic heart failure in the adult: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Writing Committee to Update the 2001 Guidelines for the Evaluation and Management of Heart Failure): developed in collaboration with the American College of Chest Physicians and the International Society for Heart and Lung Transplantation: endorsed by the Heart Rhythm Society. Circulation 112, e154–e235. 23. McMurray, J.J., Pfeffer, M.A., Swedberg, K., and Dzau, V.J. (2004) Which inhibitor of the renin-angiotensin system should be used in chronic heart failure and acute myocardial infarction? Circulation 110, 3281–3288. 24. Pfeffer, J.M., Pfeffer, M.A., and Braunwald, E. (1985) Influence of chronic captopril therapy on the infarcted left ventricle of the rat. Circ Res 57, 84–95. 25. Pfeffer, M.A., Pfeffer, J.M., Steinberg, C., and Finn, P. (1985) Survival after an experimental myocardial infarction: beneficial effects of long-term therapy with captopril. Circulation 72, 406–412. 26. Pfeffer, M.A., Lamas, G.A., Vaughan, D.E., Parisi, A.F., and Braunwald, E. (1988) Effect of captopril on progressive ventricular dilatation after anterior myocardial infarction. N Engl J Med 319, 80–86. 27. Sharpe, N., Smith, H., Murphy, J., and Hannan S. (1988) Treatment of patients with symptomless left ventricular dysfunction after myocardial infarction. Lancet 1, 255–259. 28. Pfeffer, M.A., Braunwald, E., Moyé, L.A., et al. (1992) Effect of captopril on mortality and morbidity in patients with left ventricular dysfunction after myocardial infarction. Results of the survival and ventricular enlargement trial. The SAVE Investigators. N Engl J Med 327, 669–677. 29. Acute Infarction Ramipril Efficacy (AIRE) Study Investigators. Effect of ramipril on mortality and morbidity of survivors of acute myocardial infarction with clinical evidence of heart failure. (1993) Lancet 342, 821–828. 30. Køber, L., Torp-Pedersen, C., Carlsen, J.E., et al. (1995) A clinical trial of the angiotensinconverting-enzyme inhibitor trandolapril in patients with left ventricular dysfunction after myocardial infarction. Trandolapril Cardiac Evaluation (TRACE) Study Group. N Engl J Med 333, 1670–1676. 31. Ambrosioni, E., Borghi, C., and Magnani, B. (1995) The effect of the angiotensin-convertingenzyme inhibitor zofenopril on mortality and morbidity after anterior myocardial infarction. The Survival of Myocardial Infarction Long-Term Evaluation (SMILE) Study Investigators. N Engl J Med 332, 80–85. 32. Flather, M.D., Yusuf, S., Køber, L., et al. (2000) Long-term ACE-inhibitor therapy in patients with heart failure or left-ventricular dysfunction: a systematic overview of data from individual patients. ACE-Inhibitor Myocardial Infarction Collaborative Group. Lancet 355, 1575–1581.

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33. Gruppo Italiano per lo Studio della Sopravvivenza Nell’Infarto Miocardico (GISSI)-3. Effects of lisinopril and transdermal glyceryl trinitrate singly and together on 6-week mortality and ventricular function after acute myocardial infarction. (1994) Lancet 343, 1115–1122. 34. Fourth International Study of Infarct Survival (ISIS-4): A randomised factorial trial assessing early oral captopril, oral mononitrate, and intravenous magnesium sulphate in 58,050 patients with suspected acute myocardial infarction. (1995) Lancet 345, 669–685. 35. Chinese Cardiac Study Collaborative Group. (1995) Oral captopril versus placebo among 13,634 patients with suspected acute myocardial infarction: interim report from the Chinese Cardiac Study (CC-1). Lancet 345, 686–687. 36. Antman, E.M., Anbe, D.T., Armstrong, P.W., et al. (2004) ACC/AHA guidelines for the management of patients with ST-elevation myocardial infarction–executive summary: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Writing Committee to Revise the 1999 Guidelines for the Management of Patients With Acute Myocardial Infarction). Circulation 110 , 588–636. 37. Dickstein, K., and Kjekshus, J. (2002) Effects of losartan and captopril on mortality and morbidity in high-risk patients after acute myocardial infarction: the OPTIMAAL randomised trial. Optimal trial in myocardial infarction with angiotensin II antagonist losartan. Lancet 360, 752–760. 38. Pfeffer, M.A., McMurray, J.J., Velazquez, E.J., et al. (2003) Valsartan, captopril, or both in myocardial infarction complicated by heart failure, left ventricular dysfunction, or both. N Engl J Med 349, 1893–1906. 39. Dargie, H.J. (2001) Effect of carvedilol on outcome after myocardial infarction in patients with left-ventricular dysfunction: the CAPRICORN randomised trial. Lancet 357, 1385–1390. 40. Pitt, B., Remme, W., Zannad, F., et al. (2003) Eplerenone, a selective aldosterone blocker, in patients with left ventricular dysfunction after myocardial infarction. N Engl J Med 348, 1309–1321. 41. McMurray, J.J.V., Pitt, B., Latini, R., et al. (2008) Effects of the oral renin inhibitor aliskiren in patients with symptomatic heart failure. Circ Heart Fail 1, 17–24. 42. Desai, A.S., Swedberg, K., McMurray, J.J., et al. (2007) Incidence and predictors of hyperkalemia in patients with heart failure: an analysis of the CHARM Program. J Am Coll Cardiol 50, 1959–1966.

Chapter 9

Left Ventricular Hypertrophy and Treatment with Renin Angiotensin System Inhibition Edward D. Frohlich and Javier Díez

Abstract Hypertensive left ventricular hypertrophy can be considered as the macroscopic result of the exaggerated growth response of the cardiomyocyte to the mechanical stress imposed on the left ventricle by the progressively increasing arterial pressure. Besides cardiomyocyte hypertrophy exaggerated cardiomyocyte apoptosis and alterations in the extracellular matrix and the microcirculation also develop, which lead to the structural remodeling of the myocardium. These changes may help to explain why left ventricular hypertrophy represents not only an adaptation to increased pressure load but also an independent risk factor and a marker of risk of cardiovascular complications in hypertensive patients. Experimental evidence support the notion that angiotensin II contributes in a significant way to the development of hypertrophy and remodeling of the hypertensive left ventricle. In accordance with this, data from a large number of clinical studies have shown that long-term antihypertensive treatment with drugs inhibiting the renin agiotensin system, namely angiotensin-converting enzyme inhibitors and angiotensin type 1 receptor antagonists, is associated with regression of left ventricular hypertrophy, and this is associated with improvement in outcome and with the decrease of the risk of cardiovascular morbidity and mortality, even independently from changes of other risk factors, including blood pressure.

9.1 Introduction Hypertensive heart disease can be defined as the response of the heart to the afterload imposed on the left ventricle by the progressively increasing arterial pressure and total peripheral resistance [1]. Hypertensive heart disease is characterized by increased left ventricular mass (LVM) leading to left ventricular hypertrophy (LVH) in the absence of aortic stenosis or hypertrophic cardiomyopathy [1]. J. Díez (B) Centre of Applied Medical Research, University of Navarra, Pamplona, Spain e-mail: [email protected] W.C. DeMello, E.D. Frohlich (eds.), Renin Angiotensin System and Cardiovascular Disease, Contemporary Cardiology, DOI 10.1007/978-1-60761-186-8_9,  C Humana Press, a part of Springer Science+Business Media, LLC 2009

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Hypertension-induced LVH consists of a constellation of molecular and structural abnormalities of myocardial tissue that result in alterations of LV function, abnormalities of myocardial perfusion, and disturbances of cardiac rhythm in hypertensive patients [2]. As a consequence, LVH is an independent cardiovascular risk factor related to cardiovascular complications in these patients. In fact, considered as a categorical variable, LVH significantly increases the risk of congestive heart failure, cardiac arrhythmia, and sudden death, as well as of coronary artery disease and stroke [3]. In addition, when LVM is considered as a continuous variable, a direct and progressive relationship exists between cardiovascular risk and the absolute amount of LVM. During 4 years of follow-up in the Framingham Heart Study [4], each 50 g/m2 increase in LVM was associated with a 1.49 increase in relative risk of cardiovascular disease for men and a 1.57 increase for women. The effect on cardiovascular mortality was even more striking, with a 1.73 and 2.12 relative risk for each 50 g/m2 for men and women, respectively. More recently, it has been reported that the continuous relation between LVM and cardiovascular risk in patients with hypertension remains significant after control for cardiovascular risk factors, including ambulatory blood pressure [5]. Finally, it has been shown that the cardiovascular risk decreases significantly in hypertensive patients in whom LVH regresses with antihypertensive treatment compared to patients in whom LVH persists and patients who develop de novo LVH in spite of similar hemodynamic effectiveness of the treatment [6, 7]. Therefore, there is an important need for physicians to understand the pathophysiology of hypertensive LVH and become fluent in treatment options available. This chapter details the pathways that are involved in the development of LVH in hypertension, with a focus on the renin angiotensin system (RAS). Pharmacological interventions designed to inhibit the RAS and prevent or regress LVH in hypertensive patients are subsequently discussed.

9.2 Role of the RAS in the Development of Hypertensive LVH 9.2.1 General Mechanisms of LVH The heart is very sensitive to physiological stimuli or pathological states, and even slight perturbations can lead to severe cardiac changes, eventually with detrimental outcomes. For instance, in conditions of pressure overload due to systemic hypertension, the left ventricular myocardium undergoes extensive hypertrophy because of increased size of cardiomyocytes [8]. Functionally, hypertensive LVH is characterized by an initial compensatory process that helps the heart in sustaining cardiac output despite increased afterload imposed by systemic hypertension [8]. However, this process is only an initial ‘adaptive’ response, and chronic exposure to biomechanical stress associated with hemodynamic load eventually leads to impaired inotropic/lusitropic function that, in many cases, progresses to HF [8]. This maladaptive change is accompanied by two types of events that facilitate the transition from LVH to HF [9]: (i) a switch in the cardiomyocyte gene program that leads to

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altered expression of a number of genes, and (ii) changes in the composition of the myocardium that result in its structural remodeling. The changes in genetic expression characteristic of the cardiomyocyte hypertrophic response involve isogenic shifts, which result in the re-expression of a foetal gene program, as well as the repression of other post-developmental genes [9, 10]. The long-held views are that these morphological and genetic changes, in response to pressure overload, serve to restore cardiac muscle economy back to normal, and counteract myocardial dysfunction. However, there are experiments showing that a blunting of cardiomyocyte hypertrophy and an attenuation of the foetal gene reexpression did not result in dysfunction/failure despite pressure overload. Therefore, a shift in paradigm is occurring in the sense that genetic reprogramming associated with cardiomyocyte hypertrophy is no longer considered as an adaptive process [11, 12]. In fact, a detailed analysis of the genetic changes that accompany cardiomyocyte hypertrophy allows to conclude that they will translate into derangements in energy metabolism, contractile cycle and excitation-contraction coupling, cytoskeleton and membrane properties determining mechanical dysfunction, and autocrine functions which, in turn, will provide the basis for the cardiomyocyte malfunctioning that is associated with LVH and that predisposes to diastolic and/or systolic dysfunction [13]. Changes in the composition of cardiac tissue develop in hypertensive LVH that lead to structural remodeling of the myocardium. Myocardial remodeling involves increased rates of cardiomyocyte cell death responsible for the decrease in cardiomyocyte number, exaggerated accumulation of collagen fibers within the interstitium and surrounding intramural coronary arteries and arterioles leading to myocardial fibrosis, and structural alterations in the wall of the small intramyocardial vessels that compromises myocardial perfusion (Fig. 9.1). Historically, there

A

B

C

D

Fig. 9.1 Microscopic view of an apoptotic cardiomyocyte (panel A, arrow), the two patterns (interstitial, panel B, arrow, and perivascular, panel C, arrow) of collagen deposition leading to myocardial fibrosis, and an intramyocardial artery presenting wall thickening and lumen narrowing (panel D, arrow) (right picture) present in a human hypertensive hypertrophied left ventricle (left picture)

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are three types of cell death: apoptosis, autophagy, and necrosis. Although the three types of cardiomyocyte death have been observed simultaneously enhanced in human hearts as a consequence of pressure overload or an ischemic insult, apoptosis is the most thoroughly characterized form of cardiomyocyte death in the hypertensive myocardium [14]. Myocardial fibrosis present in LVH is suggested to be the result of both increased collagen type I and type III synthesis by fibroblasts and phenotypically transformed fibroblast-like cells or myofibroblasts and unchanged or decreased collagen degradation by matrix metalloproteinases [15]. Hyperplasia and/or hypertrophy and altered alignment of vascular smooth muscle cells due to changes in extracellular matrix lead to encroachment of the tunica media into the lumen and cause both an increase in the medial thickness/lumen ratio and a reduction in the maximal cross-sectional area of intramyocardial arteries [16].

9.2.2 Role of Angiotensin II in Cardiomyocyte Hypertrophy and Myocardial Remodeling Hypertension-induced mechanical stress of cardiomyocytes, cardiac fibroblasts, and vascular smooth muscle cells elicits paracrine and autocrine signaling by inducing synthesis and secretion of neurohumoral factors and cytokines that are responsible for both cardiomyocyte hypertrophy and myocardial remodeling occurring in the hypertensive left ventricle. A number of experimental and clinical evidence suggest that angiotensin II may contribute to cardiomyocyte apoptosis [17], myocardial fibrosis [18], and alterations of the wall composition and geometry of intramyocardial arteries [19] in the hypertrophied left ventricle. A recent review of the literature suggests that the involvement of angiotensin II in the development of LVH is the result not just of its ability to increase blood pressure but mainly of its capacity to alter directly the biophysiology of cardiac cells [20] (Fig. 9.2). Several experimental and clinical evidences support this notion. On the one hand, recent studies have demonstrated that salt excess in spontaneously hypertensive rats (SHRs) produces a modestly increased arterial pressure while promoting marked myocardial fibrosis and structural damage associated with altered coronary hemodynamics and ventricular function [21, 22]. In addition, the blockade of the angiotensin type 1 (AT1 ) receptor performed concomitantly with dietary salt excess ameliorated salt-related structural and functional cardiac abnormalities in SHRs without reducing arterial pressure [23]. On the other hand, it has been reported that old hypertensive patients with high plasma angiotensin II concentrations in relation to sodium excretion exhibited a greater LVM, posterior wall thickness, and septal wall thickness than those with low angiotensin II levels in relation to sodium excretion, with no differences in blood pressure between the two groups [24]. In addition, it has been shown that plasma angiotensin II concentration at high sodium intake correlated with LVM independently of ambulatory

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ANG IIE

AT AT11

Cm

Fb

AT AT11

Ec

AT AT11

Vsmc

AT AT11

ANG III I

Hypertrophy

Apoptosis

Alterations of the cardiomyocyte compartment

>Collagen

<MMP-1

>TIMP-1

Alterations of the extracellular matrix

Hypertrophy Hyperplasia

>ECM

Alterations of the intramyocardial vasculature

Myocardial hypertrophy and remodeling

Fig. 9.2 Schematic representation of the actions of extracellular (ANG IIE ) and intracellular (ANG III ) angiotensin II in cardiac cells that result in structural alterations of the myocardium responsible for myocardial hypertrophy and remodeling that characterize hypertensive left ventricular hypertrophy. (Cm, means cardiomyocyte; Fb, fibroblast; Ec, endothelial cell; Vsmc, vascular smooth muscle cell; AT1 , angiotensin II type 1 receptor; MMP-1, matrix metalloproteinase-1; TIMP-1 tissue inhibitor of matrix metalloproteinases-1; ECM, extracellular matrix)

blood pressure in young hypertensive individuals [25]. Therefore, locally acting myocardial angiotensin II can be considered as a major determinant of LVH in hypertension. In fact, interacting with its specific G protein-coupled AT1 receptor, angiotensin II elicits Gaq pathways in the cardiomyocyte that result in activation of several Ca2+ -dependent signaling molecules (i.e., Ca2+ -dependent protein kinases and mitogen-activated protein kinases, as well as the Ca2+ -calmodulin-dependent phosphatase calcineurin) which, in turn, participate in the transduction of hypertrophic stimuli to the nuclei [26, 27]. This will activate gene expression and promote protein synthesis, protein stability, or both, with consequent increases in protein content and in the size and organization of force-generating units (sarcomeres) that, in turn, will lead to increased size of individual cardiomyocytes [28]. Of interest, the AT1 receptor itself is directly activated by mechanical stress and invokes the aforementioned signaling routes that lead to cardiomyocyte hypertrophy, which can be blocked by an inverse agonist of the receptor [29]. In addition, angiotensin II binding of AT1 receptors triggers apoptosis by a mechanism involving stimulation of p38 MAP kinase activity, activation of p53 protein and subsequent decrease of the Bcl-2-to-Bax protein ratio, activation of caspase-3, stimulation of calcium-dependent DNase I, and internucleosomal DNA fragmentation [30]. Although angiotensin II has been shown to induce apoptosis in other cardiovascular cells through stimulation of the AT2 receptor, several findings suggest that it is unlikely that this receptor is a strong signal to induce cardiomyocyte apoptosis in vivo. In fact, apoptosis is not increased in the heart of

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transgenic mice overexpressing AT2 receptors in the myocardium [31]. In addition, Ikeda and colleagues [32] have reported that blockade of AT1 receptors with losartan is accompanied by normalization of cardiac apoptosis in rats with angiotensin II-induced hypertension that exhibit increased expression of AT2 receptors in the heart. On the other hand, increasing evidence supports the notion that angiotensin II influences both fibrillar collagen synthesis and degradation [33]. In vitro studies of rat and human cardiac fibroblasts and myofibroblasts have shown that angiotensin II stimulates cell proliferation and fibrillar collagen synthesis via the AT1 receptor. The proliferative response of fibroblasts to angiotensin II might well be mediated by stimulation of the synthesis of growth or inflammatory substances like plateletderived growth factor (PDGF) and cytokines, by integrin activation due to secreted extracellular matrix proteins (e.g., osteopontin), or by a combination of these mechanisms [34]. A number of studies provide strong evidence that angiotensin II stimulates collagen synthesis by cardiac fibroblasts via specific growth factors [35]. For instance, angiotensin II has been shown to induce collagen I gene expression via activation of both MAP/ER kinase pathway and TGF-β1 signaling pathways (e.g., connective tissue growth factor - CTGF - and Smad proteins). Finally, angiotensin II stimulation of the AT1 receptor has been shown to inhibit collagen degradation by attenuating interstitial matrix metalloproteinase-1 (MMP-1) or collagenase synthesis and secretion in cardiac fibroblasts [36] and by enhancing TIMP-1 production in rat heart endothelial cells [37]. Studies of human vascular smooth muscle cells and of vessels from experimental animals have demonstrated that angiotensin II binding to the AT1 receptor leads to activation of receptor tyrosine kinases, such as epidermal growthfactor receptor (EGFR), platelet-derived growth factor receptor (PDGFR) and insulinlike growth factor-1 receptor (IGF-1R), and nonreceptor tyrosine kinases, such as c-Src [38]. In addition, AT1 receptor binding by angiotensin II induces activation of NAD(P)H oxidase resulting in intracellular generation of reactive oxygen species, which influence redox-sensitive signaling molecules, such as mitogen-activated protein (MAP) kinase p38MAP kinase, JNK, ERK1/2, and ERK5), and transcription factors (NF-κB, AP-1, and hypoxia-inducible factor-1) [39]. These signaling events stimulate vascular smooth muscle cell growth and extracellular matrix production. Some of the cardiac effects of angiotensin II seem to be independent of the AT1 receptor but the result of the ability of the peptide to operate in the intracellular space. As early as 1971, it was reported that tritiated angiotensin II is internalized and rapidly localized to the nuclei and mitochondria of cardiomyocytes [40]. Subsequently, Re and colleagues [41] demonstrated the presence of specific, high-affinity nuclear receptors for angiotensin and showed that nuclear binding of angiotensin II enhanced gene transcription. In 2004, Baker and colleagues [42] demonstrated that the transfection of cardiac myocytes with a construct encoding a nonsecreted type of angiotensin II led to the rapid induction of cell hypertrophy. In fact, when a plasmid encoding the nonsecreted angiotensin II, the expression of which was driven by the α-myosin heavy chain promoter, was injected into mice, marked LVH developed within 96 h [42].

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9.3 Effects of Pharmacological Inhibition of the RAS on Hypertensive LVH 9.3.1 General Aspects Antihypertensive drugs are effective in reducing LVM. In fact, Mosterd and colleagues [43] analyzed recently the data from 10333 participants in the Framingham Heart Study, and reported that the increasing use of effective antihypertensive therapy has caused a decrease in the prevalence of both hypertension and LVH in the general population. A large number of trials and meta-analyses have attempted to compare the effects of different antihypertensive agents on LVM, but flawed study designs and methodological problems have limited the utility of these studies. Nevertheless, a recent meta-analysis by Klingbeil and colleagues [44] including 80 double-blind, randomized controlled trials with 146 active treatment arms (n = 3767 patients) and 17 placebo arms (n = 346 patients) showed that after adjustment for treatment duration and change in diastolic blood pressure, there was a significant difference among medication classes in regressing LVH. In fact, the decrease in LVM (indexed by body surface area or LVM index) induced by the different classes was as follows: AT1 receptor antagonists>calcium channel blockers>angiotensin-converting enzyme (ACE) inhibitors>diuretics>beta-blockers (Fig. 9.3). In pair-wise comparisons, AT1 receptor antagonists, ACE inhibitors, and calcium channel blockers were more effective at reducing LVMI than were diuretics and beta-blockers. Why might AT1 receptor antagonists and ACE inhibitors decrease LVMI more effectively than would the other antihypertensive agents? The findings from the meta-analysis by Klingbeil and colleagues [44] indicate that blockade of the RAS reduces LVM beyond the effects of blood pressure reduction. This has been clearly demonstrated in two studies. A substudy of the HOPE trial showed that compared with patients given placebo, ramipril-treated patients without LVH at baseline had lower rates of subsequent LVH [45]. In addition, ramipril-treated patients with LVH at baseline had higher rates of regression of LVH, which was associated with

Reduction of LVMI (%)

20 16 12

ARAs CCBs

ACEIs

8

Ds 4

BBs

0

Fig. 9.3 Reduction (expressed as 95% confidence interval) of the left ventricular mass index (LVMI) by different classes of antihypertensive drugs (ARAs, angiotensin receptor antagonists; CCBs, calcium channel blockers; ACEIs, angiotensin converting enzyme inhibitors; Ds, diuretics; BBs, beta-blockers) (Adapted from [44])

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improved prognosis and was independent of blood pressure reduction. Devereux and colleagues [46] conducted an echocardiographic substudy of the LIFE trial, in which LVM was measured yearly up to 5 years. Patients randomly assigned to receive losartan-based therapy had a significantly greater reduction in LVMI compared with patients receiving atenolol-based therapy. The larger relative reduction in LVMI in the losartan group was detected after 1 year of treatment and persisted to year 5. Consistent with the clinical findings of the LIFE trial, regression of LVH was greater in losartan-treated patients than in atenolol-treated patients even though blood pressure reductions were similar for the two groups. Therefore, the high effectiveness of pharmacological inhibition of the RAS to reduce LVM and regress LVH seems to be related to the reduction of the direct pro-hypertrophic and pro-remodeling actions of angiotensin II on the myocardium.

9.3.2 Emerging Clinical Aspects The time has come to revisit the current management of hypertensive LVH simply focused on controlling blood pressure and reducing LVM. In fact, it is necessary to pay attention also to the correction of alterations in LV function and coronary microcirculation that associate with LVH. Although several trials have been performed to analyze these aspects, methodological problems of design and the confounding influence of factors such as the antihypertensive and antihypertrophic effects of treatment make difficult to evaluate the available information. Nevertheless, from the available data it has been proposed that the use of either ACE inhibitors or AT1 receptor antagonists provides a higher benefit than the use of other agents [47, 48]. Similarly, it is now the time to develop new approaches in the treatment of hypertensive LVH aimed to repair myocardial structure (i.e., cardiomyocyte apoptosis and myocardial fibrosis). The in vivo effects of antihypertensive drugs on cardiac apoptosis in SHRs have been reviewed elsewhere [49]. Collectively, the available findings suggest that the ability of antihypertensive drugs to inhibit cardiomyocyte apoptosis is independent of their antihypertensive efficacy, but can be related to their capacity to interfere with the pro-apoptotic actions of humoral factors such as angiotensin II. This is further supported by clinical findings showing that despite an identical antihypertensive efficacy, the AT1 receptor antagonist losartan, but not the calcium channel blocker amlodipine, reduced cardiomyocyte apoptosis in hypertensive patients with LVH after 1 year of treatment [50] (Table 9.1). Brilla and colleagues [51] showed that treatment with the ACE inhibitor lisinopril, but not with the diuretic hydrochlorotiazide, reduced myocardial fibrosis in hypertensive patients, independently from blood pressure control and LVH regression, and that this was associated with improved left ventricular diastolic function. We have shown recently that treatment with the AT1 receptor antagonist losartan was associated with inhibition of collagen type I synthesis and regression of myocardial fibrosis in hypertensive patients with LVH [52] (Table 9.1). In contrast, hypertensive patients treated with the calcium-channel blocker amlodipine did not show significant changes in collagen type I metabolism or myocardial fibrosis [52] (Table 9.1). Interestingly,

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Table 9.1 Effect of antihypertensive treatment on hemodynamic, left ventricular mass and myocardial histological parameters in hypertensive patients Losartan group

Study 1 N Systolic BP (mm Hg) Diastolic BP (mm Hg) LVMI (g/m2 ) CVF (%) Study 2 N Systolic BP (mm Hg) Diastolic BP (mm Hg) LVMI (g/m2 ) AI (TUNEL positive nuclei/106 nuclei)

Amlodipine group

At baseline

After treatment

At baseline

After treatment

21 173±6 95±2 131±6 5.65±0.44

137±2∗∗ 81±2∗∗ 105±4∗∗ 3.96±0.32∗

16 162±11 95±3 134±12 4.93±0.27

137±4∗∗ 79±2∗∗ 119±11 4.31±0.38

14 173±2 94±3 134±9 2843±730

136±2∗∗ 78±3∗∗ 105±6∗∗ 1118±176

14 176±7 96±3 127±13 1658±244

139±3∗∗ 82±2∗∗ 124±11 3211±639

Study 1 adapted from [52]. Study 2 adapted from [50]. N means number of subjects in each group. BP means blood pressure. LVMI means left ventricular mass index. CVF means myocardial collagen volume fraction. AI means cardiomyocyte apoptotic index. Data are expressed as mean±SEM. ∗ P<0.05 vs values at baseline. ∗∗ P<0.01 vs values at baseline.

the effect of the two compounds on blood pressure was similar all along the treatment period. We have reported also that the ability of losartan to induce regression of severe myocardial fibrosis is independent of its capacity to reduce blood pressure or LVM, but it is associated with a diminution of myocardial stiffness in hypertensive patients with LVH [53]. These data support experimental studies in SHRs where pharmacological interference with the production and actions of angiotensin II has proved to be effective on reversing cardiac fibrosis beyond the antihypertensive efficacy [54-56].

9.3.3 Emerging Pharmacological Aspects Although targeting the ACE and the AT1 receptor initially inhibits the RAS, the Randomized Evaluation of Strategies for Left Ventricular Dysfunction (RESOLVD) trial clearly demonstrated the concept of aldosterone escape. The authors studied a cohort of 768 patients with a depressed ejection fraction (<40%) and New York Heart Association II-IV class heart failure treated with candesartan, enalapril, or both for 43 weeks. At 17 weeks there was a significant reduction in aldosterone levels from baseline, but by 43 weeks this improvement disappeared [57]. Mechanisms of aldosterone escape are speculative and include ACTH as a secretagogue and chymase generation of angiotensin II by a non-ACE inhibitor pathway [58].

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Abundant experimental evidence suggests that aldosterone may facilitate LV growth and myocardial remodeling, beyond the ability of the hormone to facilitate sodium retention and increase blood pressure, involving the activation of mineralocorticod receptor-dependent pathways in cardiac cells that result in hypertrophy and fibrosis [59]. Recent clinical data by Muiesan and colleagues [60] add strong support to this notion. The authors evaluated the appropriateness of observed LVM to the theoretical value of LVM predicted by sex, body size, and stroke work in 125 patients with a diagnosis of primary aldosteronism (PA) and in 125 age-, sex-, and blood pressure-matched patients with essential hypertension. The prevalence of inappropriate LVM (defined by a ratio of observed:predicted LVM >135%) was greater in PA patients than in essential hypertensive patients, irrespective of the presence or absence of traditionally defined LVH. In addition, direct correlations were observed between the ratio of observed:predicted LVM and the ratio of aldosterone:renin or the post-saline infusion aldosterone concentration in PA patients. Thus, an excess of aldosterone may induce an additional increase of LVM beyond hemodynamic load. In this conceptual framework, recent data suggest the benefit of aldosterone receptor blockade in addition to the other anti-RAS therapies. In fact, Pitt et al. [61] studied 202 hypertensive patients with LVH treated for 9 months with the aldosterone blocker eplerenone, the ACE inhibitor enalapril, or both. LVM was determined by cardiac magnetic resonance imaging. Despite all groups achieving the same degree of blood pressure lowering, the combination group had the largest degree of LVH reversal. Blockade of the RAS leads to a feedback increase in renin synthesis and secretion. In the case of ACE inhibitors, large increases in the activity of renin in plasma (referred to as PRA) and angiotensin I can be observed [62, 63]. With the AT1 receptor antagonists, increases in PRA and angiotensin II are observed [64, 65]. Diuretics also enhance PRA [66]. As reviewed recently [67], several studies show an association between PRA and LVM in hypertensive patients. Therefore, it seems reasonable to propose that more complete RAS inhibition may afford greater protection from LVH. Aliskiren is the first orally active direct renin inhibitor to be approved for the treatment of arterial hypertension. Clinical studies in hypertensive patients have shown that aliskiren is effective and safe in lowering blood pressure [68]. Concerning the mode of action, direct renin inhibition decreases PRA and inhibits the formation of angiotensin I and II and most likely also angiotensin peptides [69]. There is scarce experimental information concerning the impact of direct renin inhibition on hypertensive LVH. Recently, the effects of 9 months of treatment with aliskiren, losartan, and their combination were compared on the reduction of LVMI in hypertensive patients with increased LV wall thickness, and body mass index >25 kg/m2 . LVMI was reduced significantly from baseline in all treatment groups. The reduction in LVMI in the combination group was not significantly different from that with losartan alone. Aliskiren was as effective as losartan in reducing LVMI. Therefore, aliskiren was as effective as losartan in promoting LVH regression [70].

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Recently, it has been reported that aliskiren suppresses PRA in combination with a thiazide diuretic, an ACE inhibitor, or an AT1 receptor antagonist in hypertensive patients [71]. Thus, renin inhibition offers the prospect of optimize PRA suppression in patients with hypertension. It remains to be seen whether this effect will translate in greater cardiac protection. This aspect requires specific investigation taking into account that with aliskiren, the increase in renin secretion and circulating renin concentration are greater than with an ACE inhibitor or an AT1 receptor antagonist [72]. High extracellular levels of renin could conceivably interact with the prorenin/renin receptor located at the plasma membrane level [73] and as a consequence activate cardiac fibrotic signaling pathways independent of angiotensin II [74, 75]. In addition, it must be considered the possibility that intracellular renin originated from extracellular renin internalization, a nonsecreted isoform of renin or the enhanced expression of the renin gene elicited by different pathological conditions involving stretch may also interact with the perinuclear prorenin/renin receptor [76] reducing cell communication in the heart [77].

9.3.4 Emerging Molecular Aspects The knowledge of polymorphisms in genes that potentially influence pharmacodynamic mechanisms would allow the identification of individuals who are likely to have beneficial responses to treatment with a particular drug. LVM is a complex phenotype influenced by the interacting effects of multiple genetic and environmental factors. Genetic variation probably contributes to inter-individual differences in the LVM by virtue of effects on blood pressure level as well as via pathways that are not captured by measurements of blood pressure. It is possible that identification of genes that influence LVM may enhance the ability to detect those patients that deserve early treatment to prevent the development of LVH. In this regard, a recent meta-analysis of case-control studies and association studies has shown that the D allele of the insertion/deletion (I/D) polymorphism of the ACE gene behaved as a marker for LVH in untreated hypertensive patients [78], thus making patients with the DD genotype susceptible to be treated with either ACE inhibitors or AT1 receptor antagonists to prevent LVH. Based on evidence suggesting that angiotensin II may participate in the development of myocardial fibrosis in hypertensive LVH via activation of AT1 receptors, we investigated the potential role of the A1166C polymorphism of the AT1 receptor gene in predicting the antifibrotic effect of antihypertensive drugs [79]. The antifibrotic effect was assessed by measurement of the serum concentration of the carboxy-terminal propeptide of procollagen type I or PICP, a peptide that is cleaved from procollagen type I during the synthesis of fibril-forming collagen type I and that has been shown to be associated with the volume of myocardial tissue occupied by collagen fibers in the human hypertensive heart [80]. In the study, hypertensive patients with LVH were studied before and after 1-year treatment with the AT1 receptor antagonist losartan or the beta-blocker atenolol. Baseline PICP was significantly increased in AA hypertensives compared with AC/CC

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hypertensives. Confounding factors were similar in the two subgroups of hypertensives. Administration of losartan was associated with significant reduction in PICP in AA hypertensives but not AC/CC hypertensives. Treatment with atenolol did not change PICP in either subgroup of hypertensives. Blood pressure was reduced to the same extent in the four treatment subgroups. If these results were confirmed by larger, prospective, double-blind studies, the genotype of the A1166C polymorphism of the AT1 receptor gene could be a useful indicator for antihypertensive drug strategy aimed to reduce myocardial fibrosis in hypertensive patients with LVH.

9.4 Concluding Remarks Hypertensive LVH is the response of cardiac muscle cells to increased hemodynamic load. The increase in ventricular wall thickness normalizes increased wall stress and, therefore, LVH is initially beneficial. However, sustained and progressive mechanical load is associated with additional responses of the cardiomyocyte and noncardiomyocyte compartments of the myocardium that result in its structural remodeling and the deleterious long-term consequences on myocardial function, electrical activity, and perfusion that significantly increase the risk of mortality in hypertensive patients with LVH. Angiotensin II plays a major role in the detrimental response of the myocardium to chronic mechanical overload imposed by systemic hypertension on the left ventricle. Therefore, treatment of hypertensive patients with LVH with drugs inhibiting the RAS will take advantage of its beneficial features while removing the deleterious impact of angiotensin II on the myocardium. Whereas ACEIs and ARAs have proven their efficacy in regressing LVH and reducing the associated risk in hypertensive patients, more studies are necessary to ascertain whether aldosterone blockers and renin inhibitors also possess cardioprotective properties in terms of LVH regression.

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26. Kang, M., Chung, Y., and Walker, J.W. (2007) G-protein coupled receptor signaling in myocardium: not for the faint of heart. Physiology 22, 174–184. 27. Sirker, A., Zhang, M., Murdoch, C., and Shah, A.M. (2007) Involvement of NADPH oxidases in cardiac remodelling and heart failure. Am J Nephrol 27, 649–660. 28. LeWinter, M.M., and VanBuren, P. (2005) Sarcomeric proteins in hypertrophied and failing myocardium: an overview. Heart Fail Rev 10, 173–174. 29. Zou, Y., Akazawa, H., Qin, Y., Sano, M., Takano, H., Minamino, T., Makita, N., Iwanaga, K., Zhu, W., Kudoh, S., To, H., Tamura, K., Kihara, M., Nagai, T., Fukamizu, A., Umemura, S., Iiri, T., Fujita, T., and Komuro, I. (2004) Mechanical stress activates angiotensin II type 1 receptor without the involvement of angiotensin II. Nat Cell Biol 6, 499–506. 30. Leri, A., Claudio, P.P., Li, Q., Wang, X., Reiss, K., Wang, S., Malhotra, A., Kajstura, J., and Anversa, P. (1998) Stretch-mediated release of angiotensin II induces myocyte apoptosis by activating p53 that enhances the local renin-angiotensin system and decreases the Bcl-2-toBax protein ratio in the cell. J Clin Invest 101, 1326–1342. 31. Sugino, H., Ozono, R., Kurisu, S., Matsuura, H., Ishida, M., Oshima, T., Kambe, M., Teranishi, Y., Masaki, H., and Matsubara, H. (2001) Apoptosis is not increased in myocardium overexpressing type 2 angiotensin II receptor in transgenic mice. Hypertension 37, 1394– 1398. 32. Ikeda, S., Hamada, M., Qu, P., Hiasa, G., Hashida, H., Shigematsu, Y., and Hiwada, K. (2002) Relationship between cardiomyocyte cell death and cardiac function during hypertensive cardiac remodelling in Dahl rats. Clin Sci 102, 329–335. 33. González, A., López, B., Querejeta, R., and Díez, J. (2002) Regulation of myocardial fibrillar collagen by angiotensina II. A role in hypertensive heart disease? J Mol Cell Cardiol 34, 1585–1593. 34. Bouzegrhane, F., and Thibault, G. (2002) Is angiotensin II a proliferative factor of cardiac fibroblasts? Cardiovasc Res 53, 304–312. 35. Dostal, D.E. (2001) Regulation of cardiac collagen. Angiotensin and cross-talk with local growth factors. Hypertension 37, 841–844. 36. Stacy, L.B., Yu, Q., Horak, K., and Larson, D.F. (2007) Effect of angiotensina II on primary cardiac fibroblast matrix metalloproteinase activities. Perfusion 22, 51–55. 37. Chua, C.C., Hamdy, R.C., and Chua, B.H. (1996) Angiotensin II induces TIMP-1 production in rat heart endothelial cells. Biochim Biophys Acta 1311, 175–180. 38. Higuchi, S., Ohtsu, H., Suzuki, H., Frank, G.D., and Eguchi, S. (2007) Angiotensin II signal transduction through the AT1 receptor: novel insights into mechanisms and pathophysiology. Clin Sci 112, 417–428. 39. Touyz, R.M. (2000) Oxidative stress and vascular damage in hypertension. Curr Hypertens Res 2, 98–105. 40. Robertson, A.L., and Khairallah, P.A. (1971) Angiotensin II: rapid localization in nuclei of smooth and cardiac muscle. Science 172, 1138–1139. 41. Re, R.N., and Cook, J.L. (2007) Mechanisms of disease: intracrine physiology in the cardiovascular system. Nat Clin Pract Cardiovasc Med 4, 549–557. 42. Baker, B,M, Chernin, M.I., Schreiber, T., Sanghi, S., Haiderzaidi, S., Booz, G.W., Dostal, D.E., and Kumar, R. (2004) Evidence of a novel intracrine mechanism in angiotensin IIinduced cardiac hypertrophy. Regul Pept 120, 5–13. 43. Mosterd, A., D Agostino, R.B., Silbershatz, H., Sytkowski, P.A., Kannel, W.B., Grobbee, D.E., and Levy, D. (1999) Trends in the prevalence of hypertension, antihypertensive therapy, and left ventricular hypertrophy from 1950 to 1989. N Eng J Med 340, 1221–1227. 44. Klingbeil, A.U., Schneider, M., Martus, P., Messerli, F.H., and Schmieder, R.E. (2003) A meta-analysis of the effects of treatment on left ventricular mass in essential hypertension. Am J Med 115, 41–46. 45. Mathew, J., Sleight, P., Lonn, E., Johnstone, D., Pogue, J., Yi, Q., Bosch, J., Sussex, B., Probstfield, J., and Yusuf, S. (2001) Reduction of cardiovascular risk by regression of electro-

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80. Querejeta, R., Varo, N., López, B., Larman, M., Artiñano, E., Etayo, J.C., Martínez-Ubago, J.L., Gutierrez-Stampa, M., Emparanza, J.I., Gil, M.J., Monreal, I., Pardo Mindán, J., and Díez, J. (2000) Serum carboxy-terminal propeptide of procollagen type I is a marker of myocardial fibrosis in hypertensive heart disease. Circulation 101, 1729–1735.

Chapter 10

Angiotensin-(1-7), Angiotensin-Converting Enzyme 2, and New Components of the Renin Angiotensin System Aaron J. Trask, Jasmina Varagic, Sarfaraz Ahmad, and Carlos M. Ferrario

Abstract The discovery of angiotensin-(1-7) [Ang-(1-7)] in 1988 represented the first deviation from the traditional biochemical cascade of forming bioactive angiotensin peptides. Prior to that time, the biological actions of angiotensin II (Ang II) were being investigated as it relates to cardiovascular function, including hypertension, cardiac hypertrophy and failure, as well as biological actions in the brain and kidney. We now know that Ang II elicits a whole host of actions both within and outside of the cardiovascular system. Furthermore, the discovery of Ang-(1-7) by our laboratory was also the first indication of a biologically active angiotensin peptide that further studies revealed served to counter-balance the actions of Ang II. This chapter reviews the data demonstrating the role of the vasodepressor axis of the renin angiotensin system in the regulation of cardiovascular function and the new data that shows the existence of angiotensin-(1-12) as a novel alternate substrate for the production of angiotensin peptides. The ultimate role of this discovery, as well as the continuing elucidation of mechanisms pertaining to RAS physiology, will likely be clarified in the coming years, in hopes of improving the treatment of cardiovascular disease. Keywords Angiotensin-(1-7) · Angiotensin-(1-12) · Hypertension · Mas receptor · Blood pressure regulation

Angiotensin

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10.1 Introduction The existence of the renin angiotensin system (RAS) as a major physiological regulator has been known since Tigerstedt and Bergman first discovered the enzyme renin over a century ago [1]. Nearly 60 years after the initial discovery of renin, A.J. Trask (B) Hypertension and Vascular Research Center, Department of Physiology & Pharmacology, Wake Forest University School of Medicine, Winston-Salem, NC e-mail: [email protected]

W.C. DeMello, E.D. Frohlich (eds.), Renin Angiotensin System and Cardiovascular Disease, Contemporary Cardiology, DOI 10.1007/978-1-60761-186-8_10,  C Humana Press, a part of Springer Science+Business Media, LLC 2009

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Irvine Page from the United States and Braun Menendez from Argentina independently discovered a pressor hormone, “angiotonin” or “hypertensin,” which was later agreeably called “angiotensin” (Ang). We now know this hormone to be the octapeptide pressor hormone, Ang II, which is produced from the sequential cleavage of the protein (Aogen) into Ang I by renin and Ang I into Ang II by angiotensinconverting enzyme (ACE). This linear hydrolysis cascade was undisputed for many years until studies from our laboratory in 1988 showed that a previously considered inert metabolite of Ang II, Ang-(1-7), caused the release of vasopressin from the rat brain hypothalamus [2]. This study was the first demonstration of biological activity of a peptide within the RAS that was not Ang II-mediated. The discovery of Ang-(1-7) expanded our knowledge about the complexities of the RAS and has garnered increasing support for a potential target for the therapeutic treatment of diseases such as hypertension, heart disease, and even cancer [3, 4]. This chapter focuses on the functional role of Ang-(1-7) in the heart, as well as the important contribution that angiotensin-converting enzyme 2 (ACE2) plays in degrading Ang II into Ang-(1-7). While the evidence for a protective role for this counterbalancing arm of the RAS continues to accumulate, we also comment on the identification of a new angiotensin peptide upstream of Ang I, called angiotensin-(1-12), and how it may function in tissues as an alternate precursor for angiotensin peptide production. A diagram of the most current view of the renin angiotensin system is shown in Fig. 10.1.

Fig. 10.1 Current view of the renin angiotensin system. Abbreviations: ADAMs, tumor necrosis factor-α convertases, such as ADAM17; sACE2, secreted ACE2

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10.2 Angiotensin-(1-7): Gaining Favor in the 21st Century The discovery of Ang-(1-7) in the late 1980s did not lend itself to ready acceptance [2, 5–7]. However, studies investigating the role of Ang-(1-7) are on the rise, and a whole new array of data has been emerging on this bioactive peptide since the turn of the 21st century. Most of the biological effects of Ang-(1-7) that are discussed below have been attributed to the mas receptor, which was identified as a functional receptor for Ang-(1-7) [8].

10.2.1 Angiotensin-(1-7) and the Regulation of Cardiac Dynamics The presence of Ang-(1-7) in the heart and the ability of the heart to produce Ang(1-7) were not known for some time. Ang-(1-7) was identified in cardiomyocytes of the heart, but not cardiac fibroblasts, and Averill et al. [9] further showed that its expression was augmented after coronary artery ligation. Several studies have shown that Ang-(1-7) can be synthesized by the heart, and we showed a direct conversion of Ang II into Ang-(1-7) in isolated hearts from normal and hypertensive rats [10]. Early studies investigating a direct role for Ang-(1-7) actions in the heart showed that it was protective against ischemia-induced cardiac dysfunction [11–13], which may be due in part to the activation of the sodium pump [14]. Additional studies in the cardiomyopathy hamster showed that the anti-arrhythmic effects of Ang-(1-7) are mediated through hyperpolarization of the heart cell [15]. Further evidence that Ang-(1-7) is a direct positive effector in the heart stems from data showing its antifibrotic and anti-hypertrophic actions [16–20]. Because Ang-(1-7) is a peptide and thus has a short half-life, several studies have investigated a more stable analog of Ang-(1-7), called AVE0991. The administration of this Ang-(1-7) analog is associated with improvement of cardiac function in diabetic rats [21], improved baroreceptor sensitivity [22], and potentiation of the vasodilator actions of bradykinin [23]. The mas receptor mediates the signaling mechanisms produced by Ang-(1-7) [8]. We further showed that transfection of cultured myocytes with an antisense oligonucleotide to the mas receptor blocked the Ang-(1-7)-mediated inhibition of serum-stimulated mitogen-activated protein kinase (MAPK) activation, whereas a sense oligonucleotide was ineffective [19]. In keeping with these findings, chronic mas deficiency leads to impaired Ca2+ handling in cardiomyocytes in culture [24].

10.2.2 Salt and the ACE2/Ang-(1-7)/mas Axis A clear relationship between salt intake and prevalence of hypertension has been shown in abundant epidemiological and interventional studies [25–28], giving support for the current recommendation for sodium intake of 2,400 mg per day by the American Heart Association. On the other hand, despite numerous studies suggesting that interruption of ACE2/Ang-(1-7)/mas receptor axis may lead to hypertension and cardiac dysfunction [29–31], little is known about its response to

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altered sodium intake with respect to blood pressure changes or target organ damage. Our laboratory was among the first one to report the importance of a tonic depressor activity of Ang-(1-7) to the maintenance of blood pressure in the spontaneously hypertensive rats, with endogenous RAS activation induced by chronic salt depletion [32]. Furthermore, in the face of unchanged plasma Ang-(1-7), an enhanced vascular sensitivity to endogenous Ang-(1-7) in salt-restricted state suggests significant amplification in Ang-(1-7) receptor–signaling interaction. Subsequent studies also revealed that, under the condition of increased renal Ang II due to salt depletion [33] or 2K1C Goldblatt hypertension [34], endogenous Ang-(1-7) counterbalanced the effects of Ang II to maintain a glomerular filtration rate and renal plasma flow [34]. Thus, could it be possible that insufficient synthesis or activity of ACE2/Ang-(1-7)/mas may be a critically important mechanism in saltsensitive hypertension? Indeed, it has been shown that in female Dahl salt-sensitive rats fed highsalt diet, chronic Ang-(1-7) supplementation reduced increase in blood pressure and improved aortic and renal blood flow by increasing prostacyclin and prostaglandinE2 release. It was believed that an increase in plasma levels of nitric oxide following Ang-(1-7) infusion was responsible for this observed vasodilatory effect [35]. It has also been shown that acute vasodilation by Ang-(1-7) was augmented in rats fed high-sodium versus low-salt diet due to an increase in vasodilatory and a decrease in vasoconstrictor prostanoids [36]. But the antagonistic and nitric oxide-independent effect of Ang-(1-7) on Ang II-induced vasoconstriction in aortic rings from the rats fed high-sodium diet was abolished in rats fed low-sodium diet [37]. Thus, further studies are warranted to define precisely a fine-tuning mechanism of Ang-(1-7) in the regulation of blood pressure and flow as well as vascular reactivity in different status of sodium intake. In this context, it is important to note that in salt-sensitive hypertensive patients, omapatrilat, a dual ACE and neprilysin inhibitor, effectively reduced blood pressure and increased urinary excretion of Ang(1-7) [38]. This study clearly pointed out that besides the inhibition of Ang II production and degradation of atrial natriuretic peptide and bradykinin, an Ang-(1-7) of renal origin may contribute to the hypotensive effect of omapatrilat in the patients whose blood pressure is sensitive to sodium intake. Finally, having in mind the anti-hypertrophic and anti-fibrotic effects of Ang(1-7), it is intriguing to hypothesize that salt-induced left ventricular remodeling and renal injury observed in different forms of experimental and human hypertension [39–43] may be, at least in part, governed by alteration of the ACE2/Ang-(17)/ mas axis. In fact, in Dahl salt-sensitive rats fed high-salt diet, cardiac enlargement and fibrosis were associated with an increased cardiac angiotensinogen but reduced cardiac ACE2 mRNA. Treatment with AT1 receptor antagonist, but not mineralocorticoid receptor blocker, reversed the effect of salt on ACE2 gene expression [44]. Importantly, both therapies ameliorated salt-induced cardiac remodeling along with a reduction in angiotensinogen and ACE mRNA. Therefore, it seems that the effects of salt-intake variation or RAS blockade may ultimately depend on their net effects on the two opposing arms of the RAS. Furthermore, low sodium intake in Wistar rats reduced renal ACE, but not ACE2 mRNA and

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activity; this effect was not amplified during ACE inhibition [45]. Neither plasma Ang II nor Ang-(1-7) were affected by low sodium intake, but ACE inhibition increased plasma Ang-(1-7) shifting the balance between the two opposing peptides toward Ang-(1-7) more effectively during a low sodium intake. Moreover, blood pressure was the lowest in the group treated with ACE inhibitor and lowsalt intake. The findings from these studies corroborate well with previous conclusion that anti-hypertensive and cardio-renal protective effects of RAS blockade stemmed, at least in part, from Ang-(1-7) pathway activation [46–48] and that these effects may be more pronounced if followed by dietary sodium restriction [49]. Further studies are clearly necessary to explore whether the beneficial effects of dietary sodium alteration and/or pharmacological intervention indeed depend on preferable ACE/ACE2 and ultimately Ang II/Ang-(1-7) balance in the target organs.

10.3 ACE2: A Critical Enzyme Regulator in the Heart The discovery of the biological effector peptide, Ang-(1-7) in 1988 represented the first expansion of the classical RAS cascade in that it was the only other known peptide member of the RAS to elicit some physiological function. However, the formation of Ang-(1-7) remained elusive for several years. Welches and colleagues [50] first showed that Ang-(1-7) could be formed from the traditional RAS precursor peptide, Ang I, by endopeptidases including prolyl oligopeptidase (POP, E.C. 21.26), neprilysin (NEP, E.C. 24.11), and thimet oligopeptidase (TOP, E.C. 24.15). While it was known that prolyl oligopeptidase could cleave the Pro7 -Phe8 bond of Ang II, the studies were not supported by convincing in vivo evidence. Moreover, studies by Yang et al. [51] found that prolyl carboxypeptidase (PCP, E.C. 16.2), a lysosomal enzyme with an acidic pH optimum, could cleave Ang II into Ang-(1-7). The hydrolysis appeared to be an intracellular cleavage, and the observation that the acidic pH optimum of PCP of 5.0 provided some doubt as to the physiological role for this enzyme in producing Ang-(1-7). It was not until 2000, when two independent research groups described a homolog of ACE, called angiotensin-converting enzyme 2 (ACE2) [52, 53], that a viable Ang-(1-7)-forming enzyme from Ang II was discovered. Shortly after its discovery, Vickers et al. [54] showed that ACE2 could cleave Ang II into Ang-(1-7) with high affinity. ACE2 also cleaved apelin, des-Arg9 -bradykinin, and the opioid peptide dynorphin A 1-13 with similar affinities, but its involvement in modulating these peptides in vivo remains to be clarified. Subsequent studies revealed the generation of Ang-(1-7) in human failing heart tissue, which was dependent on Ang II [55], suggesting that ACE2 was required for the cleavage of Ang II into Ang(1-7). Studies from our laboratory represented the first direct in vivo evidence for ACE2’s participation in hydrolyzing Ang II into Ang-(1-7) in hearts isolated from both normal and hypertensive rats [10]. We further showed that the hypertrophied hearts from hypertensive rats were almost completely reliant on ACE2 for the

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production of Ang-(1-7) from Ang II, whereas ACE2 in the normal heart was of less importance. ACE2 is widely expressed in many tissues in humans [56] and rodents [56, 57], including the heart. In addition, work from this laboratory first demonstrated that cardiac expression of ACE2 mRNA was regulated by the actions of Ang II via an AT1 receptor pathway [58]. A more recent study showed that the negative actions of Ang II on cardiac ACE2 mRNA could be mimicked by the addition of endothelin1 and that both effects could be blocked by inhibitors of mitogen-activated protein (MAP) kinase kinase 1, suggesting that Ang II or endothelin-1 activate ERK1/ERK2 to reduce ACE2 [59]. The importance of ACE2 in the regulation of cardiac function was determined when Crackower and colleagues [29] demonstrated that genetic inactivation of ACE2 in mice resulted in severe blood-pressure-independent systolic impairments in cardiac function, which was associated with significant accumulation of circulating and cardiac Ang II. The concomitant genetic inactivation of ACE completely rescued the ACE2-null cardiac phenotype, further implicating elevated Ang II in cardiac dysfunction observed in the ACE2-null mice. These studies were the first to support the in vivo importance of ACE2 in regulating cardiac function and Ang II metabolism. In addition to these findings, two additional ACE2-null mice strains were generated by separate groups [60, 61]. One strain exhibited cardiac dysfunction only in response to pressure overload, which was also associated with increased cardiac Ang II [61]. The impairments in cardiac dysfunction were abrogated with the co-administration of the AT1 receptor antagonist, candesartan. In contrast, Gurley et al. [60] reported that genetic inactivation of ACE2 in 129/SvEv, C57BL/6, or mixed mouse backgrounds did not induce any functional impairments in the heart, suggesting that the importance of cardiac ACE2 may be dependent on the genetic background of the animal model [62]. In this context, Mercure et al. [63] reported that an eightfold increase in Ang(1-7) in the heart of transgenic animals was associated with less ventricular hypertrophy and fibrosis than their nontransgenic littermates in response to a hypertensive challenge. A view from an opposite approach to determine the physiological importance of ACE2 further favors a cardioprotective role for the enzyme. Indeed, the overexpression of ACE2 protects the heart from Ang II-induced cardiac hypertrophy and myocardial fibrosis in rats [64]. Moreover, elevations in cardiac ACE2 exhibited a partial rescue of the cardiac functional deficits induced by coronary artery ligation in rats [65]. The cardioprotective persona given to ACE2 is further illustrated by its regulation in pathological conditions. Three very important independent studies showed that cardiac ACE2 was upregulated in both humans and rodent models of heart failure [66–68]. Moreover, we first showed that secreted ACE2 (sACE2) was elevated in the cardiac effluent of hypertrophied hearts, suggesting that the enzyme was attempting to protect the heart from progressing toward overt failure as is known in the Ren-2 transgenic rats [10]. These data were very recently supported by human studies that measured sACE2 activity in human plasma, and the authors showed that the sACE2 was indeed markedly increased in patients diagnosed with heart failure [69]. Intriguingly, Lambert et al. [70] recently demonstrated that the tumor necrosis

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factor convertase, ADAM17, participated in the shedding of ACE2 from the membrane, and other studies have reported that ADAM17 is upregulated in heart failure [71]. Collectively, the data on the physiological importance of cardiac ACE2 are clear: it exerts a cardioprotective role from the early stages of cardiac hypertrophy through overt heart failure, although its efforts may be insufficient to overcome the progression of heart disease. However, further studies are required to determine the importance of the discovery of an endogenous ACE2 inhibitor [72], as well as the emerging data that ACE2 is a functional receptor for the SARS coronavirus [73], as these may provide alternative therapeutic targets for the treatment of cardiovascular disease.

10.4 Angiotensin-(1-12) Over the years, questions have been raised regarding the capability of cardiac and vascular tissue to synthesize Ang II [74–78]. The heart remains a critical example. Although a large body of evidence suggests the existence of local tissue RAS in the regulation of cardiac function and remodeling, most studies revealed low levels of gene expression for both cardiac renin and Aogen [79]. Neither the identification of renin in cardiac mast cells [80] excludes an uptake mechanism from the blood compartment nor does the finding of renin activation by binding of prorenin to the prorenin/renin receptor [81–83] can be construed as evidence for local production of cellular renin. Likewise, Aogen gene expression in cardiac tissue has been reported at very low expression levels while the question of how much of the Aogen mRNA is due to its presence in the endothelial cells of intracoronary vessels and cardiac fibroblast has not been answered. Our current view of the RAS as a complex system entailing several levels of regulation and processing is now further expanded with the identification of proangiotensin-12 [angiotensin-(1-12), Ang-(1-12)] as an upstream propeptide to Ang I [84]. These investigators first isolated this novel Aogen-derived peptide from the rat small intestine. Consisting of 12 amino acids, this peptide was termed proangiotensin-12 based on its possible role as an Ang II precursor. Ang-(1-12) constricted aortic strips and, when infused intravenously, raised blood pressure in rats. The vasoconstrictor responses to Ang-(1-12) were abolished by either captopril or the AT1 blocker CV-11974. Current studies from this laboratory now demonstrate the existence of Ang-(1-12) in both the heart and the kidneys of spontaneously hypertensive rats (SHR), primarily restricted to cardiac myocytes and renal tubular cells [85]. In addition, the cardiac content of Ang-(1-12) was significantly augmented in the heart of SHR compared to Wistar-Kyoto (WKY) controls [85]. Moreover, an insight into the processing of Ang-(1-12) into Ang I, Ang II, and Ang-(1-7) was accomplished by studying the effects of exogenously administered Ang-(1-12) in isolated hearts from both normotensive and hypertensive rat strains [86]. In these studies, we showed processing of Ang-(1-12) into Ang I, Ang II, and Ang-(1-7). Moreover, in the group of WKY

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and SHR investigated in this study, the addition of a specific renin inhibitor to the preparation in no manner altered the production of angiotensins from Ang-(1-12). These data showed that Ang-(1-12) is processed into the active angiotensin peptides by a non-renin mechanism. While further work will be required to ascertain the biological role of Ang-(1-12), these data expand on our knowledge of the mechanisms by which the RAS regulates the expression of angiotensins in tissues [87–90].

10.5 Conclusions The discovery of the counter-balancing ACE2/Ang-(1-7)/mas arm of the RAS has expanded knowledge of the intrinsic mechanisms by which the system regulates homeostasis and tissue perfusion in both physiology and pathology [91]. Rapid advances in this field now suggest alternate approaches to suppress the pathological actions of Ang II by enhancing the counter-regulatory actions of Ang-(1-7), augmenting the activity of ACE2, or both. Moreover, the discovery of Ang-(1-12) in multiple tissues including the heart may provide additional mechanistic insights that could lead to the better treatment and management of hypertension and heart failure.

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Chapter 11

Kinin Receptors and ACE Inhibitors: An Interrelationship Ervin G. Erdös, Fulong Tan, and Randal A. Skidgel

Abstract The beneficial effects of angiotensin I-converting enzyme (ACE) inhibitors are due in part to augmenting the actions of bradykinin (BK) and LysBK on their receptors (R). They inhibit kinin inactivation and thereby stimulate the release of mediators such as prostaglandins, nitric oxide (NO), and others. In addition to inhibiting an enzyme, ACE inhibitors affect BK Rs as allosteric effectors in cultured cells, such as human endothelial cells. ACE inhibitors can potentiate BK and ACE-resistant BK analog’s actions on B2 Rs. They elevate arachidonic acid and NO release as indirect allosteric enhancers acting on a heterodimer formed by human ACE and B2 R. This has been shown by co-immunoprecipitation, immunohistochemistry and fluorescence resonance energy transfer (FRET). After carboxypeptidase N or M removes the C-terminal Arg of kinins, the resulting desArg9 -BK and des-Arg10 -Lys1 -BK are inactive on B2 Rs, but are agonists of B1 Rs. Activation of this R leads to prolonged release of NO, synthesized by iNOS. ACE inhibitors are also agonists of the B1 R at a Zn-binding sequence of this heptahelical G protein-coupled R. The site of activation is different from that of the orthosteric peptide ligands; it is on the extracellular loop 2 at residues 195–199. Thus, ACE inhibitors act as direct allosteric agonists. B1 Rs are present mainly in endothelial and other cells after an inflammatory process or induced by cytokines, which also bring about iNOS expression. While constitutively expressed eNOS activation via B2 R results in a short burst of NO, the longer lasting NO release initiated by peptide or ACE inhibitor ligands of the B1R may help to alleviate some detrimental effects in the failing heart. When we received the invitation to contribute to this volume on angiotensinconverting enzyme (ACE) and kinins, we quickly found that an all-encompassing, comprehensive review would be impossible; the number of abstracts dealing with this topic and its ramifications, published during the past 5 years, is 10,677. In a E.G. Erdös (B) Departments of Pharmacology, University of Illinois College of Medicine, Chicago, IL, USA e-mail: [email protected]

W.C. DeMello, E.D. Frohlich (eds.), Renin Angiotensin System and Cardiovascular Disease, Contemporary Cardiology, DOI 10.1007/978-1-60761-186-8_11,  C Humana Press, a part of Springer Science+Business Media, LLC 2009

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Gordon Conference some years ago, I (EGE) surmised that the relationship between bradykinin (BK) and angiotensin (Ang) reminded me of an old, bad marriage, where the association is sometimes antagonistic, other times supportive, but mostly indifferent. However, with changing morals and ACE inhibitors, this association became a ménage à trois, then, when adding receptors, a ménage à quatre or more. Initially, researchers worked on the renin angiotensin and kallikrein-kinin systems happily oblivious of each other. This ended when we showed that kininase II and ACE are one and the same protein, having the dual action of releasing a Cterminal His-Leu dipeptide from Ang I and Phe-Arg from BK [1–4] (Fig. 11.1). ACE was discovered as a factor in horse plasma by Skeggs and associates [5], and we found kininase II in a kidney microsomal fraction [1] and isolated it from human plasma when looking for another enzyme, carboxypeptidase N [3].

ACE Inhibitors

Bradykinin (Kallidin) (Lys)

BK

CPN (plasma)

(1–7)

(Inactive)

des-Arg-bradykinin g y ((Kallidin))

ACE BK

(Lys)

BK

(1–9)

(1–8)

ACE IInhibitors hibit

CPM

B2 Receptor

B1 Receptor Induction: Injury Cytokines Endotoxin

Fig. 11.1 B1 and B2R agonists and their regulation by ACE, carboxypeptidase N and carboxypeptidase M. ACE inactivates BK to BK (1-7), inactive on both B1 and B2Rs. The action of plasma carboxypeptidase N or membrane carboxypeptidase M on BK is required to generate des-Arg-kinin B1R agonists and inactivate BK or kallidin as B2R agonists. B2 Rs are constitutively expressed whereas B1R expression is induced by injury, cytokines, or endotoxin. ACE inhibitors enhance B1R signaling in two ways: (1) they block degradation of BK/kallidin by ACE and thereby enhance substrate levels for carboxypeptidase M/carboxypeptidase N generation of B1R agonists. (2) They can directly bind B1Rs and activate signaling

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Other common links between the two systems were observed later, and at least some of the effects of the kallikrein-kinin system appear to dampen the hypertensive effects of the renin angiotensin system by activating hypotensive responses. It follows that when ACE inhibitors are employed, the primary beneficial effects will result from blocking Ang II release and BK inactivation. However, many laboratory experiments [6, 7] and the treatment of tens of millions of patients with ACE inhibitors have revealed numerous effects [8–12] that could not be explained by only blocking the enzymatic hydrolysis of the two main peptide substrates. Some publications suggested that ACE inhibitors may act somehow independent of enzyme inhibition, also directly on the receptors of BK [13, 14]. Later, it was reported that ACE inhibitors transmit a signal via the intracellular tail of ACE to enhance some kinase activities, which yield elevated COX 2 expression [15].

11.1 ACE Inhibitors and Kinin B1 Receptors We found in experiments done first with bioassays of surviving tissues and in cultured cells that ACE inhibitors affect the two kinin receptors B1 and B2 (B1R and B2R) in different ways [16–18]. The B1R is usually expressed after noxious stimuli, or by adding cytokines to cultured cells [19] or by transfection [20], but some cells (bovine lung endothelial or human fibroblasts) express constitutive B1Rs. The endogenous ligands of B1R are desArg9 -BK and desArg10 -Lys1 -BK (des-Arg10 kallidin). When plasma carboxypeptidase N or cell membrane carboxypeptidase M cleave BK, they remove the C-terminal basic amino acid arginine and thereby liberate the ligands of B1 [19, 21, 22] (Fig. 11.1). ACE inhibitors directly activated B1Rs and released NO in our experiments, most consistently in cultured human endothelial cells, via iNOS [18, 23]. The real-time NO liberation measured with a porphyrinic electrode was in the same range as that of the B1 ligands, desArg10 Lys1 BK and desArg9 -BK, in concentrations ranging from 1 nM to 1 μM assessed at an embarrassing n = 284 times [23] (Brovkovych et al., to be published). Enalaprilat, quinaprilat, captopril, or ramiprilat were active, while lisinopril was not at the same concentrations. This is attributed to structural differences, for example, to the positively charged ε-NH2 group in lisinopril [23–25]. Although des-Arg10 Lys1 -BK had higher affinity for the B1R than desArg9 -BK, the mistaken notion that affinity is equivalent to potency led to statements that des-Arg10 Lys1 -BK is much more potent than desArg9 -BK [26]. We and others [27–29] found that the two peptide agonists are equally active on human B1Rs. Thus, even if their affinities to the human receptor are different, the des-Arg nona- and octa-peptide derivatives have about the same efficacy [27]. The human B1R is directly activated by ACE inhibitors, even in the absence of ACE expression, to generate NO and other mediator release [23–25]. A zincbinding motif (HEMGH) in the active site of ACE is required for its activity and is an important site of attachment of ACE inhibitors [30]. The human B1R contains in its second extracellular loop (residues 195–199) a similar HEAWH sequence, a

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canonical Zn2+ -binding pentamer, which represents the target for B1R activation by ACE inhibitors [18, 25]. Since the orthosteric peptide agonists of B1Rs and the allosteric ACE inhibitor agonists activate the receptor at different sites, the ACE inhibitor activity is blocked by agents or mutations that do not affect des-Arg-kinin activity [23–25, 27]. For example, a synthetic undecapeptide (LLPHEAWHFAR; residues 192–202 of the human B1R), which includes this pentamer, blocked B1R activation by enalaprilat but not by des-Arg-kinin [25, 27]. When the H195 was mutated to Ala in the human receptor, the activation by peptide agonist was unaffected, but that by enalaprilat was much reduced [25] (Tan et al., to be published). The increased enzymatic NO synthesis via B1R activation may also contribute to ACE inhibitors’ therapeutic effects [31] even after an MI [32]. We found that ACE inhibitor/B1R-mediated NO inhibited protein kinase Cε (PKCε) (Fig. 11.2) [27], which could also have beneficial effects on the failing heart [33]. The two endogenous peptide agonists of the B1R are also equally active in stimulating this response, and inhibition of PKCε [27] is independent of blocking Ang II generation or BK metabolism by ACE. ACE inhibitors can enhance B1R activation by another mechanism as well, by elevating its ligand concentrations. Since ACE is the major kinin inactivator, its inhibitors will raise intact kinin levels and consequently the substrate

Fig. 11.2 Stimulation of B1Rs by peptide agonist or ACE inhibitor inhibits PKCε. Human lung microvascular endothelial cells were pretreated with cytokines IL-1β (5 ng/ml) and IFN-γ (200 U/ml) to upregulate B1R and iNOS expression. Cells were then stimulated with either des-Arg10 kallidin (DAKD; 10 nM) or the ACE inhibitor enalaprilat (EPT; 10 nM) for 20 min or pretreated with the B1R antagonist des-Arg10 -Leu9 -kallidin (DALKD; 100 nM). Both DAKD and enalaprilat inhibited PKCε; inhibition was abolished by a B1R antagonist. Ordinate: PKCε relative activity. Data are expressed as mean ± SE (n = 3; done in triplicate). Reproduced from Stanisavljevic et al. [27] with permission from the American Society for Pharmacology and Experimental Therapeutics

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concentration for carboxypeptidases M and N to deliver more B1R agonists [22, 25, 34] (Fig. 11.1). In fact, without carboxypeptidase expression, B1R signaling could not occur, indicating the B1R is a “peptidase-activated” receptor. Our recent studies have shown carboxypeptidase M and B1Rs interact on the cell membrane as determined by co-immunoprecipitation, crosslinking, and FRET analysis [20]. Furthermore, carboxypeptidase M and B1R are co-localized in lipid raft/caveolinenriched membrane fractions and disruption with methyl-β-cyclodextrin reduced the B1R response. A monoclonal antibody to the C-terminal β-sheet domain of carboxypeptidase M also reduced the B1R response to native kinins without inhibiting carboxypeptidase M [20]. The intimate association of carboxypeptidase M and B1R is thus required for efficient delivery of agonist from carboxypeptidase M to the B1R. In bovine or human endothelial cells, B2R agonists stimulate a calcium signal [20] or nitric oxide production [34] that are B1R- and carboxypeptidase M-dependent. Based on the crystal structure of carboxypeptidase M and modeling [35], the glycosylphosphatidylinositol anchor and positively charged residues on one face of carboxypeptidase M likely mediate its interactions with the membrane phospholipids, resulting in the proper orientation of its C-terminal β-sheet domain in relation to the B1R to allow proper interaction and efficient delivery of agonist. Although carboxypeptidase M is expressed constitutively in many cell types [36, 37], its expression can be increased about two- to threefold in cytokine-treated human endothelial cells [34], thus further enhancing B1R signaling in inflammatory or pathological responses.

11.2 ACE Inhibitors and Kinin B2 Receptors The B2R, compared with the B1R, is the more ubiquitous receptor of intact BK and Lys-BK as it is constitutively expressed [19, 38]. Besides protecting BK from enzymatic breakdown by ACE (kininase II), ACE inhibitors can enhance BK actions by potentiating the activation of B2R and by resensitizing the receptor desensitized by the agonist BK [17, 39, 40] (Fig. 11.3). To differentiate the two effects, we tested several ACE-resistant analogs [40–42]. These either had a different covalent bond between Pro7 -Phe8 of BK (Bkan) to make it resistant to ACE [43] or are peptides we synthesized with an enlarged BK N-terminus either coupled to soluble dextran [41] or dansylated at the α- and ε-NH2 in Lys1 -BK (kallidin) [42]. The crystal structure of ACE revealed features that restrict the size of the peptide substrates it can hydrolyze [44] while the B2R still reacts to the enlarged analogue ligands. When Phe8 of BK was substituted with Tyr(Me), the resulting peptide was about 50% resistant to human ACE [45]. These peptide agonists, especially BKan, which we used frequently, could clearly separate the protection against enzymatic breakdown from an indirect augmentation of BK activity at its B2R by acting on ACE [46]. With BK analogs, we found that ACE inhibitors are indirect allosteric enhancers of B2R activity [47, 48], yielding increased mediator (e.g., NO) release; some beneficial effects of ACE inhibitors on the failing heart can involve the activation of both B1 and B2Rs [49]. When human ACE or human B2Rs were singly and

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Fig. 11.3 Schematic diagram illustrating the allosteric activation of B2Rs by ACE inhibitors mediated by conformational change of ACE. ACE has two active-site domains (N-or C-domain) attached by a bridge section, and the C-domain is followed by a “stalk” region, a hydrophobic transmembrane helix, and a short cytosolic tail. Based on the crystal structure, ACE has a deep active-site cleft largely closed to the exterior by a “lid” region (left) that would require conformational change to allow access of substrates or inhibitor (right). Thus, binding of ACE inhibitor to the N- or C-domain would alter the conformation of ACE, which is constitutively associated with B2Rs in a heterodimer. This conformational change would then result in movement of the B2R, likely mediated by the C-domain of ACE, resulting in enhanced mediator release or resensitization of the receptor

separately transfected into cultured cells, which otherwise lacked the two proteins, ACE inhibitors ceased to be allosteric enhancers. They worked as such only when both enzyme and receptors were present on the same cells. The inhibitors, acting through ACE, can increase B2R signaling, resulting in enhanced release of arachidonic acid or Ca2+ [40, 42, 50] (Fig. 11.3). Others have noticed that various agents, including some so-called BK potentiating snake venom peptides [51], related to the first clinically employed ACE inhibitor teprotide [52], can enhance BK effects on isolated tissues, even if they do not block BK inactivation [53, 54]. We obtained convincing initial pieces of evidence by using “ancient” bioassay techniques. BK contracts the isolated guinea pig ileum isotonically [17, 55]. When we added an ACE inhibitor at the peak of the contraction into the tissue bath, within seconds it doubled the magnitude of contraction (Fig. 11.4). This happened despite the very low kininase activity of the guinea pig preparation (t 1/2 = 12–16 min for BK), thus it could not have been due to blocking BK inactivation. Using an isolated guinea pig atrial preparation, enalaprilat augmented the positive inotropic effects of BK, also independent of its inactivation [46].

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Fig. 11.4 Resensitization of B2R causing contraction of guinea pig ileum by ACE inhibitor. Bradykinin (BK; 50 nM) contracts an isolated segment of the guinea pig ileum. BK is immediately potentiated by the ACE inhibitor enalaprilat (EPT; 0.2 μm) which, given at the height of isotonic contraction, further enhanced it. Reproduced from Erdös et al. [17] with permission from Elsevier

BK as an agonist induces phosphorylation of Ser residues in B2Rs [56]. ACE inhibitors diminish the phosphorylation [48], but not by acting directly on the B2R in cultured cells. From these and other experiments with specific inhibitors, we concluded that activation of B2R by BK [46, 48] and its potentiation or reactivation of the desensitized B2R by ACE inhibitors initiate different signal transduction pathways [57]. In our experiments, othosteric agonists (BK) reduce receptor signal to a second dose of agonist, which is tachyphylaxis or desensitation [57]. However, added allosteric modifiers modulate this effect. Resensitization by ACE inhibitors [58] restores sensitivity to B2R agonist BK. ACE inhibitor elevates the efficacy of B2Rs, and allosteric interactions between ACE and the receptor stabilize it in a more favorable conformation (Fig. 11.3). To determine what regions of human 150–180 kDa ACE participate in the potentiation of BK effects [47], mutated and chimeric ACE were synthesized [40] and N- and C-domain-specific reagents were used. ACE inhibitors potentiated BK by acting through either the N- or C-domain. The active center of ACE was involved in ACE effects because monoclonal antibodies [47] or sequestering agent binding the Zn2+ cofactor also potentiated the peptide agonists. But adding enalaprilat (1 μM) to the treated and inhibited enzyme further augmented BK effects [46]. For ACE inhibitors to be allosteric enhancers of B2R, both ACE and B2R would have to be expressed close enough on the plasma membrane to transmit allosteric effects induced indirectly by ACE inhibitors via ACE to the B2R [48] (Fig. 11.3). We approached the problem using several techniques [40, 42]. First, we investigated whether ACE and B2R interact by co-immunoprecipitation with polyclonal antibodies against human ACE. We tagged B2R at its C-terminal end with green fluorescent protein (GFP), to use an antibody elicited to GFP. ACE and the B2R fused with GFP were coprecipitated from lysed CHO cells, either with antibody to ACE or GFP as shown in Western blot. Next, we employed immunohistochemistry of fixed cells and located ACE with antibody to human ACE (stained red) and the B2R by green fluorescence, owing to the fused GFP. The enzyme and the receptor colocalized as indicated by the appearance of intense yellow coloring of the merged images [42].

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Fig. 11.5 FRET between ACE and B2 receptors. CHO cells co-expressing human ACE-CFP and B2-YFP were analyzed, and linear unmixing of CFP and YFP emission spectra was done with the Zeiss LSM-510 META detector. YFP fluorescence was photobleached in the region of interest by scanning with a 514 nm laser, and post-bleach images were collected. The increase in donor (ACE-CFP) intensity concomitant with the decrease in acceptor (B2R-YFP) fluorescence following bleaching indicates energy transfer (FRET). This shows that ACE and B2Rs colocalize within 10 nm on the cell membrane

We then employed fluorescence resonance energy transfer (FRET) to gain an estimate of the closeness of ACE and B2R on the membrane. For this we fused yellow fluorescent protein (YFP) with B2R as acceptor and cyan fluorescent protein (CFP) with ACE as donor. We measured FRET as enhanced fluorescence of the donor after acceptor photobleaching. The significant increase in the ACE-CFP signal after photobleaching B2R-YFP (Fig. 11.5 ) indicates that the fluorophores on ACE and B2R were within 10 nm on the cellular plasma membrane. Thus, besides the well-studied phenomena that many receptors can form homo- or heterodimers or oligomers [59], human ACE and B2R can also be considered a heterodimer. This complex formation by ACE and B2 should be a bimolecular reaction, which depends on the concentration of the reactants. If ACE is present in excess, ACE inhibitors could accelerate the activation of B2R by kinins more because the reaction would proceed as a pseudo-first order one. It also follows that when cells express many more B2Rs than ACE, ACE inhibitors would not as effectively potentiate BK as an agonist of B2R. These experiments indicate that ACE inhibitors acting on ACE induce a conformational change in B2R and thus become indirect allosteric enhancers of B2R agonists’ activity [42]. In the FRET assays above, ACE and B2Rs were tagged with CFP or YFP at their C-termini. B2R is a heptahelical G protein-coupled receptor whereas ACE is anchored to the plasma membrane by a short transmembrane sequence and a cytosolic tail, as a continuation of its C-domain [30] (Fig. 11.1). The N-domain of ACE is connected to the C-domain by a so-called bridge-section, susceptible to some proteases [60–62] which can liberate an intact N-domain. ACE is released from

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the plasma membrane by trypsin or secretase cutting at a stalk section [30, 63]. The ratio of extracellular amino acid sequence in ACE compared to the cell-bound transmembrane and cytosolic portions is about 24:1. The distribution for human B2R is quite the opposite. Its free N-terminus and extracellular loops, compared to the transmembrane plus cytosolic sequences, yield a 1:13 ratio of “free” extracellular to “bound”/intracellular portions. Thus, to establish whether the N-terminal fluorescent tags would be close enough in the heterodimer to generate FRET. To express B2R with a fluorescent protein at the N-terminus presented additional technical difficulties as attempts to express a simple fusion protein were unsuccessful. To overcome this difficulty, we (Skidgel, Tan, Chen, Erdös, to be published) fused the coding sequence for a signal peptide [20] to the N-terminus of the fluorescent protein, which facilitated the expression of B2R on the membrane. Experiments carried out as above yielded FRET between N-terminally tagged ACE and B2R, confirming our results with the C-terminal tags. As a negative control, one protein was tagged at the N-terminus and the other at the C-terminus, yielding only a low level of FRET through the membrane. The precise mechanism by which ACE inhibitors allosterically modify B2R conformation via ACE is still being explored, but our hypothesis is that ACE inhibitors induce a conformational change in ACE, which is then transmitted to the B2R by virtue of their close association on the membrane (Fig. 11.3). This is supported by several lines of evidence. First, ACE inhibitors induce phosphorylation of ACE’s Cterminal tail and activate signal transduction pathways that increase the expression of proteins such as COX-2 [15]. Second, the two active domains of ACE exhibit negative cooperativity so that binding of an ACE inhibitor to one domain alters the conformation of the second domain, making it no longer accessible to a second molecule of inhibitor [64, 65]. Third, the crystal structure of either the C- or N- domains of ACE revealed the presence of two subdomains surrounding a deep active site cleft largely closed to the exterior by a “lid” that would require conformational change to allow access of substrates or inhibitor [44, 66]. Fourth, normal mode analysis of the ACE structure showed intrinsic flexibility around the active site, and a hinge mechanism was proposed to explain opening of the subdomains to allow substrate/inhibitor binding [66]. Taken together, these data support our finding that ACE inhibitors act as indirect allosteric enhancers of B2R signaling by binding to ACE, which causes a conformational change in ACE transmitted to the receptor via the heterodimer, enhancing receptor function (Fig. 11.3).

11.3 Other Considerations The two BK receptors have different agonists and antagonists, and B2 agonists activate eNOS while B1R ligands activate iNOS [23, 34, 67]. B1 and B2Rs also have a rather complex interrelationship. While both B1R and B2R contribute to the maintenance of normal blood pressure, in B2R knockout mice constitutive B1R expression is upregulated [68]. Vice versa, B2Rs can compensate for a lack of B1Rs in mice, with myocardial infarction [69].

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Carboxypeptidase M, which generates B1R agonist peptides, is closely associated with B1Rs on plasma membranes, and in this complex, B1Rs responded to B2 agonists [20, 34]. ACE inhibitors can increase B1R agonist (Des-Arg9 -BK, DesArg10 -Lys1 -BK) release by carboxypeptidase N or M [20, 22, 70]. Hypothetically ACE inhibitor could decrease Ang 1-7 appearance by blocking Ang II liberation by ACE. ACE 2 releases Ang 1-7 mainly from Ang II [71], which is also a substrate of prolylcarboxypeptidase [72]. But the conversion of Ang I to Ang 1-7 by human neprilysin (neutral endopepeptidase 24.11) may be enhanced by ACE inhibitors, which elevate Ang I levels [73]. Although neprilysin concentration is low in plasma and in endothelial cells [74, 75], it occurs abundantly in other tissues and cells, for example, in the renal proximal tubules [76], fibroblasts [77] and leukocytes [78].

11.4 Similarities in the Development of Concepts: Kinins and Angiotensin In addition to ACE (kininase II) and its inhibitors linking the renin and kallikrein systems, more interactions were found and some novel concepts introduced recently. For example, how does blood-borne renin, an aspartic protease having only one well-defined substrate in plasma, angiotensinogen, become active when it is needed to restore blood pressure after a precipitous drop or when it is not really needed, causing clinical hypertension? [79, 80] Human renin circulates as 92% inactive prorenin [81] with little evidence for a significant activation in circulation and, anyhow, human renin does not cleave its plasma substrate human angiotensinogen efficiently at blood pH. The discovery of renin–prorenin receptors and the concept of dual Ang receptors answered unasked questions and shifted the paradigm, as described elsewhere in this volume. Our concepts dealing with the kallikrein-kinin system developed in some ways comparable to the renin angiotensin system. A good part of the actions of ACE inhibitors are attributed to protecting BK or Lys-BK against enzymatic breakdown. This requires that a free kinin be present to act on its receptor, usually B2, leading to enhanced mediator release, e.g., NO, prostaglandins, endothelium-derived hyperpolarizing factor (EDHF) [82]. This process starts with the activation of the proenzyme, prekallikrein, by an activator [83]. Then kallikrein cleaves plasma kininogen to release a kinin, which is a substrate of kininases. This complex cascade, instead of being the only process available to deliver a BK agonist, may be bypassed or “shunted.” As shown in cultured cells [45] and in rats genetically depleted of kininogen (kallikrein substrate) [84], kallikrein can directly activate the B2R even in the absence of kinin release. This can be enhanced [85] by ACE inhibitors if ACE is also expressed in the same cells. Thus, besides the major function of ACE inhibitors to block the kininase activity, as allosteric enhancers they can augment the activities of some proteases acting on BK B2R directly without the intermediate release of a peptide [84, 86].

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11.5 Epilogue The beneficial effects of ACE inhibitors are due in part to augmenting BK and Lys1 BK effects on their receptor. They inhibit their inactivation, potentiate mediator release after receptor activation, and resensitize the B2R desensitized by peptide agonist. On the B2Rs, ACE inhibitors can also be considered as indirect allosteric enhancers via ACE. After their C-terminal Arg is cleaved by carboxypeptidase M or N, the resulting desArg9 -BK and desArg10 -Lys1 -BK lose affinity to the B2R and become ligands of the B1R (Fig. 11.1). ACE inhibitors can also directly activate the B1R to cause a prolonged NO release via iNOS [23–25, 27]. Such an activation of iNOS via B1R [23–25, 34] can aid the failing heart. Overexpression of iNOS in hearts of transgenic mice decreased infarct size and reduced the detrimental effects of reperfusion [87]. Besides Ang I and BK, ACE also cleaves the metabolites of the two peptides further. Ang 1-9 and Ang 1-7 also are endogenous allosteric enhancers of B2R activation via ACE, at least in cultured cells [47, 50, 57]. Ang 1-9 is converted to Ang 1-7, but very slowly, and then Ang 1-7 is also broken down by ACE to Ang 1-5. The reaction rates for these substrates are much lower, the specificity constants are 1/4th–1/10th of that of Ang I. BK is rapidly inactivated by ACE, and the split product BK 1-7 is cleaved further by ACE to BK 1-5. This pentapeptide is considered to be the final BK metabolite in human blood [83, 88] with an intact N-terminal Arg1 -Pro2 bond, in contrast to publications emphasizing an aminopeptidase (cleaving N-terminal Arg) as a main BK inactivator in human blood [89]. Our conclusions are more self-evident than strikingly revolutionary. Receptors are like humans; they usually do not work singly and have immediate consequences, which are mainly local.

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Chapter 12

Kinins and Cardiovascular Disease Oscar A. Carretero, Xiao-Ping Yang, and Nour-Eddine Rhaleb

Abstract Autocrine, endocrine, and neuroendocrine hormonal systems are important factors that regulate cardiovascular and renal function. Alteration of the balance among these systems may result in hypertension and target organ damage. Changes in this balance could be due to (a) genetic factors and/or (b) environmental factors. Endocrine and neuroendocrine vasopressor hormonal systems, such as the renin angiotensin system, aldosterone, and catecholamines, play a well-established and important role in the regulation of blood pressure and the pathogenesis of some forms of hypertension and target organ damage. The role of vasodepressor autacoids such as kinins is less well established. However, there is increasing evidence that vasodepressor hormones not only play an important role in the regulation of blood pressure and renal function but may also oppose remodeling of the cardiovascular system. Here we will primarily review the role of kinins, which are oligopeptides containing the sequence of bradykinin. They are generated from precursors known as kininogens by enzymes such as glandular (tissue) and plasma kallikrein. Some of the effects of kinins are mediated via autacoids such as eicosanoids, nitric oxide (NO), endothelium-derived hyperpolarizing factor (EDHF), and/or tissue plasminogen activator (tPA). Acting via these mediators, kinins play an important role in the regulation of cardiovascular and renal function as well as some of the cardiovascular and renal effects of angiotensin-converting enzyme (ACE) and angiotensin type 1 receptor antagonists (ARB). A study of Utah families revealed that a dominant kallikrein gene expressed as high urinary kallikrein excretion was associated with a decreased risk of essential hypertension. Also, a restriction fragment length polymorphism (RFLP) that distinguishes the kallikrein gene family in one strain of spontaneously hypertensive rats (SHR) from normotensive Brown Norway rats has been identified; in recombinant inbred substrains derived from these SHR and Brown Norway strains, the RFLP marking the kallikrein gene family of the SHR cosegregated with an increase in blood pressure. However, humans, rats, and mice with a deficiency of one component of the kallikrein-kinin system or chronic blockO.A. Carretero (B) Hypertension and Vascular Research Division, Department of Medicine and Heart and Vascular Institute, Henry Ford Hospital, Detroit, MI, USA e-mail: [email protected] W.C. DeMello, E.D. Frohlich (eds.), Renin Angiotensin System and Cardiovascular Disease, Contemporary Cardiology, DOI 10.1007/978-1-60761-186-8_12,  C Humana Press, a part of Springer Science+Business Media, LLC 2009

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ade of the kallikrein-kinin system do not have hypertension. In the kidney, kinins participate in the regulation of papillary blood flow and water and sodium excretion. B2 -KO mice appear to be more sensitive to the hypertensinogenic effect of salt. Kinins participate in the acute antihypertensive effect of ACE inhibitors; however, in general, they are not involved in the chronic antihypertensive effects of ACE inhibitors except for the acute phase of mineralocorticoid-salt-induced hypertension. Kinins acting via nitric oxide (NO) participate in the vascular protective effect of ACE inhibitors during neointima formation. In myocardial infarction produced by ischemia/reperfusion, kinins play an important role in the reduction of infarct size induced by preconditioning or ACE inhibitors. In heart failure secondary to infarction, the therapeutic effects of ACE inhibitors are partially mediated by kinins via the release of NO. The therapeutic effect of ARB in heart failure is partly due to activation of angiotensin type 2 receptors via kinins and NO. Thus kinins could play an important role in the regulation of cardiovascular and renal function as well as in many of the beneficial effects of ACE inhibitors and ARB.

12.1 Introduction Both genetic and environmental factors acting via intermediary phenotypes participate in the regulation of blood pressure, the etiology of hypertension, and the development of target organ damage. Vasoactive systems are an important component of these intermediary phenotypes. They can act as local hormones (intracrine, autocrine, and paracrine) or as endocrine and neuroendocrine systems. We use the term intracrine to indicate hormones which act within the cells that synthesize them, such as reactive oxygen species (O2 – ) and products of proto-oncogenes. The term autocrine is used to indicate hormones which act on the cell membrane receptors where they are produced, such as growth factors. The term paracrine denotes hormones which act near the site where they are produced, such as kinins, eicosanoids, nitric oxide (NO), and endothelium-derived hyperpolarizing factor (EDHF). Endocrine refers to hormones such as aldosterone which are released into the extracellular fluid and act on distant target tissues, though they can also act in an autocrine and paracrine faction. Finally, neuroendocrine hormones such as catecholamines are released by neurons and act near to or distant from the site of release. Blood pressure is the result of a balance between vasopressor and vasodepressor systems. Alteration of this equilibrium may result in: (a) hypertension, (b) target organ damage, (c) effective antihypertensive treatment, or (d) hypotension and shock. Changes in this balance could be due to (a) genetic factors such as mutations in one of the genes of the vasoactive system and/or (b) environmental factors which alter the activity of vasoactive systems. Endocrine and neuroendocrine vasopressor systems, such as the renin angiotensin aldosterone system and catecholamines, play a well-established and important role in the regulation of blood pressure, the pathogenesis of some forms of hypertension, and target organ damage. The role of vasodepressor systems is less well established; however, evidence suggests that they play an important role in the regulation of blood flow, renal function, the pathogenesis of salt-induced hypertension and target organ damage, and the cardioprotective

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effects of angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARB) [1–5]. Vasodepressor hormones such as kinins, eicosanoids, NO, and EDHF act as local hormonal systems, opposing the effects of vasopressor systems. Some vasodepressor systems such as atrial (ANF), brain (BNP), and C-type (CNP) natriuretic peptides may act as both endocrine and local hormones. Here we will review the kinin-generating system and the role of kinins in: (1) regulation of local blood flow; (2) water and sodium excretion; (3) regulation of blood pressure and pathogenesis of hypertension; and (4) the therapeutic effects of ACE inhibitors and ARB.

12.2 The Kinin-Generating System Kininogenases such as glandular and plasma kallikreins are enzymes that generate kinins by hydrolyzing substrates known as kininogens, which circulate at high concentrations in plasma. Kinins are rapidly destroyed by a group of peptidases known as kininases (Fig. 12.1). Plasma and glandular (tissue) kallikrein are potent kininogenases and are both serine proteases. A single gene encodes for plasma kallikrein, and there is a large family of glandular kallikrein genes; however, KLK1 is the only glandular kallikrein that generates kinins (hereafter referred to as glandular kallikrein, or simply kallikrein). Plasma kallikrein, also known as Fletcher factor, is expressed mainly in the liver; in plasma it is found in the zymogen form (prekallikrein) and differs from glandular kallikrein in its biochemical, Glandular Kallikrein

Kininogenases (enzymes)

Plasma Kallikrein N-site

C-site

X-Ser-Leu-Met-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Ser-X

Bradykinin

Kininogens (substrates)

Kinins

Lys-Bradykinin

NEP-24.15

Aminopeptidase 3.4.11.2 3.4.11.9

Kininase I 3.4.11.2

Kininase II (ACE; 4.4.15.1) NEP-24.11

Kininases (peptidases)

Fig. 12.1 Site of kininogen cleavage (solid arrows) by the main kininogenases (glandular and plasma kallikrein). The broken arrows indicate sites of kinin cleavage by kininases (kininase I, kininase II, neutral endopeptidases 24.11 and 24.15, and aminopeptidases). (Modified from [1])

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immunological, and functional characteristics. It preferentially releases bradykinin from high-molecular-weight kininogen (HMWK), also known as Fitzgerald factor. Together with HMWK and Hageman factor, plasma kallikrein is involved in coagulation, fibrinolysis, and possibly activation of the complement system. The plasma kallikrein-HMWK system, acting through the release of bradykinin, could be involved in the local regulation of blood flow and in some of the effects of ACE inhibitors. On the other hand, patients with congenital deficiency of plasma HMWK (Fitzgerald trait) have normal amounts of kinins in their blood [6]. (For a review of the plasma kallikrein-HMWK system, see [7–9]) Kallikrein belongs to a family of serine proteases with very high homology; the genes encoding for these enzymes are tightly clustered and arranged in tandem on the same chromosome. The number of family members varies widely among mammals; it is estimated that the kallikrein family contains at least 3 genes in humans, 20 in the rat and 23–30 in the mouse, many of them pseudogenes [10]. Despite the highly homologous amino acid composition of the serine proteases encoded by the kallikrein gene family, most are not kininogenases and act on entirely different substrates. For example, tonin, a rat enzyme of the kallikrein family, hydrolyzes angiotensinogen and generates angiotensin II; prostate-specific antigen, a human enzyme of the kallikrein family, hydrolyzes semenogelin, a high-molecular-weight seminal vesicle protein [11, 12]. We have isolated a new member of the kallikrein family from the submandibular gland [13, 14]. This protease produces contraction of isolated aortic rings and (like tonin) also generates angiotensin II, suggesting that localized regions of variability are important in determining substrate specificity and possibly function of all enzymes of the kallikrein family. (For a review of the molecular biology of the glandular kallikrein-kininogen system, see [15–17].) True kallikrein or KLK1 is encoded by a single gene having five exons and four introns. Other members of the kallikrein gene family have a similar exonic and intronic structure, with the splice junctions completely conserved. The 5’ and 3’ flanking regions have a high homology among the various genes; however, gene regulation and site of expression are different, suggesting that small variations in the nucleotide sequence of the 5’ region are important in the regulation of expression. The kallikrein gene is expressed mainly in the submandibular gland, pancreas, and kidney; however, using the polymerase chain reaction, we have demonstrated its mRNA in vascular tissue, heart, and adrenal glands though in smaller amounts [18, 19]. Kallikrein and kallikrein-like enzymes have also been found in the arteries and veins [20], heart [21], brain [22], pituitary gland [23, 24], pancreas [25], intestine [26, 27], salivary and sweat glands [28], spleen [29], adrenal glands [30], blood cells [19], and the exocrine secretions of these structures. Some of them are probably true kallikrein, while others may be separate members of the kallikrein family. There is immunoreactive kallikrein in plasma, primarily the inactive form, with only a small portion being in the active form [31–35]. In humans [36] and rabbits [37], 50% or more of urinary kallikrein is the inactive or zymogen form, while in rats most is in the active form [38]. Kallikrein can release kinins from lowmolecular-weight kininogen (LMWK) and HMWK. In humans, kallikrein releases lys-bradykinin (kallidin), whereas in rodents it releases bradykinin [39, 40].

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Homozygous tissue kallikrein-deficient mice exhibit renal hypercalciuria and become hypocalcemic under low-calium (Ca) diet conditions as a result of defective tubular Ca reabsorption [41]. However, B2 −/− mice treated with a B1 receptor antagonist did not exhibit any change in urinary Ca excretion and adapted normally to the low-Ca diet conditions, indicating that tissue kallikrein may be a physiologic regualtor of renal tubular Ca transport via a non-kinin-mediated mechanism. In humans, a loss-of-function polymorphism in exon 3 of the tissue kallikrein gene results in the substitution of an active-site arginine at position 53 to a histidine (R53H), with substantial loss of kallikrein activity (50–60% lower than in normal) [42]. In these individuals there was increased Ca reabsorption in thick ascending limb under baseline conditions that counteracted a defect in distal tubule Ca reabsorption. However, more pharmacogenetic studies are needed to examine whether abnormal Ca regulation by the kidney in R53H individuals is linked causally to the kallikrein mutation [42]. Kininogens (kallikrein substrates) are the precursors of kinins. In plasma there are two main forms, characterized as LMWK and HMWK [43, 44]. Both are potent inhibitors of cysteine proteinases such as calpain and cathepsins H, L, and B [45– 47]. In the rat there is a third kininogen known as t-kininogen, because it releases kinins when incubated with trypsin but not with tissue or plasma kallikrein. It is one of the main acute reactants of inflammation in the rat. All kininogens also inhibit thiol proteases, such as cathepsin M, H, and calpains [48–51]. HMWK is involved in the early stages of surface-activated coagulation (intrinsic coagulation pathway) [7, 9, 52]. Kininases are peptidases found in blood and other tissues, which hydrolyze kinins and other peptidic hormones [53]. The best known is angiotensin-converting enzyme (ACE) or kininase II, which converts angiotensin I to II and inactivates kinin substance P and other peptides [53, 54]. Another important kininase is neutral endopeptidase 24.11 (NEP-24.11), also known as enkephalinase, which not only hydrolyzes kinins and enkephalins but also destroys ANF, BNP, and endothelin [55, 56]. Research performed in our laboratory suggests that it may be an important renal kininase, at least in the rat [57]. Other kininases include MEP-24.15, aminopeptidases, and carboxypeptidases; however, it is not known whether they play an important role in the degradation of kinins in vivo. After inhibition of most of these enzymes in vivo, plasma concentrations of endogenous kinins do not increase significantly and their half-life remains less than 20 s, suggesting that other peptidases are also important in kinin metabolism [58]. Kinins are oligopeptides containing the sequence of bradykinin in their structure and act mainly as local hormones, since they circulate at very low concentrations (1–50 fmol/ml) and are rapidly hydrolyzed by kininases. In tissues such as the kidney, heart, and aorta, kinin concentrations are higher (100–350 fmol/g) [59], suggesting that kinins act mainly as local hormones. Eicosanoids, NO, EDHF, tPA, and cytokines mediate at least some of the effects of exogenously administered kinins [60–64] (Fig. 12.2). At least two subtypes of kinin receptors have been well characterized using analogs of bradykinin, B1 and B2 [65, 66]. These receptors have been cloned and belong to the family of seven transmembrane receptors

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B2 - receptor

B1 - receptor

2

Eicosanoids cAMP

EDHF

NO

K++-channels cGMP

T-PA Plasmin

GLUT-1 & -4 Glucosei

Depressor / Natriuretic/Antitrophic Fibrinolysis / ROS Scavenger/ O2 consumption Improve cardiac metabolism

Inflammation Pain/Fibrosis ?

Fig. 12.2 Kinins act via the B2 and B1 receptors. Most of the known effects of kinins are mediated by the B2 receptor, which in turn acts by stimulating the release of various intermediaries: eicosanoids, endothelial derive hyperpolarizing factor (EDRF), nitric oxide (NO), tissue plasminogen activator (tPA), glucose transporter (GLU- 1 & -2 (modified from [234]))

linked to G-proteins [67]. B1 receptors are not present or only present at very low density in normal tissues, but are expressed and synthesized de novo during tissue injury, inflammation, and administration of lipopolysaccharides such as endotoxin. In some species, including rabbits, they mediate contraction of the isolated aorta and relaxation of mesenteric arteries. The main agonists for this receptor are des-Arg9 bradykinin and des-Arg10 -kallidin. B2 receptors mediate most of the effects of bradykinin and are the main receptors for the agonists, bradykinin, and kallidin (lysbradykinin). Studies using kinin analogs with agonistic and antagonistic properties in various tissues suggest the existence of other subtypes of receptors [68–72] discovered that substitution of D-phenylalanine for proline at position 7 of bradykinin converts it into a specific antagonist for B2 receptors; while the substitution of Phe8 in des-Arg9 -BK by a residue with aliphatic (Ala, Ile, Leu, D-Leu, norleucine) or saturated cyclic hydrocarbon chain (cyclohexyalanine) produced antagonists for B1 receptors [73, 74]. Further modifications have resulted in a very potent B2 receptor antagonist with long-lasting effects in vivo, DArg0 -[Hyp3 -Thi5 -DThi7 -Oic8 ]bradykinin or icatibant (Hoe-140) [75], which has become an important tool for studying the role of kinins. More recently, oral active kinin antagonists have been developed for the possible treatment of inflammation, hyperalgesia, and perhaps cancer [76–80]. In humans, it has been reported that the B2 receptor is activated by kallikreins and other serine proteases and that this effect is blocked by the kinin antagonist icatibant [81]. Some ACE inhibitors potentiate the effect of bradykinin not only by

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inhibiting its hydrolysis but also by cross-talk between ACE and the B2 receptor [82]. The B2 receptor also forms heterodimers with the angiotensin type 1 receptor (AT1 ), causing increased activation of the angiotensin receptor [83]. The stability of this heterodimer is not affected by bradykinin, angiotensin, or their respective receptor antagonists [84]. The B2 receptor also forms a complex with eNOS, inhibiting the generation of NO, and this effect is reversed by bradykinin [85]. The pathophysiological role of these interactions of the B2 receptor is not known; however, it has been reported that in preeclampsia there is an increase in AT1 and B2 heterodimers that could mediate the enhanced response to angiotensin II [86]. Thus, in some situations these interactions of the B2 receptor could play a physiopathological role.

12.3 The Kallikrein-Kinin System in the Vasculature and in the Regulation of Local Blood Flow Arteries and veins contain a kallikrein-like enzyme, and both vascular tissue and smooth muscle cells in culture contain mRNA for kallikrein [18, 20]. Vascular smooth muscle cells in culture release both kallikrein and kininogen [87]. Thus the components of the kallikrein-kinin system are present in vascular tissue, where they could play an important role in the regulation of vascular resistance. Recently, in isolated arteries of mice with deletion of the gene expressing kallikrein, blood flow-induced dilatation was found to be significantly reduced compared to controls, suggesting that the kallikrein-kinin system in the arterial wall participates in this process [88, 89]. Also, in humans, a partial genetic deficiency in tissuel kallikrein (R53H) was reported associated with an inward remodeling of the brachial artery, which is not adapted to a chronic increase in wall shear stress. This form of arterial dysfunction affects 5–7% of white population [90]. The effect of ACE inhibitors on local potentiation of kinins’ vasodilator effect appears to be partly attributable to their prevention of bradykinin degradation and subsequent increases in the production of endothelium-derived relaxing factors (EDRFs) such as NO. In SHR and in the canine coronary artery, ACE inhibitors potentiate the endothelium-dependent relaxation evoked by bradykinin [91, 92]. This vasorelaxation appears to be associated primarily with increased release of NO. When the arterial endothelium is removed, the smooth muscle cells begin to proliferate in the media; they then migrate across the internal elastic lamina into the intima, where they cause neointimal hyperplasia, mimicking some of the vascular changes that occur in atherosclerosis. ACE inhibitors have been shown to inhibit neointima formation [93, 94]. Blocking kinins or inhibiting NO synthesis lessens the protective effect of the ACE inhibitor, suggesting that it may be mediated by a local increase in kinins which stimulates the release of NO [95, 96]. Kinins play an important role in the local regulation of blood flow in organs rich in kallikrein, such as the submandibular gland, uteroplacental complex, and kidney [97–100]. In rats nephrectomized 48 h earlier to exclude the renal renin angiotensin system, use of an angiotensin I-converting enzyme (kininase II) inhibitor significantly increased blood flow in the submandibular gland but did not affect

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blood pressure. In contrast, 10 min after sympathetic stimulation of the gland to increase kallikrein secretion in the vascular compartment, the ACE inhibitor markedly decreased blood pressure and increased kinin concentrations in arterial blood [35, 101]. Changes in both blood flow and blood pressure were blocked by antibodies to kinins and kallikrein. The effect of the ACE inhibitor on basal glandular blood flow was also blocked by a kinin antagonist [97]. At low doses the antagonist caused no significant change in blood flow when the ACE inhibitor was not administered, whereas at high doses basal blood flow decreased significantly. These data suggest that in organs rich in kallikrein, kinins play a role in the regulation of basal blood flow. Studies using kinin antibodies and antagonists clearly indicate that kinins act as paracrine hormones, regulating blood flow within the gland. These studies also indicate that the effect of ACE inhibitors on blood flow is mediated by kinins [97]. In nephrectomized pregnant rabbits infused with an angiotensin antagonist to block the uterine renin angiotensin system, ACE inhibitors increased both uterine and placental blood flow and immunoreactive PGE2 , whereas these effects were blocked by a kinin antibody [98]. This suggests that endogenously generated kinins play a role in the regulation of uterine blood flow, either directly or through the release of prostaglandins. In conclusion, in organs rich in glandular kallikrein, such as the submandibular gland and uteroplacental complex, kinins appear to play an important role in the regulation of blood flow, especially when ACE is inhibited. In addition, the local arterial kallikrein-kinin system also participates in the regulation of blood flow and in the vascular protective effect of ACE inhibitors.

12.4 Kinins in the Regulation of Renal Blood Flow Kinins may also play an important role in the regulation of renal blood flow. Blocking renal kinins by infusing low doses of a kinin antagonist into the renal artery of sodium-depleted dogs decreased renal blood flow and autoregulation of the glomerular filtration rate (GFR) without changing blood pressure [102]. The changes in renal blood flow were blocked by prior inhibition of ACE, suggesting that either those changes were mediated by renin release due to an agonistic effect of the kinin antagonist or renal kinins may have increased when ACE was inhibited, thereby competing more effectively with the antagonist. The changes in GFR autoregulation were not altered by the ACE inhibitor and may have been due to a change in either the relationship between afferent and efferent glomerular arteriolar resistance or the coefficient of filtration [102]. We have recently shown that the vasodilator effect of bradykinin in the renal efferent arteriole is mediated by cytochrome P450 metabolites of arachidonic acid called epoxyeicosatrienoic acids (EETs) and that bradykinin stimulates the glomeruli to release another cytochrome P450 metabolite, the vasoconstrictor eicosanoid 20-hydroxyeicosatetraenoic acid (20-HETE), and also an unidentified vasodilator prostaglandin, which together participate in regulation of the downstream glomerular circulation and perhaps in the regulation of GFR [103, 104].

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We examined the role of kinins in the regulation of renal blood flow distribution using a laser-Doppler flowmeter [99]. The kinin antagonist lowered papillary blood flow without altering outer cortical blood flow, suggesting that intrarenally formed kinins are important in regulating blood flow in the inner medulla. This study also showed that renin angiotensin system (RAS) plays an important role in the regulation of papillary blood flow, since after kinins were blocked, enalaprilat increased flow significantly. We also found that when we inhibited both ACE and neutral endopeptidase-24.11 (NEP-24.11), papillary blood flow increased by 50%, compared to 25% when they were inhibited separately. These increases were blocked by the kinin antagonist, indicating that the augmented papillary blood flow induced by both ACE and NEP-24.11 inhibitors is mediated by increased kinin concentrations in the interstitial space. We observed no consistent effect on water or sodium excretion; however, water excretion tends to decrease in animals treated with a kinin antagonist. In anesthetized rats, blocking kinins decreased renal blood flow [100]. In dogs, when kallikrein excretion was stimulated by sodium deprivation, a kinin antagonist (given intrarenally) partially blocked the effect of enalaprilat on renal blood flow [105]. This suggests that although blockade of the renin angiotensin system accounted for a significant portion of the increase in renal blood flow caused by the ACE inhibitor, a substantial component was contributed by endogenous kinins. Similar results were reported in rats in which the kallikrein-kinin system was stimulated by deoxycorticosterone [106]. In the kidney, kinins play a minor role in the regulation of blood flow; however, when the kallikrein-kinin system is stimulated by low sodium intake or mineralocorticoids, or when endogenous kinin degradation is inhibited, kinins appear to participate in the regulation of renal blood flow [107, 108]. In addition, the data suggest that kinins play an important role in the regulation of papillary blood flow, and that during reduction of renal perfusion pressure, kinins may aid in the regulation of the GFR [109].

12.5 Kinins in the Regulation of Water and Electrolyte Excretion Renal kallikrein is located in the connecting cells of the connecting tubule; it is released in significant amounts in this segment of the nephron and excreted in the urine (Fig. 12.3) [37, 110, 111]. Kallikrein releases kinins into the lumen of the distal nephron, either from filtered kininogen or kininogen produced in the principal cells of the distal nephron [112, 113]. Kinin receptors are also present in the collecting duct [114]. In addition, kallikrein is released on the basolateral side of the nephron, where it may liberate kinins from plasma kininogens [115]. The interstitial renal fluid contains a high concentration of kinins [116]. The role of kinins in the regulation of water and sodium excretion has been studied by increasing intrarenal kinins, blocking kinins, or deleting the genes expressing kinin receptors or tissue kallikrein [41, 117–120]. Infusion of kinins into the late proximal nephron doubled excretion of simultaneously administered 22 Na [117], and that part of this effect was

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Fig. 12.3 Localization of the kallikrein-kinin system, renin, and prostaglandin in the nephron (right brackets), anatomical subdivisions or the nephron (outer left brackets), and type of cells found in the distal nephron (inner left brackets). PGE2 = prostaglandin E2 ; PGI2 = prostacyclin

mediated by prostaglandins [121], while infusion of a kinin antagonist into the late proximal nephron reduced 22 Na recovery significantly [122]. After systemic administration of phosphoramidon, an inhibitor of NEP-24.11 (a major kininase in the nephron), urinary excretion of kinins doubled; diuresis increased by 15% and natriuresis by 37% [57]. Although these data support the hypothesis that increased kinins in the nephron participate in intrarenal control of water and electrolyte excretion, it is also possible that the effect of this peptidase inhibitor is mediated by blocking hydrolysis of other peptides such as atrial natriuretic factor (ANF) [123]. Infusion of aprotinin inhibited the enzymatic activity of urinary kallikrein but did not affect acute water or electrolyte excretion in euvolemic and sodium- or water-expanded rats (124]. A transient decrease in sodium excretion has been observed during aprotinin administration in mineralocorticoid-treated rats [125]. Infusion of kinin antibodies into saline-expanded rats decreased sodium excretion [118]; however, caution should be used in interpreting this finding, since antibodies may stimulate release of histamine, cause an anaphylactoid reaction, or form a high-molecular-weight complex with kininogen, which is then deposited in the nephron, any of which might alter water and sodium excretion. To avoid these problems, we use Fab fragments of kinin antibodies, which are rapidly distributed in the extracellular fluid and excreted by the kidney; moreover, they do not form

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high-molecular-weight complexes or activate complement and other proteolytic systems in plasma, thus reducing the risk of anaphylactoid reactions. In unanesthetized rats, the Fab fragments blocked 70% of the effect of an injection of 100 ng bradykinin on blood pressure and appeared rapidly in the urine, suggesting that they block the effect of kinins not only in the vascular and interstitial spaces but also in the lumen of the distal nephron. Using these Fab fragments and a kinin antagonist, we studied a model in which the renal kallikrein-kinin system is stimulated, namely, DOCA-salt-treated rats. Both the Fab fragments and kinin antagonist significantly decreased urine volume and increased urinary osmolarity; however, only the Fab fragments significantly lessened urinary sodium excretion, but without affecting blood pressure, renal blood flow, or GFR [108]. The antidiuretic effect of the Fab fragments and kinin antagonist may be due to blockade of kinins in the vascular interstitial space of the kidney, since the antagonist is likely hydrolyzed in the proximal tubule and does not reach the lumen of the distal nephron. On the other hand, the antinatriuretic effect of Fab fragments of kinin antibodies on sodium excretion may be due to blockade of kinins in both the vascular/interstitial and the urinary compartments, and only the latter compartment, since the antibody appeared in the urine and the antidiuretic effect was not observed with the antagonist. Thus kinins may aid in the regulation of water and sodium excretion when the kallikrein-kinin system is stimulated. In normal non-anesthetized rats, inhibition of kinin release in the lumen of the nephron by Fab fragments of monoclonal antibodies to kallikrein causes urinary PGE2 , UV, and UNa V to decrease. The changes in UV and UNa V mimic those of PGE2 , suggesting that the natriuretic and diuretic effects of kinins are mediated in part by PGE2 [119]. Tissue kallikrein-deficient mice were reported to lack the 70-kDa form of γENaC (epithelial sodium channel) consistent with reduced renal ENaC activity in tissues that normally express tissue kallikrein, such as in cortical collecting duct. However, in mice lacking B2 receptors, the abundance of the 70-kDa form of γ-ENaC was increased, indicating that its absence in tissue kallikreuin-deficient mice is not kinin-mediated [126]. In vitro, stimulation of the release of EDRF from endothelial cells by bradykinin or acetylcholine increases cGMP content and inhibits Na+ transport by cortical collecting duct cells [127]. In vivo, stimulation of EDRF release by bradykinin induces natriuresis and diuresis without affecting the GFR [128]. In conclusion, kinins acting as local hormones play a role in the regulation of renal hemodynamic and excretory function, either directly or via the release of PGE2 and EDRF.

12.6 Kinins as Regulators of Blood Pressure and Pathogenesis of Hypertension The development of antibodies to kinins and kallikrein, kinin antagonists, and kininase inhibitors, as well as gene knockout (KO) models of the kallikrein-kinin system and the discovery of kininogen-deficient rats and humans with various spontaneous mutations of the system, has allowed us to study the role of kinins

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in various physiological and pathological conditions. The role of the kallikreinkinin system in the pathogenesis of hypertension has been studied by (1) measurements of the various components of the system, (2) the use of bradykinin B2 receptor antagonists, (3) using mice in which the B1 , B2 , or both B1 and B2 receptors have been deleted by homologous recombination, (4) deletion of the tissue kallikrein gene, and (5) using rats deficient in kininogen. Decreased activity of the kallikrein-kinin system may play a role in hypertension. Low urinary kallikrein excretion in children is one of the major genetic markers associated with a family history of essential hypertension, and children with high urinary kallikrein excretion have less probability of a genetic background of hypertension [129–132]. Also, a restriction fragment length polymorphism for the kallikrein gene family in spontaneously hypertensive rats has been linked to high blood pressure [133], and urinary kallikrein excretion is decreased in several models of genetic hypertension. Urinary and/or arterial tissue kallikrein are also decreased in renovascular hypertension and genetically hypertensive rats [134–137]. Although these reductions may be secondary to increases in blood pressure, decreased urinary kallikrein in normotensive children of patients with essential hypertension and in genetically hypertensive and Dahl salt-sensitive rats prior to the development of hypertension [138–142] suggest that the cause of these decreases may be different. Kinins circulate in concentrations of approximately 5–50 pg/ml of blood [6]. These concentrations need to be increased to at least 100 pg in humans [143] and 1,000 pg in rats [144] to cause acute decreases in blood pressure. Although blood kinin concentrations may increase in some physiological and pathological situations, they seldom reach levels that could explain changes in blood pressure, save for exceptional experimental conditions such as stimulation of the sympathetic nerve of the submandibular gland in animals treated with ACE inhibitors (see section on blood flow regulation). Thus kinins would have to act as paracrine hormones, regulating local vascular resistance and organ function. In early studies, acute administration of a kinin antagonist at high doses increased blood pressure in most rats tested, while a vasodepressor effect was observed in some [145]. Using a more potent antagonist [146], also at high doses, we found that it produced a transient biphasic response: first a small pressor effect, followed by a depressor effect [147]. At smaller doses, though still sufficient to block exogenous bradykinin, the same antagonists did not alter normal blood pressure. These studies appear to be somewhat compatible with the hypothesis that kinins play a role in the regulation of blood pressure. However, in order to demonstrate the pressor effect, the kinin antagonist has to be used at much higher doses than those needed to block the vasodepressor effect of exogenous bradykinin. High doses may be needed to displace kinins bound to tissue receptors. We must be cautious in interpreting these data, since we cannot rule out the possibility that these kinin antagonists have a vasopressor effect, which is unrelated to kinin-blocking activity. Studies by our group using kinin antibodies or their Fab fragments showed that although they partially block the vasodepressor effect of kinins, they do not cause acute changes in blood pressure.

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Normal blood pressure and cardiovascular are often observed in HMWKdeficient rats, B1 −/− or B2 −/− mice [148–150]. However, in mice lacking tissue kallikrein, despite having normal blood pressure, experience very obvious abnormal structure and function of the heart [89]. Chronic blockade of B2 kinin receptors with a potent and selective B2 antagonist, icatibant, did not increase blood pressure under normal conditions or under conditions that favor the development of hypertension in rats, such as (1) chronic infusion of a subpressor or pressor dose of Ang II, (2) a high-salt diet, (3) or mineralocorticoids and salt [149, 151]. However, these results are not universal [152–155]. Mice with the bradykinin B2 receptor deleted by homologous recombination (gene knockout) have normal blood pressure (Fig. 12.4). However, they develop hypertension when fed a high-sodium diet (8%) for at least 2 months [156, 157]. Thus low kinin activity may be involved in the development and maintenance of saltsensitive high blood pressure. However, in these mice hypertension induced by mineralocorticoids (renin independent) or aortic coarctation (renin independent) was not exacerbated [158]. Also, it has been reported that as these mice grow older, they also develop hypertension and left ventricular hypertrophy even on a normal sodium diet [159]. However, we were unable to confirm that ablation of B2 kinin receptors renders mice spontaneously hypertensive [148]. Others were also unable to confirm the hypothesis that B2 kinin receptors are a major component in the maintenance of normal blood pressure and cardiac structure [120, 157, 158, 160]. Mice deficient in kinin B1 , B2, or B1 /B2 receptors or tissue kallikrein had similar blood pressure to wild-type controls, confirming that kinins are not an important determinant of blood pressure [160]. Moreover, mice lacking both B1 and B2 receptors did not present

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Fig. 12.4 Diurnal and nocturnal mean blood pressure of bradykinin B2 receptor knockout (–/–) and wild type (+/+) mice. Blood pressure was measured 24 h by a telemetric system (Rhaleb N-E and Carretero O.A. unpublished data)

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any significant change in blood pressure after an increase in dietary salt intake [161]. The latter contrasts with previous observations, demonstrating a chronic hypertensive effect of high-salt diet in B2 −/− mice [156, 157]. High-salt diet duration could play an important factor in these differences. Indeed, Alfie et al. [156] could not observe hypertension until after 8 weeks of high-salt diet, and Cervenka et al. [157] started high-salt diet during gestation period and continued for up to 4 months of age. On the other hand, B1 B2 −/− were subjected to high salt for only 5 weeks [161]. It is unknown whether B1 −/− develop hypertension when given high salt. Furthermore, wild-type mice or mice in which the gene expressing tissue kallikrein has been deleted exhibited similar increase in blood pressure when submitted to renovascular hypertension [162], further supporting the minor role of kallikreinkinin system in the control of blood pressure under basal conditions or during hypertension. In kininogen-deficient Brown Norway Katholiek rats (BNK), administration of mineralocorticoids and salt or angiotensin II reportedly causes blood pressure to increase similarly to rats with a normal kallikrein-kinin system [149]. This contradicts with other reports [152–154]. In conclusion, these studies suggest that kinins do not play an important role in the regulation of normal blood pressure or in the pathogenesis of hypertension, although they may be involved in the pathogenesis of salt-induced hypertension. Overall, chronic blockade of the kallikrein-kinin-system does not appear to cause hypertension or potentiate hypertensinogenic stimuli, though the data are inconsistent.

12.7 Role of Kinins in the Antihypertensive Effect of ACE Inhibitors Inhibition of kinin and degradation of other oligopeptides may contribute to the antihypertensive effect of ACE inhibitors. While blockade of angiotensin II formation appears to be important in this regard, the role of kinins is less well established. Orally active ACE inhibitors are effective antihypertensive agents, not only in highrenin hypertension but also in clinical and experimental models in which the renin angiotensin system has not been pathogenetically implicated [163, 164]. Thus some effects of ACE inhibitors may be mediated by a local renin angiotensin system, kinins, or some other undetermined mechanism, since ACE can hydrolyze other peptides (Fig. 12.5). ACE inhibitors may also potentiate the effect of kinins by a direct interaction with the kinin B2 receptor [82]. Blood kinins are unchanged or moderately increased after the administration of ACE inhibitors [1, 165, 166] (for review, see [167, 168]). Kinins in the urine reportedly increase more consistently after the administration of ACE inhibitors, indicating that their concentration in renal tissue likewise increases [57, 169–172]. This in turn may contribute to the antihypertensive effect of ACE inhibitors by altering renovascular resistance and increasing sodium and water excretion.

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HYPOTHETICAL MECHANISMS OF ACTION OF ANGIOTENSIN CONVERTING ENZYME INHIBITORS ACE INHIBITORS

Ang-II

• Direct • Aldosterone • Sympat. Transm. • TxA2 /PGH2 • Growth Factors

Kinins

• Direct • PGI2 • NO • EDHF • Glucose upt. • tPA

Ac-SDKP

• Direct? • Inh fibrob. proliferat. • Inh collagen deposition

Other Substrate

Ang-I / Ang 1-7 • Opioid Pept. • Substance P • Neurotensin • Chemot. Pept. • LHRH, etc

Direct Effect

• Bk receptor • Scavenger O–2 • NO • Conductance

O. A. Carretero, HFH, Detroit, MI

Fig. 12.5 ACE has multiple substrates, and inhibition of their hydrolysis may explain the cardioprotective effect of ACE inhibitors

Many studies have assessed the role of kinins in the acute antihypertensive effect of ACE inhibitors. In various experimental models of hypertension, the acute antihypertensive effect of ACE inhibitors is attenuated by blocking kinins with either high titers of kinin antibodies [173–175, 174, 176] or with a B2 kinin receptor antagonist [165, 166, 177]. Kinin antagonists also partially reverse the antihypertensive effect of ACE inhibition in rats with renovascular hypertension [176]. However, deficiency in B2 kinin receptors did not affect the antihypertensive effect of ACE inhibitors in mice with renovascular (2 kidney-1 clip) hypertension (Fig. 12.6). This is not surprising since it is well established that renin angiotensin system plays a major role in the development of renovascular hypertension. We assessed the influence of kinins on the acute antihypertensive effect of enalaprilat in rats with severe hypertension induced by aortic ligation between the renal arteries [166]. In this model, renin plays an important role in the pathogenesis of hypertension [163]; however, acute and severe hypertension can produce endothelial damage that may lead to activation of plasma kallikrein and increased kinin formation. We found that enalaprilat lowered mean blood pressure by 48 ± 6 mm Hg in the controls and 21 ± 4 mm Hg in the kinin antagonist group, which is a significant difference (p < 0.01); however, kinin concentrations in arterial plasma were not significantly altered by the ACE inhibitor (41 ± 10 vs. 68 ± 20 pg/ml) (Fig. 12.7). As indicated earlier, if mean blood pressure in the unanesthetized rat is to be decreased, kinins in arterial blood must reach at least 1,000 pg/ml [144]. Thus the effect of the ACE inhibitor may be due to an increase in tissue kinins, which could act as a paracrine hormonal system regulating vascular resistance. Cachofeiro et al. [165] demonstrated that pretreatment with either a bradykinin antagonist or a NO synthesis inhibitor attenuated the acute antihypertensive effect of both captopril and ramipril in SHR, but pretreatment

166 Sham (n = 5) 2K-1C (n = 4) 2K-1C + ramipril (n = 5)

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Fig. 12.6 Antihypertensive effect of ACE inhibitor in bradykinin B2 receptor knockout (–/–) mice. Mice with 2 K-1C hypertension were given plain water (vehicle) or water mixed with ACE inhibitor, ramipril (4 mg/kg/day) to drink 5 weeks after blood pressure was increased. ACE inhibitor normalized blood pressure in B2 −/− hypertensive mice. ∗ p < 0.001, 2 K-1C versus sham;∗∗ p < 0.001, 2 K-1C + ramipril versus untreated 2 K-1C (Rhaleb N-E and Carretero O.A. unpublished data)

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Fig. 12.7 Role of kinins in the acute antihypertensive effects of an ACE inhibitor (enalaprilat) in rats with severe hypertension. Top: Blood kinin concentrations before (C) and after administration of the ACE inhibitor. Bottom: Mean blood pressure before and after ACE inhibition, open and closed circles represent rats pretreated with a kinin antagonist or vehicle, respectively. Values are mean ± SEM (bottom) (reprinted from Carbonell et al. [166])

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with a prostaglandin synthesis inhibitor failed to alter the effects of ACE inhibitors, suggesting that this acute antihypertensive effect is due to bradykinin acting via the release of NO. However, in the dog, kinins may play a role in the acute hypotensive effect of ACE inhibitors through the release of prostaglandins [178]. In humans, it has been demonstrated that an ACE insertion (I)/deletion (D) polymorphism in intron 16 of the ACE gene could be an important determinant for bradykinin metabolism [179]; ACE activity is higher in subjects with ACE D and is associated with a high rate of bradykinin degradation. In normotensive subjects and hypertensive patients with low and normal renin, aprotinin (an inhibitor of kallikrein and other proteases) blocked part of the acute antihypertensive effect of captopril [180]. The influence of aprotinin could be due to inhibition of kinin formation or other effects. However, using a specific B2 kinin receptor antagonist (icatibant), the short-term blood pressure effects of ACE inhibitors were attenuated in both normotensive and hypertensive subjects [181]. In conclusion, these studies suggest that part of the acute effect of ACE inhibitors on blood pressure is mediated by kinins, which affect local and peripheral vascular resistance either directly or through release of prostaglandins and NO. The contribution of kinins to the chronic antihypertensive effects of ACE inhibitors is more controversial. In renovascular hypertension (2K1C), chronic blockade of kinin receptors interferes with the blood pressure-lowering activity of ramipril [182]. In mineralocorticoid hypertension, in which kallikrein-kinin and ACE activity are reportedly increased [183], chronic ACE inhibitors have a small but significant antihypertensive effect; blocking the B2 receptor with icatibant blunted this chronic antihypertensive action [149, 184], suggesting that in this model kinins may play a role in the antihypertensive effect of ACE inhibitors. However, they do not appear to contribute to the chronic antihypertensive effects of ACE inhibitors in SHR (182] or in hypertension induced by aortic coarctation [165, 185, 186]. Therefore, the role of kinins in the long-term antihypertensive effect of ACE inhibitors depends on the model. To our knowledge, no studies of chronic blockade of the kallikrein-kinin system have been conducted in humans.

12.8 Role of Kinins in the Cardiac Antihypertrophic Effect of ACE Inhibitors ACE inhibitors have been shown to reverse LV hypertrophy in essential hypertension and in various experimental models. This decrease is partly due to reduced afterload; however, it has been postulated that this antihypertrophic effect may be independent of the decrease in blood pressure. A decrease in angiotensin II formation, which stimulates various proto-oncogenes and growth factors, may participate in the antihypertrophic effect of ACE inhibitors acting independently of its effect on blood pressure. The cardiac kallikrein-kinin system may also participate in the effect of ACE inhibitors on the heart. Doses of ACE inhibitors that do not decrease blood pressure reverse LV hypertrophy in rats with hypertension due to aortic coarctation [187]. However, to be certain that blood pressure does

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not decrease, direct 24-h measurements are needed. The antihypertrophic effects of ACE inhibitors have been reported to be reversed by a kinin antagonist [188]. However, using a very similar protocol, we have not been able to confirm this [186]. Further studies are needed to determine whether doses of ACE inhibitors which do not decrease blood pressure (24-hour blood pressure monitoring) reverse cardiac hypertrophy, and whether kinins participate in this effect. Capillary length and density increase in hearts of SHR treated with an ACE inhibitor at both “antihypertensive” and “non-antihypertensive doses”. There is strong evidence that angiotensin II also has significant angiogenic effects [189]; however, the effect of an ACE inhibitor on capillary growth could not be attributed to the inhibition of angiotensin production alone, since that effect is blocked by concomitant treatment with a selective B2 receptor antagonist, icatibant, suggesting that this effect of the ACE inhibitor may be due to kinins [190].

12.9 Role of Kinins in Myocardial Ischemia and in the Protective Effect of Ischemic Preconditioning and ACE Inhibitors Both human and animal studies have demonstrated that kinins are released from the heart and their release is rapidly increased during ischemia. This release of kinins could have a cardioprotective effect [191–193]. Indeed, experimental studies have shown that intracoronary infusion of bradykinin significantly limited infarct size, reduced the incidence of ventricular arrhythmias, improved cardiac performance, and normalized myocardial metabolism [194–196]. Tissue kallikrein deficiency was reported to aggravate cardiac remodeling and survivaval rate after ischemiareperfusion injury in mice [197]; whereas B2 −/− mice or Brown Norway Katholiek rats (HMWK-deficient) responded similarly to ischemia-reperfusion injury compared to their respective wild types [3], indicating that others than kinins participate in the cardioaprotective effecst of tissue kallikrein. It has also been suggested that kinins are an important mediator of ischemic preconditioning, in which repeated brief coronary occlusions render the myocardium more resistant to injury from subsequent prolonged ischemia. In patients undergoing angioplasty, balloon inflation for 1 min (which mimics ischemic preconditioning) increased kinin concentrations in the coronary sinus 50-fold compared to pre-inflation values [2]. Preconditioning almost doubled cardiac interstitial kinin concentrations compared to nonpreconditioned hearts subjected to ischemia [193]. The role of kinins in ischemic preconditioning was further demonstrated in our laboratory using animals genetically lacking B2 kinin receptors or deficient in kinins. We found that in B2 kinin receptor knockout mice as well as rats deficient in HMWK, the cardioprotective effect of preconditioning was abolished or significantly blunted [3]. During myocardial ischemia followed by sympathetic nerve stimulation, kinins in coronary sinus blood increase significantly [198]. An ACE inhibitor was shown to reduce myocardial infarct size after ischemia/reperfusion, whereas an angiotensin II antagonist (losartan) did not [199, 200]. In nephrectomized dogs in which infarction was induced by occlusion of the coronary artery for 90 min, blockade of local angiotensin II formation with protease inhibitors had no significant effect on

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myocardial infarct size despite decreased angiotensin II release. Captopril did not alter local angiotensin II formation but did increase bradykinin and reduce infarct size, suggesting that kinins were responsible for the effect of the ACE inhibitor on infarct size [192]. Similarly, when low doses of the ACE inhibitor ramiprilat (which had no systemic effect) were infused into the left coronary artery in dogs, they reduced the infarction caused by ligation of the descending branch of the left coronary artery [194]. This cardioprotective effect of ramiprilat was mimicked by bradykinin and abolished by co-administration of a kinin antagonist. ACE inhibitors have been shown to reduce ischemia/reperfusion injury, including infarct size and reperfusion arrhythmias. Recently, it was recognized that the cardioprotective effect of ACE inhibitors is due to inhibition of not only angiotensin II formation but also kinin degradation [4, 5, 199, 201–203]. In animal models of ischemia/reperfusion injury, we and others have shown that ACE inhibitors reduced infarct size and ventricular arrhythmias and these effects of ACE inhibitors were abolished or attenuated by co-administration of a B2 kinin antagonist [194, 199, 200, 204]. We further showed that the infarct-limiting and anti-arrhythmia effects of ACE inhibitors were also blocked by inhibition of NO or prostaglandin synthesis [200] and diminished in eNOS gene knockout mice [205]. Moreover, deleting the gene-expressing tissue kallikrein was also associated with decreased cardioprotective effects of ARB in mice subjected to ischemia-reperfusion injury [206]. The cardioprotective effect of kinins may be mediated in several ways. Release of NO from the endothelium may be stimulated either directly or via prostaglandins. It has been shown that myocardial ischemia increases kinin release, accompanied by increased release of cGMP (an indicator of NO production) and 6-keto-PGF1α (a metabolite of prostacyclin) [207, 208], whereas inhibiting NO or prostaglandin synthesis diminishes or blocks the cardioprotective effect of kinins [209, 210]. Kinins improve cardiac metabolism by increasing high-energy phosphate production and glucogen content in the heart, which could be mediated by facilitating translocation of intracellular glucose transporters (GLUT1 and GLUT4), thereby increasing glucose uptake [211, 212]. This is important because during ischemia the source of energy production is shifted from the oxidation of fatty acids to glycolysis. Also, activation of protein kinase C (PKC) has been shown to be involved in the protective mechanism of preconditioning [213–215]. Activation of kinins causes further phosphorylation of a secondary effector, presumably the ATP-sensitive potassium channels (KATP ). Kinins have been shown to activate PKC, thereby stimulating opening of KATP and leading to cardioprotection [216, 217]. Such responses may favorably influence functional and metabolic events during ischemic episodes and protect against ischemia/reperfusion injury.

12.10 Role of Kinins in the Cardioprotective Effect of ACE Inhibitors in Heart Failure Post-MI There is overwhelming evidence that ACE inhibitors reduce morbidity and mortality, improve cardiac function, regress LV remodeling, and prolong life in patients with heart failure (HF). We showed that in a rat model of HF due to surgically

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Fig. 12.8 Two-dimensional M-mode echocardiographs of B2−/− mice and B2+/+ mice with sham coronary ligation (sham) or HF. IS indicates interventricular septum; DD, LV diastolic dimension; and PW, LV posterior wall (Modified from [5])

induced myocardial infarction (MI), ACE inhibitors improved cardiac function and attenuated remodeling, as evidenced by increased ejection fraction and decreased LV dilatation, myocyte hypertrophy, and interstitial fibrosis. Furthermore, these beneficial cardiac effects of ACE inhibitors were diminished by blockade of kinins [203]. The role of kinins in the cardioprotective effect of ACE inhibitors was confirmed by the fact that in B2 kinin receptor knockout mice (Fig. 12.8) and kininogendeficient rats post-MI, the effect of ACE inhibitors was significantly diminished or absent [5, 202]. Although the precise mechanism by which kinins protect the heart is not yet well defined, accumulated evidence suggests that kinin-stimulated release of NO and/or prostaglandins may be largely responsible. Bradykinin stimulates the release of NO from the mouse myocardium and decreases myocardial oxygen consumption; these effects are blocked by a B2 kinin antagonist and absent in B2 receptor knockout mice [218]. We have shown that the effect of the ACE inhibitor was almost abolished in endothelial NO synthase (eNOS) knockout mice with HF postMI [219]. Taken together, these findings may suggest that kinins acting on the B2 receptor via the release of NO play an important role in the cardioprotective action of ACE inhibitors. More recently, we have demonstrated that not only B2 but also B1 kinin receptors contribute to the cardiac therapeutic effect of ACE inhibitor [150]. We found that while kinin B1 R does not appear to play an essential role in cardiac

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hemodynamics and function either under normal conditions or during development of heart failure, it may be involved in maintaining morphological integrity, since mice with targeted deletion of B1 R had increased LV mass and chamber dimension at basal conditions. Furthermore, the cardioprotective effect of ACE inhibitor is reduced in mice lacking the gene expressing B1 receptors. In conclusion, in patients with heart failure, ACE inhibitors have been shown to not only improve cardiac function and increase survival but also decrease the rate of myocardial re-infarction [220]. The mechanism of this decrease is not known; however, since ACE inhibitors may block kinin degradation in the coronary circulation, one hypothesis is that kinins stimulate the release of EDRF and PGI2 , which are important inhibitors of platelet aggregation. Since kinins are potent stimulators of the release of tPA [64, 221], it is also possible that this potentiation of tPA release may in turn activate plasmin and fibrinolysis. Although the exact mechanism of action of ACE inhibitors in re-infarction is not known, these hypotheses open up an exciting new area of cardiovascular research.

12.11 Role of Kinins in the Cardioprotective Effect of Angiotensin Receptor Blockers (ARB) Two subtypes of angiotensin II (Ang II) receptors, AT1 and AT2 , have been identified. Most biological actions of Ang II are known to be mediated by the AT1 receptor, whereas little is known about the function of AT2 receptors. In cultured endothelial cells, Ang II stimulates the release of NO and this effect was blocked by either an AT2 or B2 receptor antagonist, indicating that Ang II-stimulated NO release is mediated via activation of the AT2 receptor and a kinin-dependent mechanism [222]. Mice overexpressing the AT2 receptor were reported to have increased kininogenase activity in the vasculature [223]. Since blockade of the AT1 receptor increases angiotensin II levels, which in turn may activate the AT2 receptor, it is rational to hypothesize that the cardioprotective effect of ARB is mediated in part by kinins via activation of the AT2 receptor. In fact, we found that ARB improved cardiac function and ameliorated remodeling in rats with CHF post-MI, and that these effects of ARB were significantly attenuated by an AT2 or B2 receptor antagonist [203] or in mice lacking the AT2 receptor [224]. Using B2 kinin receptor knockout mice and kininogen-deficient rats, as well as eNOS knockout mice, we confirmed that lack of kinins or endothelium-derived NO diminished the cardioprotective effect of ARB [4, 5, 219], indicating that increased release of kinins and NO due to activation of the AT2 receptor is an important mediator of the cardioprotective effect of ARB. In addition, we recently reported B1 R mediate part of the cardioprotective effects of ARB in mice with HF post-MI [150]. Because angiotensin II also plays a critical role in the regulation of blood pressure and in the pathogenesis of many models of hypertension, the interaction of the renin angiotensin aldosterone system and the kallikrein-kinin system and the contribution of these two systems on the effects of ACE inhibitors and ARB should not be underestimated. Figure 12.9 illustrate some of these interactions. Also, the AT1 receptor

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KININOGEN

RENIN KININOGENASE

ANG I

ACE inh INACTIVE FRAGMENTS

( __)

\\

KININS

ANG 1–7/III /IV

ANG II

ACE inh

Cytokines

AT1 ant B2/1 -receptor Ac-SDKP

NO/EDHF Eicosanoids/t-PA

Vasodilation/Natriuretic Cardiovascular Protection

AT2/n-receptor

AT1-receptor

iNOS Aldosterone / Cathechol Endothelin / Adhes Molec Growth Factors / PAI-1

O2– + NO

Pressor/Anti-Natriuretic Cardiovascular Remodel

Cell Death

Liu, Y-H., Yang X-P., and Carretero O.A. J. Clin. Invest. 1997; 99:1926–1935

Fig. 12.9 The renin angiotensin and kallikrein-kinin systems. In both systems, a substrate is cleaved by an enzyme of restricted specificity, releasing a peptide which is either already active (lys-bradykinin, bradykinin) or inactive (angiotensin I). Upon further processing by a specific peptidase,angiotensin I is converted to a vasoactive peptide (angiotensin II). In turn, vasoactive peptides are inactivated by peptidases. Angiotensin-converting enzyme is common to both systems, but has different roles: it processes angiotensin I to angiotensin II and is the main kinin-inactivating peptidase (Modified from [1])

and the bradykinin B2 receptor form stable heterodimers, causing increased activation of G[alpha]q and G[alpha]i (the two major signaling proteins triggered by AT1 ). Also, the endocytotic pathways of both receptors change with heterodimerization. This appears to be the first reported example of signal enhancement triggered by heterodimerization of two different vasoactive hormone receptors [84]. The interaction of the AT1 and B2 receptors potentiates the pressor effect of angiotensin II. On the other hand, Ang (1-7) interacting with bradykinin has emerged as an endogenous antihypertensive/antitrophic mechanism, opposing many of the Ang II that are mediated by the AT1 receptor [225–227]. It has been demonstrated that Ang (1-7), acting via receptors other than AT1 or AT2 , induced bradykinin-mediated hypotension in SHR and normal rats [228] and dilatation of porcine coronary arteries [229, 230]. Ang I and II are cleaved to Ang (1-7) by various endopeptidases [231, 232]. This constitutes another mechanism by which kinins could contribute to the beneficial effects of ACE inhibitors or AT1 antagonists. Bradykinin also appears to play an important role in mediating the counterregulatory protective effect of AT2 receptors, which oppose the effect of the AT1 receptor [233, 203, 224]. Therefore, there seems to be a close interaction between kinins and angiotensins in the regulation of cardiovascular and renal function.

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In conclusion, kinins appear not to play a fundamental role in the pathogenesis of hypertension since in humans, rats, and mice with a deficiency of one component of the kallikrein-kinin-system or with chronic blockade of the kallikrein-kinin system do not have hypertension. In the kidney, kinins participate in the regulation of papillary blood flow and water and sodium excretion. B2 -KO mice appear to be more sensitive to the hypertensinogenic effect of salt. Kinins participate in the acute antihypertensive effect of ACE inhibitors; however, in general, they are not involved in the chronic antihypertensive effects of ACE inhibitors save for mineralocorticoidsalt-induced hypertension. Kinins acting via nitric oxide (NO) participate in the vascular protective effect of ACE inhibitors during neointima formation. In myocardial infarction produced by ischemia/reperfusion, kinins play an important role in the reduction of infarct size induced by seen after preconditioning or treatment with ACE inhibitors. In heart failure secondary to infarction, the therapeutic effects of ACE inhibitors are partially mediated by kinins via the release of NO. The therapeutic effect of ARB in heart failure is partly due to activation of angiotensin type 2 receptors, which act via kinins and NO. Thus kinins play an important role in the regulation of cardiovascular and renal function as well as in many of the beneficial effects of ACE inhibitors and ARB.

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Chapter 13

CMS and Type 2 Diabetes Mellitus: Bound Together by the Renin Angiotensin Aldosterone System Deepashree Gupta, Guido Lastra, Camila Manrique, and James R. Sowers

Abstract The recent epidemic of obesity has led to an increasing incidence of the cardiometabolic syndrome defined by the NCEP ATP III guidelines as a cluster of abdominal obesity, low HDL, high triglycerides, HTN, and impaired fasting glucose. Obesity predisposes the body to a state of inflammation, insulin resistance, and hyperinsulinemia, and individuals with the metabolic syndrome are at increased risk for developing CAD, stroke, PVD, CKD, and T2DM. There are various mechanisms by which these complications of the metabolic syndrome occur, and activation of systemic and local renin angiotensin aldosterone system (RAAS) and resultant oxidative stress in different organ systems is probably the most important one. Finally, based on recent trials, the approach toward management of the metabolic syndrome is usually multifactorial and multiagent and studies are still being performed to assess the efficacy of newer drugs. Keywords Cardiometabolic syndrome · Type 2 diabetes mellitus · Low HDL · High triglycerides · CVD and CKD

13.1 Introduction Type 2 diabetes mellitus (T2DM) is one of the fastest growing diseases across the globe and its prevalence has reached epidemic proportions. From 1980 through 2005, the number of Americans with diabetes increased from 5.6 million to 15.8 million, with people aged 65 years or older accounting approximately for 38% of the population with diabetes (Fig. 13.1). Currently, there are close to 30 million people in the United States with diabetes and about 8 million of these have not been D. Gupta (B) Diabetes and Cardiovascular Center, University of Missouri School of Medicine, and VA Medical Center, One Hospital Drive, Columbia, MO, USA e-mail: [email protected]

W.C. DeMello, E.D. Frohlich (eds.), Renin Angiotensin System and Cardiovascular Disease, Contemporary Cardiology, DOI 10.1007/978-1-60761-186-8_13,  C Humana Press, a part of Springer Science+Business Media, LLC 2009

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Fig. 13.1 Graph representing the increasing prevalence of T2DM in the American population

diagnosed (www.diabetes.org). Obesity, related to both genetic and environmental factors, and sedentary lifestyle are the leading driving forces behind the dramatic increase in T2DM in this country. The prevalence of T2DM is on the rise in developing countries, as well as in industrialized countries including the United States. Further, the burgeoning burden imposed by diabetes and its complications, including cardiovascular, renal, and neurological, as well as the medical cost of its treatment are indeed dramatic. Risk factors for cardiovascular disease (CVD) and chronic kidney disease (CKD) tend to cluster with endothelial dysfunction and a litany of metabolic abnormalities [1]. This cluster of metabolic, CVD, and CKD risk factors constitute the cardiometabolic syndrome (CMS). Key elements of the CMS are abdominal obesity, insulin resistance, atherogenic dyslipidemia (high LDL, low HDL and hypertriglyceridemia), hypertension (HTN), a prothrombotic and chronic low-grade inflammatory state, CKD, and proteinuria [1] (Table 13.1). Individuals meeting criteria for diagnosis of the CMS are at increased risk for developing coronary artery disease (CAD), stroke, peripheral vascular disease (PVD), and T2DM [1]. About 47 million Americans are currently thought to have CMS according to the latest American Heart Association (AHA) guidelines [1]. Central to the pathophysiology of the CMS are truncal obesity and insulin resistance (IR) [2]. In this review, we cover potential mechanisms by which IR leads to the activation of systemic and tissue renin angiotensin aldosterone system (RAAS) and associated end organ damage [3]. Further, we will discuss various alternatives that can be used for prevention and treatment. Indeed, with the extremely high prevalence of CMS, the prevention of its CVD and CKD complications is of paramount importance.

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Table 1 NCEP ATP III Guidelines for the diagnosis of metabolic syndrome Risk factor Abdominal obesity (waist circumference) Men Women TG HDL-C Men Women Blood pressure Impaired fasting glucose

Defining level >102 cm (>40 in) >88 cm (>35 in) ≥150 mg/dl <40 mg/dl <50 mg/dl ≥130/≥85 mm Hg ≥110 mg/dl

Diagnosis is established when three or more above-mentioned risk factors are present.

13.1.1 The Renin Angiotensin Aldosterone System (RAAS) RAAS plays an essential role in homeostasis of fluids, sodium and potassium. When the juxtaglomerular cells (JG) of the kidney are stimulated by decreased renal perfusion or decreased sodium content in blood or increased beta 1 adrenergic activity, they become activated and stimulate the production of rennin [3]. Renin is a protease that processes angiotensinogen in the liver, which results in the formation of the decapeptide angiotensin I. Angiotensin I is in turn converted by angiotensinconverting enzyme (ACE) to the active octapeptide angiotensin II (Ang II) mainly in the pulmonary circulation by removing two amino acids from the carboxy terminus of angiotensin I. ACE is a membrane-bound enzyme anchored to the endothelium of many vascular beds, with the highest concentrations found on the vascular epithelium of the lung. Angiotensin II (Ang II) in turn stimulates the production and secretion of aldosterone from the zona glomerulosa of the adrenal gland. Aldosterone is a mineralocorticoid that acts by signaling through the mineralocorticoid receptor (MR) to increase tubular reabsorption of sodium and thus salt and water retention with attendant increases blood pressure [3]. Ang II, which is a potent vasoconstrictor, also contributes to the elevation in blood pressure and, working in concert with aldosterone, promotes tissue remodeling and injury [3]. The effects of Angiotensin II are mediated through two primary receptors – AT1 R and AT2 R. AT1 R has two subtypes – AT1 A and AT1 B, but most of the pressor, growth, and tissue remodeling effects of Ang II are mediated via the AT1 A receptor. In addition to circulating RAAS, there is accumulating evidence supporting the importance of local RAAS in several tissues. These have been described in the brain, kidney, adrenal, testis, and arterial wall in animals and in humans [2]. As angiotensinogen is the precursor of angiotensin peptides, local synthesis of angiotensinogen is required to demonstrate the existence of a local RAAS. Angiotensinogen mRNA has been described in several different extrahepatic tissues in mice, including kidney, brain, spinal cord, aorta, mesentery, adrenal, atria, lung,

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stomach, large intestine, spleen, and ovary. Renin mRNA has also been detected in many extrarenal tissues (adrenal, heart, testes, and submaxillary gland) [3]. Although RAAS is important in maintaining homeostasis in the body, inappropriate activation of circulating and/or local RAAS can trigger excessive oxidative stress in numerous tissues. Increased RAAS activity in the CMS is related to abdominal/truncal obesity [4] (obesity per se is a salt-retaining and volume expansion state), fatty acid (FA) elevation [5], insulin resistance (IR)/compensatory hyperinsulinemia, HTN, hyperglycemia [6] and atherogenic dyslipidemia. Indeed, a local RAS system [5] has been well delineated in adipose tissue, and this local RAS plays an important role in adipocyte differentiation, as well as oxidative stress, inflammation, and the production and secretion of adipocyte cytokines.

13.1.2 The Role of Adipose Tissue and Adipokines Adipose tissue is an active endocrine organ responsible for the production several adipokines, which have numerous autocrine, paracrine, and endocrine actions. In obesity and the CMS, the adipose tissue is dysfunctional and is characterized by an imbalance between pro-inflammatory and insulin-sensitizing adipokines, which contribute to the development of a chronic low-grade inflammatory environment and insulin resistance [7]. There is increased secretion of FA originating in dysfunctional adipose tissue, leading to increased hepatic gluconeogenesis and impaired intracellular insulin signaling. IR in turn results in increased lipolysis and further increase in FA levels creating a vicious cycle [8]. Resistin is produced in white adipose tissue in mature adipocytes as well as during differentiation of adipocytes. Under experimental conditions its levels decline with prolonged fasting and increase with food intake and when injected into normal mice, it can induce hepatic and skeletal muscle insulin resistance [9, 10]. However, animal and human trials have yielded controversial results, and additional studies are needed to ascertain the role of this hormone in the pathophysiology of IR and CMS. TNF-α, mainly through a paracrine effect, causes elevation of FA levels and inhibits tyrosine phosphorylation of Insulin Receptor Substrate (IRS-1), which is required for insulin intracellular signal transduction; it does this by promoting serine phosphorylation of IRS-1. In addition, TNF-α contributes to activation and persistence of chronic inflammation in the adipose tissue by activation of Th1 lymphocyte subpopulation, increased production of monocyte chemotactic protein 1 (MCP1), monocyte colony-stimulating factor (M-CSF) in activated macrophages as well as superoxide dismutase and proinflammatory interleukins (particularly IL-1 and IL-6). TNF-α also activates the nuclear factor kB (NF-kB) pathway in endothelial and vascular smooth muscle cells (VSMCs), hence promoting endothelial dysfunction and eventually atherogenesis [11, 12]. Finally, the insulin-sensitizing adipokine, adiponectin, is inhibited by TNF-α [13].

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IL-6, unlike TNF-α, is found in high levels in blood and has mainly an endocrine action. Levels in blood are directly proportional to BMI, insulin resistance, and impaired glucose tolerance [14, 15]; conversely, weight loss leads to reduction in circulating and adipose levels of IL-6 [9]. IL-6 causes insulin resistance, hyperglycemia, and dyslipidemia via the expression of SUPPRESSOR of cytokines 3 (SOCS-3), which impairs intracellular transduction of both insulin and leptin [14]. Similar to TNF-α, IL-6 inhibits adiponectin secretion and action [15]. Leptin is another adipokine implicated in the pathophysiology of the CMS. It is produced by adipose tissue and its circulating levels are proportional to the fat body mass [16]. Leptin acts on the arcuate nucleus of the hypothalamus and decreases appetite in concert with increasing energy expenditure, hence resulting in decreased adipose mass and weight loss [17]. When injected into the CNS of mice genetically unable to produce it (Ob/Ob mice), leptin reverts hyperglycemia and hyperinsulinemia, most likely through activation of adrenergic pathways [18, 19]. Leptin also acts on peripheral tissues like skeletal muscle where it improves insulin sensitivity. It has been proposed that this action is via 5 AMP-activated protein kinase (AMPK) pathway, which inhibits anabolic processes involving consumption of ATP and stimulates catabolic pathways like cellular glucose transport and glucose and FA oxidation that produce ATP, the net effect being energy expenditure and reduction of energy stores, i.e., adipose tissue mass [20–22]. It has been demonstrated that selective leptin resistance and hyperleptinemia result in inability to lose weight and preservation of sympathoneural outflow and sodium resorption ability, which contribute to both HTN and obesity in the CMS. Finally, adiponectin is an insulin-sensitizing and anti-inflammatory adipokine, which as has been mentioned above, is inhibited by both TNF-α and IL-6, more so in obesity and dysfunctional adipose tissue. The mechanism of action of this peptide remains to be fully uncovered, but it is thought to implicate FA oxidation in skeletal muscle by stimulating the AMPK activity and decreasing the hepatic glucose output [23]. It also prevents endothelial damage and has antiatherogenic activity, through inhibition of the expression of adhesion molecules like ICAM-1, VCAM1, E-selectin, andTNF-α [24, 25]. Some studies have shown that adiponectin suppresses macrophage migration and their transformation into foam cells, decreases vascular intimal proliferation and via AMPK stimulation induces NO synthesis [25].

13.1.3 Hyperinsulinemia in CMS It is well known that in obesity, glucose uptake to peripheral tissues, especially to skeletal muscle, in response to insulin via GLUT-2 and GLUT-4 receptors becomes impaired. IR and compensatory hyperinsulinemia (HI) impair the homeostatic actions of insulin and results in enhanced activity of the sympathetic nervous system, increased sodium and water reabsorption in proximal renal tubules, decreased urinary sodium excretion, activated systemic RAAS, increased arterial tone and pressure by increased membrane transport of calcium, and increased number of

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AT1 R. IR also stimulates vascular smooth muscle cell proliferation, migration, and vascular extracellular matrix remodeling [26, 27]. It is thought to do so by the activation of local RAAS and via adverse effects of Ang II, which are oxidative stress from reactive oxygen species (ROS) (discussed in detail later), impaired insulin metabolic pathway (P13K/Akt), and increased proliferative and remodeling pathways (MAP kinase pathway). All these intracellular and intravascular changes in IR lead to increased blood volume and HTN [28]. In fact, the existence of insulin resistance and/or compensatory hyperinsulinemia in patients with essential HTN has been extensively documented [29], and hyperinsulinemia as a surrogate of insulin resistance has also been shown to be an independent predictor of HTN [30–33]. IR and/or compensatory hyperinsulinemia have also been shown to predict T2DM in several prospective studies [34–37].

13.1.4 HTN and The CMS HTN is a condition of multifactorial origin, in which genetic predisposition is a predominant feature. The European Prospective Investigation into Cancer Norfolk (EPIC) study in which more than 20,000 participants were included showed that both systolic and diastolic blood pressures increased in a directly proportional manner to the waist-to-hip ratio in men as well as women [38]. The mechanisms involved in obesity-related HTN are complex and involve derangements in multiple systems. They include activation of the RAAS, increased sympathetic nervous system (SNS) activity, insulin resistance, increased renal sodium reabsorption, impaired pressure natriuresis, and vascular volume expansion. In addition, obesity may also cause marked structural changes in the kidneys, which will eventually lead to CKD and further increases in blood pressure [28, 39, 40]. A direct correlation has been demonstrated between blood pressure and blood glucose levels in essential HTN. Uncontrolled HTN is associated with both elevated fasting and post prandial blood glucose levels [41]. It has been proposed that about 50% of patients with essential HTN will develop T2DM over 10–15years [42–44].

13.1.5 Dyslipidemia Atherogenic dyslipidemia consisting of high triglycerides (TGs) and low-density lipoprotein cholesterol (LDL-C) and low high-density cholesterol (HDL-C) is closely associated with obesity. In addition, elevated TG/HDL-C ratio is associated with elevated blood pressure and is also an independent risk factor for IR [45].

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13.2 Oxidative Stress There are various systems in the mammalian cells that can produce ROS and cause excessive oxidative stress. These include the NADPH oxidase enzymatic complex, nitric oxide synthase, cytochrome p450 enzymatic complex, the mitochondrial electron transport system, and the xanthine oxydase system [46]. Most of the work done on animal models implicates the NADPH oxidase system in causing the maximum oxidative stress-induced tissue damage in studied tissues such as cardiovascular, renal, and skeletal muscle. The NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase) is a membrane-bound electron transport complex that catalyzes the production of superoxide from oxygen and NADPH. It is a multisubunit enzyme composed of three cytosolic (p40phox , p47phox and p67phox ) and two membrane-bound components (Nox 2 (P91phox ) and p22phox ) plus the small proteins Rac 1 and Rac 2, which are essential to NADPH oxidase assembly. “Phox” stands for phagocytic oxidase and Rac 1 or Rac 2 (Rac stands for Rho-related C3 botulinum toxin substrate) is a Rho guanosine triphosphatase (GTPase). Activation of the oxidase involves the assembly in the plasma membrane of membrane bound and cytosolic components of the NADPH oxidase system, which are disassembled in the resting state. Activation starts with the phosphorylation of one of the cytosolic components and their translocation to the plasma membrane where electron transfer between gp 91 and O2 molecules leads to the formation of superoxide (O2- ) and ROS [47]. Ang II via AT1 R can promote this pathway by stimulating intracellular pathways that result in translocation of cytosolic subunits to the plasma membrane, direct phosphorylation of membrane bound subunit p22 phox via PKC activation and activation of Rac 1 by association with caveolin 1 [48, 49]. ROS cause multiple structural and functional changes in numerous tissues, and in particular to endothelial cells. ROS can induce the inflammatory NF-kB pathway and hence increase the expression of Vascular Adhesion Molecule 1(VCAM1) [50]. They can also activate tyrosine kinase pathways like extracellular signalregulated kinase 1 and 2 (ERK1 and ERK2) and cause transactivation of growth factor receptors like EGFR; the end results of these pathways being influence on vascular cell growth and proliferation. ROS can also trigger the Jak-STAT pathway leading to increased IL-6 production, which as mentioned above causes inflammation [51]. The gap in our knowledge between local RAAS activation, oxidative stress, and insulin resistance is bridged in the adipose tissue. As described in detail above, local RAAS activates adipokines in the adipose tissue contributing to insulin resistance, which in turn leads to chronic inflammation. Current research indicates that angiotensinogen messenger RNA expression is higher in abdominal fat compared to subcutaneous fat, a finding that correlates with the differences observed in insulin resistance between the two tissues [52].

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13.3 Effects of RAAS on Various Organs Local RAAS is thought to be responsible for the production of reactive oxygen species and endothelial dysfunction, which in turn cause end organ damage in multiple organs. In addition, Ang II is directly responsible for structural modification of organs like the heart. In the next section, we will try to describe the various changes taking place at tissue levels in the different end organs involved in the pathophysiology of the CMS and T2DM.

13.3.1 Heart Both insulin and IGF-1 via their receptors in cardiovascular tissue (53) result in vasorelaxation (54) and myocardial glucose uptake (41, 55–57). Ang II receptors have been characterized in cardiomyocytes and cardiac fibroblasts as well as in the endothelial lining of coronary arteries. Ang II, via e AT1 R, and especially AT1A R, causes peripheral vasoconstriction, increasing the peripheral vascular resistance and hence maintains blood pressure in the face of decreased cardiac output (58). Ang II, in addition to its vasoconstriction effects, attenuates the metabolic actions of insulin and IGF-1 in cardiovascular tissue, via the generation of ROS and activation of small molecular weight proteins such as RhoA and Rac1 (41, 53, 59). It also stimulates the release of cathecholamines from noradrenergic nerve endings and salt and water retention via aldosterone synthesis from the adrenal gland. Many of the detrimental effects of both Ang II and aldosterone are mediated via the activation of membrane NADPH oxidase as well as mitochondria-generated ROS. This Ang II-induced oxidative stress affects cell signaling responses and facilitates marked cardiac hypertrophy, interstitial fibrosis, and left ventricular dysfunction [58, 60, 69].

13.3.2 Endothelium Insulin/IGF-1 stimulates PI3K/PDK-1/Akt phosphorylation of human eNOS at Ser1177, resulting in enhanced eNOS; eNOS in turn mediates endothelial cell production of NO. Insulin and IGF-1 also increase vascular smooth muscle cell (VSMC) production of NO and attenuate Ang II-induced increase in cytosolic calcium and myosin light chain (MLC) kinase activity; which result in vascular relaxation [58]. Ang II promotes vascular growth/remodeling, apoptosis, and fibrosis via increased generation of ROS. These markedly reactive ROS molecules oxidize lipids, protein, and DNA and cause cellular injury. They cause vasoconstriction by converting NO to peroxynitrite (ONOO- ). ROS activate the transcription of factors such as TNF-α, monocyte chemoattractant protein (MCP)-1, IL-6, and C-reactive

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protein (CRP), and TNF-α impedes insulin- and IGF-1-mediated eNOS activation as well as their antiapoptotic actions [58]. Recently, new studies have reported insulin receptor expression in macrophages and implicate protein CD36 to play an important role in atherogenesis [61]. Protein CD 36 is a glycoprotein in the platelet membrane and also a class B scavenger receptor on macrophages that plays an important role in recognition, uptake, and internalization of oxidized LDL molecules in the early steps of atherogenesis [62, 63]. In obese, insulin-resistant Ob/Ob mice models, there is an increase in oxidized LDL uptake in macrophages and this event is associated with a simultaneous increase in CD36 concentrations. Increased CD36 concentrations adversely affected the tyrosine kinase activity of the insulin receptor and intracellular insulin signaling, further contributing to insulin resistance [63, 64].

13.3.3 Kidney As previously discussed, renin stimulates the synthesis of Ang II and aldosterone, which in turn acts on the mineralocorticoid receptor in the collecting duct, leading to salt and water retention. Obesity and IR/hyperinsulinemia have been shown to have deleterious effects on the renal hemodynamics, causing reduced-pressure natriuresis and increased salt retention. These changes in turn result in hyperfiltration and increased glomerular filtration rate (GFR, andurinary albumin excretion [65] in experimental conditions [66] as well as hypertensive individuals [67]. The effects of inappropriately activated RAAS on the kidney have been extensively studied in the TG (mRen2)27 (Ren2) transgenic rat model, which overexpress the mouse renin gene and exhibit increased tissue Ang II levels in glomerular mesangial cells. Activation of AT1 R by Ang II increases oxidative stress, inflammation, and endothelial dysfunction which in turn cause HTN, glomerular injury, loss of filtration barrier, and albuminuria. In the Ren2 rat model, electron microscopy (EM) measurements have demonstrated podocyte foot process effacement, loss of slit pore diaphragm integrity, and widening of the bases of the podocyte foot process [45]. The podocyte, which is the most differentiated cell type within the glomerular complex, is an integral component of glomerular basement membrane and slit pore diaphragm and hence plays a pivotal role in maintaining glomerular filtration barrier. Podocyte injury therefore causes destabilization of the foot process/slit pore diaphragm complex, with resultant loss of glomerular permeability leading to albuminuria. These structural changes are associated with increased NADPH oxidase activity in renal cortical tissue [45]. T2 DM and HTN are the most common causes of end-stage renal disease [68–70]. The earliest clinical manifestation of nephropathy is the presence of microalbuminuria (MAU), defined as urine albumin of 30–300 mg/day or 30– 300 mg/g of creatinine in a spot urine collection. MAU has been integrated as a diagnostic criterion in the WHO criteria for definition of the CMS [71, 72]. MAU is considered to be an early marker of endothelial dysfunction and kidney impairment,

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and its presence heralds the progressive loss of renal function [73–75]. In addition, it is considered an independent risk factor for the development of CVD in this patient group. This could probably be explained by the fact that elevated BP and poor glycemic control in T2DM that contribute to MAU are both associated with CVD and CKD [28]. ACEI and ARBs decreased MAU as well as overt proteinuria in addition to their other effects, and are the frontline medications used in HTN and proteinuria management in patients with T2DM.

13.3.4 Pancreas It has recently been identified that the pancreatic islet beta cell expresses pro-renin, renin, Ang II, and AT1 R [76, 77]. It is thought that the physiological role of the pancreatic RAS in mice models seems to involve islet blood flow regulation, which would affect glucose-stimulated insulin secretion and homeostasis of carbohydrate and fat metabolism [78]. The Ren2 rat model has increased islet Ang II and AT1 R. This activation of local tissue RAS in the rat pancreas leads to NADPH oxidasemediated generation of ROS. Pancreatic islets are highly vulnerable to oxidative stress since they have a low intrinsic antioxidant capacity [79, 80]. Changes induced by excessive oxidative stress include disordered islet architecture, increased fibrosis at the islet-exocrine interface, pericapillary fibrosis, and increased structurally abnormal mitochondria in both the endocrine and the exocrine pancreas. These changes are associated with significantly impaired islet blood flow and decreased insulin release from mouse islets in response to high glucose; thus oxidative stress may play an important role in the early stages of insulin resistance and T2DM [79, 81]. Ang II-mediated increased levels of NADPH oxidase in the islets is thought to stimulate the mitochondria to generate ROS via the citrate synthase and electron transport chain. This could explain the increased numbers of structurally abnormal mitochondria seen in the endocrine and exocrine pancreas as described above [80, 82, 83]. One of the ROS is superoxide, which by activating uncoupling protein 2 (UCP2), diverts energy away from ATP synthesis and hence decreases the ATP/ADP ratio. This in turn leads to less efficient glucose-dependent insulin secretion from the pancreas [82, 83]. Superoxide can also adversely affect β cell neogenesis as it irreversibly decreases one of their important transcription factors PDX-1 [80]. ROS, by increasing the IRS-1 serine (Ser) phosphorylation, leads to proteosomal degradation. Oxidative stress in the islet triggers endoplasmic stress [84], which causes more Ser phosphorylation of IRS-1 and further contributes to β cell dysfunction [85]. In the exocrine pancreas, angiotensinogen, AT1 R, and AT2 R are localized in the pancreatic ducts, blood vessels, and acinar cells [2, 76, 86, 87]. This local RAS system appears to regulate the pancreatic microcirculation, acinar enzyme secretion, and pancreatic pericyte and stellate cell function [86–89]. Oxidative stress can cause pancreatic exocrine inflammation and fibrosis in an experimental model of pancreatitis. These in vivo treatment with aliskiren, a direct

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renin inhibitor, normalized systemic insulin resistance and islet insulin, decreased islet Ang II, NADPH oxidase activity/subunits and nitrotyrosine, and improved total IRS-1 and Akt phosphorylation as well as islet/exocrine structural abnormalities [90].

13.3.5 Adipose Tissue Ang II, via AT1 R activation, inhibits preadipocytes differentiation into adipocytes. Decreased capacity of adipose tissue to store FA can lead to FA deposition/accumulation in other tissues including skeletal muscle and liver, leading to insulin resistance and contributing to the development of T2DM [8, 14, 91]. In addition, dysfunctional adipose tissue produces proinflammatory cytokines such IL-1, TNF-α, and resistin, which in turn induces synthesis and release of chemoattractant factors in the stromal adipose tissue. These factors mediate macrophage infiltration of adipose tissue, endothelial dysfunction, and atherogenesis. Thus, adipose tissue plays a role as both cause and target of a low-grade inflammatory state and provides a direct relationship to atherosclerosis, leading to generalized vascular damage, HTN, and CVD [92].

13.3.6 Therapeutic Approach for CMS The treatment of the CMS and its individual components require a multifactorial intervention. Insulin resistance and impaired fasting glucose/impaired glucose tolerance (termed “prediabetes” by some authors) predisposes a person to developing T2DM in the next 10 years and increases their risk for CVD [93, 94]. Therapeutic lifestyle modifications are the cornerstone of the multifactorial therapeutic approach to the CMS. Of paramount importance are a balanced low-fat diet, light to moderate regular exercise, and smoking cessation. From a pharmacologic standpoint, use of aspirin, reduction of blood pressure, reduction of glycated hemoglobin to less than 6.5% in diabetics, and control of hyperlipidemia have proven their efficacy. After a mean follow-up of 7.8 years, this approach results in up to 20% reduction in the risk of CVD [95]. The reduction in CVD risk is higher compared with previous trials targeted at control of isolated risk factors like hyperglycemia, HTN or dyslipidemia, mainly by means of pharmacological intervention [96–98]. As previously discussed, HTN increases the risk of T2DM in the next 10 years. Antihypertensives that produce RAAS blockade, in particular ACEIs and ARBs, have been related to a reduced incidence of T2DM. The Prospective Randomized Open Blinded End point (PROBE) trial, which compared the effect of captopril versus conventional therapy (beta-blockers and diuretics) on CVD morbidity and mortality, failed to demonstrate a significant difference in overall CVD morbidity and mortality in the ACEI-treated group, but a significant reduction of 30% in the

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incidence of DM2 was observed after a follow-up of 6.1 years [99]. The PROBE study suggests that RAAS inhibition is particularly beneficial in patients with DM2, and that the reduction observed in the incidence of DM2 could lead to a further reduction of CVD in the long term. The HOPE trial also demonstrated similar outcomes with the incidence of newonset diabetes being 34% lower in the ramipril-treated group as compared to placebo [100]. Other studies that have demonstrated decreased incidence of new onsetdiabetes with the use of ACE inhibitors and Ang II-receptor blockers include ALLHAT [101, 102], SOLVD [103], and LIFE [104]. The CHARM-Added trial demonstrated that the combination of ACEIs and ARBs was associated with a more profound reduction in the incidence of T2DM as compared with the use of either agent alone [105]. ACEIs have been demonstrated to delay the progression of microalbuminuria in T2DM patients without HTN, probably through blood pressure control, reduction in intraglomerular pressure, control of mesangial proliferation and of transmembrane protein leakage. Importantly however, in these studies, the primary outcome was not the effect of ACEIs and ARBs on the incidence of DM2, but cardiovascular outcomes. On the other hand, the Diabetes Reduction Assessment with Ramipril and Rosiglitazone Medication (DREAM) trial, which actually targeted the incidence of T2DM, demonstrated that the use of ramipril was not associated with a significant reduction in the incidence of T2DM but resulted in significantly increased regression to normoglycemia relative to placebo, thus suggesting a beneficial effect of RAAS blockade on glucose homeostasis [106]. Possible mechanisms responsible for the reduced incidence of diabetes in these trials include improved insulin sensitivity, enhanced endothelial function, increased nitric oxide activation, reduced inflammatory response, and increased bradykinin levels [107]. A recent study has shown that treatment with low dose of losartan (50 mg) significantly elevated the serum concentrations of total adiponectin in patients with essential hypertension [108]. However, in the previously diabetic group, captopril was associated with a reduced incidence of fatal and nonfatal CVD [109]. In numerous studies, statins have been reported to reduce the incidence of T2DM. The Heart Protection Study (HPS) [110] showed that simvastatin 40 mg/d significantly decreased (approximately 25%) major cardiovascular events in persons with T2DM relative to placebo regardless of the baseline LDL-C level. These findings were reproduced in the Collaborative Atorvastatin Diabetes Study (CARDS) [111] and the Atorvastatin Study for Prevention of Coronary Heart Disease Endpoints in Non-insulin-Dependent Diabetes Mellitus (ASPEN) [112]. A step further were the CORALL [113] and Pravastatin or Atorvastatin Evaluation and Infection Therapy (PROVE IT) [114] studies, which showed that high-dose statins and more aggressive LDL-C lowering were more effective in lowering the incidence of CVD. However, it is thought that the benefits of statins go beyond their LDL-C lowering action and include improvement of endothelial function and anti-inflammatory actions by inhibition of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. The inhibition of this enzyme in turn causes decreased isoprenylation of signaling

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molecules like Ras, Rho, and Rac, which are involved in smooth muscle proliferation, inflammation, oxidative stress, and cellular remodeling [115]. Dyslipidemia contributes to the progression of glomerulosclerosis and diabetic nephropathy [116, 117]. Treatment with statin therapy reduces MAU and delays the progression of diabetic nephropathy [68–70, 118–120] in experimental animals [121, 122] and in diabetic patients [123, 124]. Experimental data suggest that statins do this by reducing mesangial cell proliferation and fibrogenesis and also by decreasing the generation of ROS, macrophages, and inflammatory cytokines [121, 122, 125]. Statins have also been shown to reduce podocyte injury and effacement [121, 125]; both these processes are thought to be involved in the development of proteinuria. These effects can be explained by the fact that podocytes have lipoprotein receptors, and that these receptors are highly expressed in proteinuric states [126]. As the RAAS has been found to be the culprit in inducing oxidative stress in various organ systems, medications that block this system at various levels are being studied in detail. In addition to the already discussed role of ACEIs and ARBs, aliskiren is being used to control HTN and has been seen to reverse the effects of ROS in rat pancreas. On the other hand, hyperaldosteronism is also associated with insulin resistance. The underlying mechanisms leading to this impaired insulin sensitivity remain to be fully elucidated, but involve increased production of ROS and oxidative stress. It has recently been found that mineralocorticoid receptor (MR) antagonism can reduce oxidative stress and improve insulin sensitivity in skeletal muscle in Ren2 rats independently of any effect on blood pressure [127]. In human clinical studies, Catena et al. confirmed increased insulin resistance as well as impaired glucose utilization in primary aldosteronism (PA) patients but not in essential hypertensive individuals [128]. Fallo et al. recently reported a 41.1% prevalence of CMS in PA patients compared to 29.6% in essential hypertensives (p < 0.05), again underscoring the fact that hyperaldosteronism is an independent cause of insulin resistance [129]. The RALES and EPHESUS trials have demonstrated beneficial effects of spironolactone in decreasing cardiovascular morbidity and mortality. The RALES (Randomized Aldactone Evaluation Study) was a double-blind trial, which included patients who had severe heart failure and left ventricular ejection fraction below 35%. It was seen that participants who received 25 mg of spironolactone vs placebo plus conventional treatment with an ACEI, a loop diuretic, and in most cases digoxin had a 35% reduction in the relative risk of death; this was attributed to a lower risk of both death from refractory heart failure and sudden cardiovascular death. Patients who received spironolactone also had a significant improvement in the symptoms of heart failure [109]. In the EPHESUS (Eplerenone Post–Acute Myocardial Infarction Heart Failure Efficacy and Survival Study) trial, there was a significant reduction in cardiovascular mortality and the rate of death from any cause or hospitalization among patients receiving eplerenone [109]. These effects could be due to MR blockers’ action against HTN, oxidative stress, inflammation, apoptosis, and fibrosis in the cardiovascular and renal tissue [130].

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To summarize, the demonstration of cardiovascular injury and impaired insulin signaling in the setting of increased aldosterone as well as clinical studies showing improvement in HTN and glucose homeostasis through pharmacological blockade of the MR support a direct correlation between mineralocorticoids and CMS. The mechanisms underlying these relationships still remain to be fully elucidated; however, RAAS-mediated increased oxidative stress appears to play a key role.

13.3.7 Conclusions and Perspectives The cluster of HTN, T2DM, HLD, and MAU define that the CMS is growing dramatically, driven in large part by the excess weight. Obesity results in dysfunctional adipose tissue, chronic low-grade inflammation, insulin resistance, and hyperinsulinemia, which as demonstrated above, activate systemic and local RAAS in diverse tissues such as heart, endothelium, kidneys, pancreas, and adipose tissue. Local RAAS activation is associated with the production of ROS and oxidative stress, which has various deleterious effects on the morphology and function of these organ systems, leading to T2DM, CKD, atherogenesis, and CVD. A multifactorial approach has been recommended for the management of CMS with diet, exercise, and weight loss still being the therapeutic cornerstone. There is also consistent evidence in numerous experimental and clinical studies demonstrating a paramount role of inappropriate activation of RAAS, subsequent increased oxidative stress and insulin resistance in the pathophysiology of the CMS, and development of T2DM. Different strategies to block RAAS, in particular the use of ACEIs and ARBs, have emerged as important alternatives, which are now used ubiquitously to prevent complications of CMS, have shown benefits in terms of CVD morbidity and mortality. In addition, there is promising evidence about drugs like aliskiren, statins and MR antagonists. These agents block the RAAS at different levels, and statins are thought to ameliorate the oxidative stress induced by insulin resistance. The multifactorial approach to the management of CMS, in concert with exciting new research, will contribute to the prevention and management of the CMS and to reduce its burden on healthcare worldwide.

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Chapter 14

Renin Angiotensin Aldosterone System and Cardiovascular Disease Swynghedauw Bernard, Milliez Paul, Messaoudi Smail, Benard Ludovic, Samuel Jane-Lise, and Delcayre Claude

Abstract Aldosterone, aldo, is a rather minor component of the adrenal gland production with complex activity. Aldo and glucocorticoid are in competition and the specificity of aldo action is due to a cellular component the 11-HSD2. Aldo at high concentrations has pronounced and deleterious cardiovascular effects and causes pro-inflammatory reaction followed by myocardial and vascular fibrosis. Fibrosis modifies cardiac performances and has proarrhythmogenic consequences and is prevented by spironolactone and/or eplerenone. At low concentrations aldo inhibits BKCa potassium channel and causes coronary dysfunction without major alterations in myocardial function. Besides its classical mode of action on MR, aldo modulates intracellular Ca and cAMP concentrations, phosphorylates several kinases of major importance, and activates the EGFR signaling pathways. Aldo and angiotensin II are partners; on one hand, aldo is able to activate the transcription of components of angiotensin II activity; on the other hand, the angiotensin II-dependent increase in collagen is in part dependent on aldo. Keywords Aldosterone · Angiotensin II · Glucocorticoid · Eplerenone · Spironolactone · Transgenic mice · Aldosynthase

14.1 Introduction The RALES [1] and EPHESUS [2] clinical studies have demonstrated the important benefit of MR antagonists in patients with heart failure or with left ventricular dysfunction after myocardial infarction. A short-minded conclusion would therefore be that aldosterone plays a generally evil role and that it is important to block it in all circumstances. This is obviously not so simple, and to better understand its S. Bernard (B) Centre de Recherches Cardiovasculaires INSERM Lariboisière, PARIS, France e-mail: [email protected]

W.C. DeMello, E.D. Frohlich (eds.), Renin Angiotensin System and Cardiovascular Disease, Contemporary Cardiology, DOI 10.1007/978-1-60761-186-8_14,  C Humana Press, a part of Springer Science+Business Media, LLC 2009

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mechanisms and better anticipate the effects of treatment, several points should be highlighted. The classical role of aldosterone is to adjust the hydro-mineral balance in the body, and thus to decisively intervene in blood pressure control. Meanwhile, experimental studies have demonstrated that aldosterone induces structural and functional changes in the heart, kidneys, and blood vessels, with pronounced cardiac and renal fibrosis, inflammation, vascular remodeling, and changes in fibrinolysis. These damages are said to be mediated by aldosterone and are prevented or minimized by MR antagonists as spironolactone or eplerenone. It cannot be excluded that potassium and hypertension may also play a key role in these damages. Nevertheless above all, it is important to stress that these effects were observed at very high aldosterone concentrations, too high to complain for the body salt requirements [3]. In addition, a distinction must be made between inhibition of MR and antagonism of the effects of aldosterone. Indeed, the presence of glucocorticoids in concentrations much higher than those of aldosterone in plasma, the structural similarity of these two hormones and of their receptors, the hormone– receptor affinities measured in vitro that conclude to the possible binding of cortisol (or corticosterone in rodents) on the aldosterone receptor (and vice versa) complicate the understanding of the mode of action of aldosterone in the cardiovascular system.

14.2 Biosynthesis of Aldosterone A substance secreted by the adrenal glands and having the ability to retain salt has been evidenced for the first time in the 1930s by the teams of Kendall and Reichstein (for an historical review on the discovery of aldosterone, see [4]). From the crystallization of glucocorticoids and mineralocorticoids, researchers have discovered that a part of the fraction extracted from adrenals was not crystallized. This fraction called “amorphous” had an important mineralocorticoid activity, albeit different from that of deoxycorticosterone or other steroids. It was not until the 1950s and the improvement of biochemical techniques that some researchers (convinced of the existence of a mineralocorticoid different from desoxycorticosterone) were interested in this fraction. Advances in the techniques for determining the sodium/potassium ratio using radioactive compounds, and of chromatography from urine of adrenalectomized rats or from extracts of beef adrenal gland, have finally allowed to purify the active compound. This active fraction, first called electrocortin because of its properties on the electrolytes metabolism, will then be crystallized. The hormone was then better characterized under the name of aldosterone. That this hormone was secreted by the adrenal gland was then evidenced by Tait’s team, showing that the hormone extracted from beef or dog adrenal perfusate were identical. Such a rapid retrospective highlights the opposition that existed between the supporters of cortisol seen as the genuine adrenal hormone (aldosterone being an artifact of the synergistic action of steroids), and the supporters of aldosterone who thought it was a hormone dealing specifically with the electrolyte metabolism. In the pioneer work of Selye, the administration of deoxycorticosterone

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improved the survival of adrenalectomized rats, but adverse effects (namely a cardiac necrosis) were also observed [5]. This early observation has triggered, much later, the interest of mineralocorticoid hormones in cardiovascular diseases. The identification of aldosterone, a minor hormone of adrenal gland, was difficult for several reasons, including the fact that plasma glucocorticoids concentrations was much higher than those of aldosterone, that both the hormones and their receptors have very similar structure, and finally that both types of receptors have significant, while different, affinities for the two hormones.

14.3 Vascular Effects of Aldosterone Aldosterone has deleterious effects on both the vascular structure and the function. Several groups of investigators have observed the induction of a peri-inflammatory phenotype in the heart of rats treated with high dose of aldosterone + salt-enriched diet (review in [6]). The increase of the inflammation markers such as Cox-2 and MCP-1 is seen from the first week on, making the proliferation of inflammatory cells around the coronary arteries among the first events leading to fibrosis. The causal role of oxidative stress is suggested by the fact that spironolactone and antioxidants prevent these changes independently in the coronary [7] or peripheral [8] arteries, and aldosterone stimulates the expression of the NADPH oxidase in macrophages [9]. Again, a cooperation between aldosterone and Ang II was found in the release of free radicals that can lead to a deterioration of arterial smooth muscle cells [10]. The target of the deleterious effects of high concentrations of aldosterone is clearly the vessels. Nevertheless, it remains to understand the earliest stages. Weber and his colleagues described an early drop of intracellular magnesium and calcium concentrations in monocytes and lymphocytes of rats treated with aldosterone-salt [11]. Several markers of oxidative stress were increased in plasma (alpha-1-antiproteinase activity) and in heart (gp91phox subunit of NADPH oxidase and 3-nitrotyrosine) of these animals. If such ionic changes were not observed in cardiac cells, this work suggests that they may also exist and induce the release of free radicals, coronary lesions, perivascular, and finally interstitial fibrosis. Finally, in the transgenic mice model overexpressing the aldosterone synthase within the heart, original effects have been observed on coronary vasomotricity. In this model characterized by a moderate increase (1.7 times) of intra-cardiac aldosterone with unchanged plasma level, the vasodilatory response to acetylcholine is abolished in male transgenic mice [12]. The mechanism of the damage is the inhibition of the BKCa potassium channels of coronary smooth muscle cells. Interestingly, the cardiac structure and function remain normal, and the only potentially harmful event discovered to date is this coronary alteration. In fact, the results of this transgenic study suggests that a slightly increased concentration of aldosterone (reaching a level observed in pathological situations) can induce a coronary dysfunction, which is silent in resting conditions but that may make these animals vulnerable to an increase in cardiac work.

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14.4 Fibrogenic and Arrhythmogenic Effects of Aldosterone One of the best-documented deleterious effect of aldosterone is cardiac fibrosis, with adverse consequences on the pump function and an arrhythmogenic effect. Besides the experimental works, a relationship between mortality and the initial cardiac fibrosis and a reduced cardiac fibrosis by spironolactone treatment is observed in a subgroup of patients of the RALES study [13]. Ang II is probably also involved in the genesis of fibrosis since aldosterone increases cardiac AT1 receptors density [14], and the expression of angiotensin-converting enzyme, ACE, in rat cardiomyocytes [15]. A pro-arrhythmogenic effect of aldosterone (which might partly depend on fibrosis) is suggested by several observations. In hypertensive patients, for the same level of hypertension, atrial fibrillation is much more frequent among those with primary aldosteronism [16]. In failing rats, spironolactone significantly reduces fibrosis and atrial ventricular excitability [17]. Transgenic mice overexpressing the MR in cardiomyocytes have a normal heart function, but show arrhythmia and sudden death [18]. Other effects can be evoked, which can also affect the cardiac function. Finally, Vassort et al. observed an increase in the slow iCaL calcium current and a decrease of the Ito transitory potassium current in isolated cardiomyocytes, which could change the electrical characteristics of these cells [19].

14.5 The Signaling Pathways of Aldosterone As every steroid hormones, aldosterone binds to a cytoplasmic receptor, the MR. The hormone–receptor complex dimerizes, migrates into the nucleus, and binds to a specific DNA sequence, which triggers transcription of target genes. In epithelial cells (kidney, colon, salivary glands, skin, etc.), the induced genes as the amiloridesensitive sodium channel ENaC, Na,K-ATPase, and SGK kinase are key factors in the control of sodium reabsorption. The aldosterone–MR complex binds to the glucocorticoid responsive element (GRE). The existence of tissue-specific proteins able to modulate the response of the GRE according to the bound hormone (aldosterone or cortisol) has been postulated, but not evidenced to date. In vascular cells, some target genes are identified, such as the endothelial NO synthase (NOS3) downregulated by aldosterone (review in [20]), or the smooth muscle cell BKCa repolarizing potassium channel whose coronary expression is reduced by aldosterone, inducing a decrease in their response to acetylcholine and thus a decrease of the coronary reserve [21]. Besides this mode of action involving the classical MR, aldosterone induces cellular responses within minutes that modulate the concentration of intracellular Ca2+ and cAMP, the activity of the Na–H exchanger, and the phosphorylation of molecules such as the PKC, the EGF receptor (EGFR), and several MAP kinases. These responses are independent of the MR activation, as suggested by the rapid onset of the action, and a membrane receptor has been unsuccessfully sought. But there is also a third mode of action of aldosterone, activated by binding of aldosterone on the MR and triggering in some minutes (i.e., without synthesis of

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proteins) the activation of the EGFR signaling pathway together with an increased phosphorylation of ERK1/2 and JNK 4 kinases. All these mechanisms are associated with the inflammation and vascular remodeling leading to fibrosis.

14.6 The Receptors of Corticosteroid Hormones The MR and GR belong to the nuclear hormone receptors superfamily, and they have a pronounced sequence homology. The two receptors bind glucocorticoids (cortisol in humans and corticosterone in rats and mice) with a strong affinity. However, aldosterone binds to MR with a strong affinity, while its affinity for the GR is much lower. Because plasma aldosterone levels are 3 orders of magnitude lower than those of cortisol and corticosterone, glucocorticoids should occupy most of the MRs. However, this theoretical excess is decreased by a 10 factor by the important binding of glucocorticoids to plasma transcortin (only 3% of cortisol is free in plasma), while the binding of aldosterone to albumin is lower (30% of plasma aldosterone is free). In addition, transfection studies have shown that cortisol has a transactivation activity of MR 10 times lower than that of aldosterone despite identical binding affinities. In addition, the cortisol–MR complex is less stable than the complex aldosterone–MR, because there are differences in the conformation changes of MR induced by the hormone binding. This leads to a 2–4 time faster dissociation of the cortisol–MR complex than that of the aldosterone–MR complex. Finally, the exact mechanism of entry of cortisol and aldosterone in the cell is not clear, and there might be other differences between these steroids due to the aldosterone 11-18 hemi-acetal group. A first conclusion is that aldosterone seems disadvantaged compared to cortisol to bind to MR, but probably not as much as the ratio of plasma concentrations suggests. But in these conditions, how does aldosterone have a specific action? The answer depends on the cell type. In epithelial cells and in endothelial and smooth muscle cells, which express the MR, the binding of aldosterone on the MR is made possible by the presence of the enzyme 11-HSD2 (11-beta-hydroxysteroid dehydrogenase type II), which metabolizes cortisol and corticosterone in their inactive cortisone and 11-dehydro-corticosterone metabolites. In contrast, in cells such as cardiomyocytes which express the MR but not the 11-HSD2, the MR is probably mostly occupied by glucocorticoids. In this view, aldosterone can probably have no significant MR-dependent action. But, as outlined above, there are possible other mechanisms that allow the binding of aldosterone on the MR, even in the absence of 11-HSD2. The studies on isolated cardiomyocytes evidence effects of aldosterone on calcium or potassium currents, but these effects are observed using high concentrations of aldosterone in a milieu containing few or no glucocorticoids. The situation is different for the cortisol–mineralocorticoid receptor complex. Under physiological conditions, this complex is inactive, but under pathophysiological conditions it may be activated and function like the aldosterone– mineralocorticoid receptor complex [22]. It would be interesting to compare the effects of aldosterone and those of glucocorticoids in the presence of a GR inhibitor

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in order to identify the MR-dependent effect alone on isolated cardiomyocytes. One might think they are identical, since both hormones are able to link the MR with the same affinity, and indeed corticosterone activates the MR in smooth muscle cells and triggers rapid responses of MAP kinase and ERK1/2 pathways, which can have adverse consequences on the vessel [23]. To complicate the matter, several studies have shown that cortisol may block the action of aldosterone, suggesting that in many cases cortisol binds the MR and acts as a MR antagonist. Transgenic mice overexpressing the MR in cardiomyocytes [18] or in other cell types are a powerful and elegant means to explore these mechanisms. It is therefore important to realize that MR antagonists inhibit the effetcs of aldosterone in cells containing the 11-HSD2, but may also inhibit the action of cortisol in cells which do not express this enzyme. In the case of the few specific MR antagonist spironolactone, the actions mediated by the GR may also be partially inhibited.

14.7 Interferences Between Aldosterone and Angiotensin II One of the difficulties in interpreting the effects of aldosterone depends on the interactions between the signaling pathways activated by other hormones or receptors, namely Ang II and MR (review in [24]). Several laboratories have demonstrated that aldosterone stimulates the transcription of the AT1 receptor (AT1R) of Ang II, and ACE, which results in an increased local production of Ang II. On the other hand, the Ang II-dependent increase in collagens is at least in part an aldosteronedependent effect [25]. It has been recently shown in hamster-isolated cardiomyocytes that eplerenone inhibits the intracrine action of Ang II on inward calcium current and reduces drastically the effect of extracellular Ang II on the ICa current [26]. Since aldosterone reverse eplerenone effetcs, these results show that the MR is an essential component of the intracrine renin angiotensin aldosterone system. Interestingly, the proliferation of vascular smooth muscle cells is stimulated by a combination of low doses of aldosterone and of Ang II, while aldosterone or Ang II alone have no effects [27]. Similarly, aldosterone increases neovascularization in an in vivo model of ischemia secondary to right femoral artery ligature in mice [28]. Inhibition of these effects by valsartan shows that the pro-angiogenic action of aldosterone involves the AT1R. Finally, Ang II can directly activate the MR in the coronary and aortic CML [29]. So there are interactions between the effects of aldosterone and those of Ang II, reinforcing the therapeutic interest of combining MR and AT1R inhibitors in cardiovascular diseases.

14.8 Conclusion There are still many avenues to explore. In addition to the mechanisms, it is pertinent to determine whether the increase in aldosterone in common diseases such as diabetes, hypertension, and left ventricular hypertrophy is an additional risk factor.

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For example, in metabolic syndrome plasma aldosterone is increased [30]. In rat cardiomyocytes, the local production of aldosterone modulates potassium currents and increases oxidative stress, but only in male diabetic animals [31]. Preliminary results from our laboratory show that in the mouse heart, a slight increase in aldosterone exerts a protective role against the deleterious effects of type 1 diabetes. This is another example of the complexity of the effects of aldosterone which appear to vary depending on the concentration of the hormone, of gender, and of the cellular environment.

References 1. Pitt, B., Zannad, F., and Remme, W.J., et al. (1999) The effect of spironolactone on morbidity and mortality in patients with severe heart failure. Randomized Aldactone Evaluation Study Investigators. N Engl J Med 341(10), 709–717. 2. Pitt, B., Remme, W., and Zannad, F., et al. (2003) Eplerenone, a selective aldosterone blocker, in patients with left ventricular dysfunction after myocardial infarction. N Engl J Med 348(14), 1309–1321. 3. Adler, G.K., and Williams, G.H. (2007) Aldosterone: villain or protector? Hypertension 50(1), 31–32. 4. Tait, S.A., Tait, J.F., and Coghlan, J.P. (2004) The discovery, isolation and identification of aldosterone: reflections on emerging regulation and function. Mol Cell Endocrinol 217(1–2), 1–21. 5. Bois, P., and Selye, H. (1956) 2-Methyl-9(alpha)-chlorocortisol, a new synthetic mineralocorticoid with unusually intense nephrotoxic actions. Can Med Assoc J 75(9), 720–724. 6. Delcayre, C., Swynghedauw, B. (2002) Molecular mechanisms of myocardial remodeling. The role of aldosterone. J Mol Cell Cardiol 34(12), 1577–1584. 7. Sun, Y., Zhang, J., Lu, L., Chen, S.S., Quinn, M.T., and Weber, K.T. (2002) Aldosteroneinduced inflammation in the rat heart: role of oxidative stress. Am J Pathol 161(5), 1773–1781. 8. Virdis, A., Neves, M.F., Amiri, F., Viel, E., Touyz, R.M., and Schiffrin, E.L. (2002) Spironolactone improves angiotensin-induced vascular changes and oxidative stress. Hypertension 40(4), 504–510. 9. Keidar, S., Kaplan, M., and Pavlotzky, E., et al. (2004) Aldosterone administration to mice stimulates macrophage NADPH oxidase and increases atherosclerosis development: a possible role for angiotensin-converting enzyme and the receptors for angiotensin II and aldosterone. Circulation 109(18), 2213–2220. 10. Mazak, I., Fiebeler, A., and Muller, D.N., et al. (2004) Aldosterone potentiates angiotensin II-induced signaling in vascular smooth muscle cells. Circulation 109(22), 2792–2800. 11. Gerling, I.C., Sun, Y., and Ahokas, R.A., et al. (2003) Aldosteronism: an immunostimulatory state precedes proinflammatory/fibrogenic cardiac phenotype. Am J Physiol Heart Circ Physiol 285(2), H813–H821. 12. Garnier, A., Bendall, J.K., and Fuchs, S., et al. (2004) Cardiac specific increase in aldosterone production induces coronary dysfunction in aldosterone synthase-transgenic mice. Circulation 110(13), 1819–1825. 13. Zannad, F., Alla, F., Dousset, B., Perez, A., and Pitt, B. (2000) Limitation of excessive extracellular matrix turnover may contribute to survival benefit of spironolactone therapy in patients with congestive heart failure: insights from the randomized aldactone evaluation study (RALES). Rales Investigators. Circulation 102(22), 2700–2706. 14. Robert, V., Heymes, C., Silvestre, J.S., Sabri, A., Swynghedauw, B., and Delcayre, C. (1999) Angiotensin AT1 receptor subtype as a cardiac target of aldosterone: role in aldosterone-saltinduced fibrosis. Hypertension 33(4), 981–986.

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15. Harada, E., Yoshimura, M., and Yasue, H., et al. (2001) Aldosterone induces angiotensinconverting-enzyme gene expression in cultured neonatal rat cardiocytes. Circulation 104(2), 137–139. 16. Milliez, P., Girerd, X., Plouin, P.F., Blacher, J., Safar, M.E., and Mourad, J.J. (2005) Evidence for an increased rate of cardiovascular events in patients with primary aldosteronism. J Am Coll Cardiol 45(8), 1243–1248. 17. Milliez, P., Deangelis, N., and Rucker-Martin, C., et al. (2005) Spironolactone reduces fibrosis of dilated atria during heart failure in rats with myocardial infarction. Eur Heart J 26(20), 2193–2199. 18. Ouvrard-Pascaud, A., Puttini, S., and Sainte-Marie, Y., et al. (2004) Conditional gene expression in renal collecting duct epithelial cells: use of the inducible Cre-lox system. Am J Physiol Renal Physiol 286(1), F180–F187. 19. Benitah, J.P., Perrier, E., Gomez, A.M., and Vassort, G. (2001) Effects of aldosterone on transient outward K+ current density in rat ventricular myocytes. J Physiol 537(Pt 1), 151–160. 20. Cachofeiro, V., Miana, M., and de Las Heras, N., et al. (2008) Aldosterone and the vascular system. J Steroid Biochem Mol Biol 109, 331–335. 21. Ambroisine, M.L., Favre, J., and Oliviero, P., et al. (2007) Aldosterone-induced coronary dysfunction in transgenic mice involves the calcium-activated potassium (BKCa) channels of vascular smooth muscle cells. Circulation 116(21), 2435–2443. 22. van den Meiracker, A.H., and Batenburg, W.W. (2008) Corticosteroid-dependent, aldosteroneindependent mineralocorticoid-receptor activation in the heart. J Hypertens 26(7), 1307–1309. 23. Molnar, G.A., Lindschau, C., and Dubrovska, G., et al. (2008) Glucocorticoid-related signaling effects in vascular smooth muscle cells. Hypertension 51(5), 1372–1378. 24. Lemarie, C.A., Paradis, P., and Schiffrin, E.L. (2008) New insights on signaling cascades induced by cross-talk between angiotensin II and aldosterone. J Mol Med 86, 673–678. 25. Neves, M.F., Amiri, F., Virdis, A., Diep, Q.N., and Schiffrin, E.L. (2005) Role of aldosterone in angiotensin II-induced cardiac and aortic inflammation, fibrosis, and hypertrophy. Can J Physiol Pharmacol 83(11), 999–1006. 26. De Mello, W.C., and Gerena, Y. (2008) Eplerenone inhibits the intracrine and extracellular actions of angiotensin II on the inward calcium current in the failing heart. On the presence of an intracrine renin angiotensin aldosterone system. Regul Pept [Epub ahead of print]. 27. Min, L.J., Mogi, M., Li, J.M., Iwanami, J., Iwai, M., and Horiuchi, M. (2005) Aldosterone and angiotensin II synergistically induce mitogenic response in vascular smooth muscle cells. Circ Res 97(5), 434–442. 28. Michel, F., Ambroisine, M.L., Duriez, M., Delcayre, C., Levy, B.I., and Silvestre, J.S. (2004) Aldosterone enhances ischemia-induced neovascularization through angiotensin II-dependent pathway. Circulation 109(16), 1933–1937. 29. Jaffe, I.Z., and Mendelsohn, M.E. (2005) Angiotensin II and aldosterone regulate gene transcription via functional mineralocortocoid receptors in human coronary artery smooth muscle cells. Circ Res 96(6), 643–650. 30. Krug, A.W., and Ehrhart-Bornstein, M. (2008) Aldosterone and metabolic syndrome: is increased aldosterone in metabolic syndrome patients an additional risk factor? Hypertension 51(5), 1252–1258. 31. Shimoni, Y., Chen, K., Emmett, T., and Kargacin, G. (2008) Aldosterone and the autocrine modulation of potassium currents and oxidative stress in the diabetic rat heart. Br J Pharmacol 154, 675–687.

Chapter 15

Renin Angiotensin System and Atherosclerosis Changping Hu and Jawahar L. Mehta

Abstract Renin angiotensin system (RAS) regulates a host of biological functions in the body, including maintenance of vascular tone. All components of RAS have been identified in atherosclerotic tissues and are believed to regulate oxidation of LDL-cholesterol, endothelial function, formation of foam cells, smooth muscle cell proliferation, and collagen deposition that covers the atherosclerotic plaque. In keeping with the concept of the pathogenic role of RAS activation in atherosclerosis, inhibition of renin formation, angiotensin-converting enzyme and angiotensin II type 1 receptor activation, all have been shown to inhibit atherogenesis, primarily via the inhibition of oxidative stress. Recent studies from our laboratory show that delivery of angiotensin II type 2 receptor cDNA with adeno-associated virus as vector can inhibit the process of atherogenesis in the LDL receptor-knockout mice. This is associated with a marked increase in the expression of endothelial constitutive nitric oxide synthase, heme-oxygenase-1, and Akt activation and a dramatic reduction in the expression of LOX-1 and activity of NADPH oxidase and the redox-sensitive transcription factor NF-kB as well as the pro-inflammatory activity of p38 component of MAP kinase. This new information has the potential to lead to the development of novel therapeutic strategies directed at different components of RAS either in combination or as stand-alone therapy. Keywords Angiotensin II · Atherogenesis · LDL receptor-knockout mice · LOX-1

15.1 A Brief Overview of Renin Angiotensin System As recognized by all [1], renin is a 40,000-dalton glycoprotein that is expressed, stored, and released in a regulated manner by the juxtaglomerular cells of the kidneys. Renin, initially synthesized as an inactive zymogen known as pro-renin, C. Hu (B) Division of Cardiovascular Medicine, University of Arkansas for Medical Sciences and the Central Arkansas Veterans Healthcare System, Little Rock, AR, USA e-mail: [email protected] W.C. DeMello, E.D. Frohlich (eds.), Renin Angiotensin System and Cardiovascular Disease, Contemporary Cardiology, DOI 10.1007/978-1-60761-186-8_15,  C Humana Press, a part of Springer Science+Business Media, LLC 2009

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has exquisite substrate specificity, and its only known substrate is angiotensinogen. Angiotensinogen, mainly synthesized in the liver, is the only precursor of angiotensin peptides. Renin cleaves the N-terminus of circulating angiotensinogen to form angiotensin (Ang) I, which is an inactive peptide for which no specific receptor has so far been established. Ang I, in turn, is cleaved by the endothelial cell-associated or soluble dipeptidyl carboxypeptidase angiotensin-converting enzyme (ACE) to form the biologically active Ang II. Among many receptors that Ang II acts on, the two main receptors relevant to cardiovascular system are type 1 (AT1) and type 2 (AT2); both stem from single genes found on chromosome 3 and X, respectively. Both AT1 and AT2 receptors are seven-transmembrane, G-protein-coupled receptors. Both are expressed endogenously at very low levels. The AT1 receptors are mainly expressed in blood vessels, adrenal cortex, liver, and brain. The AT2 receptors are found primarily in fetal tissues, adrenal medulla, and uterus. While AT2 receptors are present in large numbers at birth, their number declines rapidly so that in the adult the AT1 receptors predominate [2, 3]. Most of the known physiologic functions of Ang II appear to be mediated by the activation of AT1 receptors, whereas the AT2 receptor activation appears to mediate apoptosis and growth inhibition as well as fetal growth regulation [4, 5]. Activation of AT1 receptors by Ang II stimulates a variety of intracellular signal pathways, including those typically activated by G-protein-coupled receptors, growth factor receptors, and cytokines, as well as events leading to the regulation of receptor function, such as phosphorylation and internalization of the receptor [6]. AT1 receptor activation also regulates gene expression, leading to a variety of growth-related responses, including activation of receptor and non-receptor protein tyrosine kinases [1, 2]. In addition, AT1 receptor activation by Ang II stimulates signal transducers and activators of transcription pathway, small G proteins, and expression of other important regulatory enzymes, such as phospholipase D, phospholipase A2, and NAD(P)H oxidase [1, 2]. Ang II binding to AT1 receptors also stimulates the internalization and processing of the ligand–receptor complex [1, 2]. Ang II exerts most of the physiologic functions of the renin angiotensin system by activating membrane-bound receptors, whereas angiotensinogen and Ang I are inactive peptides for which no function has been described so far [1, 2].

15.2 Renin Angiotensin System, Oxidative Stress, and LOX-1 Expression Ang II activates NADPH oxidase system, perhaps the most powerful system in mammalian species, resulting in the generation of reactive oxygen species (ROS), including superoxide anions and hydrogen peroxide [7]. Many of the effects of Ang II are thought to be related to the release of ROS. When the effect of ROS cannot be adequately countered by innate antioxidant system, this state is often termed as oxidative stress [8]. Oxidative stress is present in a variety of cardiovascular disease states, such as atherosclerosis, hypertension, and myocardial ischemia [8]. While some amount of oxidative stress is necessary for survival, release of large amounts

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of oxidants is clearly pathologic. Studies from our and others’ laboratories have shown that Ang II via redox-sensitive NF-κB pathway leads to the expression of a lectin-like oxidized low-density lipoprotein (LDL) receptor (now commonly known as LOX-1) in endothelial cells as well as smooth muscle cells [9]. LOX-1 activation by oxidized LDL-cholesterol (ox-LDL) also via the activation of NF-κB and Oct-1 pathways leads to the transcription of AT1 receptors [10, 11]. Thus, there is a positive feedback loop between Ang II and dyslipidemia involving redox-sensitive intracellular pathways [12]. This aspect of renin angiotensin system biology and its relevance will be discussed in detail later in this chapter.

15.2.1 Oxidative Stress in Atherosclerosis Atherosclerosis is the most common aging-associated disease in the developed countries, and its incidence is rapidly rising in the developing countries. All atherosclerosis risk factors, such as diabetes, hypertension, dyslipidemia, and smoking are associated with excessive production of ROS well beyond the capability of endogenous antioxidants to scavenge them [13]. Increased oxidative stress plays an important role in various steps in atherosclerosis as exemplified by the following [12, 14]: (i) oxidative stress induces the oxidation of LDL-cholesterol, resulting in the formation of ox-LDL and its uptake by vascular wall components in large part by activation of LOX-1 and other scavenger receptors; (ii) oxidative stress activates endothelial cells, resulting in the expression of adhesion molecules which lead to the recruitment and adhesion of monocytes to the endothelium and their subsequent migration into the subendothelial space; (iii) oxidative stress is responsible for the release of metalloproteinases (MMPs) which cause the disruption of atherosclerotic plaque; (iv) oxidative stress causes the proliferation and migration of smooth muscle cells and fibroblasts, leading to the thickening of vessel wall and narrowing of the lumen; and (v) oxidative stress activates platelets and stimulates the formation of the occlusive thrombus in the narrowed arterial lumen. Both ox-LDL and Ang II facilitate the initiation and propagation of oxidative stress in the blood vessel wall [15–17]. Previous in vitro studies indicated that treatment directing at ox-LDL or renin angiotensin system have antioxidant effects in vascular wall components [18, 19]. Further, therapies targeted at hyperlipidemia (such as with statins) or renin angiotensin system blockers, especially AT1 receptor blockers, have potent antioxidant and antiatherosclerotic effects in laboratory animals and humans [20, 21]. Recent evidence supports the concept that ox-LDL and Ang II interact synergistically in the context of oxidative stress [22]. This idea is based on the following considerations: first, there are striking similarities between the effects of ox-LDL and Ang II, especially those related to oxidative stress, in vascular endothelial cells and smooth muscle cells as well as in animal tissues [15–17]; second, the blockade of AT1 receptors normalizes the activity of NADPH oxidases and reduces atherosclerotic plaque burden in animals fed a high-cholesterol diet [23]; and third, ox-LDL and Ang II together induce cumulative injurious effects in cells, and importantly, antioxidants such as vitamin E attenuate these injurious effects [24].

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15.2.2 Role of LOX-1 in Atherosclerosis LOX-1 is a 52-kD surface receptor initially identified in bovine aortic endothelial cells [25]. This receptor, upregulated by ox-LDL, is responsible for binding and uptake of ox-LDL in endothelial cells [26, 27]. The contributory role of LOX-1 in atherogenesis is supported by several lines of evidence [14]: (i) LOX-1 binds, internalizes, and degrades ox-LDL in endothelial cells; (ii) ox-LDL-induced endothelial dysfunction is mediated by LOX-1 activation; (iii) besides ox-LDL, other mediators of atherosclerosis, such as angiotensin II, cytokine TNF-α, sheer stress, ROS, and advanced glycation end-products, upregulate LOX-1; (iv) LOX-1 is dynamically upregulated by pro-atherogenic conditions, such as diabetes, hypertension, and dyslipidemia; (v) LOX-1 is present in atheroma-derived cells and in human and animal atherosclerotic lesions; (vi) LOX-1 blockade modulates endothelial function in coronary arterioles in atherosclerotic ApoE knockout mice [28] as well as in LDLreceptor knockout mice [29]; and most importantly LOX-1 deletion in the LDLR knockout mice significantly reduced atherogenesis [29]. LOX-1 is intimately involved in the generation of ROS in inflammatory cells and endothelial cells [30, 31], and in a positive feedback fashion ROS directly induces LOX-1 expression. LOX-1 has also recently been identified on the surface of platelets [32], which plays an important role in platelet aggregation and thrombus formation [33]. In recent studies, we established that fibroblasts proliferate and express collagen signal rapidly in response to Ang II [16]. Chen and colleagues [9] in our laboratory observed that NF-κB activation plays a critical role in Ang II-induced LOX-1 promoter activation. As is well known, vascular fibrosis is stimulated by the cytokine transforming growth factor β1 [34]. Hu et al. [35] observed that the transforming growth factor β1-mediated increase in collagen synthesis is markedly attenuated in fibroblasts from the LOX-1 knockout mouse, suggesting that LOX-1 is an important link in collagen formation in fibroblasts in response to growth factors such as angiotensin II and transforming growth factor β1. Ishigaki et al. [36] reported recently that LOX-1 expressed ectopically in the liver with adenovirus administration in apoE-deficient mice, another animal model of hypercholesterolemia and atherosclerosis, removed ox-LDL from circulating blood and possibly decreased systemic oxidative stress, resulting in the prevention of atherosclerosis. In keeping with this concept, LOX-1 transgenic mice display accelerated intramyocardial vasculopathy and a marked increase in atheroma-like lesion areas [37].

15.2.3 Cross-Talk Between Oxidized-LDL and Renin Angiotensin System Ox-LDL has been shown to increase the expression of ACE and AT1 receptors in cultured endothelial cells and smooth muscle cells [10, 38]. Atherosclerotic lesions in hyperlipidemic animals show activation of renin angiotensin system

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[39]. Studies in human atherosclerotic tissues have also confirmed the activation of renin angiotensin system, particularly in the regions prone to plaque rupture [40]. Conversely, there is evidence for downregulation of AT1 receptor expression with lipid-lowering reagents, particularly 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (also known as statins), in vascular smooth muscle cells and endothelial cells [38, 41], and in hyperlipidemic atherosclerotic animals and humans [41, 42]. It also should be noted that while high levels of LDL-cholesterol upregulate various components of renin angiotensin system, Ang II via AT1 receptor activation facilitates cellular cholesterol biosynthesis, oxidation of LDL, and uptake of ox-LDL by vessel wall components [12].

15.2.4 Aneurysm Formation in Atherosclerosis – Role of Lipids and Renin Angiotensin System Aneurysm is defined as a permanent dilation of the arterial wall, which is characterized by outward vascular remodeling, both in vascular dimension and in structure. Processes such as proteolysis, inflammation, and ROS formation are important during aneurysm formation. As discussed earlier, ACE and downstream regulators such as Ang II and AT1 receptor activation are also involved in this process [43]. ACE is expressed in the aneurysmal vascular wall, both in human tissues and in animal models. In aneurysmal aortic specimens obtained during operative repair in patients, ACE activity was found to be significantly increased compared to that in the normal segments [43]. ACE-positive cells, determined by immunohistochemical staining, were mainly macrophages, both in the media and in the intima [43]. Chymase activity was also significantly increased in these specimens and chymase-positive cells were mainly mast cells in the media and adventitia [43]. In a rabbit model for aneurysm formation, by elastase perfusion, ACE protein levels in the aortic wall increased during aneurysm growth over time [43]. In mice, infusion of Ang II induced aneurysm formation independent of changes in blood pressure. This was shown in a hyperlipidemic setting in ApoE knockout mice as well as in the wild-type C57BL6 mice, although aneurysm formation was smaller in the wild-type mice [43]. This implies a critical role of hyperlipidemia in the development of aneurysms in response to Ang II. In this hyperlipidemic mice model, proteolytic processes are clearly involved since the broad-spectrum matrix MMP inhibitor doxycycline reduced the severity of aneurysm formation [43]. Ang II-induced aneurysm also displays characteristic inflammatory features, which fits with the role of Ang II in inflammatory processes [43]. These studies suggest that Ang II, partly because of its inflammatory effects, is an important initiator of aneurysm formation.

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15.2.5 Studies of Angiotensin-Converting Enzyme Inhibitors and Angiotensin Type 1 Receptor Antagonists in Atherosclerosis ACE inhibitors act by inhibiting the conversion of Ang I to Ang II. In addition, ACE inhibitors decrease the breakdown of bradykinin. Furthermore, ACE inhibitors, which increase tissue and plasma levels of angiotensin (1–7), may also improve the fibrinolytic balance by decreasing plasminogen activator inhibitor-1 formation via Ang IV and/or reduce Ang II-induced activation of angiotensin type 4 receptors [44]. Since tissue ACE is highly expressed in human atherosclerotic plaques [45], where it is localized in areas of clustered macrophages, and is significantly increased in patients with unstable angina [44], it has been suggested that blocking ACE will prevent plaque fissuring, thrombosis, and rupture. A direct anti-atherogenic effect of ACE inhibitors has been shown in a variety of animal models. A large body of data on the beneficial effects of ACE inhibitors comes from landmark clinical trials such as the Heart Outcomes Prevention Evaluation (HOPE) study and the EUropean trial of Reduction Of cardiac events with Perindopril in stable coronary Artery disease (EUROPA), which included patients with documented coronary artery disease (CAD), risk factors, and preserved left ventricular function. These two, and several other, trials demonstrated convincingly that cardiovascular events could be reduced in patients who were treated with an ACE inhibitor [46, 47]. These large morbidity and mortality trials clearly support the role of ACE inhibitors in the treatment of atherosclerotic vascular diseases. The AT1 antagonists bind to the AT1 receptor with high affinity. These agents reduce the activation of AT1 receptor-mediated actions of Ang II more effectively than ACE inhibitors since the latter do not reduce alternative, non-ACE Ang II-generating pathways, such as those involving chymase, or cathepsin G [44]. In contrast to ACE inhibitors, AT1 antagonists indirectly activate AT2 receptors. The importance of AT2-mediated effects is not clearly established. Nevertheless, recent studies suggest that AT2 receptors exert anti-proliferative, pro-apoptotic, and vasodilatory actions, and may have a modest effect on promoting bradykinin release [44]. ACE inhibitors increase angiotensin (1–7) levels more than AT1 antagonists, and this may result in additional beneficial cardiac and vascular effects. Moreover, ACE inhibitors increase the levels of a number of other ACE substrates that are not angiotensin peptides, including bradykinin. The increase in bradykinin levels may also contribute to the beneficial cardiovascular effects of ACE inhibitors. Whether or not these distinct pharmacological differences between AT1 antagonists and ACE inhibitors result in significant differences in therapeutic outcomes is not known at present. Experimental studies in mice, rabbits, and primates have demonstrated convincingly that AT1 receptor antagonists decrease cardiac and arterial medial hypertrophy and reduce the development of atherosclerotic lesions [44]. A number of studies have demonstrated that AT1 antagonists, especially losartan, may exert additional anti-aggregatory and anti-inflammatory actions [44]. These effects may be of potential interest especially with regard to the treatment of patients with acute manifestations of atherosclerotic disease such as acute myocardial infarction.

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Clinical trials have shown that AT1 receptor antagonists can effectively lower blood pressure and may positively influence atherosclerosis in hypertensive patients. However, unfortunately, there are no randomized, clinical trials to date on the effects of AT1 receptor antagonists on the anatomic progression of atherosclerotic vascular disease. The Losartan Intervention for Endpoint reduction (LIFE) in hypertension study showed lower primary event rate in the losartan-based treatment group. Interestingly, most benefit was related to a 25% reduction in the rate of strokes [44]. A renoprotective effect of AT1 receptor antagonists was convincingly demonstrated in the Irbesartan Diabetic Nephropathy Trial (IDNT) [48], the Reduction of Endpoints in NIDDM (Non–Insulin-Dependent Diabetes Mellitus) with the Angiotensin II Antagonist Losartan (RENAAL) study [48], and the IRbesartan MicroAlbuminuria type 2 diabetes mellitus in hypertensive patients trial (IRMA 2) [44]. Cardiovascular end-points were pre-specified secondary outcomes in the RENAAL and IDNT studies. In the RENAAL study, the rates of fatal and non-fatal cardiovascular events did not differ significantly between the study groups, with the exception of hospitalization for heart failure, for which the risk was reduced by 32% in the losartan group. In the IDNT study, there was no significant difference in cardiovascular outcome between study groups. However, these studies, which enrolled relatively small number of patients, were statistically underpowered to show significant differences in cardiovascular outcome. It remains speculative whether larger trials may show more clear-cut cardiovascular benefits and whether the renoprotective benefits demonstrated may be at least in part mediated by vascular protective actions of AT1 receptor antagonists. To date, there is only small body of data from large-scale randomized trials with AT1 receptor antagonist in chronic atherosclerotic vascular diseases. Further, whether AT1 receptor antagonists are superior to ACE inhibitors has not been clearly established.

15.2.6 Studies with Renin Inhibitors in Atherosclerosis Renin is the rate-limiting enzyme in the production of all angiotensin peptides. It has been shown that in fat-fed LDL receptor-deficient mice administered the novel renin inhibitor aliskiren over a broad dose range, renin inhibition results in striking reductions of atherosclerotic lesion size in both the aortic arch and the root [49]. Subsequent studies demonstrated that cultured macrophages express all components of the renin angiotensin system. To determine the role of macrophage-derived angiotensin in the development of atherosclerosis, renin-deficient bone marrow was transplanted to irradiated LDL-receptor deficient mice and a profound decrease in the size of atherosclerotic lesions was observed. In similar experiments, transplantation of bone marrow deficient for Ang II type 1a receptors failed to influence lesion development. These findings suggest that renin-dependent angiotensin production in macrophages does not act in an autocrine/paracrine manner. Furthermore, in vitro studies demonstrated that co-culture with renin-expressing macrophages augmented monocyte adhesion to endothelial cells. Therefore, although previous

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work suggests that angiotensin peptides have conflicting effects, renin inhibition profoundly decreases lesion atherosclerotic lesion development in mice.

15.2.7 Reduction in Atherosclerosis with Combination of Statins and Angiotensin-Converting Enzyme Inhibitors/AT1 Receptor Antagonists The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) induce regression of atherosclerosis and reduce cardiovascular-related morbidity and mortality in patients with and without coronary artery disease [50]. Data from both primary and secondary prevention trials demonstrate that statins reduce the risk of cardiovascular events well beyond their hyperlipidemic effect. Recent evidence suggests that statins and blockers of the renin angiotensin system may share the ability to interfere with a number of key atherogenic processes, including smooth muscle cell migration/proliferation, inflammatory reactions, platelet adhesion/aggregation, macrophage activation, and mediator expression [50]. Statins reduce Ang II-induced inflammation within atherosclerotic plaques, leading to stabilization of these lesions and also to downregulation of AT1 receptor expression in isolated vascular smooth muscle cells. Statin-mediated reduction of AT1 receptor gene expression may impair the effects of Ang II on intracellular signaling, which may ultimately lead to diminished activation of NADPH oxidase. In studies by Chen et al. [51], combination of a potent statin rosuvastatin and an AT1 receptor blocker candesartan almost completely blocked atherogenesis in ApoE knockout mice. Further, there was a dramatic cumulative effect of these agents on markers of oxidative stress, inflammation, and expression of MMPs. These effects may well have been a direct effect of total blockade of LOX-1 expression. Hyperlipidemia is a frequent component of the metabolic syndrome, and pre-clinical studies show that statins in combination with ACE inhibitors or AT1 receptor antagonists may facilitate blood pressure control via a mechanism independent of lowering of plasma lipids. Nazzaro and colleagues [52] examined the individual and combined effects of ACE inhibitors and statins on blood pressure in patients with coexisting hypertension and hypercholesterolemia. Monotherapy with enalapril or simvastatin resulted in a reduction in blood pressure, but administration of both agents produced greater blood pressure reduction than with either agent alone. The propensity of lipid-lowering agents to provide additive benefit to ACE inhibitors in lowering blood pressure has also been noted in several other studies. Collectively, these studies provide strong evidence of a close interplay between dyslipidemia and the renin angiotensin system. Investigation of combined effects of statins and ACE inhibitors or AT1 receptor antagonists on management of patients with resistant hypertension is of considerable interest, because endothelial dysfunction may reduce the antihypertensive action of drugs that predominantly act via restoration of vascular endothelial function. The combination of these drugs may provide an improved approach to the management of endothelial dysfunction, inflammation, oxidative stress, and atherosclerosis.

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15.3 Angiotensin II Type 2 Receptor Upregulation in Atherogenesis There is substantial evidence that Ang II mediates atherosclerosis development mainly via the activation of AT1 receptors [1, 44]. Moreover, deletion of the AT1a receptor in ApoE knockout mice prevents atherosclerosis formation, underlining the importance of this receptor subtype in this pathology [53]. Since there is growing evidence that the activation of the AT2 receptor may act opposite to the activation of the AT1 receptor, the role of AT2 receptor in atherogenesis has been examined. Iwai et al. [54] showed that deletion of AT2 receptor exaggerated atherosclerosis in Apo-E deficiency mice. Others, however, reported that AT2 receptor deficiency had no effect on atherogenesis in LDL receptor deficient [55] and ApoE deficient mice [56]; yet AT2 receptor deficiency augmented the cellularity of atherosclerotic lesions. Johnasson et al. [57] found that administration of an AT2 receptor antagonist had no effect on Ang II-accelerated atherosclerosis in Apo-E deficiency mice. Results presented in these studies are discrepant, and, therefore, no definitive conclusion concerning the role of AT2 receptors in atherogenesis can yet be proposed. The reasons advocated for this discrepancy can be linked to sex, strain of mice, and the model of Ang II-induced atherogenesis. In fact, in all these studies, increased circulating Ang II levels were achieved by infusion of high doses of exogenous Ang II. Using this model, the physiologic regulatory mechanisms of Ang II secretion were not taken into account. In addition, it has been recently shown that wild-type mice treated with an AT1 receptor antagonist have decreased tissue Ang II, despite increased plasma levels of Ang II [1]. This suggests that blockade of the AT1 receptor rather than enhanced stimulation of AT2 receptors may account for many of the beneficial effects seen with AT1 receptor antagonists. Moreover, in clinical trials and in everyday clinical practice, no substantial difference in the beneficial effects has been seen with either ACE inhibitors or AT1 receptor antagonists, suggesting that the precise contribution of AT2 receptors still remains to be established.

15.3.1 Reduction in Atherosclerosis with Gene Therapy Directed at AT2 Receptor Overexpression As mentioned above, AT2 receptor deletion was shown to exaggerate atherosclerosis in ApoE-deficient mice [54]. Therefore, we hypothesized that AT2R (agtr2) overexpression might inhibit atherogenesis [58]. We prepared recombinant adeno-associated virus type-2 (AAV) carrying AT2 receptor cDNA (AAV/AT2R) (Fig. 15.1), and homozygous LDL receptor-deficient mice were given AAV/AT2R, AAV/Neo or saline. All mice were placed on a high-cholesterol diet. After 18 weeks, AT2 receptor was found to be overexpressed systemically in AAV/AT2Rtreated mice. Atherogenesis in aorta was reduced in the AAV/AT2R group by ≈50% compared to other LDLR knockout mice given saline of AAV/Neo. Expression of NADPH oxidase, nitrotyrosine (a marker of oxidative stress), and the

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Fig. 15.1 Construction of AAV/AT2R vector and generation of rAAV stocks. Mouse AT2R (agtr2) cDNA was generated, sequenced, and ligated into an AAV vector, dl6-95. Recombinant AAV vector is referred to as AAV/AT2R. The titer of purified virus, in encapsidated genomes per milliliter (eg/ml), was calculated by dot-blot hybridization and determined to be about 1011 eg/ml

redox-sensitive transcription factor NF-κB was increased in aortic tissues of the LDL receptor-deficient mice given saline or AAV/Neo, but not in mice with AT2 receptor upregulation. Expression of endothelial nitric oxide synthase and hemeoxygenase-1 was decreased and that of LOX-1 increased in the LDL receptordeficient mice, but not in the mice with AT2 receptor overexpression. Further, Akt-1 phosphorylation was reduced in the LDL receptor-deficient mice, but not in the mice with AT receptor 2 overexpression (Fig. 15.2). Thus, AT2 receptor upregulation can reduce atherogenesis, possibly by modulating oxidative stress and the pro-inflammatory cascade, mediated via Akt-1, and may be an important therapeutic approach in atherosclerosis. However, these data need to be replicated in other experimental animals. If this approach proves successful, use of strategies designed at overexpression of AT2 receptors alone or with AT1 receptor antagonist therapy might be a useful approach in the treatment of atherosclerosis and related disorders.

15.3.2 Reduction in Collagen Deposition with AT2 Receptor Upregulation We also found that LDL receptor-deficient mice treated with saline or AAV/Neo exhibited extensive collagen accumulation in the aortic wall, which was reduced

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Fig. 15.2 (A) Representative examples of reduction in atherogenesis in LDLR-deficient mice given AAV/AT2R. The administration of AAV/Neo had no effect on the extent of atherosclerosis. (B) The administration of AAV/AT2R altered the expression of proteins assisted with atherogenesis, such as LOX-1, nitrotyrosine, eNOS, phos-Akt, and HO-1. This alteration most likely occurred as a result of reduction in NADPH oxidases (p47phox and p22phox ) and activation of NF-κB

by about 50% with AT2 receptor overexpression [59]. Further, AT2 receptor upregulation completely blocked the alterations in the expression of procollagenI, osteopontin, fibronectin, CD68, and MMPs (MMP-2 and MMP-9), as well as phosphorylation of p38 and p44/42 MAPKs. Activity of superoxide dismutase was reduced in the LDL receptor-deficient mice and it increased with AT2 receptor upregulation (Fig. 15.3). This study for the first time showed that AT2 receptor overexpression reduces enhanced collagen accumulation and MMP expression and activity in atherosclerotic regions via inhibition of pro-oxidant signals. Whether the reduction in collagen reflects merely a reduction in atherosclerosis or if it would soften the plaque leading to complications such as plaque rupture cannot be discerned from the present study.

15.3.3 Mechanisms of the Efficacy of AT2R Upregulation Enhanced expression of collagen appears to be an inherent part of the atherosclerotic process. Ang II is present in the atherosclerotic regions and activates NADPH oxidase system. The intense oxidant stress in the atherosclerotic regions stimulates mitogen-activated protein kinases and the redox-sensitive transcription factors, such as NF-κB, followed by upregulation of genes, such as fibronectin, osteopontin, collagens, and MMPs, which result in the formation of collagen. Interestingly, excessive collagen deposition is associated with enhanced release of MMPs. While ox-LDL and Ang II-stimulated LOX-1 activation enhances oxidative stress and inflammation, and oxidative stress per se upregulates LOX1 expression. This process may self-amplify leading to intense collagen deposition in atherosclerotic regions over time. These events are summarized in Fig. 15.4.

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Fig. 15.3 (A) Representative examples of reduction in collagen deposition in atherosclerotic regions in LDLR-deficient mice given AAV/AT2R. Collagen was estimated by tissue staining with Trichrome and Picro-serius red and the results were consistent. The administration of AAV/Neo had no effect on collagen deposition in atherosclerotic regions. (B) The administration of AAV/AT2R altered the expression of proteins assisted with atherogenesis, such as pro-collagen-1, osteopontin, fibronectin, extracellular superoxide dismutase (ecSOD), metalloproteinases (MMP-9 and MMP-2), and the inflammatory marker CD68. This alteration most likely occurred as a result of reduction in NADPH oxidases (p47phox and p22phox ) shown in Fig. 15.2 and activation of phosp38MAPK

15.4 Summary, Questions, Future Perspective, and Clinical Implications There is accumulating clinical and experimental evidence that the pathways by which hypertension and dyslipidemia lead to vascular changes may overlap and that Ang II is involved in restructuring (or remodeling) of the arterial wall in both atherosclerosis and hypertension. Activation of the renin angiotensin system seems to be a central pathophysiologic event in the progression of endothelial cell dysfunction to atherosclerosis and subsequent development of clinical syndromes. The pivotal role of the renin angiotensin system in atherogenesis is highlighted by studies in animal models as well as in humans indicating that inhibition of the renin angiotensin system with ACE inhibitors or AT1 receptor antagonists retards the development of atherosclerotic lesions. In light of a causal and central role of Ang II in atherogenesis, blockade of the renin angiotensin system has come to represent an important therapeutic consideration in the prevention and treatment of atherosclerotic disease.

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Fig. 15.4 Hypothesized pathways of collagen and MMPs changes modulated by LOX-1 activation (by angiotensin II) and the inhibition of the cascade by AT2R upregulation

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Chapter 16

Renin Angiotensin System and Aging León F. Ferder

Abstract We advance herein the hypothesis that aging and its consequences, including cardiac hypertrophy, result from actions of Angiotensin II (AII). Experimental findings indicate that hypertension and aging have similar effects on the structure and function of blood vessels, the heart, and, presumably, the kidney. Keywords Mitochondria · ROS · Aging · Angiotensin II (AII) · AII blockade AII has been proposed to be a mediator of oxidative stress through the induction of reactive oxygen species (ROS) in aging and in normal animals. Mitochondria are a major source of ROS in aging. AII blockade decreases ROS at the mitochondrial level, thus protecting against both age-related mitochondrial dysfunction and ultrastructural alterations, underscoring the role of renin angiotensin system (RAS) in the aging process. Diminished density of AII receptors leading to enhanced sensitivity to AII could explain partly the development of the aging process. This hypothesis is supported by our findings that ACE inhibitors and AII receptor antagonists decrease tissue oxidative state and improve mitochondrial number and function, leading to higher animal survival and prolonged lifespan. Organ malfunction is an eventual result of the natural process of aging, which all species undergo. Modifications in organ structure directly related to aging cause in humans functional deterioration of varying degrees by, among other things, leading to replacement of functional parenchyma by fibro-connective tissues and induction of arteriosclerosis in the blood vessels of several organs. Changes in kidney function, including its role as an endocrine organ, related to age have been recognized since the seventies. These changes include alterations in the regulation of the renin angiotensin system (decreased renin production and secretion) and in the endocrine and paracrine functions of their product angiotensin II (AII), both of which are L.F. Ferder (B) Departments of Physiology, Pharmacology and Medicine, Ponce School of Medicine, Ponce, PR e-mail: [email protected]

W.C. DeMello, E.D. Frohlich (eds.), Renin Angiotensin System and Cardiovascular Disease, Contemporary Cardiology, DOI 10.1007/978-1-60761-186-8_16,  C Humana Press, a part of Springer Science+Business Media, LLC 2009

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dependent on angiotensin II receptors [1, 2]. We advance herein the hypothesis that aging, and possibly hypertension and its consequences, including cardiac hypertrophy, results from actions of AII.

16.1 Angiotensin Receptors The AT1 receptor belongs to the G-protein-coupled receptor superfamily and has been cloned and characterized [3, 4]. AT1 receptor is considered to be the mediator for the cardiovascular and renal effects of AII in normal and, possibly, aging subjects. In contrast, the role, if any, of the AT2 receptor, which is highly expressed in growing tissues, in the cardiovascular changes in normal and aging subjects, remains unclear [5–7]. AII receptors show a great variability and can be detected in a wide variety of tissues; therefore, it is difficult to relate them to primary hypertension and their role has not been extensively explored in the aging process. Two AT1 receptor subtypes appear to exist in rodents, the AT1A and AT1B , which exhibit highly homologous sequences and similar binding and functional characteristics [8]. AT1A expression is by far the dominant form in liver, kidney, vasculature, and heart, whereas the AT1B is expressed predominantly in the adrenal gland, uterus, and anterior pituitary gland [9]. Both are downregulated by AII. Complementary mechanisms exist for AII downregulation of the cardiac AT1 gene in vitro via calcium- and cAMP-dependent mechanisms. Such a downregulation of the receptor gene by its agonist is consistent with other members of the G-protein-coupled family of receptors. The AT2 receptor, however, does not appear to be G-protein-coupled and possibly signals through phosphotyrosine phosphatase [10]. AII receptor subtypes have been characterized in cardiac and renal tissue. The AT1A and AT1B receptors present highly homologous sequences, and similar binding and functional characteristics [3, 8, 11]. In the rat heart, levels of both AT1 and AT2 receptor expression are increased during the neonatal period and decrease with maturation [6, 12]. This family of receptors typically responds to long-term agonist binding with a decrease in receptor number and mRNA levels [13–16]. AII treated mesangial cells also demonstrate a dose-dependent decrease in AT1 mRNA levels similar to that seen in cardiocytes and fibroblasts, whereas in some studies AII infused in vivo had no effect on the AT1 mRNA levels in the rat kidney or aorta, although mRNA levels were increased in the adrenal gland [17, 18]. By contrast, in models of experimental ureteral obstruction [19, 20], which leads to high intrarenal levels of AII, AT1 mRNA levels are downregulated, and this downregulation is partially reversed by angiotensin I converting inhibitors (ACEI) and angiotensin II receptor antagonists (AIIRA), suggesting that AII participates in gene regulation and the underlying pathophysiological conditions may determine the nature of the relationship between the peptide and the expression of the gene for its receptor. Moreover, in ureteral obstruction, both glomerular and tubular cells exhibit the changes in AT1 mRNA levels. This tissue- and cell-specific regulation of the AT1 gene likely speaks to the potential differences in the role of AII in different segments of various tissues [21].

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The high susceptibility of cardiomyocytes to the effects of AII and aging may be the result of the density of receptors at this site. Quantitative PCR studies in the rat have found that both AT1 receptor subtypes are present in the heart with AT1A mRNA levels threefold higher than AT1B [22]. Analysis of both the number and binding affinity of specific receptors appears to be important in the understanding of the pathophysiological role of the renin angiotensin system and in the potential role of AII in the heart and kidney in hypertension and aging. Nevertheless, as already mentioned, variation in AII receptor expression complicates the design of studies relating it to primary hypertension, while studies of its potential role in aging are just beginning to emerge [23, 24].

16.2 Angiotensin II and Reactive Oxygen Species in Aging Experimental findings indicate that hypertension and aging have similar effects on the structure and function of blood vessels, the heart, and, presumably, the kidney. Both conditions also lead to endothelial dysfunction, decreased vascular compliance, left ventricular hypertrophy, and stiffness. In the normal kidney, despite no change in blood pressure, AII leads to the expression of genes of sclerosisgenerating cytokines such as transforming growth factor beta (TGFβ1), plateletderived growth factors (PDGF), and osteopontin (OPT), all which enhance tissue fibrosis [25]. Despite the fact that aging is associated with decreased production and secretion of renin, kidneys are more sensitive to the renal vasoconstrictive effects of exogenous AII [26], suggesting an increase in receptor number, greater binding affinity of the peptide, or diminished metabolism of the peptide–receptor complex. Similar changes might be expected in the aging kidney, which could explain why ACEI or AIIRA mitigate some of the functional and architectural renal changes observed as age advances. AII has been proposed to be a mediator of oxidative stress through the induction of reactive oxygen species (ROS) in aging and in normal animals [27]. Oxidative stress is known to induce apoptosis and activation of the pro-inflammatory transcription factor nuclear factor kappa B (NFκB), whose biologic effects may play a role in the renal damage associated with arterial hypertension [28]. Oxidative stress, fueled in part by AII, upregulates the expression of adhesion molecules, chemoattractant compounds, and cytokines [29]. Mitochondria are a major source of ROS in aging because their mechanisms of defense from normally formed ROS and superoxide become less effective with the aging process. Moreover, excessive respiratory chain superoxide can combine with nitric oxide (NO) produced from mitochondrial nitric oxide synthase (NOS) to induce apoptosis through the formation of peroxynitrate. This potent oxidant can promote cytochrome C release by increasing the permeability of mitochondrial membranes by opening up permeability transition pores [30]. AII contributes to ventricular remodeling by promoting both cardiac hypertrophy and apoptosis; however, the mechanism underlying the latter phenomenon is poorly understood. One

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possibility that has been advanced is that AII activates NADPH oxidase, generating free radicals that trigger DNA damage and apoptosis [31]. AII blockade can protect against both age-related mitochondrial dysfunction and ultrastructural alterations, underscoring the role of RAS in the aging process [27]. It has been suggested that the adaptor protein p66Shc may also be a target for ROS [32]. When this protein is phosphorylated on Ser36 by oxidative stress, it markedly sensitizes cells to apoptosis, because it participates in the phosphorylation-induced repression of Forkhead transcription factors that regulate the expression of various antioxidant enzymes [32]. Furthermore, activation of a mitochondrial pool of p66Shc leads to enhanced generation of ROS [33]. Although the study did not examine the effect of AII on this potential pathway of oxidative damage in aging, it is conceivable that the same mechanism may apply to aging tissues, including the kidney. It is of great interest that p66Shc knock-out mice were resistant to the deleterious cardiac effects (remodeling) of sub-pressor doses of AII [34]. It remains to be studied whether a similar effect of p66Shc occurs in renal tissue.

16.3 Aging Response to AII Blockade It has long been recognized that plasma renin and aldosterone level fall with advancing age. Studies in aging animals indicate that both renal renin formation and release are reduced, and contribute to the fall in plasma renin concentration. The significance of this finding has remained obscure and its complexity is enhanced by the fact that elderly patients develop hypertension, which could be a factor in the reduced activity of the RAS. Even more intriguing, in attempting to explain the importance of falls in circulating levels of renin and angiotensin, are the findings reported by experiments in one of our laboratories. It was found that CF1 mice treated with the angiotensin I-converting enzyme blocker Enalapril and Wistar rats treated with an AIIRA exhibited a reduction of age-associated cardiovascular and renal changes, and had increases in the mitochondrial number within cells. This was associated with an increase in survival and lifespan of the animals. The beneficial effect of RAS blockade occurs in spite of the fact that, as reported, plasma renin concentration and intrarenal renin mRNA is reduced in older animals [35]. It is, however, consistent with an increase in angiotensin II formation or action within the kidney [36], and supports the concept that the intrarenal RAS is regulated independently of the circulating system in normal and also in aging animals. Our studies revealed that plasma levels of angiotensin I and angiotensin II were low in control (untreated) aged animals and that, as expected in non-aged normal animals, aged rats receiving AIIRA exhibited elevated levels of plasma renin, AI and AII, an indication that the angiotensin–renin feedback mechanism is intact in aged rats. Aging was accompanied by high glomerular angiotensin II receptor density as demonstrated by autoradiography. By contrast, receptor density was much lower in the interstitium of untreated aged animals, and was lower in both glomeruli and interstitium in the losartan-treated group [37].

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Ang II

Receptors AT1 and AT2

Redox Imbalance

Permeability Transition Pore (PTP) Activation

OXIDATIVE STRESS

Oxidative Damage to MtDNA

NF-кβ Defective Electron Transport Chain Profibrotic Cytokines

Release of Cytochrome C

Fibrosis Energy Deficit

Apoptosis

AGING, KIDNEY AND CARDIOVASCULAR DISEASES

Fig. 16.1 This figure shows how Angiotensin II (All), through oxidative stress, regulates apotosis, inflammation, and energy balance

These results strongly suggest that the renin angiotensin system plays an important role in the mechanisms by which aging affects renal function and that an abnormal regulation of angiotensin II receptors may be responsible for the functional alterations of the kidney. Moreover, AII effects through the production of reactive oxygen species, and by damage of mitochondrial function can be responsible for these alterations. This hypothesis is supported by our findings that ACE inhibitors and AIIRA decrease tissue oxidative state, improve mitochondrial number and function, leading to higher animal survival and prolonged lifespan (Fig. 16.1).

16.4 Vascular and Cardiac AII AT1 and AT2 Receptors The percentages of AT1 and AT2 receptors vary depending on the cell type or tissues considered: in rat aorta 60% of the receptors are AT1 , whereas in total rat heart, the percentage of AT1 reaches 90% [38] of the angiotensin receptors. Cardiomyocytes express exclusively the AT1 subtype [39], whereas fibroblasts express AT2 receptor subtype as well [40]. Although it has been suggested that AT2 receptor activation is involved in the control of cell differentiation, proliferation, and

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apoptosis [41, 42], the possible roles of AT2 receptors in vivo are poorly understood. In addition, their contribution to the pathophysiology of aging remains obscure (see below). Moreover, limited knowledge exists of the state of function and genetic regulation of AT receptors and their subtypes in renal tissues of aging animals. AII-induced hypertension and associated cardiac hypertrophy are mediated mainly via the AT1 receptor subtype. That AII mediates vascular smooth muscle cell (VSMC) trophic effect via AT2 receptor subtype and independently of a pressuredependent mechanism can be inferred from the finding that AT2 receptor blockade with PD123319 infusion in AII-treated rats had no significant effect on blood pressure but prevented the development of vascular hypertrophy of both aorta and coronary arteries. In addition, in normotensive rats, treatment with losartan alone had no effect on blood pressure but induced a medial hypertrophy that was prevented by an additional treatment with PD123319, suggesting that the increase in systemic AII concentration, as a result of AT1 receptor blockade [43] activates the AT2 receptors and, as a consequence, unmasks their trophic effect. The trophic effect of the AT2 receptor subtype appears to be specific to VSMCs and independent of whether it is found in conductive (aorta) or resistance (coronary artery) vessels. Also, it has been shown that AT2 receptors play a major role in myointimal formation after arterial injury [44] and that the expression of AT2 receptors is increased in hypertrophied left ventricle [45]. The role, if any, of this interactive regulation between AT1 and AT2 receptors in aging tissues (whether accompanied or not by hypertension), particularly the kidney, remains to be established. The relative abundance of AT2 subtype receptors in many fetal tissues supports a role of AT2 during development [46]. Furthermore, a key role for the AT2 subtype receptor is suggested by several studies that correlate enhanced AT2 receptor expression with cardiovascular system disease states such as diabetes, hypertension, and senescence [47–49]. The ability of a tissue to change the expression of AT1 receptors to AT2 has been described in experimentally induced vascular injury [21], suggesting that AII may also play a role, through AT2 receptors, in smooth muscle cell differentiation and proliferation. In addition, Brilla et al. [40] reported that in cultured adult rat cardiac fibroblasts AII stimulates collagen synthesis by both AT1 and AT2 receptors, and that AII inhibition of collagenase activity is specifically mediated by the AT2 subtype receptor. Chronic pharmacological blockade of AT2 subtype AII receptors has no systemic hemodynamic (arterial pressure, cardiac output, heart rate) either in normotensive rats or in AII-induced hypertensive rats, and does not affect the hypertensioninduced cardiac hypertrophy [50]. Furthermore, plasma angiotensin II level and aortic reactivity to AII are not affected after chronic AT2 receptor blockade. Chronic blockade of AT2 receptors antagonizes the vascular growing effects related to longterm AII injection, whereas blockade of AT1 receptors does not. The vasotrophic effect of AII is at least partially mediated via AT2 receptor subtype in some experimental model of hypertension. Considering AII’s role in the induction of cardiac hypertrophy and possibly fibrosis of tissues, such a mechanism to decrease receptor number could be essential for maintaining normal cardiac structure and function. A similar proposal could be

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marshaled for the kidney. The protective effects of AII blockade, by either diminishing its production or its actions, on fibrosis and proliferation is indirect evidence that, despite low angiotensin II production, increased sensitivity of the kidney to the effects of the hormone could mediate the development of fibrosis and tissue damage in aging, hypertension, or aging accompanied by hypertension.

16.5 Aging and Enhanced Cardiac Expression of Angiotensin II Receptor Subtypes In the heart and the kidney, the presence of angiotensinogen (ANG), renin, and angiotensin-converting enzyme (ACE) suggests local synthesis of AII [51, 52]. In young adult rats, the myocardium is able to synthesize AII from angiotensinogen and angiotensin-converting enzyme [51], but it has been shown that a several-fold increase in both ANG and ACE mRNA occurs in the aged LV but not in the RV. This suggests that during aging, an activation of cardiac AII synthesis may compensate in part for the depression of the circulating RAS. The possibility cannot be discarded that a similar mechanism exists to maintain normal intrarenal resistances and glomerular filtration fraction in the aging kidney. Although less is known relative to the kidney, senescence is associated with marked changes in cardiac structure and morphology such as cardiomyocyte loss, hypertrophy of the remaining cells, and the development of fibrosis [53, 54]. These changes may account for the functional characteristics of the senescent myocardium such as impaired myocardial perfusion [55], altered diastolic compliance [53], and arrhythmias [56]. Vascular structures are also modified, and aging is associated with increased arterial wall stiffness, as shown by a significant decrease in systemic and local arterial compliance and an increase in aortic impedance [53, 57], which may induce mild left ventricular hypertrophy. There is now evidence that senescence is associated with increased cardiac ANG and ACE gene expression, suggesting increased cardiac AII synthesis, whereas plasma RAS activity, as already mentioned, is largely depressed [58]. The presumed mechanistic pathways involved in the increase in cardiac AII receptor gene expression are multiple. Upregulation could be intrinsic to the developmental gene reprogramming often associated with senescence. Cardiac senescence is characterized by the re-expression of fetal proteins, such as the contractile protein isoform b-MHC and the atrial natriuretic peptide gene [59, 60]. On the other hand, several studies in rat ventricular tissue have demonstrated developmental regulation of cardiac AII receptor subtype densities and gene expression, which are abundant during the neonatal period and decrease with maturation [61–63]. Secondly, humoro-hormonal status is modified during senescence and is characterized by a large decrease in plasma AII synthesis and an increase in plasma cortisol level [58]. A number of circulating factors, such as vasoactive substances, growth factors, and steroids, modulate AT1 expression via their effects on the transcriptional activity of AT1 gene [18, 64, 65]. However, the differential pattern of AT1 gene expression observed in the LV and RV of aged rats makes a major role of these hormones in the regulation of AT1 gene expression unlikely.

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Much less is known about the hormonal regulation of AT2 mRNA level. However, based on the upregulation of AT2 gene expression in both ventricles of aged rats, hormonal and humoral factors might be one of the triggers for the increase in cardiac AT2 gene expression during senescence. Mechanical factors are repeatedly proposed as triggers for the regulation of genetic expression during the development of cardiac hypertrophy. Activation of AT1 and/or AT2 gene expression has been demonstrated in ventricular tissue during hemodynamic overload [66, 67]. Mechanical stretch has also been recently shown to upregulate AT1 and AT2 gene expression in neonatal rat cardiac myocytes, the increase in AT1 gene expression being mainly due to increased transcription, whereas that of AT2 results from stabilization of AT2 mRNA metabolism [68]. Even though cardiac output and ejection fraction of both aged human and rat hearts are unaltered, the increase in vascular stiffness and aortic impedance during aging result in a moderate increase in LV afterload [69, 70]. These changes in LV properties might therefore account, at least in part, for the upregulation of AT1 gene expression in the LV of aged rats. Heymes and collaborators [49] have demonstrated increased density of both AT1 and AT2 receptors in the LV myocardium of senescent rats and activation of the intracardiac RAS in aging, associated with suppression of plasma AII synthesis. Such local and independent regulation of intracardiac AII synthesis and receptor subtype expression could account for both autocrine and paracrine actions [71] and support the concept of intracardiac AII production as a regulator of cardiac hypertrophy and collagen accumulation, a condition that could also prevail in renal tissues. A strong correlation between AT2 gene expression and fibrosis in both ventricles, compared with the correlation of AT1 and ACE mRNA levels with LV fibrosis only, has suggested [49] that age-associated cardiac fibrosis is more closely related to the AT2 than the AT1 receptor subtype. This suggestion is given credence by studies of Lorell et al. [72], who demonstrated that, both AT1 inhibition and decrease in ACE gene expression affected neither cardiac fibrosis nor hypertrophy in a pressureoverload rat model. Upregulation of the cardiac RAS or the intrarenal RAS may, in part, compensate for the large age-related fall in plasma AII synthesis. However, activation of local RAS systems and increased expression of AII receptor subtypes might have detrimental effects when other pathological manifestations often associated with senescence, such as hypertension, heart failure, tubulointerstitial or glomerular damage, are superimposed. AII receptor subtype antagonists could therefore be therapeutically useful in normal elderly people or elderly people with renal disease.

16.6 Kidney AII Receptors as a Function of Age Studies by Correa and collaborators [73] in immature (1-week-old) and adult (12week-old) normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) revealed that AII receptors and ACE binding sites, measured by quantitative autoradiography for quantification of AII receptors in both neonatal and adult

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animals of either strain, were of AT1 subtype. In all kidney segments of 1-week-old rats, AII receptor density was higher in SHR than WKY. Binding density increased with age in WKY rats; thus, in the glomeruli and the outer stripe of the outer medulla of 12-week-old WKY, binding was significantly higher than that present in age-matched SHR. By contrast, [125 I]351A (an iodotyrosyl derivative of the ACE inhibitor lisinopril that binds specifically to the active site of ACE [74]) was highest in the outer medulla and not detectable in glomeruli. In 1-week-old rats, binding to ACE was higher in WKY than in SHR strain. Differences in ACE binding between adult SHR and WKY rats were inexistent, with the exception of the inner stripe of the outer medulla, where no binding was detected in SHR. These studies suggest that the renal RAS is developmentally regulated and is involved in the genesis and maintenance of genetic hypertension in SHR as aging proceeds. The evolution of the regulation of ACE and the AT1 and AT2 receptor beyond 12 weeks of aging and how this evolves in relation to hypertension and renal damage remain to be studied. An influence of age on the RAS can also be gleaned from studies in male heterozygous transgenic hypertensive rats TGR (mREN2)27 (TGR) [75]. As compared to Sprague-Dawley control rats, receptor density was significantly lower in TGR. Measurement of density and affinity of AII receptors in glomeruli of animals 11 weeks old as compared to 18–20 weeks old rats revealed that receptor number increased with aging. In renal arteries, the AII receptor mRNA of the main receptor subtype AT1A was neither strain- nor age-dependent, AT1B - and AT2 -receptor mRNAs were significantly lower in TGR than SPRD rats. This study provide evidence that an overactive renin angiotensin system in TGR rats led to a downregulation of glomerular angiotensin II receptors that was not accompanied by a downregulation of the mRNA of the dominant AT1A -receptor subtype, particularly as age advanced. Diminished density of AII receptor leading to enhanced sensitivity to AII could explain in part the development of nephrosclerosis and the tubulointerstitial damage seen in these rats, particularly as they age.

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Subject Index

A ACE2 and cardiac arrhythmias, 3, 85 and enzyme regulation in heart, 123–125 ACE inhibitors and B2 kinin receptors, 161, 163, 164, 166 and kinin B1 receptors, 133–135 Adipose tissue, 16, 193 and adipokines, 186–187, 189 Adrenal RAS, 29 Aging ang Ang II blockade, 227, 230–231, 233 and cardiac expression of Ang II receptors, 233–234 and kidney Ang II receptors, 234–235 and RAS, 227–235 and reactive oxygen species, 227, 229–230, 231 Aldosterone and Ang II, 205 and arrhythmogenic effects, 206 biosynthesis, 60, 204–205 and fibrogenic effects, 206 receptor, 87, 112, 204 signaling pathways, 37, 38, 206–207, 208 and vascular effects, 41, 205 Ang (1-12), 125 Ang (1-7) and cardiac arrhythmias, 85, 121 and cardiac dynamics, 121 and cell volume, 85–87 and ischemia/reperfusion, 85, 121 Ang II and aldosterone, 36, 42, 47, 190, 191 and oxidative stress, 38, 39, 41, 47, 71, 83, 84–85, 188, 189, 190, 191, 192, 205, 212–218, 221, 231

Ang II receptor blockade clinical efficacy in hypertension, 62, 69–70 pleiotropic actions, 71–72 AT1 receptor blockade and activation of autoantibodies, 63–65, 66 ang II-independent activation of AT1 receptor, 62–63 and cross-talk, 67–68 and dimerization, 67–68 and G protein interacting proteins, 65–67 and hypertensive disease, 59–74 renal AT1 receptors and blood flow, 68 AT1 receptors, 3, 35, 36, 37, 38, 39, 40, 45, 46, 47, 59–74, 83–84, 88, 106, 107, 108, 109, 110, 111, 112, 113, 114, 122, 124, 167, 168, 206, 208, 212, 213, 214, 215, 216, 217, 218, 219, 220, 222, 228, 229, 232, 234 chronic blockade, 83–84, 232 AT2 receptor activation, 212, 231 and AT1 receptor blockade, 232 Atherosclerosis and ACE, 40, 46, 153, 216 and AT1 receptor blockers, 213, 218 and AT2 receptor upregulation, 220, 221 and collagen deposition, 221, 222 and gene therapy/AT2 overexpression, 219–220 and renin inhibitors, 217–218 C Cell volume, 3, 85–87 and RAS, 85–87 Chymase, 2, 26, 27, 29, 72, 95, 111, 215, 216

W.C. DeMello, E.D. Frohlich (eds.), Renin Angiotensin System and Cardiovascular Disease, Contemporary Cardiology, DOI 10.1007/978-1-60761-186-8,  C Humana Press, a part of Springer Science+Business Media, LLC 2009

245

246 CMS and HTN, 184, 186, 187, 188, 191, 193, 195, 196 and hyperinsulinemia, 186, 187–188, 196 and therapeutic approach, 193–196 Components of RAS, 1, 2, 3, 28, 31 D Diabetes and RAAS and endothelium, 190–191 and heart, 190, 194, 195 and kidney, 191–192 and pancreas, 192–193, 195 Dyslipidemia, 184, 186, 187, 188, 193, 195, 213, 214, 218, 222 E Eplerenone and AT1 receptors, 3, 46, 88, 208 and intracrine RAS, 88 F Failing heart connexins, 82 remodeling, 81–88 reprogramming, 82, 85 I Intracellular Ang II on calcium current, 2, 3 on cell communication, 86 Intracrine renin angiotensin aldosterone system (RAAS) and cardiovascular diseases, 208 and diabetes, 99, 183–196 and uterus and pregnancy, 11 K Kallikrein-kinin system and angiotensin II receptor blockers, 167–169 and blood pressure, 140, 154, 157, 158, 160, 163, 167 and heart failure, 165–167, 169 and local blood flow, 153–154 and LV hypertrophy, 163 and myocardial ischemia, 164–165 and renal blood flow, 154–155, 157 Kininases, 132, 135, 136, 140, 149, 151, 153, 156, 157 Kininogens, 140, 149, 150, 151, 152, 153, 155, 156, 157, 158, 160, 166, 167, 168

Subject Index Kinins and ACE inhibitors, 131–141, 148, 149, 153, 154, 155, 158, 160–167, 168, 169 and cardiovascular diseases, 147–169 and NO, 133–135, 140, 148, 149, 151, 152, 153, 161, 163, 165, 166, 167, 168, 169 and water and electrolytes, 155–157 L Local renin angiotensin system and clinical inclications, 7–12 and ventricular hypertrophy, 103–114 O Oxidative stress and insulin resistance, 186, 188, 189, 195, 196 and local RAAS, 186, 188, 189, 196 P Prorenin and diabetes, 3, 16, 17, 19, 20, 22, 26, 27, 28, 30 and retinopathy, 3, 16, 17 (Pro) Renin receptor biochemistry, 18–20 and cardiovascular disease, 20, 22 conformation change, 18 intracellular renin binding protein, 18 M6P/IGF2 receptor, 18 and MAP kinases, 17–22 ontogeny, 21–22 and renal disease, 20 R RAAS inhibition and heart failure, 45, 46, 99 and myocardial infarction, 46, 99 RAAS, see Intracrine renin angiotensin aldosterone system RAS, 1, 2, 3, 20, 21, 25, 26, 27, 28–31, 41, 42, 46, 47, 59, 61, 62, 63, 64, 65, 68, 69, 71, 72, 73, 74, 82, 83, 84, 86, 88, 104–108, 109–114, 119, 120, 122, 123, 125, 126, 155, 186, 192, 227, 230, 233, 234, 235 and atherosclerosis, 211–223 Renal RAS, 29–30 Renin, 1–3, 7–12, 15–22, 25–31, 35–48, 59, 60, 64, 68, 69, 73, 74, 82, 83, 84, 85–87, 93–100, 104, 112, 113,

Subject Index 114, 119–126, 131, 133, 140, 148, 153, 154, 155, 156, 159, 160, 161, 163, 167, 168, 183–196, 203–209, 211–223, 227–235 gene expression, 26, 28 Renin transcript, 2, 29, 31 overexpression, 2 Renin uptake, 29

247 S Salt, 9, 10–11, 27, 35, 36, 39, 42, 43, 71, 106, 121–123, 148, 157, 158, 159, 160, 169, 185, 186, 190, 191, 204, 205 and ACE2/Ang (1–7)/mas Axis, 121–123 Salt-loading, 10–11 and cardiovascular mortality, 10, 11