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Role of Angiotensin-(1-7) in Cardiovascular Physiology

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THE ROLE OF ANGIOTENSIN-(1-7) IN CARDIOVASCULAR PHYSIOLOGY By JUSTIN L. GROBE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2006

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Copyright 2006 by Justin L. Grobe

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This document is dedicated to my wife, my family, my mentors, and my friends.

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ACKNOWLEDGMENTS I would first like to thank my graduate mentor, Michael J. Katovich, for his guidance over the last five years. His continual support, guidance, encouragement, and direction have been invaluable. I truly appreciate all the friendship he has shown me, and all the attention and effort he has poured into my education. Along with my graduate mentor, I would like to thank my undergraduate mentor, Christopher C. Barney. His guidance and friendship are the primary reasons for my success as an undergraduate, and my interest in animal physiology. Next, I would like to thank my graduate committee members, Mohan K. Raizada, Maureen Keller-Wood, William J. Millard, and Jeffrey A. Hughes, for their input into my education and this document. Their direction over the last five years has shaped me into a very capable modern physiologist. I would also like to thank Joanna Peris for her continuous encouragement, and for filling in during my final defense. I have been blessed with many other wonderful teachers and mentors at all levels of my education, but a few deserve particular attention. I would like to thank Marjorie and James Bullerdick, for teaching me patience and compassion. I would like to thank Trena Thornburg for nurturing my abilities and teaching me to appreciate thinking outside the box. Jeff Ehlers and David Dodendorf were wonderful coaches, and I thank them for encouraging me and promoting my interest in the sciences. The faculty and staff of the Hope College Biology Department welcomed me from the day I entered college, and I am grateful for their continuing support and encouragement. Matthew J. Huentelman, iv

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both my peer and teacher, also deserves my gratitude for his excitement and uncompromising drive. Many people have blessed me with their friendship, and have made my years in college and graduate school truly enjoyable. Many should be named, but Bill Murdoch and Lane Blanchard deserve particular mention for sticking with me in both my joys and sorrows throughout my undergraduate and graduate years. I would like to thank my parents, Edward and Loretta Grobe, for their continual support, energy, and encouragement. From childhood they have recognized my interests, and provided me every conceivable opportunity to follow my dreams. Finally, my thanks go to my wife, Connie, who continually provides me with support, encouragement, inspiration, and direction as she also pursues her Ph.D. I look forward to sharing my life and work with my best friend. v

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TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................iv LIST OF TABLES .............................................................................................................ix LIST OF FIGURES .............................................................................................................x ABSTRACT ......................................................................................................................xii CHAPTER 1 GENERAL INTRODUCTION....................................................................................1 Heart Failure.................................................................................................................1 Cardiac Remodeling.....................................................................................................2 The Renin-Angiotensin System....................................................................................4 Angiotensin Converting Enzyme 2.....................................................................10 Angiotensin-(1-7)................................................................................................13 Estrogenic Modulation of Heart Failure.....................................................................13 Estrogenic Modulation of the Renin-Angiotensin System.........................................14 Aims............................................................................................................................15 2 CARDIAC FIBROSIS IS PREVENTED BY CHRONIC ANGIOTENSIN-(1-7) DURING ANGIOTENSIN II INFUSION.................................................................16 Abstract.......................................................................................................................16 Introduction.................................................................................................................17 Methods......................................................................................................................19 Animals................................................................................................................19 Indirect Blood Pressure.......................................................................................19 Cardiac Remodeling............................................................................................20 Statistics...............................................................................................................20 Results.........................................................................................................................20 Blood Pressure.....................................................................................................20 Cardiac Hypertrophy...........................................................................................20 Cardiac Fibrosis...................................................................................................22 Discussion...................................................................................................................22 vi

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3 CHRONIC ANGIOTENSIN-(1-7) PREVENTS CARDIAC FIBROSIS IN THE DOCA-SALT MODEL OF HYPERTENSION.........................................................27 Abstract.......................................................................................................................27 Introduction.................................................................................................................28 Methods......................................................................................................................30 Animals................................................................................................................30 Chemicals............................................................................................................30 DOCA-salt Model...............................................................................................30 Indirect Blood Pressure.......................................................................................31 Direct Blood Pressure/Acute Angiotensin II Responses.....................................31 Cardiac Remodeling............................................................................................32 Statistics...............................................................................................................33 Results.........................................................................................................................33 Indirect Blood Pressure.......................................................................................33 Direct Blood Pressure/Acute Angiotensin II Responses.....................................34 Cardiac Remodeling............................................................................................34 Discussion...................................................................................................................36 4 PROTECTIVE EFFECTS OF ANGIOTENSIN-(1-7) DURING ISOPROTERENOL-INDUCED CARDIAC REMODELING..................................45 Abstract.......................................................................................................................45 Introduction.................................................................................................................46 Methods......................................................................................................................47 Animals................................................................................................................47 Group A Males with isoproterenol at 1 mg/kg..........................................47 Group B Ovariectomized and intact females with isoproterenol at 1 and 2 mg/kg.....................................................................................................47 Group C Males with isoproterenol at 2 mg/kg..........................................48 Indirect Blood Pressure.......................................................................................48 Cardiac Remodeling............................................................................................49 Results.........................................................................................................................49 Cardiac Hypertrophy...........................................................................................49 Blood Pressure.....................................................................................................52 Discussion...................................................................................................................52 5 ALTERATIONS IN AORTIC VASCULAR REACTIVITY TO ANGIOTENSIN-(1-7) IN 17--ESTRADIOL-TREATED FEMALE SD RATS.................................56 Abstract.......................................................................................................................56 Introduction.................................................................................................................57 Methods......................................................................................................................59 Animal Housing and Surgery..............................................................................59 Reagents..............................................................................................................60 Aortic Vascular Reactivity Assay.......................................................................60 Plasma Steroid Measurement..............................................................................61 vii

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Statistics...............................................................................................................61 Results.........................................................................................................................62 Estradiol Levels and Effects on Body, Heart and Uterine Growth.....................62 Aortic Ring Properties.........................................................................................62 Aortic Relaxation to Angiotensin-(1-7)...............................................................63 Aortic Ring Integrity...........................................................................................65 Discussion...................................................................................................................65 6 PRELIMINARY CELL CULTURE EXPERIMENTS..............................................70 Abstract.......................................................................................................................70 Introduction.................................................................................................................70 Methods......................................................................................................................72 Isolation and Culturing of Neonatal Rat Cardiac Myocytes...............................72 Isolation and Culturing of Neonatal Rat Cardiac Fibroblasts.............................72 Myocyte-Fibroblast Cross-Talk Paradigm..........................................................73 Real-time RT-PCR..............................................................................................73 Lentiviral ACE2 Gene Transfer..........................................................................73 Hypoxia-Reperfusion Paradigm..........................................................................74 ACE2 Activity.....................................................................................................74 Soluble Collagen.................................................................................................75 Transforming Growth Factor-1.........................................................................75 Results.........................................................................................................................75 Cardiac Myocyte-Fibroblast Cross-Talk is Inhibited by Ang-(1-7) at the Myocyte...........................................................................................................75 Lentiviral Delivery of EF1-ACE2 Results in Increased ACE2 Activity and Protection from Hypoxia..................................................................................77 ACE2 Gene Delivery Attenuates Hypoxia-Reperfusion Induction of Transforming Growth Factor-1......................................................................78 Discussion...................................................................................................................78 7 GENERAL DISCUSSION.........................................................................................82 Summary and Conclusions.........................................................................................82 Pitfalls and Shortcomings...........................................................................................84 Future Directions........................................................................................................91 Perspectives................................................................................................................94 APPENDIX A LOADING OSMOTIC MINIPUMPS........................................................................95 B MASSONS TRICHROME STAINING AND ANALYSIS.....................................96 LIST OF REFERENCES...................................................................................................98 BIOGRAPHICAL SKETCH...........................................................................................119 viii

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LIST OF TABLES Table page 5-1. Effects of steroid treatments on body parameters in female Sprague-Dawley rats..62 5-2. Aortic ring properties...............................................................................................62 ix

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LIST OF FIGURES Figure page 1-1. The renin-angiotensin system.....................................................................................5 1-2. Ventricular hypertrophy with chronic Ang II is attenuated by lentiviral delivery of ACE2....................................................................................................................11 1-3. Ang II-induced cardiac interstitial fibrosis is prevented by lentiviral delivery of ACE2........................................................................................................................12 1-4. Lentiviral delivery of ACE2 has no effect on the blood pressure increase from chronic Ang II infusion............................................................................................12 2-1. Chronic Ang-(1-7) has no effect on blood pressure increases with chronic Ang II infusion.....................................................................................................................21 2-2. Chronic Ang-(1-7) prevents myocyte hypertrophy due to chronic Ang II infusion21 2-3. Chronic Ang-(1-7) prevents interstitial fibrosis due to chronic Ang II infusion.....22 3-1. Chronic Ang-(1-7) has no effect on blood pressure increases with DOCA-salt treatment...................................................................................................................33 3-2. Chronic Ang-(1-7) has no effect on acute pressor responses to Ang II in animals treated with DOCA-salt............................................................................................34 3-3. Chronic Ang-(1-7) has no effect on cardiac hypertrophy due to DOCA-salt treatement.................................................................................................................35 3-4. Chronic Ang-(1-7) prevents interstitial fibrosis with DOCA-salt treatment............36 3-5. Chronic Ang-(1-7) prevents perivascular fibrosis with DOCA-salt treatment........37 4-1. Isoproterenol (1 mg/kg/day) induces cardiac hypertrophy in male rats...................50 4-2. Ovariectomy potentiates the isoproterenol-mediated induction of cardiac hypertrophy in female rats........................................................................................50 4-3. Chronic Ang-(1-7) prevents the hypertrophic, but not fibrotic effects of isoproterenol (2 mg/kg/day) in male rats.................................................................51 x

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4-4. Isoproterenol and Ang-(1-7) had no chronic ffects on blood pressure in male rats.52 5-1. Maximal contraction to angiotensin II in aortic rings from ovariectomized rats with estrogen replacement........................................................................................63 5-2. Estradiol attenuates the aortic relaxation response to acute Ang-(1-7)....................64 6-1. Ang-(1-7) attenuates myocyte-derived pro-fibrotic signaling..................................76 6-2. Ang-(1-7) attenuates expression of TGF 1 mRNA in cardiac myocytes.................76 6-3. Lentiviral delivery of ACE2 attenuates the collagen production response to hypoxia in cultured cardiac fibroblasts....................................................................77 6-4. Lentiviral delivery of ACE2 attenuates hypoxia-induced expression of transforming growth factor-1 protein.....................................................................78 xi

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy THE ROLE OF ANGIOTENSIN-(1-7) IN CARDIOVASCULAR PHYSIOLOGY By Justin L. Grobe May, 2006 Chair: Michael J. Katovich Major Department: Pharmacodynamics Heart failure (HF) is a devastating condition that impacts our society emotionally, physiologically, and fiscally, and it is a growing problem in the United States. Aberrant activity of the renin-angiotensin hormone system (RAS) has been implicated as a causative factor in HF. Recently, a new angiotensin converting enzyme 2 (ACE2) was recognized, which catalyzes the degradation of the active angiotensin II (Ang II) hormone to angiotensin-(1-7) (Ang-(1-7)). Studies into the actions of ACE2 have led to the concept that Ang-(1-7) may act as an active, endogenous inhibitor of the RAS. Interestingly, investigations into the mechanisms of action of ACE inhibitors and AT 1 R antagonists have revealed that both of these treatments result in increased circulating levels of Ang-(1-7). Some have therefore hypothesized that pharmacological inhibitors of the RAS may provide cardioprotective effects through the actions of Ang-(1-7). The present studies were designed to characterize the cardioprotective actions of Ang-(1-7) in various models of cardiac remodeling. The protective effect of Ang-(1-7) was evaluated in Ang II-infusion, mineralocorticoid, and -adrenergic models of cardiac xii

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remodeling. Further, due to gender differences in the development and progression of heart failure, the protective actions of Ang-(1-7) were evaluated in intact and ovariectomized female rats. Cardiac remodeling was inhibited by Ang-(1-7), with specific effects determined by the type of stimulus, and estrogens were determined to play an important role in regulating tissue sensitivity to Ang-(1-7). From these findings, it was concluded that Ang-(1-7) has specific actions in the cardiovascular system, some of which oppose the actions of its precursor, Ang II. In addition, our results lead us to hypothesize that estrogen replacement therapy during menopause may predispose women to cardiovascular diseases through decreased tissue sensitivity to the protective Ang-(1-7) hormone. Future studies should more directly characterize the effects of estrogens on cardiac tissue, and further elucidate the tissue-level and intracellular signaling mechanisms of Ang-(1-7). Our results indicate that Ang-(1-7) may represent a new, unexploited class of targets for pharmacological intervention before and during HF. xiii

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CHAPTER 1 GENERAL INTRODUCTION Heart Failure Congestive heart failure (CHF) is one of the most debilitating cardiovascular pathologies, having both tremendous economic and emotional impacts on our society. Approximately five million Americans, or 2.3% of the entire population of the United States, suffers from CHF (Thom et al., 2006). The lifetime risk of developing CHF in the fourth decade of life is 20%, and this risk doubles for individuals with blood pressure >160/90 (Thom et al., 2006). Congestive heart failure is a disease characterized by inadequate movement of blood by the heart. Inadequate blood flow results in poor perfusion of critical organs, including the brain and the heart itself, thereby leading to cellular hypoxia and death. It was estimated that in 2005, 80% of men and 70% of women under age 65 diagnosed with CHF will die within eight years of diagnosis. After diagnosis, survival is poorer in men than women, but fewer than 15% of women will survive longer than eight to twelve years. Twenty percent of people diagnosed with CHF will die within one year of diagnosis (Thom et al., 2006). There are two major forms of CHF, which are defined by the morphological and functional changes that lead to inadequate blood movement. Systolic heart failure is characterized by insufficient contraction of the ventricles (which are typically dilated), usually resulting from idiopathic dilated or ischemic cardiomyopathies. Diastolic heart failure is characterized by thickened, noncompliant ventricular walls and small 1

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2 ventricular volumes, usually resulting from hypertension, stenotic valvular disease, or primary hypertrophic cardiomyopathies. Clinically, patients typically present with a combination of both systolic and diastolic heart failure, in that the heart is too weak to maintain cardiac output (systolic dysfunction) despite thick, stiff walls (diastolic morphology). Thus, conventional therapy is targeted at relieving symptoms of inadequate blood flow rather than reversing or preventing the myocardial damage which leads to CHF. Cardiac Remodeling Whereas systolic heart failure is typically the result of myocardial ischemia and cellular death (which leads to a loss of muscle and force-generating ability), diastolic heart failure is the result of inappropriate myocyte growth accompanied by a large increase in extracellular matrix deposition. Together, myocyte hypertrophy and net increases in extracellular matrix components are referred to as cardiac remodeling. Cardiac remodeling can be normal and compensatory, as is observed in the myocardium of athletes engaged in aerobic sports. In this normal remodeling, the process results from increased peripheral demand for cardiac output, which provides a mechanical stimulus for myocyte hypertrophy. Cardiac fibroblasts, which produce the proteins of the extracellular matrix, retain relatively normal growth patterns and function. This type of remodeling differs from pathological remodeling primarily due to the difference in the response of the cardiac fibroblast to the stimulus (Weber, 2000; Miner and Miller, 2006). Pathological remodeling, in contrast, can take many forms, which are differentiated primarily by stimulus and mechanism. Mineralocorticoids can elicit direct effects on cardiac remodeling. Aldosterone levels are elevated during heart failure, and this elevation is related to increased mortality

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3 (Swedberg et al., 1990), and inhibition of mineralocorticoid receptors by spironolactone or eplerenone significantly reduces mortality in heart failure patients (Pitt et al., 1999, Pitt et al., 2003). Spironolactone treatment also results in decreased extracellular matrix remodeling (Zannad et al., 2000). In vitro, cardiac fibroblasts are stimulated by aldosterone to increase collagen production, and to change the relative pattern of collagen types expressed (Zhou et al., 1996; Brilla, 2000; Rombouts et al., 2001). Angiotensin II also directly promotes cardiac remodeling. In vitro, angiotensin II promotes proliferation of cardiac fibroblasts, collagen production, and expression of adhesion receptors (Kim and Iwao, 2000; Schnee and Hsueh, 2000; Lijnen et al., 2001). Inhibition of angiotensin converting enzyme improves the outcomes for heart failure patients (Khalil et al., 2001), as does antagonism of the AT 1 subtype angiotensin II receptor (Frigerio and Roubina, 2005), thereby implicating angiotensin II in cardiac remodeling. Catecholamines can act upon the myocardium to cause remodeling. Epinephrine and norepinephrine are elevated during heart failure and correlate to prognosis (Francis et al., 1993). Inhibition of -adrenergic receptors decreases the symptoms of heart failure, and improves survival (Shibata et al., 2001). The actions of norepinephrine on the extracellular matrix are not well described at the present time, though it is known that norepinephrine induces the production of growth factors, such as transforming growth factor 1, which are potent mediators of fibrosis (Bhambi and Eghbali, 1991; Barth et al., 2000; Akiyama-Uchida et al., 2002; Miner and Miller, 2006). Cytokines such as transforming growth factor-1 are produced by cardiac cells during heart failure, and are well-established mediators of cardiac remodeling (Sun et al.,

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4 1998; Lijnen et al., 2000; Iwata et al., 2005; Tallant et al., 2005). In vitro, transforming growth factor-1 acts in an autocrine/paracrine fashion to modulate collagen and fibronectin production in the cardiac fibroblast (Roberts et al., 1986). Because it is upregulated by angiotensin II and norepinephrine, this cytokine may represent a common mediator of the fibrotic effects of these hormones (Bhambi et al., 1991; Yoo et al., 1998). The Renin-Angiotensin System In 1836, Richard Bright observed a link between left ventricular hypertrophy and renal disease, and he suggested a link among hypertrophy, increased resistance to blood flow in small vessels, and an altered condition of the blood (Basso and Terragno, 2001). George Johnson in 1868 and F.A. Mahomed in 1872 also reported links between renal diseases and left ventricular hypertrophy. Finally, in 1898, Tigerstedt and Bergman determined that extracts from rabbit renal tissue contained a pressor agent which mediated the relationship between renal disease and cardiac hypertrophy. Tigerstedt and Bergman named this vasoactive substance renin. Subsequent attempts to isolate and fully characterize this and related blood-borne substances resulted in simultaneous discovery of the compound hypertensin by Braun-Menendez et al. (1939), and angiotonin by Page et al. (1939). These two would later agree to compromise on standard nomenclature for the various components of the developing system, including the name angiotensin as a combination of the two earlier names (Braun-Menendez and Page, 1958). Many more components of the renin-angiotensin system (RAS) have been discovered, as have the relationships among the various components. The system is present both systemically (in the blood plasma) and in the interstitium (as an autocrine or paracrine system), and it is the presence or absence of the various components at the site

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5 of action which determines the specific outcome of RAS activation in that location. Figure 1-1 summarizes the presently recognized components of the RAS. Angiotensinogen Figure 1-1. The renin-angiotensin system. (ACE angiotensin converting enzyme; ACE2 angiotensin converting enzyme 2; Endopeptidases neprilysin, prolyl endopeptidase, and thimet oligopeptidase; AP-A aminopeptidase A; AP-B aminopeptidase B; AT1 angiotensin II type 1 receptor; AT2 angiotensin II type 2 receptor; AT1-7? possible receptor for angiotensin-(1-7), which may be the Mas receptor; AT4 angiotensin type 4 receptor). Release of renin from the kidney is the rate limiting step in the generation of circulating angiotensin II (Ang II). Renin, an aspartyl protease, is synthesized and released by juxtaglomerular cells located in the walls of the afferent arterioles of the nephron just as the vessel enters the glomerulus. Renin is synthesized as the 406 amino acid preprorenin, then cleaved to form prorenin which is a 373 amino acid mature, but Angiotensin I Angiotensin II Angiotensin-(1-9) Angiotensin-(1-7) Angiotensin III Angiotensin IV Angiotensin-(1-5) ACE2 Pro-Renin Renin ACE2 ACE E ndoACE p eptidases ACE AT 1 AT 2 AT 1-7 ? AP-A AP-B AT 4 Vasoconstriction Anti-AT 1 effects Hypertrophy Vasodilation Vasoconstriction Anti-AT 1 effects Fibrosis Differentiation Memory effects? Vasodilation

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6 inactive, enzyme. Final activation of renin by an as yet uncharacterized enzyme involves removal of the 43 amino terminal residues (Goodman and Gilman, 2001). The only known substrate for renin is angiotensinogen. This highly abundant globular glycoprotein is synthesized as a 452-amino acid preangiotensinogen by liver, fat, certain nervous tissues, and the kidney. Angiotensinogens synthesis is stimulated by inflammation, insulin, estrogens, glucocorticoids, thyroid hormone, and angiotensin II. Due to the circulating levels of angiotensinogen and the K m of renin for its substrate (approximately 1 M), the rate of synthesis of angiotensinogen can modulate the levels of circulating angiotensin II. Indeed, increased circulating levels of angiotensinogen are associated with essential hypertension (Jeunemaitre et al. 1992; Goodman and Gilman, 2001). The mature peptide is only fourteen amino acids in length. Angiotensin I represents the first ten amino acids of the amino terminus of angiotensinogen, and is the result of renin action upon angiotensinogen. This peptide is inactive until converted to angiotensin II by angiotensin converting enzyme. Angiotensin converting enzyme (ACE) is also known as Kininase II and Dipeptidyl Carboxypeptidase. This ectoenzyme is comprised of 1277 amino acids, and displays two homologous zinc-dependent enzymatic domains. The large extracellular domain of this enzyme can be cleaved by secretase (Beldent et al., 1995; Goodman and Gilman 2001) to produce circulating ACE enzyme, but it has been suggested that intact, membrane-bound ACE may act as a serine-threonine kinase extracelluar receptor (Fleming et al., 2005). ACE is a nonspecific peptidase that removes two amino acids from substrates with different sequences (although it prefers substrates with one free carboxyl group in the carboxyl-terminal residue, and proline can not be the penultimate amino acid therefore

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7 it can not act on angiotensin II). Substrates for ACE include bradykinin (ACE inactivates bradykinin and other potent vasodilators), angiotensin I, angiotensin-(1-9), and angiotensin-(1-7). An important genetic variation of ACE exists, in which an insertion/deletion polymorphism in intron 16 of the gene significantly contributes to circulating ACE levels (Rigat et al., 1990). The deletion version of the polymorphism results in higher levels of circulating ACE and may be a risk factor for the development of heart disease (Cambien et al., 1992; Gardeman et al., 1995; Mattu et al., 1995), vascular endothelial dysfunction (Butler et al., 1999), left ventricular hypertrophy (Iwai et al., 1994; Schunkert et al., 1994), and hypertension (Fornage et al., 1998; ODonnell et al., 1998). It is interesting to note that humans who live past 100 years of age display a high frequency of the deletion polymorphism (Schachter et al., 1994), which may be a result of the strong association of the deletion allele with protection from Alzheimers disease (Kehoe et al., 1999). ACE-mediated cleavage of the two carboxyl amino acids of angiotensin I results in the production of the eight-amino acid angiotensin II (Ang II). Ang II is the most active component of the RAS, and its actions are receptor-dependent. The subtype of receptor that is activated by Ang II determines the cellular response to this ligand. Other enzymatic pathways exist, which can produce active angiotensin peptides without involvement of renin and/or ACE. Nonrenin proteases can convert angiotensinogen to angiotensin I, and cathepsin G and tonin can convert angiotensinogen directly to angiotensin II. Angiotensin I can be converted to angiotensin II by cathepsin G, chymostatin-selective angiotensin II-generating enzyme, and heart chymase (Dzau et al., 1993; Goodman and Gilman, 2001). In humans, chymase is an important enzyme for

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8 the conversion of angiotensin I to angiotensin II at the tissue level, especially in cardiac (Wolny et al., 1997; Wei et al., 1999) and renal tissue (Hollenberg et al., 1998). Regardless of its formation pathway, angiotensin II exerts its effects through two major subtypes of G-protein coupled receptors. The first subtype of receptor, the angiotensin II type 1 (AT 1 ) receptor, is nearly ubiquitous, and is responsible for most of the overall actions of angiotensin II. This 359-amino acid cell-surface receptor can activate a large number of signal transduction pathways. These pathways can include calcium release and influx, phospholipases, mitogen-activated protein kinase pathways, Janus kinase pathways, serine/threonine protein kinases, nonreceptor tyrosine kinases, small GTP-binding proteins, inducible transcription factors, factors affecting translational efficiency, and the production of reactive oxygen species (Griendling et al., 1997; Berk, 1999; Blume et al., 1999; Inagami, 1999; Goodman and Gilman, 2001). Activation of AT 1 receptors results in endothelial dysfunction, vasoconstriction, vascular smooth and cardiac muscle cell growth, enhanced coagulation and sympathetic nerve activity, all of which can be detrimental to cardiovascular function and ventricular structure (Yasunari et al., 2005). AT 1 receptors on cardiac myocytes cause hypertrophy, increased protein synthesis, overall gene reprogramming, increased production of atrial natriuretic factor, and can induce apoptosis (Yasunari et al., 2005). These same receptors, on cardiac fibroblasts, lead to proliferation, increased DNA and protein synthesis, and increased gene expression including transforming growth factor 1 and fibronectin (Yasunari et al., 2005). Pharmacological inhibition (Yasunari et al., 2005) and genetic modulation (Raizada et al., 2000) of the AT 1 receptor show great promise for inhibiting cardiac remodeling.

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9 The second subtype of angiotensin II receptor is the angiotensin II type 2 (AT 2 ) receptor. Much less is known about the AT 2 receptor, but its actions are generally considered to oppose those of the AT 1 receptor. The AT 2 receptor is highly expressed in fetal tissues, but levels are low in adults until cardiac insult, such as myocardial infarction, which results in increased AT 2 expression (Nio et al., 1995). This G-protein coupled receptor is 363-amino acids long, and is known to activate phosphatases, potassium channels, and nitric oxide production, while inhibiting calcium channels (Horiuchi et al., 1999). Most of the effects of the AT 2 receptor are mediated by the G i binding protein (Goodman and Gilman, 2001). AT 2 receptors on cardiac myocytes inhibit cell growth and decrease protein synthesis, while their activation on cardiac fibroblasts inhibits AT 1 -mediated effects, and decreases DNA and protein synthesis (Yasunari et al., 2005). Overexpression of this receptor subtype has been shown to prevent cardiac remodeling (Metcalfe et al., 2004). In addition to angiotensin II, there are multiple other angiotensin peptides that are endogenously produced by the RAS. For example, the alanine residue at the amino terminus of angiotensin II can be removed by aminopeptidase A, thus producing angiotensin-(2-8), also known as angiotensin III. The role of angiotensin III is under debate, as this peptide has been shown to have similar pharmacological actions at the AT 1 and AT 2 receptor as angiotensin II (Wright and Harding, 1997). Angiotensin III is subsequently digested by aminopeptidase B to remove the arginine residue at the amino terminus. This digestion produces angiotensin-(3-8), which is commonly known as angiotensin IV. Angiotensin IV selectively binds the AT 4 receptor, which is also known as the insulin-regulated aminopeptidase. In the heart, this

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10 receptor stimulates protein synthesis in cardiac fibroblasts and inhibits mechanically-stimulated expression of immediate early genes cfos and egr-1, although the predominantly recognized role of the AT 4 receptor at this time is in learning and memory (Chai et al., 2004). Finally, angiotensin IV can be cleaved by aminopeptidase N or dipeptidylaminopeptidase to form angiotensin-(4-8), angiotensin-(5-8), or angiotensin-(6-8), all of which are inactive fragments of the RAS (von Bohlen and Halbach, 2003). Angiotensin Converting Enzyme 2 Recently a second angiotensin converting enzyme (ACE2) was discovered by Tipnis (2000) and Donoghue (2000). Although this enzyme shares approximately 40% homology to ACE within its catalytic domain, ACE2 differs from ACE in substrate specificity and is not inhibited by classic ACE inhibitors (Tipnis et al., 2000, Guy et al., 2003). ACE2 has been shown to play a role in heart development and function (Boehm and Nabel, 2002; Crackower et al., 2002; Donoghue et al., 2003; Oudit et al., 2003). Genetic knockout of the ACE2 gene causes increased circulating angiotensin II, impaired cardiac function, and induction of hypoxia-response genes (Crackower et al., 2002; Donoghue et al., 2003). Overexpression of this gene in rats delays the onset of cardiac hypertrophy (Santos et al., 2004), and transgenic overexpression in mice results in decreased systolic blood pressure (Crackower et al., 2002). Several animal models of hypertension exhibit depressed ACE2 levels, which suggests a relationship between ACE2 and hypertension (Crackower et al., 2002; Danilczyk et al., 2003; Garcia et al., 2003). Finally, the ACE2 gene maps to a defined quantitative trait locus that has been associated with hypertension (Crackower et al., 2002). It has been suggested (Donoghue

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11 et al., 2000; Tipnis et al., 2000; and Turner and Hooper, 2002) that the balance of actions between ACE and ACE2 may determine the overall physiological / pathophysiological (blood pressure and structural) balance within various organ systems. To examine the hypothesis that increased activity of ACE2 would result in protection from cardiac remodeling, we previously delivered the murine ACE2 gene to rats by use of a lentivirus. Neonatal delivery of lenti-mACE2 to Sprague-Dawley rats provided these animals protection from cardiac remodeling with angiotensin II infusion during adulthood (Huentelman et al., 2005). ACE2 overexpression by these animals attenuated cardiac hypertrophy (Figure 1-2), and completely prevented the interstitial fibrosis (Figure 1-3) caused by angiotensin II infusion. It is interesting to note that these effects were not mediated by changes in blood pressure (Figure 1-4), thus indicating that ACE2 can selectively protect the myocardium from remodeling. Figure 1-2. Ventricular hypertrophy with chronic Ang II is attenuated by lentiviral delivery of ACE2. (Reprinted with permission from Huentelman MJ, Grobe JL, Vazquez J, Stewart JM, Mecca AP, Katovich MJ, Ferrario CM, Raizada MK. Protection from angiotensin II-induced cardiac hypertrophy and fibrosis by systemic lentiviral delivery of ACE2 in rats. Experimental Physiology. 90(5):783-90. 2005.)

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12 A B Figure 1-3. Ang II-induced cardiac interstitial fibrosis is prevented by lentiviral delivery of ACE2. A) Representative sections of left ventrile wall, and B) quantified collagen density in left ventricle wall sections. (Reprinted with permission from Huentelman MJ, Grobe JL, Vazquez J, Stewart JM, Mecca AP, Katovich MJ, Ferrario CM, Raizada MK. Protection from angiotensin II-induced cardiac hypertrophy and fibrosis by systemic lentiviral delivery of ACE2 in rats. Experimental Physiology. 90(5):783-90. 2005.) Figure 1D. Lentiviral delivery of ACE2 has no effect on the systolic blood pressure increase from chronic Ang II infusion. (Reprinted with permission from Huentelman MJ, Grobe JL, Vazquez J, Stewart JM, Mecca AP, Katovich MJ, Ferrario CM, Raizada MK. Protection from angiotensin II-induced cardiac hypertrophy and fibrosis by systemic lentiviral delivery of ACE2 in rats. Experimental Physiology. 90(5):783-90. 2005.)

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13 The mechanism by which ACE2 can provide cardioprotection during hypertension is unknown, but it is likely that increased formation of the enzymes product, angiotensin-(1-7), may be important. Angiotensin-(1-7) ACE2 has been shown to be one of several enzymes that catalyzes the conversion of angiotensin I and angiotensin II to the newly recognized hormone angiotensin-(1-7) (Figure 1-1, and Ferrario and Chappell, 2004), but in the heart, ACE2 and neprilysin have been shown to have the most significant roles in the generation of angiotensin-(1-7) (Zisman et al., 2003). ACE, which is a key enzyme in the RAS for the conversion of angiotensin I to angiotensin II, is the primary enzyme involved in the degradation of angiotensin-(1-7) to angiotensin-(1-5) (Yamada et al., 1998; Ferrario and Chappell, 2004). Estrogenic Modulation of Heart Failure Gender differences exist in the susceptibility, onset, progression, and outcome of heart failure. Approximately 15% of women and 20% of men have hypertension, which is a major risk factor for the development of heart failure (Regitz-Vagrosek and Lehmkuhl, 2005). Blood pressure tends to increase with age in both sexes, although at menopause, the prevalence of hypertension increases greatly in women. Between the ages of 65 and 75, approximately 45% of women and 41% of men are hypertensive (Hayes and Taler, 1998; Gasse et al., 2001). It is important to note that systolic blood pressure disproportionately increases in women compared to men, and the subsequent rise in pulse pressure represents an important and independent predictor of cardiovascular outcome (Burt et al., 1995; Regitz-Vagrosek and Lehmkuhl, 2005).

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14 The effects of hypertension in women are more pronounced than in men. Hypertension is by far the major causative factor for the development of heart failure in women, whereas myocardial infarction is the most causative factor in men. In the Framingham Heart Study, hypertension was the primary causative factor for the development of heart failure in 59% of women, compared to only 39% in men (Levy et al., 1996). Left ventricular hypertrophy (which results from chronic hypertension) also carries a greater risk for mortality in women than in men (Liao et al., 1995). Roles of estrogen and its receptors in the development and progression of heart failure are fairly well established. In many animal models of cardiovascular abnormalities, phenotypes are worse in male and ovariectomized females, and administration of estrogens can attenuate or reverse the phenotype (Regitz-Vagrosek and Lehmkuhl, 2005). Estrogen has been shown to reduce the size of myocardial infarcts, and reduce apoptosis (Patten et al., 2004). Estrogen also antagonizes cardiac myocyte hypertrophy in vitro (Van Eickels et al., 2001). Many of the cardioprotective effects of estrogen have been attributed to the estrogen receptor (ER)subtype (Zhai et al., 2000), although both and estrogen receptor subtypes are known to be present in cardiac myocytes and fibroblasts from humans and rats (Grohe et al., 1997; Taylor and Al-Azzawi, 2000). In humans, both receptor subtypes show increased expression during cardiac hypertrophy (Nordmeyer et al., 2004). Estrogenic Modulation of the Renin-Angiotensin System Estrogens are known to modulate many hormonal systems involved in the regulation of cardiac structure and function. The RAS is regulated by estrogens at several points. Angiotensinogen and the AT2 receptor are increased with estrogen treatment (Healy et al., 1992; Armando et al., 2002), while prorenin, ACE, neprilysin,

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15 aminopeptidases, and the AT1 receptor are all decreased with estrogen treatment (Szilagyi et al., 1995; Pinto et al., 1999; Nickenig et al., 2000; Seli et al., 2001; Turner and Hooper, 2002). Recent studies by Brosnihan et al. (2003) have determined that during pregnancy, renal ACE2 expression is increased, as are circulating levels of angiotensin-(1-7). Together, these findings indicate that estrogen decreases the production and effectiveness of angiotensin II, while it has positive effects on the generation of angiotensin-(1-7). Aims It was the goal of these studies to elucidate the role, if any, of angiotensin-(1-7) in protecting the myocardium from pathological remodeling. First, the protective effects of angiotensin-(1-7) will be examined in both angiotensin IIdependent and independent forms of hypertension-induced cardiac remodeling. Second, modulation of normotensive cardiac remodeling by angiotensin-(1-7) will be studied. Third, modulation of the effects of angiotensin-(1-7) by estrogen will be examined. Finally, preliminary studies into the cellular mechanism of action of angiotensin-(1-7) will be presented. Together, these studies will demonstrate the cardioprotective actions of angiotensin-(1-7), establish the modulatory role of estrogen on this hormone, and point the direction for future studies into the actions of angiotensin-(1-7).

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CHAPTER 2 CARDIAC FIBROSIS IS PREVENTED BY CHRONIC ANGIOTENSIN-(1-7) DURING ANGIOTENSIN II INFUSION Abstract Aberrant activity of the renin-angiotensin system is well established to cause hypertension and associated cardiac remodeling, and chronic infusion of angiotensin II (Ang II) can be used to model these effects in animal models. Recent studies have indicated that the angiotensin-(1-7) (Ang-(1-7)) breakdown product of angiotensin II may act as an endogenous buffer against the actions of Ang II. Here, we examine the hypothesis that chronic infusion of Ang-(1-7) provides cardioprotection during hypertension due to chronic infusion of Ang II. Sprague-Dawley rats between 320-470 grams were implanted with osmotic minipumps which chronically delivered Ang II (100 ng/kg/min). A subset of these animals was also implanted with osmotic minipumps that chronically delivered Ang-(1-7) (100 ng/kg/min). Control animals underwent sham implantation surgery. Blood pressure was monitored by an indirect tail cuff method, and after four weeks of treatment, hearts were harvested, sectioned and stained using Massons Trichrome. Chronic infusion of Ang II resulted in significant increases in blood pressure, ventricular and myocyte hypertrophy, and interstitial fibrosis. Chronic infusion of Ang-(1-7) significantly decreased this myocyte hypertrophy and interstitial fibrosis. These results lead us to conclude that Ang-(1-7) selectively attenuates cardiac remodeling during chronic infusion of Ang II, without effect on blood pressure. These findings complement and extend our previous findings that lentiviral delivery and 16

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17 subsequent overexpression of the murine ACE2 gene prevents cardiac remodeling in Ang II-infused rats. Introduction The renin-angiotensin system (RAS) is well documented as an important endogenous system that regulates cardiac structure and function. An aberrant RAS has been shown to be integrally involved in the development and maintenance of hypertension and cardiac remodeling (Kim et al., 1995; Alderman et al., 1991; Dzau, 1993; Parmley, 1998; Bader et al., 2001; Ruiz-Ortega et al., 2001; Unger, 2002; Zaman et al., 2002). Further, use of RAS inhibitors (both angiotensin converting enzyme inhibitors and angiotensin II type 1 receptor blockers) has been shown to decrease blood pressure, attenuate cardiac remodeling, and prevent cardiac function loss (Frigerio and Roubina, 2005). Chronic infusion of angiotensin II (Ang II) has long been used in animal models to mimic the onset and progression of hypertension and hypertension-induced pathophysiologies. Chronic infusion of Ang II induces these changes through direct vasoconstriction of the vasculature (Rajagopalan et al., 1996), water and salt reabsorption at the kidney (Zou et al., 2002; Lopez et al., 2003; Rugale et al., 2003), and altered sympathetic tone, both centrally and peripherally (Zanzinger and Czachurski, 2000; Zimmerman et al., 2002). Within the heart, Ang II induces structural changes both directly through actions at cardiac cells and indirectly through changes in arterial blood pressure. In culture, Ang II stimulates growth of cardiac myocytes, fibroblasts, and smooth muscle cells (Baker et al., 1992; Corda et al., 2000). Ang II also increases production of the extracellular matrix proteins, including collagen and fibronectin, and inhibits the activity of collagenases

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18 (Baker et al., 1992; Corda et al., 2000). These results are paralleled in vivo during Ang II-induced hypertension and following myocardial infarction in the rat (Brilla et al., 1990; Weber and Brilla, 1991; Lorell, 1997; Swynghedauw, 1999; Corda et al., 2000). Low (non-pressor) doses of Ramipril, and angiotensin converting enzyme (ACE) inhibitor, can regress cardiac fibrosis, thus highlighting the direct effects of the local RAS in regulating cardiac structure independent of blood pressure (Nagasawa et al., 1995; Corda et al., 2000). The mechanism by which low-dose ACE inhibition can attenuate cardiac remodeling is likely three-fold. First, ACE inhibition can result in decreased levels of Ang II. Second, ACE inhibition may result in increased levels of bradykinin, as ACE is known to degrade this protective hormone (Goodman and Gilman, 2001). Third, ACE inhibition has been shown to result in increased levels of the newly recognized hormone angiotensin-(1-7) (Ang-(1-7)) (Tom et al., 2003), likely due to the fact that ACE is the principle degradation pathway for Ang-(1-7) (Ferrario and Chappell, 2004). We have recently demonstrated that increased expression of angiotensin converting enzyme 2 (ACE2), the primary Ang-(1-7) production enzyme in the heart (Zisman et al., 2003; Ferrario and Chappell, 2004), prevents Ang II-induced cardiac remodeling in rats (Huentelman et al., 2005). Specifically, lentiviral transfer of the mouse ACE2 gene to prevented cardiac hypertrophy (Figure 1-2) and cardiac fibrosis (Figure 1-3) without effect on blood pressure (Figure 1-4). At least two possible mechanisms involving the renin-angiotensin system may be at work in ACE2-mediated cardioprotection during Ang II-induced hypertension and cardiac remodeling. First, it is possible that ACE2 simply decreases the levels of its reactant, Ang II. Second, it is possible that the increased levels

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19 of Ang-(1-7) resulting from ACE2 enzymatic activity provide some active protection from cardiac remodeling. Therefore, the following study was undertaken to examine the hypothesis that chronic infusion of Ang-(1-7) would prevent cardiac remodeling in the Ang II-infusion model of hypertension. Methods Animals Eighteen sprague-Dawley rats, between 320-470 grams, were divided into three groups. Animals were anesthetized by halothane inhalation before implantation of subcutaneous osmotic minipumps (Alzet model 2004). Eight animals received pumps that delivered Ang II at 100 ng/kg/min. Four animals received an additional pump that delivered Ang-(1-7) at 100 ng/kg/min. Six animals underwent sham surgery. For a detailed method for loading osmotic minipumps, please see Appendix A. Indirect Blood Pressure Systolic blood pressure was determined weekly using an indirect tail cuff method as previously described (Katovich et al., 2001). Briefly, animals were heated using a 200-watt heat lamp for 5 minutes before restraint in a heated Plexiglas restrainer to which the animals had been previously conditioned. A pneumatic pulse sensor was then attached to the tail distal to a pressure cuff under the control of a Programmed Electro-Sphygmomanometer (Narco). Voltage outputs from the pressure sensor bulb and inflation cuff were recorded and analyzed electronically using a PowerLab signal transduction unit and associated Chart software (ADInstruments). At least three separate indirect pressures were averaged for each animal.

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20 Cardiac Remodeling Following four weeks of chronic treatment, animals were sacrificed by overdose of halothane inhalant, and hearts were removed by blunt dissection. Heart ventricles were cleaned of atria and other adherent tissue before wet weight was determined. Cross-sections of the heart ventricles were then cut at the level of the papillary muscles, embedded in paraffin, and sectioned at 4 microns. Sections were then stained using Massons Trichrome, and collagen content was determined using the ImageJ program from NIH (Rasband, 1997), as previously described (Grobe et al., 2006 Chapter 3). For a detailed method, please see Appendix B. Statistics Indirect blood pressure data were analyzed by one-way repeated measures ANOVA. Cardiac hypertrophy measurements were analyzed by one-way ANOVA. Interstitial fibrosis was analyzed by nonparametric analyses, namely an overall Kruskal-Wallis test followed by post-hoc Mann-Whitney U tests. For all analyses, P<0.05 was considered statistically significant. Results Blood Pressure Angiotensin II (100 ng/kg/min) infusion resulted in significant increases in indirect systolic blood pressures (Figure 2-1). Cardiac Hypertrophy Ventricular size was significantly increased in animals infused with Ang II (Figure 2-2A). Angiotensin-(1-7) had no significant effect on this increase in ventricle size, but a trend toward reversal was observed. Ang-(1-7) did, however, significantly prevent the Ang II-induced increase in individual cardiac myocyte diameter (Figure 2-2B).

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21 1101201301401501601700123WeeksSystolic BP (mmHg) Ang II Ang II + Ang-(1-7) Sham Figure 2-1. Chronic Ang-(1-7) has no effect on blood pressure increases with chronic Ang II infusion. Analysis by one-way repeated measures ANOVA reveals a significant increase in BP with Ang II (P=0.0053), and Ang II + Ang-(1-7) was not different from Ang II alone (P=0.4579). Data presented as mean SE. 2.72.82.93.03.13.23.33.4ShamAng IIAng II +Ang-(1-7)Heart Mass (g/kg)*A 12131415161718ShamAng IIAng II +Ang-(1-7)Myocyte Diameter (microns)*B Figure 2-2. Chronic Ang-(1-7) prevents myocyte hypertrophy due to chronic Ang II infusion. (A) Heart weight, normalized to body weight, is significantly increased by chronic Ang II. Chronic Ang-(1-7) causes a non-significant (P=0.09) trend toward reversing this effect. (B) Chronic infusion of Ang II significantly increases myocyte cross-sectional diameter, and chronic Ang-(1-7) significantly prevents this effect. (* indicates P<0.05 vs. Sham, and indicates P<0.05 vs. Ang II) Data presented as mean SE.

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22 Cardiac Fibrosis Interstitial collagen deposition was significantly increased in animals with chronic Ang II infusion, and this increase was prevented by simultaneous Ang-(1-7) infusion (Figure 2-3). 97100104107ShamAng IIAng II +Ang-(1-7)Interstitial Fibrosis (% Sham)*A Ang II + Ang-(1-7) Ang II B Sham Figure 2-3. Chronic Ang-(1-7) prevents interstitial fibrosis due to chronic Ang II infusion. (A) Infusion of Ang II significantly increased interstitial fibrosis in the left ventricle wall. Infusion of Ang-(1-7) significantly prevented this effect. (* indicates P<0.05 vs Sham, and indicates P<0.05 vs. Ang II) Data presented as mean SE. (B) Representative 250x-magnification images illustrate changes in myocyte size and interstitial collagen. Discussion In this study, chronic Ang II infusion resulted in increased blood pressure, hypertrophied heart ventricles and cardiac myocytes, and increased interstitial collagen

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23 deposition. Simultaneous infusion of Ang-(1-7) at a nearly equal molar rate prevented cardiac remodeling, both in terms of hypertrophy and fibrosis, without effect on blood pressure. These findings are parallel to those observed with ACE2 overexpression during Ang II infusion (Huentelman et al., 2005). The finding that chronic Ang-(1-7) provides the same cardioprotection as overexpression of ACE2 supports the concept that Ang-(1-7) mediates the cardiovascular actions of ACE2. Further, these findings indicate that Ang-(1-7) is an active hormone (rather than simply a degradation product of the trophic Ang II peptide). There are several possible mechanisms by which Ang-(1-7) may inhibit pathophysiologies induced by Ang II. First, it has been proposed that Ang-(1-7) may antagonize the AT 1 receptor (Gironacci et al. 1999; Caruso-Neves et al., 2000; Ferrario et al., 2005; Ferrario et al., 2005; Igase et al., 2005). Second, some evidence exists that Ang-(1-7) selectively activates the AT 2 receptor (Santos et al., 2000; De Souza et al., 2004; Gironacci et al., 2004; Roks et al., 2004), and thereby opposes the effects of AT 1 receptor activation. Third, Ang-(1-7) may inhibit ACE (Donoghue et al., 2000), and therefore inhibit endogenous production of Ang II. Fourth, Ang-(1-7) may act through its own receptor to effect changes in cellular function directly (Santos et al., 2003; Tallant et al., 2005). Several groups have proposed that Ang-(1-7) may bind the AT 1 receptor. For example, Ang-(1-7) stimulates the Na + -ATPase, but not the Na + ,K + -ATPase activity present in pig kidney proximal tubules, and this stimulation is attenuated by losartan (a selective AT 1 antagonist), but not PD-123,319 (a selective AT2 antagonist) or D-Ala-Ang-(1-7) (also known as A779, an Ang-(1-7) receptor binding antagonist) (Caruso

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24 Neves et al., 2000). Similarly, others have shown that radiolabeled Ang II binds to AT 1 receptors in rat renal cortex, and that Ang-(1-7) displaces the Ang II with high affinity (Gironacci et al., 1999). Clearly it is possible that Ang-(1-7) may bind to the AT1 receptor in target tissues. If Ang-(1-7) did interfere with Ang II binding to AT 1 receptors in the present study, though, one would hypothesize that the Ang-(1-7) would interfere with pressor effects of chronic Ang II (which are well established to be AT 1 dependent). Indirect pressure data over time (Figure 2-1), however, fail to support this hypothesis. Therefore, Ang-(1-7) antagonism of AT 1 receptors seems unlikely as the cardioprotective mechanism in the current study. Activation of the AT 2 receptor is another possible mechanism by which Ang-(1-7) prevents cardiac remodeling in the present study. Ang-(1-7) inhibits the pig renal inner cortex basolateral Na + -ATPase activity in a dose-dependent manner, and this effect is attenuated by PD-123,319 but not losartan or A779 (De Souza et al., 2004). Ang-(1-7)-mediated inhibition of the release of norepinephrine in the spontaneously hypertensive rat is also AT 2 dependent (Gironacci et al., 2004), as is the endothelium-dependent, non-competitive antagonism of Ang II contraction of aortic rings (Roks et al., 2004), and the osmotic water permeability in isolated toad skin (Santos et al., 2000). It has been shown that AT 2 receptors are cardioprotective (Metcalfe et al., 2004), and these receptors are known to be expressed in both cardiac myocytes (Matsubara, 1998) and fibroblasts (Lijnen and Petrov, 1999; Lijnen and Petrov, 2003), although their expression profiles during adulthood are debated. Therefore, it is possible that AT 2 -mediated transduction of Ang-(1-7) signaling could account for the present results.

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25 Another possible mechanism by which Ang-(1-7) may mediate its effects is through the inhibition of the ACE enzyme. This enzyme is responsible for most of the endogenous production of Ang II in rats, and is also known to degrade Ang-(1-7). It has been suggested (Tom et al., 2003) that Ang-(1-7) may work through inhibition of ACE, and therefore decreased production of Ang II. This inhibition could represent a feedback loop, with the degradation product of Ang II regulating the production of Ang II. Although this mechanism seems plausible, it is unlikely that decreased production of Ang II through ACE inhibition could account for the present results, as Ang II is chronically infused in the current paradigm. Therefore, it is unlikely that Ang-(1-7) inhibition of Ang II production through ACE would account for the present results. Inhibition of ACE could also represent a mechanism of cross-talk with the bradykinin / nitric oxide system, though, which likely could mediate the observed effects. Active (vasodilatory and cardioprotective) bradykinin is inactivated and degraded to bradykinin-(1-7) by ACE (Tom et al., 2003). In fact, more than 50% of the degradation of bradykinin in vivo is mediated by ACE (Goodman and Gilman, 2001). It is plausible that Ang-(1-7) may, by inhibiting the destruction of the cardioprotective bradykinin through inhibition of ACE, elicit the observed cardioprotective actions. One would expect, though, that increased survival of bradykinin through Ang-(1-7) inhibition of ACE would significantly impact blood pressure regulation. Therefore, although it is possible that inhibition of ACE may account for the cardioprotective actions of Ang-(1-7), the observed effects of chronic Ang-(1-7) on blood pressure do not support this hypothesis. A few years ago, Santos et al. (2003) established that Ang-(1-7) selectively binds the G-protein coupled receptor Mas, and that the anti-diuretic actions of Ang-(1-7) in the

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26 mouse kidney are Mas-dependent. Subsequent studies by others have established that Ang-(1-7) mediates anti-hypertrophic (Tallant et al., 2005) and vasodilator (Lemos et al., 2005) effects in vitro through the Mas receptor. In light of these findings, it seems possible that chronic Ang-(1-7) effects on cardiac structure in vivo are mediated through the Mas receptor. While the determination that Ang-(1-7) actively opposes the trophic, fibrotic actions of Ang II is significant, this study does not clarify the mechanism of Ang-(1-7) action, nor does it determine if the actions of Ang-(1-7) are limited to Ang II-mediated cardiac remodeling. Therefore, the next study was designed to determine if Ang-(1-7) is effective in preventing cardiac remodeling in a hypertensive animal model that displays low circulating Ang II levels.

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CHAPTER 3 CHRONIC ANGIOTENSIN-(1-7) PREVENTS CARDIAC FIBROSIS IN THE DOCA-SALT MODEL OF HYPERTENSION Note: This chapter has already been published in the American Journal of Physiology: Heart & Circulatory Physiology. Grobe JL, Mecca AP, Mao H, and Katovich MJ. Chronic angiotensin-(1-7) prevents cardiac fibrosis in the DOCA-salt model of hypertension. Am J Physiol Heart Circ Physiol. [Epub ahead of print], 2006. Abstract Cardiac remodeling is a hallmark hypertension-induced pathophysiology. In the current study, the role of the angiotensin-(1-7) fragment in modulating cardiac remodeling was examined. Sprague-Dawley rats underwent uninephrectomy surgery and were implanted with a deoxycorticosterone acetate pellet (DOCA). DOCA animals had their drinking water replaced with 0.9% saline solution. A subgroup of DOCA-salt animals was implanted with osmotic minipumps, which delivered angiotensin-(1-7) chronically (100 ng/kg/min). Control animals underwent sham surgery and were maintained on normal drinking water. Blood pressure (BP) was measured weekly using a tail-cuff method, and after four weeks of treatment, BP responses to graded doses of angiotensin II were determined by direct carotid artery cannulation. Ventricle size was measured and cross-sections of the heart ventricles were paraffin embedded and stained using Massons Trichrome to measure interstitial and perivascular collagen deposition and myocyte diameter. DOCA-salt treatment caused significant increases in blood pressure, cardiac hypertrophy, and myocardial and perivascular fibrosis. Angiotensin-(1-7) infusion prevented the collagen deposition effects without any effect on blood pressure 27

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28 or cardiac hypertrophy. These results indicate that angiotensin-(1-7) selectively prevents cardiac fibrosis independent of blood pressure or cardiac hypertrophy in the DOCA-salt model of hypertension. Introduction Cardiac fibrosis is a major facet of hypertensive cardiac disease, and it interferes with the normal function and structure of the myocardium (Brilla et al. 1991; Weber and Brilla 1991; Weber, 2000). Increased deposition of basement membrane collagen is a hallmark of the remodeling process, and results in an increase in cardiac tissue stiffness. This remodeling predisposes the patient to an increased risk of adverse cardiac events, including myocardial ischemia, infarction, arrhythmias and sudden cardiac death (Weber, 2000). Thus, prevention and reversal of cardiac fibrosis are essential in the management of hypertensive heart disease. The renin angiotensin aldosterone system (RAAS) has been suggested to participate in the development of end-organ damage in hypertensive patients (Brunner et al., 1972; Alderman et al. 1991). Support for this concept comes from clinical trials that demonstrate treatment of hypertensive patients with either ACE inhibitors (Pfeffer et al., 1992; The SOLVD Investigators, 1992) or AT 1 receptor blockers (Pitt et al., 1997; Thurmann et al. 1998) provides significant protection from, and even reversal of, end-organ damage. Animal studies have also demonstrated that ACE inhibitors and AT 1 receptor antagonists prevent cardiovascular injury (Jalil et al., 1991; Stier et al., 1991; Stier et al., 1993) as well as protect against renal (Anderson et al., 1986; Stier et al., 1991; Stier et al. 1998) and cerebral (Stier et al. 1991; Stier et al., 1993; Stier et al., 1998) injury.

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29 The use of these antagonists of the RAAS not only reduce the formation and actions of angiotensin II (Ang II), but also result in a significant elevation of another fragment of the RAAS, angiotensin-(1-7) (Ang-(1-7)) (Ferrario et al., 2005; Keidar et al., 2005). This peptide, which can be generated locally in the myocardium (Santos et al., 1990), has been reported to work antagonistically to Ang II and has been implicated in protecting against cardiac pathophysiology (Santos et al., 2004). Recently, Loot et al. (2002) demonstrated that chronic infusion of Ang-(1-7) improved endothelial function and coronary perfusion, and preserved cardiac function in an animal model of heart failure. Ang-(1-7) has also been shown to significantly increase cardiac output and stroke volume in rodents (Ferreira et al., 2002) and to improve contractile function in isolated perfused rat hearts (Sampaio et al., 2003). Using a fusion protein to generate a transgenic rat model that overproduces Ang-(1-7), Santos et al. (2004) showed that Ang-(1-7) reduced the induction of cardiac hypertrophy and the duration of reperfusion arrhythmias, and improved post-ischemic function in isolated perfused hearts. Thus, Ang-(1-7) may be an important component of the RAAS that has opposing actions to Ang II, and it may provide protective action(s) in the heart and possibly other end-organs. Angiotensin-(1-7) also recently has been reported to inhibit the growth of cardiac myocytes in vitro (Tallant et al., 2005), suggesting that elevated levels of Ang-(1-7) may also protect against cardiac hypertrophy in hypertension. Interestingly, blockade of Ang-(1-7) receptors has also been shown to reverse the antihypertensive effects of long term administration of ACE inhibitors (Iyer et al., 1998). Thus, Ang-(1-7) may play a significant role in the beneficial effects on the cardiovascular system attributed to some antihypertensive agents.

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30 Not only has Ang II been implicated in the development of cardiac fibrosis, but so has the aldosterone component of the RAAS (Funder, 2001). It has been demonstrated in experimental studies utilizing uninephrectomized rats, that deoxycorticosterone acetate supplementation (DOCA-salt) treatment results in significant cardiac and renal remodeling, including intersititial fibrosis and hypertrophy (Dobrzynski, et al, 2000; Young et al. 1995). The purpose of this study was to determine if chronically elevated levels of Ang-(1-7) alters the development of hypertension, cardiac hypertrophy and/or cardiac fibrosis in the DOCA-salt model of hypertension. Methods Animals Male Sprague-Dawley rats weighing between 170 and 250 grams were used for this study. Animals were housed in a temperature and humidity controlled room in standard shoebox cages, and maintained on a 12:12 hour light:dark schedule with free access to food and water. All procedures were approved by the University of Florida Institutional Animal Care and Use Committee. Chemicals Angiotensin-(1-7) was obtained from Bachem Bioscience, Inc. Deoxycorticosterone acetate was purchased from Sigma Aldrich (St. Louis, MO). DOCA-salt Model Animals were anesthetized using an intramuscular injection of a Ketamine, Xylazine Acepromazine, and (30, 6, and 1 mg/kg, i.m., respectively) before uninephrectomy. During the surgery, fifteen animals were implanted with a subcutaneous pellet of deoxycorticosterone acetate (40 mg). Seven of these animals were additionally implanted with a subcutaneous osmotic pump that delivered Ang-(1-7) (100

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31 ng/kg/min) for the duration of the experiment. After surgery (and for the remainder of the experiment), drinking water for these animals was replaced with 0.9% saline solution. Ten animals underwent a sham surgery, and were maintained on normal drinking water. Four additional sham animals were implanted with only an Ang-(1-7) pump. Indirect Blood Pressure Systolic blood pressures were determined weekly by an indirect method utilizing a tail-cuff and pneumatic pulse sensor as previously reported (Katovich et al., 2001). Briefly, animals were heated using a 200-watt heat lamp for 3-5 minutes before being restrained in a heated Plexiglas restrainer to which the animals had been conditioned prior to the experiment. A pneumatic pulse sensor was then attached to the tail distal to a pressure cuff under the control of a Programmed Electro-Sphygmomanometer (Narco). Voltage outputs from the pressure sensor bulb and inflation cuff were recorded and analyzed electronically using a PowerLab signal transduction unit and associated Chart software (ADInstruments). At least three separate indirect pressures were averaged for each animal. Direct Blood Pressure/Acute Angiotensin II Responses After four weeks of chronic treatment, animals underwent carotid artery and jugular cannula implantation surgery as previously reported (Lu et al., 1997). Briefly, animals were anesthetized with a mixture of Ketamine, Xylazine, and Acepromazine (30, 6, and 1 mg/kg, i.m., respectively). A polyethylene cannula (PE-50, Clay Adams) was introduced into the carotid artery for direct blood pressure measurements, while a silicone elastomer cannula (Helix Medical) was introduced into the descending jugular vein for acute intravenous injections of drug. Both cannulae were filled with heparin saline (40 U/mL, sigma), and sealed with stylets. Animals were allowed 24 hours to recover before

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32 experiments were performed. Direct blood pressure responses to acute intravenous injections of Ang II (5 to 320 ng/kg) in awake, freely moving animals were recorded by connecting the carotid cannula to a liquid pressure transducer, which was interfaced to a PowerLab (ADInstruments) signal transduction unit. Data were analyzed using the Chart program that was supplied with the PowerLab system. Cardiac Remodeling Cardiac remodeling was determined at the end of the direct blood pressure experiment by ventricular hypertrophy and cardiac fibrosis after four weeks of respective treatments. Following the acute Ang II blood pressure experiment, animals were anesthetized by inhaled halothane, then euthanized by decapitation. Ventricular hypertrophy was determined by measuring ventricle mass normalized by body mass. Cross-sections of the ventricles were then embedded in paraffin, sectioned at 4 microns, and stained for collagen deposition using Massons Trichrome. Single sections from each animal were then viewed and photographed with a Moticam 1000 digital camera (Motic, Richmond, British Columbia, Canada) under 10X (whole heart) and 250X (perivascular collagen and myocyte diameter) magnifications. The collagen content of the left ventricle wall was determined by measuring the blue stain density of the left wall (from the septum in the posterior to the septum in the anterior) and was normalized to the red stain density of the same portion of the image. The area of blue-stained collagen surrounding the left anterior descending coronary artery was measured and normalized to the area of the lumen of the vessel. Myocyte cross-sectional diameter was also measured to confirm ventricular hypertrophy results. Color density, perivascular collagen and lumen areas, and myocyte diameter were determined using the ImageJ program from NIH (Rasband, 1997).

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33 Statistics Basal direct blood pressure and ventricular hypertrophy (by mass and by myocyte diameter) were analyzed by two-way ANOVA, while indirect blood pressures and direct blood pressure responses to graded, acute doses of Ang II were analyzed by two-way ANOVA with repeated measures. Cardiac fibrosis measurements were compared using the more stringent nonparametric Kruskal-Wallis ANOVA, followed by post-hoc Mann-Whitney U tests. Data were considered significantly different with p<0.05. Results Indirect Blood Pressure Figure 3-1. Chronic Ang-(1-7) has no effect on blood pressure increases with DOCA-salt treatment. DOCA-salt treatment led to a significant increase in systolic blood pressure as measured by an indirect tail-cuff method (p=0.006). Chronic infusion of Ang-(1-7) had no effect on the systolic blood pressure increases due to DOCA-salt treatment (p=0.22). Data presented as mean +/SE. DOCA-salt treatment caused a significant increase (p=0.006) in systolic blood pressure over the course of the experiment. Ang-(1-7), when infused simultaneously, failed to prevent these increases (p=0.22) (Figure 3-1). Ang-(1-7), when infused alone,

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34 did not significantly alter blood pressure when compared to sham-implanted animals (p=0.69, data not shown). Direct Blood Pressure/Acute Angiotensin II Responses Direct pressure measurements confirmed that while DOCA-salt treatment significantly increased pressure (p=0.007), Ang-(1-7) had no effect (p=0.20) on basal systolic blood pressure (Sham, 129.2.7; DOCA, 160.0.2; DOCA + Ang-(1-7), 171.6.8; Ang-(1-7) alone, 143.8.0 mmHg). In addition, DOCA-salt treatment significantly potentiated (p=0.02), and Ang-(1-7) infusion had no effect (p=0.41), on the dose-response curves to acute, graded injections of Ang II (Figure 3-2). Figure 3-2. Chronic Ang-(1-7) has no effect on acute pressor responses to Ang II in animals treated with DOCA-salt. DOCA-salt treatment significantly potentiated the dose-response curve to acute Ang II (p=0.02). Chronic Ang-(1-7) had no effect (p=0.41) on the acute responses to Ang II. Data presented as mean +/SE. Cardiac Remodeling DOCA-salt treatment significantly increased ventricle mass (p=0.02), and Ang-(1-7) failed to prevent this effect (p=0.35) (Figure 3-3A). These effects were confirmed by

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35 measurement of myocyte diameter, which uncovered a significant increase in size with DOCA-salt treatment (p<0.0001), but no effect of Ang-(1-7) (p=0.71) (Figure 3-3B). A B Figure 3-3. Chronic Ang-(1-7) has no effect on cardiac hypertrophy due to DOCA-salt treament. (A) DOCA-salt treatment significantly increased heart mass (p=0.02). Chronic Ang-(1-7) had no effect on the hypertrophy due to DOCA-salt treatment (p=0.35). (B) Cardiac myocyte diameter was also significantly increased by DOCA-salt treatment (p<0.0001), but chronic Ang-(1-7) had no effect (p=0.71). Data are presented as mean +/SE. (* indicates p<0.05 vs. Sham) A significant increase in left ventricular wall fibrosis was observed with DOCA-salt treatment (p=0.04 vs. sham), and chronic Ang-(1-7) treatment significantly prevented this trend (p=0.04 vs. DOCA) (Figure 3-4). While the increase in ventricular wall fibrosis is only approximately 4%, this value is most likely diluted by our measurement of collagen in the entire left ventricular wall at 10X magnification (as opposed to the common method of measuring selected representative samples at high magnification). Clearly, from Figure 3-4B, analysis of representative areas within the left wall would lead to much larger relative changes.

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36 A Figure 3-4. Chronic Ang-(1-7) prevents interstitial fibrosis with DOCA-salt treatment. (A) DOCA-salt treatment caused a significant increase in collagen deposition in the left ventricle wall. Chronic Ang-(1-7) significantly prevented this increase. Data are presented as mean +/SE. (* indicates p<0.05 vs. Sham, indicates p<0.05 vs. DOCA). (B) Representative images of mid-myocardium at 250X magnification, stained with Massons Trichrome. Blue color indicates collagen fibers. DOCA-salt treatment further resulted in a significant (p=0.0002 vs sham) increase in perivascular collagen deposition around the left anterior descending coronary artery, and Ang-(1-7) completely prevented this effect (p=0.0007 vs DOCA) (Figure 3-5). Discussion In this study, DOCA-salt treatment resulted in increases in blood pressure, acute pressor responses to Ang II, cardiac hypertrophy, and mid-myocardial and perivascular fibrosis. The Ang-(1-7) fragment had no effect on the elevated blood pressure, acute B

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37 pressor responses or cardiac hypertrophy, but significantly prevented the cardiac mid-myocardial and perivascular fibrosis in the DOCA-salt hypertensive rat model. Figure 3-5. Chronic Ang-(1-7) prevents perivascular fibrosis with DOCA-salt treatment. (A) DOCA-salt significantly increased perivascular collagen around the left 5 f Deoxprevios A B anterior descending coronary artery. Chronic Ang-(1-7) significantly prevented this effect. Data are presented as mean +/SE. (* indicates p<0.0vs. Sham, indicates p<0.05 vs. DOCA). (B) Representative images operivascular collagen surrounding the left anterior descending coronary artery, stained with Massons Trichrome. Blue color indicates collagen fibers. ycorticosterone treatment in uninephrectomized rats drinking 1.0% NaCl has usly been shown to cause hypertension, cardiac hypertrophy and interstitial and perivascular fibrosis (Young et al. 1995; Dobrzynski et al., 2000). These cardiac effectare mediated via mineralocorticoid receptors (Young and Funder, 2000; Rocha and Stier,

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38 2001; Weber, 2001), and are not observed in animals on a low salt diet (Brilla et al., 1992). Others have suggested the involvement of AT 1 receptors in the fibrosis associwith DOCA-salt treatment (Kim et al. 1994; Nishikawa, 1998). Elevation of cardiac AT ated pertrophy response to DOCA treatmr than d ce cts of DOCA on cardiac hypertrophy have been postulated to involve angiotensin II or transforming growth factor (TGF) (Young et al., 1994; Takeda et al., 1 receptors associated with DOCA-salt treatment may potentiate the well described fibrotic effect of Ang II. There is evidence that the trigger for cardiac fibrosis in this animal model is coronary vasculitus, as the inflammatory cell infiltration occurs within a fewdays of DOCA treatment (Young and Funder, 2003). In a study by Young et al. (1995), the cardiac hy ent was restricted to the left ventricle, whereas the collagen deposition was indistinguishable between left and right ventricle, thus supporting a humoral, rathea hemodynamic, etiology. Further support for a humoral etiology for collagen depositioncomes from Webers (1994) laboratory, where spironolactone prevented aldosterone-induced cardiac fibrosis without lowering blood pressure. Additionally, interstitial anperivascular fibrosis can be produced by mineralocorticoid and salt without substantial hypertension or cardiac hypertrophy (Young et al., 1995). Young et al. (1995) also demonstrated that central infusion of a mineralocorticoid receptor antagonist loweredblood pressure but not the pattern of cardiac fibrosis, further suggesting an independenof fibrosis and blood pressure. Our results also demonstrate the fibrosis observed in this model was found in both the right and left ventricle walls (right ventricle data not shown), and such fibrosis appears to be independent of both blood pressure and hypertrophy. The effe

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39 2002)ot ), CA-sts ibrosis induced by coronary artery ligation in male Wistaal sly ) udy, Whether these same mechanisms are responsible for the cardiac fibrosis are nclear. Young and Funder (2003) demonstrated that animals treated for as few as 8 days with DOCA/salt treatment demonstrated a significant increase in cardiac perivascular collagen deposition, with no changes in heart weight, and the collagen formation was inhibited by a mineralocorticiod receptor antagonist. Further evidence that the cardiachypertrophy and fibrosis may be independent include results from Lekgabe et al. (2005who demonstrated that cardiac hypertrophy, and the related markers of hypertrophy thatwere elevated in the SHR compared to the WKY, were not reversed with chronic relaxin treatment, whereas the fibrosis and collagen content in the hearts of the SHR were normalized by relaxin treatment to levels observed in the WKY. In the current study, Ang-(1-7) also showed differential effects upon hypertrophy and fibrosis in the DOsalt model of hypertension, suggesting different mechanisms may exist for these two endpoints of cardiac remodeling. Nehme et al. (2005) reported that both aldosterone and Ang II receptor antagonisignificantly reduced the cardiac f r rats. The effects of the aldosterone receptor antagonist (spironolactone) were independent of changes in mean blood pressure. This form of experimental myocardiinfarction (MI) in rats has been shown to activate the RAAS (Pinto et al., 1993, Schunkert et al., 1993). Spironolactone also has been shown to reduce arterial collagen production, independent of changes in systemic blood pressure in the spontaneouhypertensive rat (Benetos et al., 1997) and in an L-NAME (nitro-L-arginine methyl esterplus Ang II with salt model of hypertension (Rocha et al., 2000). As in the current stNehme et al. (2005) also reported that these treatments did not prevent the cardiac

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40 hypertrophy induced in this MI model. This is further evidence that the mechanisms for cardiac hypertrophy and fibrosis may be independent. Generally, the actions of Ang-(1-7) are opposite of those attributed to Ang II (Santos et al., 2000). Ang-(1-7) has been reported to hav e intrinsic vasoactive effects in some., the gts no studies (Brosnihan et al., 1996; Iyer et al., 1998; Sasaki et al., 2001; Ren et al2002), whereas these effects are absent in other studies (Davie et al., 1999; Wilsdorf et al., 2001; Zhu et al., 2002; Stegbauer et al., 2003). Bayorh et al. (2002) demonstratedhypotensive effects of Ang-(1-7) were not observed in Dahl animals on a low salt diet and Eatman et al. (2001) noted a gender difference in the vasodilatory action of Ang-(1-7) in Dahl rats. Neves et al. (2004) recently reported that the vasodilatory effects of An(1-7) are modulated during the estrus cycle. Thus, the contrasting findings of Ang-(1-7) on the vasculature may be related to the differences in animal models studied, dose of Ang-(1-7), vascular bed studied, or the experimental condition (in vitro versus in vivo) in which the peptide is evaluated. In the current study, at the dose of Ang-(1-7) infused, there was no effect on the basal blood pressure of normotensive SD animals or those animals made hypertensive with DOCA-salt treatment. There are situations, such as inmodels of endothelial dysfunction (Le Tran et al., 1997), where the vasodilatory effecof Ang-(1-7) have been shown to be blunted. This may be the reason for a lack of effectin our hypertensive model. In the current study, no reduction in the pressor response to acute administration of Ang II was observed. Benter et al. (1995) reported that infusion of Ang-(1-7) did not alter the pressor responses to Ang II or phenylephrine in WKY animals; however, it did reduce the response to these agents in the SHR. A higher dose of Ang-(1-7) was utilized in the Benter study compared to ours. Although there was

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41 observable effect on blood pressure at the dose of Ang-(1-7) used in the current study, there was a significant attenuation of cardiac fibrosis. The mechanism of action of Ang-(1-7), an active peptide fragment of the renin angiotensin system, is currently controversial. It has b een reported to act as an angiond eviewed f hypertension. First, Ang-(1-7) could reduce blood pressure, whichts would ute asts. f various pro-thelin-1, as tensin receptor antagonist, an ACE inhibitor, as well as altering prostaglandins athe bradykinin-nitric oxide system. These mechanisms have been exhaustively rby Santos et al. (2000) and others. The mechanisms may involve the reported mas receptor (Tallant et al., 2005) or direct interaction with other angiotensin receptors (Santos et al., 2000). There are several mechanisms by which Ang-(1-7) may decrease collagen deposition in models o would decrease hemodynamic-dependent pro-fibrotic signaling. Our resulnot support this mechanism, as changes in blood pressures and pressor responses to acAng II were not observed by indirect or direct methods during chronic Ang-(1-7) treatment (Figures 3-1 and 3-2). Second, Ang-(1-7) could directly inhibit the secretion of collagen or the activation of the fibroblasts or their differentiation into myofibroblThird, Ang-(1-7) may increase collagen degradation by activating matrix metalloproteinases (MMPs), or by inhibiting tissue inhibitors of matrix metalloproteinases (TIMPs). Finally, Ang-(1-7) may inhibit the actions ofibrotic factors such as norepinephrine, angiotensin II, TGF and/or endoAng-(1-7) infusion has been reported to result in a reduction of cardiac Ang II levels (Mendes et al., 2005). Future studies will be aimed at dissecting the possible

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42 mechanisms by which Ang-(1-7) reduces cardiac fibrosis in hypertension and determine if this anti-fibrotic action also occurs in non-hypertensive fibrotic condThis study also supports the idea that angiotensin-(1-7) has direct physiological to itions. actionre ptor owth collagen protective effect s, rather than simply being the result of metabolized Ang II. Our in vivo results asupported by a recent in vitro study (Iwata et al., 2005). In this study, the authors examined angiotensin fragment binding in cultured adult rat cardiac fibroblasts, andshowed that Ang-(1-7) binds to two separate sites that are unaffected by the AT 1 receantagonist valsartan and the AT 2 receptor antagonist PD-123,319. Importantly, these authors showed that within the concentration ranges studied for binding, Ang-(1-7) inhibited collagen formation as measured by [ 3 H]proline incorporation, and also decreased mRNA expression of various growth factors including transforming grfactor 1 (TGF 1 ), endothelin-1 (ET-1), and leukemia inhibitory factor (LIF). Pretreatment of the cardiac fibroblasts with Ang-(1-7) inhibited Ang II-inducedprotein. Together, the current in vivo study and the in vitro study by Iwata et al. (2005) strongy support the hypothesis that Ang-(1-7) actively regulates cardiac fibrosis. These findings further indicate the significant role that Ang-(1-7) plays in the heart. Our results parallel those of Loot et al. (2002), who demonstrated cardio s of Ang-(1-7). In the current study, we utilized one fourth of the dose of Ang-(1-7)that these authors employed. We did not determine the plasma or cardiac levels of Ang-(1-7) in the current study. It is conceivable that utilization of higher concentrations of Ang-(1-7) may result in greater cardioprotection, and it may also uncover effects on hypertrophy.

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43 The results of the current study fit well with the findings of our recent study, in which systemic overexpression of ACE2 (the most important enzyme involved in angiotensin-(1-7) production) in angiotensin-infusion hypertensive rats resulted in blood pressure-independent decreases in cardiac fibrosis (Huentelman et al., 2005). This study supports the concept that ACE2 mediates many (or most) of its protective effects through the production of angiotensin-(1-7). Myocardial infarction increases ACE2 expression in rats and humans (Burrell et al., 2005). Averill et al. (2003) also reported that Ang-(1-7) immunoreactivity was significantly increased in the ventricular tissue surrounding an area of myocardial infarction and absent in the remodeled area of infarction. Thus, there is evidence suggesting that the ACE2 enzyme and the formation of Ang-(1-7) play significant roles in modifying cardiac remodeling. Recently, Keider et al. (2005) demonstrated that the mineralocorticoid aldosterone significantly increased cardiac ACE and decreased ACE2 mRNA and activity levels in both humans and mice, whereas aldosterone antagonists produced the opposite responses. This data would suggest that ACE2 levels, and the subsequent generation of Ang-(1-7) may be the mechanism by which mineralocorticoid receptor blockers improve cardiac function in myocardial infarct patients (Pitt et al., 2003). Several investigators (Abraham and Simon, 1994; Simon et al., 1995; Melargno and Fink, 1996) have demonstrated that chronic infusion of angiotensin II results in the development of hypertension, cardiac hypertrophy and fibrosis. Functional Ang II receptors have been documented in cardiac fibroblasts (Crabbos et al., 1994) and Ang II-stimulated expression and secretion of collagen is completely abolished by AT 1 and not AT 2 receptor antagonism in vivo and in vitro (Lijnen and Petrov, 1999, Lijnen et al.,

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44 2001). As Ang-(1-7) is significantly elevated with the use of AT 1 antagonists (Ferrario et al., 2005), it is possible that some of the anti-fibrotic effects attributed to AT 1 antagonism are mediated via actions of Ang-(1-7). The development of cardiac fibrosis is a major complication of hypertensive cardiac disease. The increased deposition of basement membrane collagen is a hallmark of the remodeling process. This remodeling can predispose a patient to increased risk of adverse cardiac events, and therefore any reduction or reversal of cardiac fibrosis would facilitate the management of hypertensive heart disease. Our results suggest that Ang-(1-7) may be a beneficial component of the RAAS that provides this cardioprotection during hypertension, and any maneuver that would increase cardiac levels of ACE2 and subsequently Ang-(1-7) may be used as an effective therapeutic intervention in hypertensive heart disease patients.

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CHAPTER 4 PROTECTIVE EFFECTS OF ANGIOTENSIN-(1-7) DURING ISOPROTERENOL-INDUCED CARDIAC REMODELING Abstract -Adrenergic stimulation of cardiac tissue leads to myocyte hypertrophy and can result in heart failure. A recent study by Santos et al. showed that transgenic rats overexpressing an angiotensin-(1-7) (Ang-(1-7)) fusion gene were significantly protected from cardiac hypertrophy and arrhythmias induced by administration of the -adrenergic receptor agonist isoproterenol. Here, we first characterized the effects of isoproterenol in male and female rats, then examined the hypothesis that chronic infusion of Ang-(1-7) would prevent cardiac hypertrophy and fibrosis due to isoproterenol administration. Intact male, intact female, and ovariectomized female Sprague-Dawley rats between 180-280 grams were injected with isoproterenol at either 1 or 2 mg/kg daily for fourteen consecutive days. A subset of the 2 mg/kg isoproterenol-treated male rats were implanted with osmotic minipumps which chronically delivered Ang-(1-7) (100 ng/kg/min). Hearts were harvested and weighed, and the (2 mg/kg isoproterenol) male rat hearts were sectioned and stained using Massons Trichrome. Isoproterenol injections resulted in large increases in heart size in males at both doses, but had no effect on cardiac fibrosis. While isoproterenol also induced large increases in heart size in intact females, this effect was blunted in ovariectomized animals. Chronic infusion of Ang-(1-7) in the 2 mg/kg male group resulted in significantly attenuated cardiac hypertrophy. From these studies we conclude both that Ang-(1-7) inhibits the hypertrophic response to 45

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46 isoproterenol, and that ovarian hormones are permissive to the hypertrophic effects of isoproterenol. These findings lead us to the hypothesis that ovarian hormones may alter cardiac tissue sensitivity to the protective Ang-(1-7). Introduction High blood pressure is a key risk factor for cardiac remodeling and subsequent heart failure (Regitz-Zagrosek and Lehmkuhl, 2005; Thom et al., 2006). Others have previously demonstrated that reductions in arterial pressure can prevent or even reverse the cardiac remodeling associated with high blood pressure (Diez et al., 2001). To determine whether the cardioprotective effects of Ang-(1-7) are mediated by decreased arterial pressure (or limited to hypertensive models), the following study was carried out using a normotensive model of cardiac remodeling. Isoproterenol is a pharmacological catecholamine with an N-alkyl substitution, making it almost entirely selective for -adrenergic receptors (Jacob, 1996; Goodman and Gilman, 2001). Administration of isoproterenol results in decreased peripheral resistance through stimulation of 2 receptors, which leads to a decrease in diastolic pressure. Although systolic pressure typically is normal or slightly increased, mean arterial pressure is usually depressed. Stimulation of 1 receptors in the heart results in positive inotropic and chronotropic effects, thus increasing cardiac output. Stimulation of cardiac tissue by isoproterenol may lead to sinus tachycardia, palpitations, and more serious arrhythmias, and large doses may cause myocardial necrosis (Goodman and Gilman, 2001). These properties of isoproterenol have led many investigators to use the compound to induce cardiac remodeling without increasing blood pressure. A recent study by Santos et al. (2004) found that increasing circulating Ang-(1-7) levels protected rats from isoproterenol-induced hypertrophy and associated arrhythmias.

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47 While the data are encouraging, the authors utilized transgenic rats that overexpressed an Ang-(1-7) fusion protein, which confounds interpretation of the physiological data as the role of Ang-(1-7) during development is currently unknown. Further, the authors did not examine the effect of Ang-(1-7) on isoproterenol-induced cardiac fibrosis. In the present study, daily injections of isoproterenol were used to induce cardiac remodeling. Several groups of both male and female animals were tested at various doses of isoproterenol to characterize this normotensive model of cardiac remodeling before testing the cardioprotective effects of angiotensin-(1-7) in this model. Methods Animals Group A Males with isoproterenol at 1 mg/kg Fourteen male Sprague-Dawley rats weighing 220-250 grams were divided into two groups. The first group, with eight animals, was treated with daily injections of 0.9% saline solution (1 mL/kg, s.c.). The second group, with six animals, was treated with daily injections of isoproterenol (1 mg/kg, s.c.). Daily injections were performed between 11:00 AM and 1:00 PM. Both groups were treated for fourteen consecutive days, and on the fifteenth day all animals were sacrificed without a final injection. Group B Ovariectomized and intact females with isoproterenol at 1 and 2 mg/kg Twenty-four female Sprage-Dawley rats weighing 180-210 grams were divided into two groups of twelve animals each. The first group underwent ovariectomy surgery, while the second group served as intact controls. Within each group, three subgroups were formed, including a saline control-injection subgroup and two subgroups that received daily isoproterenol injections at either 1 mg/kg or 2 mg/kg, s.c. Daily injections

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48 were performed between 11:00 AM and 1:00 PM for fourteen days. On the fifteenth day, animals were sacrificed without a final injection. Group C Males with isoproterenol at 2 mg/kg Seven male Sprague-Dawley rats weighing 240-280 grams were divided into three groups. Three animals served as saline-injection controls. Two animals received daily injections of isoproterenol (2 mg/kg, s.c.). The final two animals were implanted with subcutaneous osmotic minipumps (Alzet, model 2002), which chronically delivered angiotensin-(1-7) at 100 ng/kg/min. This final group also received daily injections of isoproterenol (2 mg/kg, s.c.). Again, daily injections were performed between 11:00 AM and 1:00 PM for fourteen days. On the fifteenth day, animals were sacrificed without a final injection. Indirect Blood Pressure Indirect blood pressures were recorded from animals in group C one day before pump implantation and isoproterenol injections, and weekly during the isoproterenol treatment using an indirect tail-cuff method as described in previous experiments. Briefly, animals were heated using a 200-watt heat lamp for 3-5 minutes before being restrained in a heated Plexiglas restrainer to which the animals had been conditioned prior to the experiment. A pneumatic pulse sensor was then attached to the tail distal to a pressure cuff under the control of a Programmed Electro-Sphygmomanometer (Narco). Voltage outputs from the pressure sensor bulb and inflation cuff were recorded and analyzed electronically using a PowerLab signal transduction unit and associated Chart software (ADInstruments). At least three separate indirect pressures were averaged for each animal.

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49 Cardiac Remodeling Following fourteen days of isoproterenol injections with or without chronic infusion of angiotensin-(1-7), animals were sacrificed by overdose of halothane inhalant. Hearts were removed by blunt dissection, weighed, and sectioned. Again, as in previous experiments, cross-sections at the level of the papillary muscles were then fixed in 10% formalin solution, embedded in paraffin, sectioned at 4 microns, and stained using Massons Trichrome. Single sections from each animal were then viewed and photographed with a Moticam 1000 digital camera (Motic, Richmond, British Columbia, Canada) with a 10X magnification. The collagen content of the left ventricle wall was determined by measuring the blue stain density of the left wall (from the septum in the posterior to the septum in the anterior) and was normalized to the red stain density of the same portion of the image. The area of blue-stained collagen surrounding the left anterior descending coronary artery was measured and normalized to the area of the lumen of the vessel. Color density and perivascular collagen and lumen areas were determined using the ImageJ program from NIH (Rasband, 1997). Results Cardiac Hypertrophy Male rats injected daily with 1 mg/kg isoproterenol (Group A) displayed marked increases in cardiac hypertrophy (Figure 4-1). The increase in size was approximately 28%, which is much larger than that observed with the Ang II (5%) or DOCA-salt (14%) models of hypertension-induced cardiac remodeling.

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50 Figure 4-1. Isoproterenol (1 mg/kg/day) induces cardiac hypertrophy in male rats. Fourteen days of isoproterenol treatment resulted in a significantly increased heart size (P<0.0001). Data presented as mean SE. Figure 4-2. Ovariectomy potentiates the isoproterenol-mediated induction of cardiac hypertrophy in female rats. Daily injections of isoproterenol resulted in differential effects on cardiac hypertrophy, depending on ovarian hormone status. Two-way ANOVA reveals a significant effect of isoproterenol treatment level (P<0.0001), but ovariectomy treatment only resulted in a non-signficant trend (P=0.0765). The presence of a significant interaction (P=0.0308), however, indicates some impact of ovary status. Data presented as mean SE.

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51 Female rats injected daily with isoproterenol (Group B) showed differential hypertrophic responses to isoproterenol injection based on ovarian hormone status (Figure 4-2). Isoproterenol significantly increased heart size in both surgery groups (P<0.0001). Ovariectomy appeared to attenuate the response to isoproterenol, and although two-way analysis of variance reveals only a non-significant trend with regard to the effect of surgery (P=0.0765), an significant interaction between surgery and isoproterenol dose was uncovered (P=0.0308). Figure 4-3. Chronic Ang-(1-7) prevents the hypertrophic, but not fibrotic effects of isoproterenol (2 mg/kg/day) in male rats. (A) Isoproterenol resulted in significantly increased cardiac hypertrophy (P=0.0007), and chronic infusion of angiotensin-(1-7) at 100 ng/kg/min significantly attenuated this effect (P=0.0082). (B) Isoproterenol and angiotensin-(1-7) had no effect on cardiac fibrosis. Data presented as mean SE. Male rats injected daily with 2 mg/kg isoproterenol (Group C) exhibited tremendous cardiac hypertrophy. As this combination of gender and drug dose resulted in the greatest effect upon cardiac hypertrophy, the effect of angiotensin-(1-7) was evaluated with this combination. Chronic infusion of angiotensin-(1-7) significantly

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52 attenuated the hypertrophic response to isoproterenol (Figure 4-3A). These hearts were then processed to determine the effect of isoproterenol and angiotensin-(1-7) on cardiac fibrosis. Isoproterenol and angiotensin-(1-7) had no significant effects on cardiac fibrosis (Figure 4-3B). Blood Pressure Figure 4-4. Isoproterenol and Ang-(1-7) had no chronic effects on blood pressure in male rats. (P=0.4207 and P=0.6615 vs. saline, respectively) Data presented as mean SE. Indirect blood pressure was also determined in the male rats with 2 mg/kg isoproterenol, as this combination of gender and dose resulted in the greatest changes in cardiac hypertrophy. Isoproterenol had no significant effect on blood pressure, and neither did chronic infusion of angiotensin-(1-7) (Figure 4-4). Discussion Daily injections of isoproterenol resulted in cardiac hypertrophy in both male and female rats. It was determined that ovariectomy significantly attenuated the effects of

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53 isoproterenol in females, and that males were slightly more sensitive to isoproterenol-induced cardiac remodeling than intact females at the 2 mg/kg dose. Angiotensin-(1-7) infusion significantly attenuated the hypertrophic response in these high-dose isoproterenol male rats. Cardiac fibrosis and blood pressure were unaffected by the 2 mg/kg dose of isoproterenol in male rats. Further, angiotensin-(1-7) had no effect on either blood pressure or cardiac fibrosis. While the lack of effect of angiotensin-(1-7) on blood pressure is unsurprising, given the results of both the Ang II and DOCA-salt models (Chapters 2 and 3, respectively), the trend toward increased fibrosis in this model (Figure 4-3B) was unanticipated. These findings appear to conflict with the results in both the Ang II and DOCA-salt models, as Ang-(1-7) completely prevented interstitial fibrosis in both of these models, but also give clues as to the site of Ang-(1-7) interference with pro-fibrotic signaling. Neves et al. (2005) recently determined that mineralocorticoid receptor activation mediates the pro-fibrotic actions of Ang II. Bos et al. (2005) found that isoproterenol-induced fibrosis could be inhibited by antagonism of mineralocorticoid receptors. Together, these findings suggest that the anti-fibrotic effects of Ang-(1-7) may work though interference with the pro-fibrotic signaling pathways of Ang II and DOCA, upstream of the convergence with isoproterenol signals. The effect of Ang-(1-7) upon ventricular hypertrophy was substantial, which is again in direct opposition to the effects observed in the Ang II and DOCA-salt models (Chapters 2 and 3, respectively). Others have determined that antagonism of the mineralocorticoid receptor (Bos et al., 2005) or AT 1 receptors (Nakano et al., 1995) in vivo can prevent isoproterenol-induced cardiac effects, thus clearly demonstrating cross

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54 talk among these signaling cascades. Ang-(1-7) likely inhibits the hypertrophy process between the convergence of signals from Ang II or isoproterenol with signals from mineralocorticoids. The difference in inhibitory roles for Ang-(1-7) with regard to hypertrophy and fibrosis in the Ang II, DOCA-salt, and isoproterenol models likely is related to the differential effects of Ang-(1-7) in the various cardiac cell types. Tallant et al. (2005) showed that Ang-(1-7) acts in vitro at the cardiac myocyte to modulate hypertrophy, and Iwata et al. (2005) also showed that Ang-(1-7) acts in vitro at the cardiac fibroblast to elicit changes in collagen turnover. Possible differences in intracellular signaling cascades in each cell type, or differences in their in vitro models versus our in vivo models, may account for the differential effects of Ang-(1-7) on hypertrophy and fibrosis with mineralocorticoid or -adrenergic stimulation. Further studies in vitro will help elucidate the intracellular signaling cascade(s) of Ang-(1-7) in myocytes and fibrosis, which should uncover the biochemical cause of the different outcomes. Differences in cardiac responsiveness by gender have previously been noted, which prompted the inclusion of intact and ovariectomized female rats in the present study. Schwertz et al., (1999) showed that isolated female rat hearts have smaller inotropic responses to isoproterenol administration than male hearts. In the present study, only small differences in cardiac hypertrophy responses between males and females were observed with 1 and 2 mg/kg isoproterenol, but ovariectomized females exhibited blunted responses to isoproterenol. This may indicate a role for ovarian hormones in regulating cardiac structure.

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55 Previous work by Tallant et al. (2005) uncovered that the anti-hypertrophic actions of Ang-(1-7) are mediated at the cardiac myocyte through the Mas receptor. Preliminary in silico analysis of the Mas receptor promoter indicates that there are several possible estrogen receptor and AP-1 binding sites within the first few kilobases 5 of the transcription start site, thus suggesting that estrogens may alter expression of this receptor (data not shown). These findings, along with our present study of cardiac hypertrophy responses in ovariectomized versus intact animals, lead us to the hypothesis that ovarian hormones may decrease tissue sensitivity to the cardioprotective angiotensin-(1-7) hormone. It has been documented that the Ang-(1-7) receptor (Mas) is found in the vasculature, and stimulation of this receptor results in a relaxation response (le Tran and Forster, 1997). Therefore, in the subsequent study we decided to evaluate estrogenic modulation of vascular reactivity to Ang-(1-7) in rat aorta.

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CHAPTER 5 ALTERATIONS IN AORTIC VASCULAR REACTIVITY TO ANGIOTENSIN-(1-7) IN 17--ESTRADIOL-TREATED FEMALE SD RATS Note: This chapter has already been published in Regulatory Peptides. Grobe, JL and Katovich MJ. Alterations in aortic vascular reactivity to angiotensin-(1-7) in 17--estradiol-treated female SD rats. Reg Pept 133:62-67, 2006. Abstract Estrogen's suggested cardio-protective effects have come into question following the results of recent clinical trials. Two major components of the reninangiotensin system (RAS) that are modulated by estrogen are angiotensin converting enzyme, and the angiotensin II type 1 receptor. Further research has revealed several new components of the RAS, including angiotensin converting enzyme 2, its peptide product angiotensin-(1-7) (Ang-(1-7)), and that peptide's receptor, Mas. These components appear to oppose the classical effects of the RAS, and may act to buffer the RAS in vivo. Recent work has shown that during pregnancy, when estradiol levels are elevated, renal and urinary Ang-(1-7) are greatly increased. This study examined the effects of estradiol on the efficacy of Ang-(1-7) in the rat aorta. Female SpragueDawley rats were ovariectomized and a subgroup was chronically treated with subcutaneous pellets of estradiol for 3 weeks. Thoracic aortas were harvested for assessment of in vitro vascular reactivity to Ang-(1-7). The results demonstrated that increased estradiol exposure attenuated the relaxation response to Ang-(1-7) in a dose-dependent manner. These findings are in contrast to recent work showing potentiated responses to Ang-(1-7) in mesenteric arteries from 56

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57 estrogen-manipulated rats, and may suggest a regional specificity in estradiol-mediated changes in the RAS. Introduction The leading cause of death in women is cardiovascular disease (CVD). Blood pressure (BP) is higher in men than in premenopausal women at similar ages, but following menopause, BP increases in women to levels higher than men (Tremollieres et al., 1999; Harrison-Bernard and Raij, 2000). In the United States, nearly 38% of postmenopausal women use some form of hormone replacement therapy (HRT) (Keating et al., 1999). For many years, it has been thought that HRT provided women some level of protection against CVD, specifically with regard to myocardial infarction (Grodstein and Stampfer, 1995; Grodstein and Stampfer, 1998; Mendelsohn and Karas, 1999; Arkhrass et al., 2003). Recent clinical trials, including the Women's Health Initiative (WHI) and the Heart and Estrogenprogestin Replacement Study (HERS), have cast some doubt on the protective effects of estrogens in cardiovascular biology, as these studies failed to show a decreased risk for stroke in postmenopausal women treated with HRT (Simon et al., 2001). The WHI was stopped 2 years early due to an increase in risk of heart disease, strokes, and blood clots (Hulley et al., 2002; Rossouw et al., 2002). Questions remain, then, as to what specific harmful and/or protective effects estrogens have on the cardiovascular system. One endogenous hormonal system that is very important in cardiovascular regulation and is altered by estrogen is the reninangiotensin system. Specifically, estrogen has been shown to stimulate the production of angiotensinogen (Healy et al., 1992) and the angiotensin II type 2 receptor (AT 2 R) (Armando et al., 2002), and to

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58 inhibit the production of prorenin (Szilagyi et al., 1995), angiotensin converting enzyme (ACE) (Turner and Hooper, 2002), neprilysin (Pinto et al., 1999), aminopeptidase N (Seli et al., 2001), and the angiotensin II type 1 receptor (AT 1 R) (Nickenig et al., 2000). These changes in the RAS have the net effect of attenuating the pressor effects of the RAS, but potentiating the depressor effects. Collectively, these effects of estrogen on components of the reninangiotensin system should reduce blood pressure and offer some cardiovascular protective effects. Angiotensin-(1-7) (Ang-(1-7)) is an endogenous product of the reninangiotensin system. Production of this peptide hormone is achieved by three distinct pathways, including breakdown of angiotensin II (Ang II) by angiotensin converting enzyme 2 (ACE2) (Donoghue et al., 2000; Tipnis et al., 2000), and breakdown of angiotensin I (Ang I) both directly by various endopeptidases and also through the intermediate formation of angiotensin-(1-9) by ACE2 followed by digestion by ACE (Ferrario and Chappell, 2004). Ang-(1-7) is then degraded by ACE to form an inactive product, angiotensin-(1-5) (Ferrario and Chappell, 2004). At least three possible physiological actions of Ang-(1-7) have been suggested. First, it is possible that Ang-(1-7) acts as a partial agonist or antagonist of the AT 1 and/or AT 2 receptors. This is supported by evidence that Ang-(1-7) can block the effects of Ang II (Brosnihan et al., 1998; Chaves et al., 2000; Machado et al., 2001; Pinheiro et al., 2004), and that in some tissues Ang-(1-7) can signal through the AT 1 R (Gironacci et al., 1998; Caruso-Neves et al., 2000) and the AT 2 R (Santos et al., 2000; De Souza et al., 2004; Gironacci et al., 2004; Roks et al., 2004). Second, it is possible that Ang-(1-7) acts through its own receptor. The Mas receptor has been suggested as one possible Ang-(1-7)

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59 receptor, and various experiments have shown changes in Ang-(1-7) signaling in Mas-altered tissues and animals (Santos et al., 2003). Third, it has been suggested that Ang-(1-7) may act as an endogenous ACE inhibitor. This is supported by experiments which examined the catalytic efficiency of ACE in the presence of Ang-(1-7) (Iyer et al., 1998; Collister and Hendel, 2003; Tom et al., 2003). Recent work by Broshinhan and colleagues (Brosnihan et al., 2003; Neves et al., 2003) has shown that both the production and the efficacy of Ang-(1-7) are altered in pregnancy. Specifically, during pregnancy, plasma and urinary Ang-(1-7) increase significantly, and mesenteric artery preparations are much more reactive to Ang-(1-7). Because circulating estrogen increases during pregnancy, it is conceivable that estrogens may control production and/or sensitivity to Ang-(1-7). The current study was undertaken to further examine the effects of estrogen on vascular reactivity to Ang-(1-7). Aortic rings were used to examine the Ang-(1-7) effects upon the rat vasculature, as it has been shown that rat aorta relax to Ang-(1-7) in an endothelium-dependent manner (Le Tran and Forster, 1997). As estrogen significantly increases in pregnancy, it was hypothesized that estrogen would cause a significant potentiation of vascular responses to Ang-(1-7). Methods Animal Housing and Surgery Twelve female SpragueDawley rats (150 g) were housed doubly in shoebox style cages with free access to standard rat chow and tap water, and were maintained on a 12 : 12 h light : dark cycle, beginning at 7:00 AM. Twenty-one days before the aortic vascular reactivity experiment, all animals were ovariectomized while under anesthesia by xylazine/acepromazine/ketamine injectable cocktail. Subsets of animals (four each

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60 group) received estradiol replacement via subcutaneous pellet (0.05 or 25 mg). All procedures involving animals were approved by the University of Florida Institutional Animal Care and Use Committee. Reagents Subcutaneous estradiol pellets were obtained from IRA (Innovative Research of America, Sarasota, FL). All chemicals and peptides for aortic vascular reactivity experiments were obtained from Sigma Aldrich (St. Louis, MO). Estradiol ELISA kit was obtained from ALPCO, Inc. (Windham, NH). Aortic Vascular Reactivity Assay Rats were decapitated and aortas were isolated using blunt dissection. The thoracic aorta was cleaned of all adherent tissue and two adjacent 3.0 mm ring sections were obtained from each animal. These rings were then placed onto pressure transducer arms and submerged in tissue baths containing a modified Krebs solution (118 mM NaCl, 4.7 mM KCl, 1.2 mM KH 2 PO 4 1.2 mM MgSO 4 25.0 mM NaHCO 3 11.1 mM Dextrose, and 2.5 mM CaCl 2 ) with 95% O 2 / 5% CO 2 compressed gas mixture bubbling through. Pressure transducers were connected to a PowerLab signal transduction unit (ADInstruments), and data was continuously recorded on a PC computer, and further analyzed using the Chart program (ADInstruments). Rings were pretensioned to 4 g over 1 h, as preliminary studies determined this to provide the optimal lengthtension ratio (data not shown). A single dose of potassium chloride was applied (60 mM final concentration) to determine smooth muscle viability. After maximal contraction was reached, the tissue baths were purged and fresh modified Krebs solution was applied. Following relaxation back to the 4 g baseline, phenylephrine (10 7 M final concentration) was applied. After maximal contraction was reached, graded doses of Ang-(1-7) were

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61 applied (10 10 M through 10 6 M final concentrations), separated by 3 min intervals. Baths were then purged, and the contractile response to angiotensin II (10 7 M) was assessed. Finally, relaxation responses to acetylcholine and sodium nitroprusside were determined in a similar manner to those of Ang-(1-7). Rings were then removed from the tissue chambers and allowed to dry overnight in a dessicating oven. Dry ring masses were then measured, and contractile responses to each drug were normalized to the mass of the dried ring. Relaxation responses were determined by dividing maximal relaxation responses (tension change from maximal contraction to phenylephrine) by total active tension (total tension minus 4 g baseline). Plasma Steroid Measurement Trunk blood was collected from the animals at decapitation into EDTA-coated collection tubes and centrifuged for 5 min at 3000 g in a Triac centrifuge (Becton, Dickinson). Plasma was removed and placed into cryogenic vials for storage at 20 C until assayed. To determine plasma estradiol levels, an ELISA kit (ALPCO, sensitivity of 4.6 pg/mL) was utilized following the manufacturer's instructions, in duplicate. Statistics Body, uterine and heart masses, plasma estradiol, ring masses and contractile responses to potassium chloride and angiotensin II were analyzed using one-way ANOVA with significance set at P<0.05. Relaxation responses to angiotensin-(1-7) were analyzed using repeated measures ANOVA with significance set at P<0.05. All post-hoc tests were performed using Fisher's method with significance set at P<0.05.

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62 Results Estradiol Levels and Effects on Body, Heart and Uterine Growth Estrogen caused significant dose-related reductions in overall body mass at all doses, but no significant effects were observed on heart size with any level of estrogen replacement. Uterine mass was significantly increased in a dose-dependent manner with estrogen replacement. Plasma estradiol was significantly (P < 0.05) increased over ovariectomized levels in the 25 mg pellet group (Ovex, 8.90 0.34; 0.05 mg pellet, 16.28 3.99; 25 mg pellet, 95818.26 3515.76 pg/mL). Body Mass (g) Heart (g/kg) Uterus (g/kg) Ovariectomized 274.48.1 3.240.14 0.610.11 Estrogen, 0.05 mg 198.4.8 3.67.24 2.67.24 Estrogen, 25 mg 171.2.5 3.25.15 5.36.80 Table 5-1. Effects of steroid treatments on body parameters in female Sprague-Dawley rats. Estrogen exposure caused significant changes in body size and uterine mass at all doses. No significant changes were observed in heart size with any treatment. Data presented as means SE. (* indicates P<0.05 versus ovariectomized). Aortic Ring Properties 60 mM KCl (g) Phenylephrine (g) Ring mass (g) Ovariectomized 3.310.09 1.960.50 1.350.02 Estrogen, 0.05 mg 3.17.16 1.68.17 1.41.11 Estrogen, 25 mg 2.83.48 3.13.43 1.27.07 Table 5-2. Aortic ring properties. Aortas were sectioned into 3 mm length rings for vascular reactivity assay. Control contractions by potassium chloride (KCl) and phenylephrine were performed to determine treatment effects on contractile responses. Following the assay, rings were dried in a dessicator and then weighed to determine effects of treatment on protein content. Data presented as means SE. Vascular smooth muscle contractions to 60 mM potassium chloride and phenylephrine were similar across treatments, as were ring masses (Table 5-2). Estradiol treatment, however, resulted in significant alterations of the vascular smooth muscle

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63 response to stimulation of components of the reninangiotensin system. Aortic rings from animals treated with estrogen showed an estrogen-dose dependent attenuation of constrictor response to angiotensin II (Fig. 5-1). Fig. 5-1. Maximal contraction to angiotensin II in aortic rings from ovariectomized rats with estrogen replacement. Aortic rings were exposed to 10 7 M angiotensin II, and the maximal contractile response was recorded. These responses were normalized by dividing each ring's response by its own maximal response to 10 7 M phenylephrine. ( indicates P<0.05 versus ovariectomized). Data presented as means SE. Aortic Relaxation to Angiotensin-(1-7) Increasing concentrations of Ang-(1-7) in preconstricted vessels from ovariectomized rats resulted in a dose-dependent relaxation response that was significantly greater than matched time controls (data not shown). This response was similar in animals treated with a low dose of estradiol. Aortic rings harvested from animals maintained on a pharmacological estradiol treatment were completely unresponsive to any dilatory effects of Ang-(1-7) (Fig. 5-2), and even appeared to display a slight contractile response to lower concentrations of Ang-(1-7), although this effect was not significant. It is interesting to note that the maximum percent-relaxation of rings

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64 from ovariectomized animals at each dose is similar in magnitude to those of the mesenteric arteries from pregnant animals in the work from Neves et al. (2003). Fig. 5-2. Estradiol attenuates the aortic relaxation response to acute Ang-(1-7). Increasing doses of estrogen caused dose-dependent attenuations of vascular reactivity to angiotensin-(1-7). Repeated measures ANOVA reveals a significant effect of steroid treatment. Post-hoc analyses reveal no differences between ovariectomized and the 0.05 mg estradiol treatment, but 25 mg estradiol replacement caused a significant attenuation of the relaxation response when compared to either ovariectomized or 0.05 mg estradiol treatments. Data presented as means SE. In a subsequent study, the dependence of aortic relaxation responses to Ang-(1-7) upon the Mas receptor was determined. At doses of 10 6 M and lower, aortic relaxation responses in endothelium-intact rings were completely abolished by 10 5 M D-Ala 7 -Angiotensin-(1-7) (A779), an antagonist to the Mas receptor. At 10 5 M, however, Ang-(1-7) relaxation responses were minimally effected by this receptor antagonist (data not

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65 shown). These observations have been reported previously for rat aortic rings (Le Tran and Forster, 1997). Aortic Ring Integrity Endothelial integrity was examined by exposing rings pre-constricted with 10 7 M phenylephrine to 10 4 M acetylcholine. Rings from all animals relaxed more than 60% (with the exception of one animal, which was dropped from all analyses), and no statistical difference exists between groups (P=0.5219). Smooth muscle integrity and responsiveness to nitric oxide was also tested by exposing rings pre-constricted with 10 7 M phenylephrine to 10 5 M sodium nitroprusside. All rings relaxed between 80% and 100%, with no differences between groups (P=0.8397). Discussion This study provides some significant insight into the effects of estradiol on cardiovascular physiology. Although it was determined that estrogen does appear to have effects on the vasodilatory responses of the aorta to Ang-(1-7), the directions of responses were unexpected. Estrogen was, in fact, detrimental to the vasodilatory responses to Ang-(1-7). Estrogen caused the expected dose-dependent attenuation of body weight, and a potentiation of uterine mass (Table 5-1). These effects are well documented in the literature (Harris et al., 2002), as is the dose-dependent attenuation of aortic ring constrictor responses to Ang II (Cheng and Gruetter, 1992). These effects of estrogen on the response to Ang II have been attributed to decreased transcription and activity of the AT 1 R (Schunkert et al., 1997; Nickenig et al., 1998; Fisher et al., 2002). Other responses to Ang II, such as the dipsogenic or in vivo pressor response are also attenuated with

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66 elevated levels of estrogen (Jonklaas and Buggy, 1984; Fregly, et al, 1985). It is possible that Ang-(1-7) works through interactions with the AT 1 and/or AT 2 receptors (Jaiswal et al., 1992; Gironacci et al., 1998; Caruso-Neves et al., 2000; Santos et al., 2000; Roks et al., 2004), and that altered levels of these receptors may account for the changes in responsiveness to Ang-(1-7) in these aortic rings. Actions of Ang-(1-7) through the AT 1 receptor seem less likely, in that the 0.05 mg estradiol pellet group displayed a significantly decreased constrictor response to Ang II (Figure 5-1), but its relaxation response to Ang-(1-7) was not different from ovariectomized (Figure 5-2). Chronic estrogen treatment did not appear to alter the in vitro vascular contractile response to a depolarizing agent (potassium chloride), or a receptor-mediated vasoconstrictor (phenylephrine). The lack of effect on vascular ring mass also suggests no significant effect of steroid treatment on aortic vessel musculature. Likewise, the similar relaxation responses of the vessels to sodium nitroprusside demonstrate that the vascular smooth muscle response to nitric oxide was not altered by estrogen treatment. Maximal responses to acetylcholine suggest that endothelial function was not compromised with estrogen treatment. Utilization of lower doses of acetylcholine may have been able to distinguish if the sensitivity to acetylcholine is altered by estrogen treatment. Other literature reports would suggest that this may be the cause, as estradiol increases endothelial and calcium-dependent nitric oxide synthase (Saito, et al, 1999; Morschl et al., 2000). Estradiol, however, does appear to alter the vascular response to components of the reninangiotensin system. Vasodilatory responses to Ang-(1-7) were hypothesized to increase with estrogen exposure, as the circulating level of estradiol significantly increases along with Ang-(1-7)

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67 levels and sensitivity during pregnancy (Neves et al., 2003). The results of the current study suggest that in the aorta, increased estrogen appears to have a dose-dependent inhibitory effect on the vasodilatory response to Ang-(1-7) (Figure 5-2). These results, obtained from aortic rings, are in contrast to the recent findings of Neves et al. (2004), who found that estrogen mediates increased vasodilatory responses to angiotensin-(1-7) in mesenteric artery preparations of female rats. These differential effects on the aorta and the mesentery may suggest a regional specificity of the effects of estrogen, which has been suggested for other estrogen-mediated physiological effects (Zubair et al., 2005). Further, methodological differences between this study and those of Brosnihan et al. may contribute to the conflicting results. Brosnihan's group utilizes the peptide endothelin-1 to preconstrict their vessels before relaxation with Ang-(1-7), whereas we have used phenylephrine. Additionally, their measurements of vascular reactivity in mesenteric arteries are performed using a pressurized myograph system, whereas our measurements are made using an isometric wire myograph system. Finally, our experiments utilized a pharmacological dose of estradiol, whereas their group has used more physiological doses. Differential effects between vascular beds may contribute to the ongoing debate regarding the cardio-protective effects of estrogen. Further studies in this area, examining the different mechanisms of action of estrogen in the aorta versus the mesenteric arteries may help to discover a mesentery-selective estrogen analog that would be able to produce the cardio-protective effects of estrogen, but not cause the detrimental aorta-specific effects.

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68 One potential shortfall to this design was the absence of an intact control. This particular group was not utilized as we anticipated that the normal estrous cycle could impact the results and confound data interpretation. An intact control could be used in future studies, provided the stage of estrous cycle was determined prior to harvesting the vascular tissue. Another confounding effect with the estrogen treatment paradigm incorporated was that a normally cycling estrogen was replaced with a chronic exposure. This chronic exposure is of interest, despite its difference from normal physiology, as it more closely approximates the estrogen dosing paradigm of women on monthly estrogen pills. Further, this chronic exposure mimics the chronically increased levels of estradiol observed during pregnancy. The doses of estradiol utilized were recommended by IRA to mimic levels achieved during pregnancy. At the completion of the physiological measurements, the results of the estradiol ELISA assay revealed much higher levels than expected, and future studies will evaluate lower levels of estradiol that more closely mimic those observed during normal pregnancy in the rat. The fact that such high estrogen levels were achieved in the current study may suggest that nonspecific effects of estrogen are mediating the observed attenuation of Ang-(1-7) responses, however the responses observed to angiotensin II are consistent with the literature (Cheng and Gruetter, 1992). In a preliminary study with human coronary artery endothelial cells, we observed that estrogen treatment significantly reduced the transcription of the Mas receptor mRNA when examined using realtime RT-PCR. This observation would further support our current demonstration that estrogen decreased relaxational responses to Ang-(1-7) in rat aorta. These observations were made in cells at a relatively late passage number, and this

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69 preliminary study will have to be repeated at early passages before a definitive effect of estrogen on the Mas receptor could be validated. In conclusion, we have found that angiotensin-(1-7) causes a dose-dependent relaxation of the female rat aorta, and that this relaxation response is attenuated by 17--estradiol. This aortic relaxation has previously been shown to be endotheliumand Mas receptor-dependent (Le Tran and Forster, 1997). These results are in contrast to findings from other studies on the effects of estradiol on the relaxation response to angiotensin-(1-7), although as these other studies have been performed in mesenteric arteries, a regional specificity of action of estradiol is suggested. Future studies will examine this regional (vessel) specificity, the doseresponse relationship of estradiol with the Mas receptor, and the influence of 17--estradiol and other estrogenic compounds on Mas receptor expression.

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CHAPTER 6 PRELIMINARY CELL CULTURE EXPERIMENTS Abstract Angiotensin-(1-7) (Ang-(1-7)) has been previously demonstrated to provide protection from cardiac remodeling in vivo. Here, the role of Ang-(1-7) in mediating the production of collagen by cultured neonatal rat cardiac fibroblasts following stimulation by both myocyte preconditioned media and by hypoxia-reperfusion injury was examined. It was determined that Ang-(1-7) inhibits myocyte-derived pro-fibrotic stimuli, possibly including transforming growth factor(TGF ). Further, it was determined that lentiviral delivery of the angiotensin converting enzyme 2 gene to fibroblasts prevented hypoxia-induced collagen and TGF production. Together, these results indicate protective roles for ACE2 and Ang-(1-7) in vitro, and may suggest future therapeutic avenues for myocardial injury. Introduction The preceding chapters have explored the beneficial effects of angiotensin-(1-7) (Ang-(1-7)) in vivo. While these studies provide an integrated-systems verification of the cardioprotective role of Ang-(1-7), they fail to examine the tissue-level mechanism(s) of these effects. The two major components of cardiac remodeling, myocyte hypertrophy and cardiac fibrosis, are primarily mediated by (and between) cardiac myocytes and fibroblasts. When these cells are cultured independently, cardiac myocyte hypertrophy can be triggered by treatment with media formerly on cardiac fibroblasts (Iwata et al., 70

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71 2005), and collagen production by cardiac fibroblasts can be potentiated by co-culture with cardiac myocytes (Sarkar et al., 2004). Together, these findings indicate that cross-talk between cardiac cell types is an important factor in the regulation of normal tissue-level regulation. Iwata et al. (2005) recently demonstrated that cultured adult rat cardiac fibroblasts show a blunted collagen-production response to various stimuli when the culture media is supplemented with Ang-(1-7). Tallant et al. (2005) also recently demonstrated that Ang-(1-7) inhibits cardiac myocyte growth in vitro through the Mas receptor. These investigators, though, did not examine the effect of Ang-(1-7) on myocyte-fibroblast cross-talk demonstrated by Sarkar et al. (2004). Therefore, the effects of Ang-(1-7) on myocyte-mediated stimulation of collagen production by cardiac fibroblasts were examined. In addition to myocyte-derived pro-fibrotic stimuli, certain pathologies can lead to aberrant activity of cardiac fibroblasts. One pathophysiological state which leads to excessive collagen production by cardiac fibroblasts is hypoxia, which can result from acute or chronic myocardial ischemia (Agocha et al., 1997). Averill et al. (2003) recently determined that Ang-(1-7) is conspicuously absent in the infarct zone of surgically-induced myocardial ischemia in Lewis rats, although intact myocardium surrounding the infarct zone stains with normal levels of the peptide. Preliminary studies from our lab have also determined that cardiac myocytes have a moderate basal activity of the angiotensin converting enzyme 2 (ACE2) enzyme, which is the major source of Ang-(1-7) in the heart (Zisman et al., 2003). Cultured cardiac fibroblasts, on the other hand, do not have detectable ACE2 activity. Together, these

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72 findings next led us to the hypothesis that the absence of ACE2 in an infarct zone is permissive for aberrant extracellular matrix production by fibroblasts that are able to survive in the hypoxic local environment. Methods Isolation and Culturing of Neonatal Rat Cardiac Myocytes Neonatal rat cardiac myocytes were isolated using a commercially available kit (Worthington Biochemical Corp., Lakewood, NJ). Briefly, hearts from 5-day old rats were removed by blunt dissection, then digested with trypsin and collagenase. Myocytes were grown in DMEM/F-12 media supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum (FBS) in a 5% CO 2 / 95% air humidified incubator. Cells were used for experiments within one week. Isolation and Culturing of Neonatal Rat Cardiac Fibroblasts Neonatal rat cardiac fibroblasts were isolated using a protocol adapted from Zhang et al. (2001). Five day old Sprague-Dawley rat pups were deeply anesthetized by inhalation of halothane. Following heart excision and mincing, heart tissue was digested for 2 hours with 1% collagenase (Worthington Biochemical Corp, Lakewood, NJ), spun down at 3000 rpm for 2 min, then digested for 1 hour with 1% trypsin (Worthington Biochemical Corp, Lakewood, NJ). Cells were then spun down for 2 min at 3000 rpm, and reconstituted in media and plated in a T-75 flask. Cells were maintained in DMEM supplemented with 1% penicillin/streptomycin, 50 g/mL ascorbic acid, and 10% fetal bovine serum. Cells were split by standard trypsin procedure, and were used within the first three passages. Cells were seeded at 3000 cells/cm 2

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73 Myocyte-Fibroblast Cross-Talk Paradigm Cultured cardiac myocytes were grown for three to five days before serum-starvation for 24 hours. Following this starvation, cells were treated with 10% FBS-supplemented media with or without angiotensin-(1-7) at 10 -7 M for 24 hours. At the end of this incubation, medias were transferred to confluent cultured fibroblasts, and some (previously 10% FBS-only) media was supplemented with angiotensin-(1-7) at this time, as indicated in Figure 6-1A. Collagen was sampled after 12 hours of incubation on the cardiac fibroblasts. An additional set of cardiac myocytes was treated in a parallel fashion, but following the 24 hour incubation with or without angiotensin-(1-7), RNA from these cells was isolated using a commercially available kit from Ambion. These RNA samples were then used to measure transcript for transforming growth factor-1. Real-time RT-PCR Real-time RT-PCR was used to determine levels of transforming growth factor-1 mRNA. Primers and probe for the gene, along with all reagents for the reaction and primers and probe for the loading control (the 18S gene), were obtained from Applied Biosystems. One-step RT-PCR reactions were carried out using the manufacturers instructions in a Prism 7000 thermocycler. Lentiviral ACE2 Gene Transfer Lentivirus harboring the gene for ACE2 under transcriptional control of the elongation factor-1 promoter (EF1) was obtained from the Raizada laboratory. This batch of virus was determined to exhibit an infectious titer of 6.68x10 9 ifu/mL. Cultured cardiac fibroblasts were grown to approximately 80% confluence before infection. The virus was diluted in serum-free media for infection. Residual media was

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74 removed from the fibroblasts, and the minimal volume of virus-containing media (200 L for a single well of a 24-well plate) was placed onto the cells. After four hours, 10% FBS media was added to the cells to bring the media volume up to normal (500 L total for a single well of a 24-well plate). Hypoxia-Reperfusion Paradigm Upon confluence, cardiac fibroblasts were serum-starved for 48 hours. Medias were then replaced with 10% FBS media immediately before cells were transferred to a hypoxia chamber (or a normoxia control chamber). Cells in the hypoxia chamber were exposed to a continual flow of 95% N 2 / 5% CO 2 gas for one hour. After one hour, cells were returned to the normoxia condition for 12 or 24 hour hours before assays for ACE2 activity, collagen or transforming growth factor-1 were performed. ACE2 Activity ACE2 activity was determined in cardiac fibroblasts as previously described (Huentelman et al., 2004). Briefly, cells were scraped into a buffer consisting of 1 M NaCl, 75 mM Tris, 0.5 mM ZnCl 2 pH 7.5. Cells were then sonicated and protein content was determined by a standard Bradford Assay. ACE2 activity was then determined by kinetic assay using the Fluorogenic Peptide Substrate VI (FPS VI, 7Mca-Y-V-A-D-A-P-K(Dnp)-OH, R&D Systems, Minneapolis, MN). Samples containing ACE2 enzyme (up to 50 l) were incubated with 100 M FPS VI, 10 M captopril (to inhibit ACE activity), and reaction buffer (1 M NaCl, 75 mM Tris, 0.5 mM ZnCl 2 pH 7.5) in a final reaction volume of 100 l at 37 C. In the assay, ACE2 removes the C-terminal dinitrophenyl moiety that quenches the inherent fluorescence of the 7-methoxycoumain group resulting in an increase in fluorescence in the presence of ACE2 activity at excitation and emission spectra of 328 and 392 nm, respectively.

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75 Soluble Collagen Soluble collagen was determined using a commercially available Sircol Soluble Collagen kit (Accurate Chemical) according to manufacturers instructions. Transforming Growth Factor-1 Total transforming growth factor-1 protein was determined by ELISA (R&D Systems) according to manufacturers instructions. Results Cardiac Myocyte-Fibroblast Cross-Talk is Inhibited by Ang-(1-7) at the Myocyte In this first series of experiments, cardiac myocytes were serum starved for twenty-four hours before treatment with normal (10% FBS) media with or without Ang-(1-7) supplementation. After twenty-four hours of incubation, this myocyte-preconditioned media was transferred to cardiac fibroblasts. A preliminary study determined that myocyte-preconditioned media caused increases in fibroblast collagen production only if the myocytes were stimulated with FBS (data not shown). In this experiment (summarized in Figure 6-1A), Ang-(1-7) supplementation caused a significant decrease in collagen production by the cardiac fibroblasts if the peptide was allowed to act at the cardiac myocyte, but had no effect if only the fibroblast was treated with Ang-(1-7) (Figure 6-1B). In a subsequent set of cultured cells, myocytes were treated with serum-free, 10% FBS, or 10% FBS media supplemented with Ang-(1-7). After twenty-four hours of incubation, cells were scraped from their growth chambers and total RNA was isolated. One-step real-time RT-PCR measurement of mRNA for TGF 1 demonstrated that while 10% FBS results in a significant increase in TGF 1 mRNA, Ang-(1-7) co-incubation significantly attenuates this effect (Figure 6-2).

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76 Figure 6-1. Angiotensin-(1-7) attenuates myocyte-derived pro-fibrotic signaling. (A) Neonatal rat cardiac myocytes were incubated 24 hours with 10% fetal bovine serum-supplemented media with or without 10 -7 M Ang-(1-7). Medias were then transferred to neonatal rat cardiac fibroblasts, and a subset of fibroblasts receiving Ang-(1-7)-free media were spiked with Ang-(1-7) at time of transfer. (B) Significant attenuation of collagen production (P=0.0320 vs. 10% FBS) was only achieved if Ang-(1-7) was allowed to act at the cardiac myocyte. Figure 6-2. Angiotensin-(1-7) attenuates expression of TGF 1 mRNA in cardiac myocytes. Neonatal rat cardiac myocytes were stimulated for 24 hours with serum-free, 10% serum, or 10% serum supplemented with 10 -5 M Ang-(1-7). Real-time RT-PCR was used to measure mRNA for the TGF 1 cytokine. Serum significantly increased transcription of TGF 1 (P=0.0009), and Ang-(1-7) significantly attenuated this effect (P=0.0282).

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77 Lentiviral Delivery of EF1-ACE2 Results in Increased ACE2 Activity and Protection from Hypoxia Preliminary studies determined that while cultured cardiac myocytes do exhibit a moderate basal level of ACE2 activity, cultured cardiac fibroblasts have no detectable basal activity (data not shown). Lentiviral delivery of the murine ACE2 gene under transcriptional control of the elongation factor-1 (EF1) promoter, though, results in a viral dose-dependent increase in ACE2 activity in cultured cardiac fibroblasts (Figure 6-3A). Figure 6-3. Lentiviral delivery of ACE2 attenuates the collegen production response to hypoxia in cultured cardiac fibroblasts. (A) Lentiviral delivery of the EF1-ACE2 construct results in a dose-dependent increase in ACE2 activity in cultured cardiac fibroblasts. (B) Lentiviral delivery of ACE2 attenuates the collagen production response to hypoxia in cultured cardiac fibroblasts. Fibroblasts, previously infected with various doses of lentivirus harboring the EF1-ACE2 construct, were exposed to 1 hour of hypoxia, before being returned to normoxic conditions for 12 hours. Two-way ANOVA indicates significant effects of hypoxia (P<0.0001) and virus (P<0.0001) on collagen production, and a significant interaction between these factors (P=0.0111). The viral dose of Low indicates 2.36x10 5 ifu/cm 2 Medium indicates 5.9x10 5 ifu/cm 2 and High indicates 11.82x10 5 ifu/cm 2

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78 Cardiac fibroblasts exhibit a strong collagen-production response to acute hypoxia followed by normoxia. Lentiviral delivery of the murine ACE2 gene caused a significant attenuation of this response to hypoxia (Figure 6-3B). ACE2 Gene Delivery Attenuates Hypoxia-Reperfusion Induction of Transforming Growth Factor-1 In addition to attenuating the collagen-production response to hypoxia, lentiviral delivery of murine ACE2 significantly attenuated both basal and hypoxia-stimulated media levels of TGF 1 protein (Figure 6-4). 10001050110011501200125013001350NoneLowMidHighViral doseTGF-beta (pg/mL) Normoxia Hypoxia Figure 6-4. Lentiviral delivery of ACE2 attenuates hypoxia-induced expression of transforming growth factor-1 protein. Two-way ANOVA indicates significant effects of hypoxia (P=0.0019) and virus (P=0.0002) on TGF-1 protein, but no interaction between these factors (P=0.5405). Viral doses are the same as above. Discussion In this preliminary set of experiments utilizing cultured neonatal rat cardiac myocytes and fibroblasts, we determined that Ang-(1-7) inhibits myocyte-fibroblast cross-talk, and that this inhibition may be due to decreased transcription of the myocyte-derived pro-fibrotic cytokine TGF

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79 In addition to examining the effects of Ang-(1-7) on myocyte-fibroblast cross-talk, the protective effects of ACE2 in fibroblasts treated with hypoxia were also examined. Here, it was determined that while cultured neonatal rat cardiac fibroblasts show no detectable basal ACE2 activity, lentiviral delivery of the murine ACE2 gene dose-dependently increases ACE2 activity. This increased activity prevents hypoxia-reperfusion induced collagen production by the fibroblasts, and this may be mediated by decreased TGF protein. Finally, it was demonstrated that increased ACE2 activity, even if temporally limited to the onset of hypoxia, is sufficient in cultured fibroblasts to provide some anti-fibrotic protection. The determination that ACE2 protects fibroblasts from hypoxia-induced metabolic changes may lead to a future therapy for myocardial infarction. During the hypoxic phase of a myocardial infarction, cardiac myocytes die and formerly quiescent fibroblasts are stimulated by the hypoxic environment to produce large quantities of collagens I and III, and these cells morphologically change into myofibroblasts (Stenmark et al., 2002). The formation of myofibroblasts is vital, as this is a necessary step in wound healing. Death of cardiac myocytes is also important, as these cells represent the endogenous cardiac source of ACE2 and Ang-(1-7) (Averill et al., 2003; Zisman et al., 2003). Future studies should be directed at determining the mechanism of ACE2-mediated protection in cultured fibroblasts. Although Iwata et al. (2005) determined that Ang-(1-7) can act upon cultured fibroblasts, our cross-talk experiment (Figure 6-1) suggests that Ang-(1-7) did not have direct effects upon cultured fibroblasts. In light of these conflicting findings, one can not simply assume that ACE2 mediates its effects in cultured fibroblasts through Ang-(1-7). In addition to increasing Ang-(1-7) levels, which

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80 may or may not be able to act upon fibroblasts, it is possible that ACE2 may work in this paradigm solely through degradation of Ang II. Agocha et al. (1997) demonstrated that during hypoxia, Ang II is a significantly more potent stimulus for cardiac fibroblasts, and decreased Ang II levels would presumably contribute to decreased stimulation of the fibroblasts. Lijnen et al. (2006) recently showed that Ang II mediates its actions in cultured cardiac fibroblasts through NAD(P)H oxidase, and Byrne et al. (2003) published a suggestive review article regarding the role of reactive oxygen species (which are produced by NAD(P)H oxidase) in the development and progression of heart failure. Okada et al. (2005) showed that viral delivery of a gene encoding a soluble TGF II receptor (thereby artificially decreasing available TGF cytokine in the myocardium) resulted in significant protection from cardiac remodeling, function loss, and apoptosis of the reparative myofibroblasts. Together with our current findings regarding the effects of Ang-(1-7) and ACE2 on TGF we conclude that Ang-(1-7) and ACE2 may mediate their cardioprotective effects through inhibition of TGF and that loss of myocytes (and therefore the source of ACE2 and Ang-(1-7)) may permit increased TGF signaling. Collectively, these preliminary studies in vitro suggest important roles for both Ang-(1-7) and ACE2. In particular, it was determined that Ang-(1-7) likely mediates myocyte-fibroblast cross-talk through actions on the cardiac myocyte, and that it may be the loss of myocytes (which are the endogenous cardiac source of ACE2, and therefore Ang-(1-7)) that permits increased TGF signaling and overactivity of cardiac fibroblasts in an infarct zone. These findings lead to the conclusion that ACE2, and possibly Ang-(1-7), may represent an as-yet unexplored therapy for myocardial infarction. Ongoing

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81 studies are currently examining the utility of viral and pharmacological stimulation of ACE2 in vivo during surgically-induced infarction. Future studies should also repeat and expand upon the examination of Ang-(1-7) modulation of myocyte-fibroblast cross-talk, both to confirm these results, and to determine whether Ang-(1-7) can act directly upon cardiac fibroblasts. Interpretation of the current findings (Figure 6-1) is hindered by the experimental design, as Ang-(1-7) was delivered in a single bolus, and therefore the rate of degradation of the peptide in cultures of cardiac fibroblasts may mask the effects of Ang-(1-7) upon these cells. Ongoing studies are designed to utilize lentiviral-mediated delivery of the gene for an Ang-(1-7) fusion protein (Santos et al., 2004) to provide a constant source of the peptide, and thereby overcome this design limitation.

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CHAPTER 7 GENERAL DISCUSSION Summary and Conclusions The studies presented here aimed to examine the cardioprotective actions of angiotensin-(1-7) (Ang-(1-7)). Lentiviral-mediated increases in expression of angiotensin converting enzyme 2 (ACE2) resulted in significant protection from cardiac remodeling during chronic infusion of angiotensin II (Ang II) in vivo. Here, it was demonstrated that chronic infusion of Ang-(1-7) in rats resulted in the same cardioprotective effects during chronic Ang II infusion, thus suggesting that Ang-(1-7) mediates the anti-trophic, anti-fibrotic actions of ACE2 on the myocardium. These findings lead us to several important conclusions. First, this provides evidence that the predominant mechanism of ACE2 cardioprotection during hypertension is to produce Ang-(1-7) rather than simply degrade Ang II or metabolize ACE2s other endogenous ligands, including apelin-13 and -32, some of the kinin metabolites (with the exception of bradykinin), neurotensin, and opioid peptides such as dynorphin (Vickers et al., 2002). Second, this establishes Ang-(1-7) as an active cardioprotective hormone. The lack of effect of Ang-(1-7) on blood pressure is also important, as this indicates a direct effect of Ang-(1-7) on cardiac tissue, and establishes that any changes in blood pressure associated with ACE2 overexpression may be a by-product of the degradation of Ang II. Next, it was determined that the cardioprotective actions of Ang-(1-7) are not limited to the Ang II-infusion model of hypertension and cardiac remodeling. In the deoxycorticosterone acetate (DOCA)-salt hypertensive model, it was observed that 82

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83 chronic infusion of Ang-(1-7) attenuated both interstitial and perivascular fibrosis, without effect on cardiac hypertrophy. The anti-fibrotic actions of Ang-(1-7) in the DOCA-salt (mineralocorticoid-dependent) model are potent but independent of effect upon hypertrophy and blood pressure, which leads to two important conclusions. First, these findings further support the notion that the signaling mechanisms of cardiac hypertrophy and fibrosis are independent. Second, Ang-(1-7) must either only oppose the effects of mineralocorticoids at the cardiac fibroblast, or act at the cardiac myocyte to alter mineralocorticoid-induced paracrine pro-fibrotic signals but not intracellular (or autocrine) hypertrophic signals. Use of the normotensive isoproterenol-injection model of cardiac remodeling further demonstrated that the cardioprotective effects of Ang-(1-7) are not mediated by decreased blood pressure. In the isoproterenol model, it was observed that infusion of Ang-(1-7) prevents cardiac hypertrophy, without effect on fibrosis. These findings, while in contrast to those of the DOCA-salt model, establish that the effects of Ang-(1-7) are model-dependent, and that Ang-(1-7) may have diverse effects across the cell types within cardiac tissue. Protective effects in this model further establish that Ang-(1-7) has an active role in the myocardium, and that this hormone is not a passive inhibitor of the RAS. A follow-up study on the effect of ovarian hormones on isoproterenol-induced cardiac remodeling indicated that ovarian hormones potentiate the hypertrophic response to isoproterenol injection. Next, it was determined that estrogen attenuates cardiovascular tissue sensitivity to Ang-(1-7). Aortic rings from ovariectomized rats showed a dose-dependent relaxation response to Ang-(1-7), while rings from ovariectomized rats with 17--estradiol

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84 replacement showed an estrogen dose-dependent attenuation of this relaxation response. Other hormones, including progesterone and testosterone were examined, but these hormones showed little effect on vascular reactivity to Ang-(1-7) (data not shown). Finally, the roles of ACE2 and Ang-(1-7) in cultured cardiac fibroblasts and myocytes were examined to begin to dissect their mechanism(s) of action in these cell types. It was determined that Ang-(1-7) inhibits myocyte-derived pro-fibrotic signaling, which may be mediated by decreased transforming growth factor(TGF ) gene transcription. Further, it was determined that ACE2 gene delivery to cultured cardiac fibroblasts, which have no detectable basal ACE2 activity, causes dose-dependent increases in ACE2 activity and provides protection from hypoxia-induced collagen and TGF production. Together with the literature, these findings suggest that within an infarct zone, where myocytes (the endogenous source of ACE2 in myocardium) are absent, lack of ACE2 (and possibly Ang-(1-7)) may be permissive for overactivity of cardiac fibroblasts. This overactivity would result in severe cardiac remodeling, and loss of function, eventually resulting in heart failure. Pitfalls and Shortcomings Few studies are ever perfect in design and execution, and the studies presented here are no exception. There are several design flaws and shortcomings in these studies, which should be noted so as not to repeat these errors in future studies. In chapter 2, chronic infusion of Ang II resulted in increased blood pressure, cardiac hypertrophy, and interstitial fibrosis. Chronic infusion of Ang-(1-7) had no effect on the increased blood pressure, but significantly attenuated the hypertrophy and fibrosis. In this study, a single dose of Ang II, and a single dose of Ang-(1-7) were utilized. The dose of Ang II (100 ng/kg/min) was selected based on a long series of previous and

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85 preliminary studies within the Katovich lab to provide a moderate increase in blood pressure and moderate cardiac remodeling over four weeks, and the dose of Ang-(1-7) was selected to represent a nearly-equimolar dose to that of the Ang II. The four-week timeframe for the study was selected for practical limitations, namely the maximum time afforded by the commercially available osmotic minipumps that were used to infuse both hormones. Timeframes longer than four weeks would require removal of original pumps and implantation of secondary pumps for each hormone after every four weeks of treatment, and it was decided that additional anesthesia and subcutaneous surgery was an unnecessary risk for the animals. Presumably, a longer treatment timecourse would lead to overt heart failure in the Ang II-infused animals, as direct effects on cardiac remodeling and indirect effects through increased vascular resistance and peripheral blood pressure would result in gross morphological changes of the myocardium. An additional problem with this first study was the use of Massons Trichrome to stain the extracellular matrix of the myocardium. While this stain provides beautiful pictures, it is not particularly well suited for the specific analysis of collagen deposition in the matrix. This treatment results in red-stained myocytes, green-stained endothelial cells, and blue-stained matrix. To analyze matrix deposition in the heart with two different dominant stain colors on each slide (red and blue), pictures of the slides must be analyzed by splitting the RGB data into three separate files (using the ImageJ program from NIH Rasband, 2005). These three individual files represent a pixel-by-pixel color-saturation matrix for each of the primary (red, green, blue) values. Sections of the left ventricle wall are then outlined in the red and blue files, and the average color saturation for red and for blue are then determined for each animals heart-slice picture.

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86 The blue saturation is then normalized to the red saturation within each animal to control for batch-to-batch differences in the stain. Together, this process, along with the analysis of the entire left ventricle wall (as described in chapter 2), artificially compresses the magnitude of matrix deposition change with treatments, as is evidenced by only an approximate 5% increase with chronic Ang II infusion (Figure 2-3). This problem is again reflected in chapters 3 (mineralocorticoid model) and 4 (isoproterenol model). This same extracellular matrix deposition, if analyzed using a different stain and analyzed through a percent-area method, may result in more impressive numerical changes with treatments. Regardless of the magnitude of the numerical values, though, the changes which are visually obvious in the pictures (especially in chapter 3) are significantly different even when analyzed by stringent non-parametric analysis when analyzed this way. Heart slices from animals in each treatment group (from at least one future experiment) should be stained by both Massons Trichrome and another more specific stain, such as Sirius Red, to confirm the reliability of Massons Trichrome and to determine the relationship between numerical results (in other words, to determine what a 5% difference when Massons Trichrome is used relates to if Sirius Red had been used). In chapter 3, where deoxycorticosterone acetate (DOCA) was delivered by subcutaneous pellet to uninephrectomized animals, similar design flaws existed. Again, a single dose of DOCA was administered, and a single dose of Ang-(1-7) was utilized. Once again, preliminary studies in the Katovich lab led to the use of the DOCA dose. The Ang-(1-7) dose of 100 ng/kg/min was used because of successful results in the Ang II study (chapter 2). Once again, four weeks was used as the timecourse, as this represented the time limit imposed by the use of osmotic minipumps. This study was

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87 also plagued by the numerically-unimpressive, yet statistically significant differences in interstitial extracellular matrix changes with DOCA treatment. Future studies certainly should be done with either the Ang II or DOCA models to determine the dose-response relationship of Ang-(1-7) and its anti-remodeling effects. Chapter 4 examined the effects of chronic Ang-(1-7) infusion on isoproterenol-induced cardiac remodeling. Again, similar flaws in design were present as were mentioned above. Several preliminary studies were carried out to determine the appropriate dose of isoproterenol and timecourse, as the literature is hyper-variable with regard to the specifics of this model. It was determined that while daily subcutaneous injections of 1 mg/kg isoproterenol for one week resulted in moderate cardiac hypertrophy in males, the variability between animals would require a very large sample size to see statistical differences. Increasing the timecourse of treatment to two weeks resulted in only slightly larger hypertrophy, and thus it was decided to increase the dose of isoproterenol to 2 mg/kg daily. This increase in dosage resulted in a much larger cardiac hypertrophy, but also greatly increased spontaneous deaths of animals. Preliminary experiments led to the conclusion that in male Sprague-Dawley rats, 2 mg/kg daily for 14 days was required to see large, reliable changes in cardiac hypertrophy. Further, to decrease the spontaneous death rate of these animals, it was determined that the daily injections of isoproterenol should be administered during the middle of the light cycle of the animals. Animals should also be allowed to recover for at least one day before the first injection of isoproterenol is administered, as the precipitous acute drop in blood pressure is potentiated by anesthesia and minipump delivery of blood pressure lowering compounds.

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88 In chapter 5, the effect of estrogen status upon aortic vascular reactivity to Ang-(1-7) was investigated. Several design flaws plagued this study, although their effects probably would not change the conclusions of the study; at most these flaws likely only limit the implications of the study. In particular, the lack of an intact (not ovariectomized) treatment group and the use of a super-physiological dose of estradiol in the high estradiol group detract from the utility of the data. An intact control group would confirm the normal aortic response to graded doses of Ang-(1-7). The super-physiological dose of estradiol in the high estradiol treatment group was the result of an error by the commercial supplier of the pellets, as we requested a low dose which should mimic a high-cycle level of estradiol, and a high dose which would mimic circulating estradiol levels in a rat near the end of pregnancy. The estradiol ELISA at the end of the study determined that the estradiol dose in the high group was approximately 1000-fold higher than desired. While this dose of estradiol was obviously too high to mimic end-pregnancy levels, it should certainly indicate the ceiling of the hypothetical estradiol / attenuation-of-Ang-(1-7)-effect dose-response curve. As this study was actually carried out first in the series of experiments in this dissertation, errors were made in the execution of the experiment despite an excellent design. While analysis of ring responses to acetylcholine and sodium nitroprusside were included in the design (to determine effects of estrogen modulation on endothelial and smooth muscle integrity, respectively), these determinations should have been done at multiple doses of each compound to more fully characterize the dose-response relationships instead of the single (maximum) doses which were administered in the current study. As executed, it is impossible to fully know how estradiol treatments may

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89 (or may not) have affected the low-dose effects of these compounds, and thus the true basal changes due to estradiol treatment. Finally, this vascular reactivity experiment was carried out using aortic rings, which limits interpretation and application of the data obtained. As was described in the discussion section of chapter 5, other groups have examined estrogen effects upon vascular reactivity to Ang-(1-7) in mesenteric artery preparations and have determined the exact opposite effect of estrogens upon Ang-(1-7) vascular reactivity. Clearly, as the goal of this work was to characterize the cardiac effects of estrogen on Ang-(1-7) actions, future studies should be performed using cardiac tissue. This could involve the vascular reactivity endpoint, but the vessel must be coronary arteries. This could also involve alternate endpoints, including myocyte hypertrophy, or interstitial or perivascular collagen deposition. Ongoing studies are currently investigating the effect of estrogen status on myocyte-derived pro-fibrotic factors in cultured cardiac myocytes. Chapter 6 focused on cultured cardiac cells to begin to tease apart tissue-level effects of ACE2 and Ang-(1-7). The first and most obvious limitation of these studies revolves around the use of a cultured tissue system, as no in vitro system can ever fully emulate in vivo physiology. Cultures of cardiac cells are particularly plagued by a lack of constant mechanical and electrical stimulation that is present in vivo, and is established to affect myocyte morphology (Kira et al., 1994). Cardiac myocyte cultures are further troublesome due to problems with cell type purity of cultures. While the isolation procedure for cardiac fibroblasts is very selective, and other cell types die due to harsh isolation conditions, cardiac myocytes are fragile cells which require serial selection steps. Myocytes do not readily attach to plastic culture dishes, and they require constant

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90 oxygenation. Because of these weak isolation conditions, cardiac fibroblasts, smooth muscle, and endothelial cells typically survive the isolation procedure, and thus contaminate myocyte cultures. Therefore, cultures of myocytes are typically a mixture of many cell types. Before the preliminary experiments described in chapter 6 are publishable in a scientific journal, a series of characterizations must be done to determine the morphology of cell types present in the myocyte and fibroblast cultures. The major endpoint for both the myocyte-fibroblast cross-talk and hypoxia series of experiements was soluble collagen. In both experiments, soluble collagen in the cell culture media was quantified using a commercially available kit that works by staining and pelleting the free-floating collagen, then resuspending the pellet and releasing the collagen-bound dye. The dye, Sirius Red, is thus present in the resuspended solution in a directly proportional ratio to the amount of collagen in the original sample. This dye is the same as used to stain heart tissue cross-sections in many in vivo experiments, and this chemical stain is very specific for collagens. The inclusion of standardized collagen solutions in the assay allows for the construction of a standard curve, and thus absolute collagen values for each sample. Despite all of the advantages of this fast and simple assay, most studies in the literature examine collagen levels through the use of a collagenase-degradable tritiated-proline incorporation assay. This procedure, while highly sensitive, does not allow for construction of a standard curve, and is much more difficult and time consuming due to the use of tritiated proline. It is possible that different results would be obtained using the proline assay versus the Sirius Red assay, as the proline assay only determines the amount (or rate) of new collagen being produced by the cells. Just as Massons Trichrome stains may result in different numerical values

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91 than Sirius Red stains of heart sections, this assay will offer different numerical values than the tritiated proline assay, but the interpretation of the results (that the positive control increases collagen, and Ang-(1-7) or ACE2 reverses that effect) is unchanged. While there are many tangential issues which could be pursued to improve the wow factor of the numerical data presentation, and various tweaks to the doses and timecourses of the experiments presented, these changes likely would have little effect on the conclusions drawn here. The fact that several chapters have already been published (data in chapter 1 in Experimental Physiology, chapter 3 in the American Journal of Physiology, and chapter 5 in Regulatory Peptides) supports this view. Future Directions While these studies have resulted in several major advancements in our understanding of the role of Ang-(1-7) in cardiovascular regulation, there are several lines of investigation which have not yet been explored. First, the mechanism of Ang-(1-7) signal transduction needs further examination. As was described in chapter 2, there are many possible mechanisms of Ang-(1-7) action, including antagonism of the AT 1 -subtype Ang II receptor, activation of the AT 2 -subtype Ang II receptor, activation of the Mas receptor, and inhibition of angiotensin converting enzyme (ACE). The current studies most strongly support actions of Ang-(1-7) at the Mas receptor, although this support is entirely based upon indirect inference. A very recent publication by Santos et al. (2006) describing the development and characterization of a Mas-receptor knockout mouse represents the type of direct study that is needed to fully characterize the mechanism of Ang-(1-7). Repeating the present studies in a Mas-receptor knockout mouse would help elucidate which of the anti-remodeling effects of Ang-(1-7) are mediated through this receptor.

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92 The search for downstream hormone targets of Ang-(1-7) action presented here was in no way exhaustive. There is a large body of literature that supports the role of TGF in cardiac remodeling, and therefore the discovery that Ang-(1-7) modulates this cytokine simultaneously solidified the importance of Ang-(1-7) in modulating cardiac remodeling, and precluded exploration of other possible targets of Ang-(1-7). Other pro-remodeling and anti-remodeling hormones may very well be modulated by Ang-(1-7). Interleukins 1 and 6, and tumor necrosis factor are all suggested to be pro-fibrotic cytokines, while interleukin 10 may represent an anti-fibrotic hormone, and future studies should be designed to determine if Ang-(1-7) modulates any of these hormones in the myocardium. While ligands, including Ang II, endothelin, catecholeamines, mineralocorticoids and the various above-mentioned cytokines may be altered by Ang-(1-7), their receptors may also be regulated by Ang-(1-7). Binding studies should be done to determine the effects of Ang-(1-7) on AT 1 and AT 2 populations in both myocytes and fibroblasts, and similar studies should be done on the receptors for the other proand anti-remodeling hormones as well. Two intermediaries of Ang-(1-7) action have been suggested in the literature. It has been suggested that Ang-(1-7) may act through increasing production of local nitric oxide, and through the production of protective prostaglandins such as prostaglandin I 2 (Ferrario and Chappell, 2004). A study examining the protective actions of Ang-(1-7) in the L-NAME model of hypertension and remodeling is currently in the planning stages, and should be completed soon. Additionally, future studies could be directed at

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93 determining the contribution of Ang-(1-7) modulation of prostaglandins on cardiac structure and function. It is possible that Ang-(1-7) may modulate not only the production of extracellular matrix proteins, but also the activities of extraceullar matrix degradation enzymes. The collagenases (including matrix metalloproteinases (MMPs)-1, -8, and -13) and the gelatinases (MMP-2 and -9) may be modulated by Ang-(1-7), and this modulation may contribute to the actions of Ang-(1-7) in vivo, especially in recovery following myocardial infarction. Ongoing studies in the laboratory are focused on investigating the utility of ACE2 overexpression following surgically-induced myocardial infarction. These studies are needed to verify the results obtained in cultured cardiac fibroblasts (chapter 6). Further, these in vivo studies are needed to examine the effect of ACE2 overexpression on the preservation of not only cardiac structure, but cardiac function. Many, or most, of the presented studies focus on Ang-(1-7) modulation of cardiac structure, and the choice of this endpoint was primarily based on the assumption that cardiac structure changes result in functional changes, in addition to availability of assays for assessing changes in cardiac function. Although no data are presented herein, a system for directly measuring left ventricular pressure is in development, through which left ventricle end diastolic pressure, ventricular contractility (positive dP/dt), and reflexive ventricular relaxation (negative dP/dt) can be measured. Future experiments should examine both structural changes and functional changes, as it will be the loss of function (which is only indirectly related to structure) that is the clinically relevant endpoint. Timecourses of future

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94 experiments will likely need to be much longer than four weeks, as a functional loss will likely not be present until severe cardiac remodeling is present. With the development of primary cardiac myocyte fibroblast and myocyte cultures described here, many avenues of investigation are now possible for our laboratory. For example, independent cultures of the various cell types represents the only direct way to examine directional cross-talk between myocytes and fibroblasts. Further, characterization of the effects of Ang-(1-7) in cultured cardiac fibroblasts during hypoxia will be much faster, easier, and less expensive than if these studies were done in vivo. Perspectives In summary, these studies have shown that angiotensin-(1-7) is an active hormone, which opposes cardiac fibrosis in two different models of hypertensive cardiac remodeling, and cardiac hypertrophy in a normotensive model. It was also determined that estrogens modulate the actions of angiotensin-(1-7). Finally, preliminary data was shown that suggests angiotensin-(1-7) can act at the cardiac myocyte to inhibit myocyte-fibroblast cross-talk, and that transforming growth factor-1 may represent one target of angiotensin-(1-7) in the cardiac myocyte. From these findings, we conclude that angiotensin-(1-7) is a cardioprotective hormone which warrants further study. As actions of this hormone may be mediated by a novel receptor class, angiotensin-(1-7) may represent a new class of anti-remodeling target that could eventually lead to an entirely new class of heart failure medication and treatment.

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APPENDIX A LOADING OSMOTIC MINIPUMPS Alzet (Cupertino, CA) osmotic minipumps were used for all studies. Model 2004 pumps with a 0.25 L/hr infusion rate were used for four-week studies (Ang II and DOCA-salt), and model 2002 pumps with a 0.5 L/hr infusion rate were used for two-week studies (isoproterenol). The concentration of drug in the filling solution for the pumps is determined by the following calculation: Target Rate 1 g Body Mass Pumps are filled with appropriate solution, then primed for 24 hours in a 0.9% saline solution for twenty-four hours at 37C before implantation. Animals are anesthetized by inhalation of metophane, halothane, or isoflurane before shaving and cleaning an area between the shoulder blades. A small incision is then made, and the pump is introduced into the subcutaneous space. Wound clips are then used to close the incision. Wound clips should be removed from the skin after 1-2 weeks. (ng/kg/min) (kg) X 1000 ng X = Filling Solution (g/L)or (mg/mL) Pump Rate 1 hr X (L/hr) 60 min 95

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APPENDIX B MASSONS TRICHROME STAINING AND ANALYSIS One cross-section of the heart ventricles is cut (approximately 2 mm thick) just above the level of the papillary muscles, and this section is immediately placed into a 10% neutral buffered formalin solution. These cross-sections (in formalin) are then processed by the University of Florida Molecular Pathology and Immunology Core. Briefly, the cross-section is paraffin embedded and sectioned at 5 microns. Sections are then mounted on glass slides and stained sequentially with Weigerts Iron Hematoxylin, Biebrich Scarlet, and Aniline Blue stains. For the presented studies, embedding, sectioning, and staining were performed by the University of Florida Molecular Pathology Core. Slides stained with Massons Trichrome are viewed under 10X (whole heart cross-section), 250X (muscle bundles and interstitial collagen) and 400X (left anterior descending coronary artery and associated perivascular collagen) magnification, and digital photographs are taken using a Moticam 1000 digital microscope camera. Images are stored and analyzed on a PC computer using the ImageJ program from NIH (Rasband, 1997). It is imperative that all slides within one study are imaged with the same lighting conditions, and therefore all images at a given magnification should be taken in one session. Images of slides at 250X magnification are analyzed for myocyte diameter using the ImageJ program. Briefly, an image of a micrometer taken at the same (250X) magnification is used to calibrate the ruler tool within the ImageJ program. At least 96

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97 fifteen individual myocytes that have been cut across their short-axis in the slide are then measured for cross-sectional diameter. Only myocytes with nearly circular shape are used to ensure measurements are made across the short-axis of the myocyte. The fifteen values are then averaged to give one myocyte diameter value per animal. Images of slides viewed at 10X magnification are used to analyze left wall fibrosis. Briefly, the digital image is split into red, blue, and green channels by the ImageJ program. The left ventricle wall is then manually outlined and red and blue color intensity within the outlined section is then determined by the program. These color intensity data are then exported to a spreadsheet, and for each heart the blue color intensity is normalized to the red color intensity. Higher ratios indicate larger blue staining, and hence more collagen deposition. Because of differences in each batch of stain, comparison of blue/red ratios between batches is inappropriate. Therefore, data presentation as % control is necessary. Images of slides viewed at 400X magnification of the left anterior descending coronary artery are used to measure perivascular collagen deposition. Briefly, the freestyle area selection tool within the ImageJ program is used to trace (A) the outline of the outside edge of the perivascular collagen, (B) the outside edge of the vessels smooth muscle layer, and (C) the lumen of the vessel. The areas enclosed within the outlines are used to calculate the normalized area of perivascular collagen as follows; [normalized area] = ( [A] [B] ) / [C]. It is imperative to only use images of nearly circular (not collapsed) blood vessels, as the lumen area is highly dependent upon the shape of the vessel.

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BIOGRAPHICAL SKETCH Justin L. Grobe was born November 3, 1979, to Edward E. Grobe and Loretta L. Grobe, Ph.D. in Mason, Michigan. From a young age, he was interested in the physical, chemical, and biological sciences. Nurtured by his parents, his caregivers Marjorie and James Bullerdick, and the school districts Gifted and Talented Coordinator, Trena Thornburg, Justin had many advanced educational opportunites from a young age. His early interests in science and mathematics were manifested in his involvement in the Mason Middle School Science Olympiad Team, coached by teachers Jeffrey Ehlers and David Dodendorf. During his four years on the team, Justin personally won twenty-two regional, fifteen state, and two national awards and shared several team honors as well. Justins aptitude toward science and mathematics earned him an invitation to a newly developed mathematics program at Michigan State University. This Cooperative Highly Accelerated Mathematics Program (CHAMP) allowed him the opportunity to complete courses in algebra I and II, geometry, trigonometry, and pre-calculus by the end of 9 th grade. Early completion of these courses prepared Justin for the Honors Calculus I and II courses at Michigan State University during his sophomore year of high school, in addition to several courses at Lansing Community College. Dual-enrollment at these higher education institutions and Mason High School allowed Justin to complete requirements for graduation after only three years. Upon graduation, Justin enrolled as an undergraduate at Hope College, in Holland, Michigan. During his first week, Justin attended a research symposium summarizing the 119

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120 previous summers findings by undergraduate researchers at Hope College. One of these presenters was Bradley Andresen, a college senior working in the laboratory of Christopher C. Barney, Ph.D. After some discussion of the project, Justin was invited to try working in Dr. Barneys laboratory to examine his interest in animal research. Over the next four years, Justin continued working in Dr. Barneys laboratory, and their work resulted in several scientific publications, and multiple poster presentations at both the Pew Midstates Undergraduate Research Symposium and the Experimental Biology annual meetings. In the summer of 1998, Justin received a National Science Foundation Research Experiences for Undergraduates (NSF-REU) Fellowship through Hope College to work in Dr. Barneys laboratory. In the summer of 1999, Justin received an individual undergraduate research fellowship from the American Heart Association to continue his work. Then, in the summer of 2000, Justin was awarded a summer NSF-REU position at the University of Florida, Department of Psychology, and he worked in the laboratories of Neil E. Roland, Ph.D. and Michael J. Katovich, Ph.D. Finally, in the spring of 2001, Justin graduated from Hope College with a Bachelor of Science in biology, and a Bachelor of Arts in chemistry. Having enjoyed working with Dr. Katovich the previous summer, Justin applied to graduate school at the University of Florida, College of Pharmacys Department of Pharmacodynamics, with the intention of pursuing his Ph.D. under the tutelage of Dr. Katovich.


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Physical Description: Mixed Material
Copyright Date: 2008

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THE ROLE OF ANGIOTENSIN-(1-7) IN CARDIOVASCULAR PHYSIOLOGY


By

JUSTIN L. GROBE













A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2006

































Copyright 2006

by

Justin L. Grobe

































This document is dedicated to my wife, my family, my mentors, and my friends.















ACKNOWLEDGMENTS

I would first like to thank my graduate mentor, Michael J. Katovich, for his

guidance over the last five years. His continual support, guidance, encouragement, and

direction have been invaluable. I truly appreciate all the friendship he has shown me, and

all the attention and effort he has poured into my education.

Along with my graduate mentor, I would like to thank my undergraduate mentor,

Christopher C. Barney. His guidance and friendship are the primary reasons for my

success as an undergraduate, and my interest in animal physiology.

Next, I would like to thank my graduate committee members, Mohan K. Raizada,

Maureen Keller-Wood, William J. Millard, and Jeffrey A. Hughes, for their input into my

education and this document. Their direction over the last five years has shaped me into

a very capable modern physiologist. I would also like to thank Joanna Peris for her

continuous encouragement, and for filling in during my final defense.

I have been blessed with many other wonderful teachers and mentors at all levels of

my education, but a few deserve particular attention. I would like to thank Marjorie and

James Bullerdick, for teaching me patience and compassion. I would like to thank Trena

Thornburg for nurturing my abilities and teaching me to appreciate thinking "outside the

box." JeffEhlers and David Dodendorfwere wonderful coaches, and I thank them for

encouraging me and promoting my interest in the sciences. The faculty and staff of the

Hope College Biology Department welcomed me from the day I entered college, and I

am grateful for their continuing support and encouragement. Matthew J. Huentelman,









both my peer and teacher, also deserves my gratitude for his excitement and

uncompromising drive.

Many people have blessed me with their friendship, and have made my years in

college and graduate school truly enjoyable. Many should be named, but Bill Murdoch

and Lane Blanchard deserve particular mention for sticking with me in both my joys and

sorrows throughout my undergraduate and graduate years.

I would like to thank my parents, Edward and Loretta Grobe, for their continual

support, energy, and encouragement. From childhood they have recognized my interests,

and provided me every conceivable opportunity to follow my dreams.

Finally, my thanks go to my wife, Connie, who continually provides me with

support, encouragement, inspiration, and direction as she also pursues her Ph.D. I look

forward to sharing my life and work with my best friend.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ...................... .. ................................................................... iv

LIST OF TABLES ........................................... .. ................ ix

LIST OF FIGURES ................................. ...... ... ................. .x

ABSTRACT ........ .............. ............. .. ...... .......... .......... xii

CHAPTER

1 GENERAL INTRODUCTION ..................................................... .....................

H heart F failure ............................................................... .................. .... .....
C cardiac R em modeling ................... .......................................... ........ ......... .. ....
The R enin-A ngiotensin System ......................................................... ................ 4
Angiotensin Converting Enzym e 2 ........................................ ............... 10
A ngiotensin-(1-7) ....................................... ...... ........ .. ........ .... 13
Estrogenic M odulation of Heart Failure .......................... ................................... 13
Estrogenic Modulation of the Renin-Angiotensin System .............. .... ..............14
A im s............... ............... ............ ................................................15

2 CARDIAC FIBROSIS IS PREVENTED BY CHRONIC ANGIOTENSIN-(1-7)
DURING ANGIOTENSIN II INFUSION ...................................... ............... 16

A b stra c t ...................................... ................................................... 1 6
In tro d u ctio n ...................................... ................................................ 17
M eth o d s .............................................................................. 19
A n im als................................. ..................................................... ............... 19
Indirect B lood Pressure ............................................... ............................ 19
C ardiac R em odeling ............................ ...................... .... ....... ...... ............20
S ta tistic s ......................................................................................................... 2 0
R e su lts ...........................................................................................2 0
B lo o d P re ssu re ............................................................................................... 2 0
C ardiac H y p ertrophy ..................................................................................... 2 0
C a rd ia c F ib ro sis ............................................................................................. 2 2
D isc u ssio n ................................ ........... .. .. .......................................................... 2 2









3 CHRONIC ANGIOTENSIN-(1-7) PREVENTS CARDIAC FIBROSIS IN THE
DOCA-SALT MODEL OF HYPERTENSION ............. .................................. 27

A b stra ct ............ ......... ... .. ......... ... .. ................................................... 2 7
In tro d u ctio n .......................... .. ......... ... .. ................................................ 2 8
M eth o d s .............................................................................. 3 0
A n im als ........................................................................ 3 0
C h em icals ........................................................................ 3 0
D O CA -salt M odel .................................. ..... .. ..... ............ 30
Indirect B lood Pressure ............... ...... ................................. ... 31
Direct Blood Pressure/Acute Angiotensin II Responses.................................31
C ardiac R em odeling ............................ ...................... .... ....... .... ..... ...... 32
S ta tistic s ......................................................................................................... 3 3
R e su lts .........................................................................................................................3 3
Indirect B lood Pressure ............... ...... ................................. ... 33
Direct Blood Pressure/Acute Angiotensin II Responses..................................34
C ardiac R em odeling ............................ ...................... .... ....... .... ..... ...... 34
D isc u ssio n ............................................................................................3 6

4 PROTECTIVE EFFECTS OF ANGIOTENSIN-(1-7) DURING
ISOPROTERENOL-INDUCED CARDIAC REMODELING..............................45

A b stra c t ...................................... ................................................... 4 5
Introduction..................................... .................................. .......... 46
M eth o d s ..............................................................................4 7
A n im a ls .................... ........................... .... ............................. 4 7
Group A Males with isoproterenol at 1 mg/kg........................................47
Group B Ovariectomized and intact females with isoproterenol at 1 and
2 m g/kg .................. ........ ....................... ....... .. .. ............... 47
Group C Males with isoproterenol at 2 mg/kg............... ...................48
Indirect B lood Pressure ............................................... ............................ 48
C ardiac R em odeling ............................ ...................... .... ....... ...... ............49
R e su lts ...........................................................................................4 9
C ardiac H y p ertrophy ........................................ ............................................4 9
B lood P pressure .................................................................................. 52
D isc u ssio n ..................................................... .................. ................ 5 2

5 ALTERATIONS IN AORTIC VASCULAR REACTIVITY TO ANGIOTENSIN-
(1-7) IN 17-P-ESTRADIOL-TREATED FEMALE SD RATS ...............................56

A b stra c t ...................................... ................................................... 5 6
Introduction..................................... .................................. ........... 57
M eth o d s .................. .......................................................... ..... 5 9
A nim al H housing and Surgery ........................................ ......................... 59
R e a g e n ts ................ ...............................................................6 0
A ortic V ascular Reactivity A ssay ............................................ ............... 60
Plasm a Steroid M easurem ent ........................................ ......................... 61









S ta tistic s ......................................................................................................... 6 1
R e su lts ............... ... .. ....... ........ ......... ....... ........ .... ...... ............... 6 2
Estradiol Levels and Effects on Body, Heart and Uterine Growth ...................62
A ortic R ing Properties ............................................................ .................. 62
Aortic Relaxation to Angiotensin-(1-7).......... .............................63
A ortic R in g Integ rity ........................................ ............................................6 5
D iscu ssio n .................................... ........ ............. ...........................................6 5

6 PRELIMINARY CELL CULTURE EXPERIMENTS...........................................70

A b stra c t ................................................................................................................. 7 0
In tro d u ctio n .......................................................................................7 0
M methods .......................... ................................. .... ..................... 72
Isolation and Culturing of Neonatal Rat Cardiac Myocytes ..............................72
Isolation and Culturing of Neonatal Rat Cardiac Fibroblasts .............................72
Myocyte-Fibroblast Cross-Talk Paradigm ................. ................. ..........73
R eal-tim e R T -P C R ........................................................................................ 7 3
Lentiviral ACE2 Gene Transfer .............................. ...............73
Hypoxia-Reperfusion Paradigm ............................... ...............74
ACE2 Activity .................................................................................. .................. 74
S o lu b le C o llag en ........................................................................................... 7 5
Transforming Growth Factor-P 1 ......................... ....................... 75
R e su lts ..... .a ................... ....... .... ......... .......... ......... ...............7 5
Cardiac Myocyte-Fibroblast Cross-Talk is Inhibited by Ang-(1-7) at the
M yocyte ........................... .............. ...... ........................ 75
Lentiviral Delivery of EF 1 a-ACE2 Results in Increased ACE2 Activity and
Protection from H ypoxia....................... ... ..........................77
ACE2 Gene Delivery Attenuates Hypoxia-Reperfusion Induction of
Transforming Growth Factor-3P1 .............. ... ...... ........... ...............78
D discussion ......... ........ ............................................. ......78

7 GENERAL DISCUSSION ......... ....... ................. ........82

Sum m ary and Conclusions .................................... ...................................... 82
Pitfalls and Shortcom ings ............. .... .......................... ............ ........ ....... 84
F utu re D direction s ................................................................ 9 1
P ersp ectiv e s ................................................................9 4

APPENDIX

A LOADING OSMOTIC MINIPUMPS .............................................. .....................95

B MASSON'S TRICHROME STAINING AND ANALYSIS ..................................96

L IST O F R EFER EN CE S .......................................................................................98

BIOGRAPHICAL SKETCH ........................................................................119



v111
















LIST OF TABLES


Table pge

5-1. Effects of steroid treatments on body parameters in female Sprague-Dawley rats..62

5-2 A ortic ring properties ....................................................................... ..................62
















LIST OF FIGURES


Figure pge

1-1. T he renin-angiotensin system ....................................................................... ........5

1-2. Ventricular hypertrophy with chronic Ang II is attenuated by lentiviral delivery
o f A C E 2 ........................................................................................1 1

1-3. Ang II-induced cardiac interstitial fibrosis is prevented by lentiviral delivery of
A C E 2. .................................................................... 12

1-4. Lentiviral delivery of ACE2 has no effect on the blood pressure increase from
chronic A ng II infusion .................. ......................................... ........ 12

2-1. Chronic Ang-(1-7) has no effect on blood pressure increases with chronic Ang II
in fu sio n ....................................................................... 2 1

2-2. Chronic Ang-(1-7) prevents myocyte hypertrophy due to chronic Ang II infusion 21

2-3. Chronic Ang-(1-7) prevents interstitial fibrosis due to chronic Ang II infusion .....22

3-1. Chronic Ang-(1-7) has no effect on blood pressure increases with DOCA-salt
treatm ent ..................................... .................................. ......... 33

3-2. Chronic Ang-(1-7) has no effect on acute pressor responses to Ang II in animals
treated w ith D O C A -salt......................................... .............................................34

3-3. Chronic Ang-(1-7) has no effect on cardiac hypertrophy due to DOCA-salt
treated ent ..................................... ................................. .......... 35

3-4. Chronic Ang-(1-7) prevents interstitial fibrosis with DOCA-salt treatment...........36

3-5. Chronic Ang-(1-7) prevents perivascular fibrosis with DOCA-salt treatment ........37

4-1. Isoproterenol (1 mg/kg/day) induces cardiac hypertrophy in male rats .................50

4-2. Ovariectomy potentiates the isoproterenol-mediated induction of cardiac
hypertrophy in fem ale rats.............................................. .............................. 50

4-3. Chronic Ang-(1-7) prevents the hypertrophic, but not fibrotic effects of
isoproterenol (2 mg/kg/day) in male rats ...................................... ............... 51









4-4. Isoproterenol and Ang-(1-7) had no chronic effects on blood pressure in male rats.52

5-1. Maximal contraction to angiotensin II in aortic rings from ovariectomized rats
w ith estrogen replace ent ......................................................................... .........63

5-2. Estradiol attenuates the aortic relaxation response to acute Ang-(1-7) ..................64

6-1. Ang-(1-7) attenuates myocyte-derived pro-fibrotic signaling ............................76

6-2. Ang-(1-7) attenuates expression of TGFp1 mRNA in cardiac myocytes. ...............76

6-3. Lentiviral delivery of ACE2 attenuates the collagen production response to
hypoxia in cultured cardiac fibroblasts ........................................ ............... 77

6-4. Lentiviral delivery of ACE2 attenuates hypoxia-induced expression of
transforming growth factor-pl protein ............................................................78















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

THE ROLE OF ANGIOTENSIN-(1-7) IN CARDIOVASCULAR PHYSIOLOGY

By

Justin L. Grobe

May, 2006

Chair: Michael J. Katovich
Major Department: Pharmacodynamics

Heart failure (HF) is a devastating condition that impacts our society emotionally,

physiologically, and fiscally, and it is a growing problem in the United States. Aberrant

activity of the renin-angiotensin hormone system (RAS) has been implicated as a

causative factor in HF. Recently, a new angiotensin converting enzyme 2 (ACE2) was

recognized, which catalyzes the degradation of the active angiotensin II (Ang II)

hormone to angiotensin-(1-7) (Ang-(1-7)). Studies into the actions of ACE2 have led to

the concept that Ang-(1-7) may act as an active, endogenous inhibitor of the RAS.

Interestingly, investigations into the mechanisms of action of ACE inhibitors and AT1R

antagonists have revealed that both of these treatments result in increased circulating

levels of Ang-(1-7). Some have therefore hypothesized that pharmacological inhibitors

of the RAS may provide cardioprotective effects through the actions of Ang-(1-7).

The present studies were designed to characterize the cardioprotective actions of

Ang-(1-7) in various models of cardiac remodeling. The protective effect of Ang-(1-7)

was evaluated in Ang II-infusion, mineralocorticoid, and P-adrenergic models of cardiac









remodeling. Further, due to gender differences in the development and progression of

heart failure, the protective actions of Ang-(1-7) were evaluated in intact and

ovariectomized female rats. Cardiac remodeling was inhibited by Ang-(1-7), with

specific effects determined by the type of stimulus, and estrogens were determined to

play an important role in regulating tissue sensitivity to Ang-(1-7).

From these findings, it was concluded that Ang-(1-7) has specific actions in the

cardiovascular system, some of which oppose the actions of its precursor, Ang II. In

addition, our results lead us to hypothesize that estrogen replacement therapy during

menopause may predispose women to cardiovascular diseases through decreased tissue

sensitivity to the protective Ang-(1-7) hormone. Future studies should more directly

characterize the effects of estrogens on cardiac tissue, and further elucidate the tissue-

level and intracellular signaling mechanisms of Ang-(1-7). Our results indicate that Ang-

(1-7) may represent a new, unexploited class of targets for pharmacological intervention

before and during HF.














CHAPTER 1
GENERAL INTRODUCTION

Heart Failure

Congestive heart failure (CHF) is one of the most debilitating cardiovascular

pathologies, having both tremendous economic and emotional impacts on our society.

Approximately five million Americans, or 2.3% of the entire population of the United

States, suffers from CHF (Thom et al., 2006). The lifetime risk of developing CHF in the

fourth decade of life is 20%, and this risk doubles for individuals with blood pressure

>160/90 (Thom et al., 2006).

Congestive heart failure is a disease characterized by inadequate movement of

blood by the heart. Inadequate blood flow results in poor perfusion of critical organs,

including the brain and the heart itself, thereby leading to cellular hypoxia and death. It

was estimated that in 2005, 80% of men and 70% of women under age 65 diagnosed with

CHF will die within eight years of diagnosis. After diagnosis, survival is poorer in men

than women, but fewer than 15% of women will survive longer than eight to twelve

years. Twenty percent of people diagnosed with CHF will die within one year of

diagnosis (Thom et al., 2006).

There are two major forms of CHF, which are defined by the morphological and

functional changes that lead to inadequate blood movement. Systolic heart failure is

characterized by insufficient contraction of the ventricles (which are typically dilated),

usually resulting from idiopathic dilated or ischemic cardiomyopathies. Diastolic heart

failure is characterized by thickened, noncompliant ventricular walls and small









ventricular volumes, usually resulting from hypertension, stenotic valvular disease, or

primary hypertrophic cardiomyopathies. Clinically, patients typically present with a

combination of both systolic and diastolic heart failure, in that the heart is too weak to

maintain cardiac output (systolic dysfunction) despite thick, stiff walls diastolicc

morphology). Thus, conventional therapy is targeted at relieving symptoms of

inadequate blood flow rather than reversing or preventing the myocardial damage which

leads to CHF.

Cardiac Remodeling

Whereas systolic heart failure is typically the result of myocardial ischemia and

cellular death (which leads to a loss of muscle and force-generating ability), diastolic

heart failure is the result of inappropriate myocyte growth accompanied by a large

increase in extracellular matrix deposition. Together, myocyte hypertrophy and net

increases in extracellular matrix components are referred to as cardiac remodeling.

Cardiac remodeling can be normal and compensatory, as is observed in the

myocardium of athletes engaged in aerobic sports. In this "normal" remodeling, the

process results from increased peripheral demand for cardiac output, which provides a

mechanical stimulus for myocyte hypertrophy. Cardiac fibroblasts, which produce the

proteins of the extracellular matrix, retain relatively normal growth patterns and function.

This type of remodeling differs from pathological remodeling primarily due to the

difference in the response of the cardiac fibroblast to the stimulus (Weber, 2000; Miner

and Miller, 2006). Pathological remodeling, in contrast, can take many forms, which are

differentiated primarily by stimulus and mechanism.

Mineralocorticoids can elicit direct effects on cardiac remodeling. Aldosterone

levels are elevated during heart failure, and this elevation is related to increased mortality









(Swedberg et al., 1990), and inhibition of mineralocorticoid receptors by spironolactone

or eplerenone significantly reduces mortality in heart failure patients (Pitt et al., 1999,

Pitt et al., 2003). Spironolactone treatment also results in decreased extracellular matrix

remodeling (Zannad et al., 2000). In vitro, cardiac fibroblasts are stimulated by

aldosterone to increase collagen production, and to change the relative pattern of collagen

types expressed (Zhou et al., 1996; Brilla, 2000; Rombouts et al., 2001).

Angiotensin II also directly promotes cardiac remodeling. In vitro, angiotensin II

promotes proliferation of cardiac fibroblasts, collagen production, and expression of

adhesion receptors (Kim and Iwao, 2000; Schnee and Hsueh, 2000; Lijnen et al., 2001).

Inhibition of angiotensin converting enzyme improves the outcomes for heart failure

patients (Khalil et al., 2001), as does antagonism of the AT1 subtype angiotensin II

receptor (Frigerio and Roubina, 2005), thereby implicating angiotensin II in cardiac

remodeling.

Catecholamines can act upon the myocardium to cause remodeling. Epinephrine

and norepinephrine are elevated during heart failure and correlate to prognosis (Francis et

al., 1993). Inhibition of P-adrenergic receptors decreases the symptoms of heart failure,

and improves survival (Shibata et al., 2001). The actions of norepinephrine on the

extracellular matrix are not well described at the present time, though it is known that

norepinephrine induces the production of growth factors, such as transforming growth

factor 31, which are potent mediators of fibrosis (Bhambi and Eghbali, 1991; Barth et al.,

2000; Akiyama-Uchida et al., 2002; Miner and Miller, 2006).

Cytokines such as transforming growth factor-p31 are produced by cardiac cells

during heart failure, and are well-established mediators of cardiac remodeling (Sun et al.,









1998; Lijnen et al., 2000; Iwata et al., 2005; Tallant et al., 2005). In vitro, transforming

growth factor-3p acts in an autocrine/paracrine fashion to modulate collagen and

fibronectin production in the cardiac fibroblast (Roberts et al., 1986). Because it is

upregulated by angiotensin II and norepinephrine, this cytokine may represent a common

mediator of the fibrotic effects of these hormones (Bhambi et al., 1991; Yoo et al., 1998).

The Renin-Angiotensin System

In 1836, Richard Bright observed a link between left ventricular hypertrophy and

renal disease, and he suggested a link among hypertrophy, increased resistance to blood

flow in small vessels, and an altered condition of the blood (Basso and Terragno, 2001).

George Johnson in 1868 and F.A. Mahomed in 1872 also reported links between renal

diseases and left ventricular hypertrophy. Finally, in 1898, Tigerstedt and Bergman

determined that extracts from rabbit renal tissue contained a pressor agent which

mediated the relationship between renal disease and cardiac hypertrophy. Tigerstedt and

Bergman named this vasoactive substance reninn." Subsequent attempts to isolate and

fully characterize this and related blood-borne substances resulted in simultaneous

discovery of the compound hypertension by Braun-Menendez et al. (1939), and

"angiotonin" by Page et al. (1939). These two would later agree to compromise on

standard nomenclature for the various components of the developing system, including

the name "angiotensin" as a combination of the two earlier names (Braun-Menendez and

Page, 1958).

Many more components of the renin-angiotensin system (RAS) have been

discovered, as have the relationships among the various components. The system is

present both systemically (in the blood plasma) and in the interstitium (as an autocrine or

paracrine system), and it is the presence or absence of the various components at the site










of action which determines the specific outcome of RAS activation in that location.

Figure 1-1 summarizes the presently recognized components of the RAS.


Angiotensinoger

Pro-Renin Renin

Angiotensin I

ACEI

Angiotensin II
AP-A

Angiotensin III

AP-B I

Angiotensin IV


ACE2
-- Angiotensin-(1-9)
Endo-
peptidases ACE

- Angiotensin-(1-7)
ACE2

-'U' ACE

,' '' Angiotensin-(l-5)

\~ s '%
II l
\ 1


Vasoconstriction
Memory effects?


Vasoconstriction
Hypertrophy
Fibrosis


Anti-AT1 effects
Vasodilation
Differentiation


Anti-AT1 effects
Vasodilation


Figure 1-1. The renin-angiotensin system. (ACE angiotensin converting enzyme;
ACE2 angiotensin converting enzyme 2; Endopeptidases neprilysin, prolyl
endopeptidase, and thimet oligopeptidase; AP-A aminopeptidase A; AP-B -
aminopeptidase B; AT1 angiotensin II type 1 receptor; AT2 angiotensin II
type 2 receptor; AT1-7? possible receptor for angiotensin-(1-7), which may
be the Mas receptor; AT4 angiotensin type 4 receptor).

Release of renin from the kidney is the rate limiting step in the generation of

circulating angiotensin II (Ang II). Renin, an aspartyl protease, is synthesized and

released by juxtaglomerular cells located in the walls of the afferent arterioles of the

nephron just as the vessel enters the glomerulus. Renin is synthesized as the 406 amino

acid preprorenin, then cleaved to form prorenin which is a 373 amino acid mature, but









inactive, enzyme. Final activation of renin by an as yet uncharacterized enzyme involves

removal of the 43 amino terminal residues (Goodman and Gilman, 2001).

The only known substrate for renin is angiotensinogen. This highly abundant

globular glycoprotein is synthesized as a 452-amino acid preangiotensinogen by liver, fat,

certain nervous tissues, and the kidney. Angiotensinogen's synthesis is stimulated by

inflammation, insulin, estrogens, glucocorticoids, thyroid hormone, and angiotensin II.

Due to the circulating levels of angiotensinogen and the K, of renin for its substrate

(approximately 1 [M), the rate of synthesis of angiotensinogen can modulate the levels of

circulating angiotensin II. Indeed, increased circulating levels of angiotensinogen are

associated with essential hypertension (Jeunemaitre et al. 1992; Goodman and Gilman,

2001). The mature peptide is only fourteen amino acids in length.

Angiotensin I represents the first ten amino acids of the amino terminus of

angiotensinogen, and is the result of renin action upon angiotensinogen. This peptide is

inactive until converted to angiotensin II by angiotensin converting enzyme.

Angiotensin converting enzyme (ACE) is also known as Kininase II and Dipeptidyl

Carboxypeptidase. This ectoenzyme is comprised of 1277 amino acids, and displays two

homologous zinc-dependent enzymatic domains. The large extracellular domain of this

enzyme can be cleaved by secretase (Beldent et al., 1995; Goodman and Gilman 2001) to

produce circulating ACE enzyme, but it has been suggested that intact, membrane-bound

ACE may act as a serine-threonine kinase extracelluar receptor (Fleming et al., 2005).

ACE is a nonspecific peptidase that removes two amino acids from substrates with

different sequences (although it prefers substrates with one free carboxyl group in the

carboxyl-terminal residue, and proline can not be the penultimate amino acid therefore









it can not act on angiotensin II). Substrates for ACE include bradykinin (ACE inactivates

bradykinin and other potent vasodilators), angiotensin I, angiotensin-(1-9), and

angiotensin-(1-7).

An important genetic variation of ACE exists, in which an insertion/deletion

polymorphism in intron 16 of the gene significantly contributes to circulating ACE levels

(Rigat et al., 1990). The deletion version of the polymorphism results in higher levels of

circulating ACE and may be a risk factor for the development of heart disease (Cambien

et al., 1992; Gardeman et al., 1995; Mattu et al., 1995), vascular endothelial dysfunction

(Butler et al., 1999), left ventricular hypertrophy (Iwai et al., 1994; Schunkert et al.,

1994), and hypertension (Fomage et al., 1998; O'Donnell et al., 1998). It is interesting to

note that humans who live past 100 years of age display a high frequency of the deletion

polymorphism (Schachter et al., 1994), which may be a result of the strong association of

the deletion allele with protection from Alzheimer's disease (Kehoe et al., 1999).

ACE-mediated cleavage of the two carboxyl amino acids of angiotensin I results in

the production of the eight-amino acid angiotensin II (Ang II). Ang II is the most active

component of the RAS, and its actions are receptor-dependent. The subtype of receptor

that is activated by Ang II determines the cellular response to this ligand.

Other enzymatic pathways exist, which can produce active angiotensin peptides

without involvement of renin and/or ACE. Nonrenin proteases can convert

angiotensinogen to angiotensin I, and cathepsin G and tonin can convert angiotensinogen

directly to angiotensin II. Angiotensin I can be converted to angiotensin II by cathepsin

G, chymostatin-selective angiotensin II-generating enzyme, and heart chymase (Dzau et

al., 1993; Goodman and Gilman, 2001). In humans, chymase is an important enzyme for









the conversion of angiotensin I to angiotensin II at the tissue level, especially in cardiac

(Wolny et al., 1997; Wei et al., 1999) and renal tissue (Hollenberg et al., 1998).

Regardless of its formation pathway, angiotensin II exerts its effects through two

major subtypes of G-protein coupled receptors. The first subtype of receptor, the

angiotensin II type 1 (ATi) receptor, is nearly ubiquitous, and is responsible for most of

the overall actions of angiotensin II. This 359-amino acid cell-surface receptor can

activate a large number of signal transduction pathways. These pathways can include

calcium release and influx, phospholipases, mitogen-activated protein kinase pathways,

Janus kinase pathways, serine/threonine protein kinases, nonreceptor tyrosine kinases,

small GTP-binding proteins, inducible transcription factors, factors affecting translational

efficiency, and the production of reactive oxygen species (Griendling et al., 1997; Berk,

1999; Blume et al., 1999; Inagami, 1999; Goodman and Gilman, 2001). Activation of

AT1 receptors results in endothelial dysfunction, vasoconstriction, vascular smooth and

cardiac muscle cell growth, enhanced coagulation and sympathetic nerve activity, all of

which can be detrimental to cardiovascular function and ventricular structure (Yasunari et

al., 2005). AT1 receptors on cardiac myocytes cause hypertrophy, increased protein

synthesis, overall gene reprogramming, increased production of atrial natriuretic factor,

and can induce apoptosis (Yasunari et al., 2005). These same receptors, on cardiac

fibroblasts, lead to proliferation, increased DNA and protein synthesis, and increased

gene expression including transforming growth factor 31 and fibronectin (Yasunari et al.,

2005). Pharmacological inhibition (Yasunari et al., 2005) and genetic modulation

(Raizada et al., 2000) of the AT1 receptor show great promise for inhibiting cardiac

remodeling.









The second subtype of angiotensin II receptor is the angiotensin II type 2 (AT2)

receptor. Much less is known about the AT2 receptor, but its actions are generally

considered to oppose those of the AT1 receptor. The AT2 receptor is highly expressed in

fetal tissues, but levels are low in adults until cardiac insult, such as myocardial

infarction, which results in increased AT2 expression (Nio et al., 1995). This G-protein

coupled receptor is 363-amino acids long, and is known to activate phosphatases,

potassium channels, and nitric oxide production, while inhibiting calcium channels

(Horiuchi et al., 1999). Most of the effects of the AT2 receptor are mediated by the Gai

binding protein (Goodman and Gilman, 2001). AT2 receptors on cardiac myocytes

inhibit cell growth and decrease protein synthesis, while their activation on cardiac

fibroblasts inhibits AT1-mediated effects, and decreases DNA and protein synthesis

(Yasunari et al., 2005). Overexpression of this receptor subtype has been shown to

prevent cardiac remodeling (Metcalfe et al., 2004).

In addition to angiotensin II, there are multiple other angiotensin peptides that are

endogenously produced by the RAS. For example, the alanine residue at the amino

terminus of angiotensin II can be removed by aminopeptidase A, thus producing

angiotensin-(2-8), also known as angiotensin III. The role of angiotensin III is under

debate, as this peptide has been shown to have similar pharmacological actions at the AT1

and AT2 receptor as angiotensin II (Wright and Harding, 1997).

Angiotensin III is subsequently digested by aminopeptidase B to remove the

arginine residue at the amino terminus. This digestion produces angiotensin-(3-8), which

is commonly known as angiotensin IV. Angiotensin IV selectively binds the AT4

receptor, which is also known as the insulin-regulated aminopeptidase. In the heart, this









receptor stimulates protein synthesis in cardiac fibroblasts and inhibits mechanically-

stimulated expression of immediate early genes cfos and egr-1, although the

predominantly recognized role of the AT4 receptor at this time is in learning and memory

(Chai et al., 2004).

Finally, angiotensin IV can be cleaved by aminopeptidase N or

dipeptidylaminopeptidase to form angiotensin-(4-8), angiotensin-(5-8), or angiotensin-(6-

8), all of which are inactive fragments of the RAS (von Bohlen and Halbach, 2003).

Angiotensin Converting Enzyme 2

Recently a second angiotensin converting enzyme (ACE2) was discovered by

Tipnis (2000) and Donoghue (2000). Although this enzyme shares approximately 40%

homology to ACE within its catalytic domain, ACE2 differs from ACE in substrate

specificity and is not inhibited by classic ACE inhibitors (Tipnis et al., 2000, Guy et al.,

2003).

ACE2 has been shown to play a role in heart development and function (Boehm

and Nabel, 2002; Crackower et al., 2002; Donoghue et al., 2003; Oudit et al., 2003).

Genetic knockout of the ACE2 gene causes increased circulating angiotensin II, impaired

cardiac function, and induction of hypoxia-response genes (Crackower et al., 2002;

Donoghue et al., 2003). Overexpression of this gene in rats delays the onset of cardiac

hypertrophy (Santos et al., 2004), and transgenic overexpression in mice results in

decreased systolic blood pressure (Crackower et al., 2002). Several animal models of

hypertension exhibit depressed ACE2 levels, which suggests a relationship between

ACE2 and hypertension (Crackower et al., 2002; Danilczyk et al., 2003; Garcia et al.,

2003). Finally, the ACE2 gene maps to a defined quantitative trait locus that has been

associated with hypertension (Crackower et al., 2002). It has been suggested (Donoghue










et al., 2000; Tipnis et al., 2000; and Turner and Hooper, 2002) that the balance of actions

between ACE and ACE2 may determine the overall physiological / pathophysiological

(blood pressure and structural) balance within various organ systems.

To examine the hypothesis that increased activity of ACE2 would result in

protection from cardiac remodeling, we previously delivered the murine ACE2 gene to

rats by use of a lentivirus. Neonatal delivery of lenti-mACE2 to Sprague-Dawley rats

provided these animals protection from cardiac remodeling with angiotensin II infusion

during adulthood (Huentelman et al., 2005). ACE2 overexpression by these animals

attenuated cardiac hypertrophy (Figure 1-2), and completely prevented the interstitial

fibrosis (Figure 1-3) caused by angiotensin II infusion. It is interesting to note that these

effects were not mediated by changes in blood pressure (Figure 1-4), thus indicating that

ACE2 can selectively protect the myocardium from remodeling.










3-0




2.6
GFP GFP ACE2 ACE2
Saline Ang Saline Ang I

Figure 1-2. Ventricular hypertrophy with chronic Ang II is attenuated by lentiviral
delivery of ACE2. (Reprinted with permission from Huentelman MJ, Grobe
JL, Vazquez J, Stewart JM, Mecca AP, Katovich MJ, Ferrario CM, Raizada
MK. Protection from angiotensin II-induced cardiac hypertrophy and fibrosis
by systemic lentiviral delivery of ACE2 in rats. Experimental Physiology.
90(5):783-90. 2005.)












A GFP-Saline



10x




20x=


GFP-Ana II ACE2-Ana 1I


GFP GFP ACE2 ACE2
Saline Ang I Saline Ang I


Figure 1-3. Ang II-induced cardiac interstitial fibrosis is prevented by lentiviral delivery
of ACE2. A) Representative sections of left ventrile wall, and B) quantified
collagen density in left ventricle wall sections. (Reprinted with permission
from Huentelman MJ, Grobe JL, Vazquez J, Stewart JM, Mecca AP, Katovich
MJ, Ferrario CM, Raizada MK. Protection from angiotensin II-induced
cardiac hypertrophy and fibrosis by systemic lentiviral delivery of ACE2 in
rats. Experimental Physiology. 90(5):783-90. 2005.)



240

220



IS -


B 140

120I

100
GFP GFP ACE2 ACE2
Saline Ang 0 Saline Ang I

Figure ID. Lentiviral delivery of ACE2 has no effect on the systolic blood pressure
increase from chronic Ang II infusion. (Reprinted with permission from
Huentelman MJ, Grobe JL, Vazquez J, Stewart JM, Mecca AP, Katovich MJ,
Ferrario CM, Raizada MK. Protection from angiotensin II-induced cardiac
hypertrophy and fibrosis by systemic lentiviral delivery of ACE2 in rats.
Experimental Physiology. 90(5):783-90. 2005.)









The mechanism by which ACE2 can provide cardioprotection during hypertension

is unknown, but it is likely that increased formation of the enzyme's product,

angiotensin-(1-7), may be important.

Angiotensin-(1-7)

ACE2 has been shown to be one of several enzymes that catalyzes the conversion

of angiotensin I and angiotensin II to the newly recognized hormone angiotensin-(1-7)

(Figure 1-1, and Ferrario and Chappell, 2004), but in the heart, ACE2 and neprilysin have

been shown to have the most significant roles in the generation of angiotensin-(1-7)

(Zisman et al., 2003). ACE, which is a key enzyme in the RAS for the conversion of

angiotensin I to angiotensin II, is the primary enzyme involved in the degradation of

angiotensin-(1-7) to angiotensin-(1-5) (Yamada et al., 1998; Ferrario and Chappell,

2004).

Estrogenic Modulation of Heart Failure

Gender differences exist in the susceptibility, onset, progression, and outcome of

heart failure. Approximately 15% of women and 20% of men have hypertension, which

is a major risk factor for the development of heart failure (Regitz-Vagrosek and

Lehmkuhl, 2005). Blood pressure tends to increase with age in both sexes, although at

menopause, the prevalence of hypertension increases greatly in women. Between the

ages of 65 and 75, approximately 45% of women and 41% of men are hypertensive

(Hayes and Taler, 1998; Gasse et al., 2001). It is important to note that systolic blood

pressure disproportionately increases in women compared to men, and the subsequent

rise in pulse pressure represents an important and independent predictor of cardiovascular

outcome (Burt et al., 1995; Regitz-Vagrosek and Lehmkuhl, 2005).









The effects of hypertension in women are more pronounced than in men.

Hypertension is by far the major causative factor for the development of heart failure in

women, whereas myocardial infarction is the most causative factor in men. In the

Framingham Heart Study, hypertension was the primary causative factor for the

development of heart failure in 59% of women, compared to only 39% in men (Levy et

al., 1996). Left ventricular hypertrophy (which results from chronic hypertension) also

carries a greater risk for mortality in women than in men (Liao et al., 1995).

Roles of estrogen and its receptors in the development and progression of heart

failure are fairly well established. In many animal models of cardiovascular

abnormalities, phenotypes are worse in male and ovariectomized females, and

administration of estrogens can attenuate or reverse the phenotype (Regitz-Vagrosek and

Lehmkuhl, 2005). Estrogen has been shown to reduce the size of myocardial infarcts,

and reduce apoptosis (Patten et al., 2004). Estrogen also antagonizes cardiac myocyte

hypertrophy in vitro (Van Eickels et al., 2001). Many of the cardioprotective effects of

estrogen have been attributed to the estrogen receptor (ER)-a subtype (Zhai et al., 2000),

although both a and 0 estrogen receptor subtypes are known to be present in cardiac

myocytes and fibroblasts from humans and rats (Grohe et al., 1997; Taylor and Al-

Azzawi, 2000). In humans, both receptor subtypes show increased expression during

cardiac hypertrophy (Nordmeyer et al., 2004).

Estrogenic Modulation of the Renin-Angiotensin System

Estrogens are known to modulate many hormonal systems involved in the

regulation of cardiac structure and function. The RAS is regulated by estrogens at

several points. Angiotensinogen and the AT2 receptor are increased with estrogen

treatment (Healy et al., 1992; Armando et al., 2002), while prorenin, ACE, neprilysin,









aminopeptidases, and the AT1 receptor are all decreased with estrogen treatment

(Szilagyi et al., 1995; Pinto et al., 1999; Nickenig et al., 2000; Seli et al., 2001; Turner

and Hooper, 2002). Recent studies by Brosnihan et al. (2003) have determined that

during pregnancy, renal ACE2 expression is increased, as are circulating levels of

angiotensin-(1-7). Together, these findings indicate that estrogen decreases the

production and effectiveness of angiotensin II, while it has positive effects on the

generation of angiotensin-(1-7).

Aims

It was the goal of these studies to elucidate the role, if any, of angiotensin-(1-7) in

protecting the myocardium from pathological remodeling. First, the protective effects of

angiotensin-(1-7) will be examined in both angiotensin II-dependent and -independent

forms of hypertension-induced cardiac remodeling. Second, modulation of normotensive

cardiac remodeling by angiotensin-(1-7) will be studied. Third, modulation of the effects

of angiotensin-(1-7) by estrogen will be examined. Finally, preliminary studies into the

cellular mechanism of action of angiotensin-(1-7) will be presented. Together, these

studies will demonstrate the cardioprotective actions of angiotensin-(1-7), establish the

modulatory role of estrogen on this hormone, and point the direction for future studies

into the actions of angiotensin-(1-7).














CHAPTER 2
CARDIAC FIBROSIS IS PREVENTED BY CHRONIC ANGIOTENSIN-(1-7)
DURING ANGIOTENSIN II INFUSION

Abstract

Aberrant activity of the renin-angiotensin system is well established to cause

hypertension and associated cardiac remodeling, and chronic infusion of angiotensin II

(Ang II) can be used to model these effects in animal models. Recent studies have

indicated that the angiotensin-(1-7) (Ang-(1-7)) breakdown product of angiotensin II may

act as an endogenous buffer against the actions of Ang II. Here, we examine the

hypothesis that chronic infusion of Ang-(1-7) provides cardioprotection during

hypertension due to chronic infusion of Ang II. Sprague-Dawley rats between 320-470

grams were implanted with osmotic minipumps which chronically delivered Ang II (100

ng/kg/min). A subset of these animals was also implanted with osmotic minipumps that

chronically delivered Ang-(1-7) (100 ng/kg/min). Control animals underwent sham

implantation surgery. Blood pressure was monitored by an indirect tail cuff method, and

after four weeks of treatment, hearts were harvested, sectioned and stained using

Masson's Trichrome. Chronic infusion of Ang II resulted in significant increases in

blood pressure, ventricular and myocyte hypertrophy, and interstitial fibrosis. Chronic

infusion of Ang-(1-7) significantly decreased this myocyte hypertrophy and interstitial

fibrosis. These results lead us to conclude that Ang-(1-7) selectively attenuates cardiac

remodeling during chronic infusion of Ang II, without effect on blood pressure. These

findings complement and extend our previous findings that lentiviral delivery and









subsequent overexpression of the murine ACE2 gene prevents cardiac remodeling in Ang

II-infused rats.

Introduction

The renin-angiotensin system (RAS) is well documented as an important

endogenous system that regulates cardiac structure and function. An aberrant RAS has

been shown to be integrally involved in the development and maintenance of

hypertension and cardiac remodeling (Kim et al., 1995; Alderman et al., 1991; Dzau,

1993; Parmley, 1998; Bader et al., 2001; Ruiz-Ortega et al., 2001; Unger, 2002; Zaman

et al., 2002). Further, use of RAS inhibitors (both angiotensin converting enzyme

inhibitors and angiotensin II type 1 receptor blockers) has been shown to decrease blood

pressure, attenuate cardiac remodeling, and prevent cardiac function loss (Frigerio and

Roubina, 2005).

Chronic infusion of angiotensin II (Ang II) has long been used in animal models to

mimic the onset and progression of hypertension and hypertension-induced

pathophysiologies. Chronic infusion of Ang II induces these changes through direct

vasoconstriction of the vasculature (Rajagopalan et al., 1996), water and salt reabsorption

at the kidney (Zou et al., 2002; Lopez et al., 2003; Rugale et al., 2003), and altered

sympathetic tone, both centrally and peripherally (Zanzinger and Czachurski, 2000;

Zimmerman et al., 2002).

Within the heart, Ang II induces structural changes both directly through actions at

cardiac cells and indirectly through changes in arterial blood pressure. In culture, Ang II

stimulates growth of cardiac myocytes, fibroblasts, and smooth muscle cells (Baker et al.,

1992; Corda et al., 2000). Ang II also increases production of the extracellular matrix

proteins, including collagen and fibronectin, and inhibits the activity of collagenases









(Baker et al., 1992; Corda et al., 2000). These results are paralleled in vivo during Ang

II-induced hypertension and following myocardial infarction in the rat (Brilla et al., 1990;

Weber and Brilla, 1991; Lorell, 1997; Swynghedauw, 1999; Corda et al., 2000). Low

(non-pressor) doses of Ramipril, and angiotensin converting enzyme (ACE) inhibitor, can

regress cardiac fibrosis, thus highlighting the direct effects of the local RAS in regulating

cardiac structure independent of blood pressure (Nagasawa et al., 1995; Corda et al.,

2000).

The mechanism by which low-dose ACE inhibition can attenuate cardiac

remodeling is likely three-fold. First, ACE inhibition can result in decreased levels of

Ang II. Second, ACE inhibition may result in increased levels of bradykinin, as ACE is

known to degrade this protective hormone (Goodman and Gilman, 2001). Third, ACE

inhibition has been shown to result in increased levels of the newly recognized hormone

angiotensin-(1-7) (Ang-(1-7)) (Tom et al., 2003), likely due to the fact that ACE is the

principle degradation pathway for Ang-(1-7) (Ferrario and Chappell, 2004).

We have recently demonstrated that increased expression of angiotensin converting

enzyme 2 (ACE2), the primary Ang-(1-7) production enzyme in the heart (Zisman et al.,

2003; Ferrario and Chappell, 2004), prevents Ang II-induced cardiac remodeling in rats

(Huentelman et al., 2005). Specifically, lentiviral transfer of the mouse ACE2 gene to

prevented cardiac hypertrophy (Figure 1-2) and cardiac fibrosis (Figure 1-3) without

effect on blood pressure (Figure 1-4). At least two possible mechanisms involving the

renin-angiotensin system may be at work in ACE2-mediated cardioprotection during Ang

II-induced hypertension and cardiac remodeling. First, it is possible that ACE2 simply

decreases the levels of its reactant, Ang II. Second, it is possible that the increased levels









of Ang-(1-7) resulting from ACE2 enzymatic activity provide some active protection

from cardiac remodeling. Therefore, the following study was undertaken to examine the

hypothesis that chronic infusion of Ang-(1-7) would prevent cardiac remodeling in the

Ang II-infusion model of hypertension.

Methods

Animals

Eighteen sprague-Dawley rats, between 320-470 grams, were divided into three

groups. Animals were anesthetized by halothane inhalation before implantation of

subcutaneous osmotic minipumps (Alzet model 2004). Eight animals received pumps

that delivered Ang II at 100 ng/kg/min. Four animals received an additional pump that

delivered Ang-(1-7) at 100 ng/kg/min. Six animals underwent sham surgery. For a

detailed method for loading osmotic minipumps, please see Appendix A.

Indirect Blood Pressure

Systolic blood pressure was determined weekly using an indirect tail cuff method

as previously described (Katovich et al., 2001). Briefly, animals were heated using a

200-watt heat lamp for 5 minutes before restraint in a heated Plexiglas restrainer to which

the animals had been previously conditioned. A pneumatic pulse sensor was then

attached to the tail distal to a pressure cuff under the control of a Programmed Electro-

Sphygmomanometer (Narco). Voltage outputs from the pressure sensor bulb and

inflation cuff were recorded and analyzed electronically using a PowerLab signal

transduction unit and associated Chart software (ADInstruments). At least three separate

indirect pressures were averaged for each animal.









Cardiac Remodeling

Following four weeks of chronic treatment, animals were sacrificed by overdose of

halothane inhalant, and hearts were removed by blunt dissection. Heart ventricles were

cleaned of atria and other adherent tissue before wet weight was determined. Cross-

sections of the heart ventricles were then cut at the level of the papillary muscles,

embedded in paraffin, and sectioned at 4 microns. Sections were then stained using

Masson's Trichrome, and collagen content was determined using the ImageJ program

from NIH (Rasband, 1997), as previously described (Grobe et al., 2006 Chapter 3).

For a detailed method, please see Appendix B.

Statistics

Indirect blood pressure data were analyzed by one-way repeated measures

ANOVA. Cardiac hypertrophy measurements were analyzed by one-way ANOVA.

Interstitial fibrosis was analyzed by nonparametric analyses, namely an overall Kruskal-

Wallis test followed by post-hoc Mann-Whitney U tests. For all analyses, P<0.05 was

considered statistically significant.

Results

Blood Pressure

Angiotensin II (100 ng/kg/min) infusion resulted in significant increases in indirect

systolic blood pressures (Figure 2-1).

Cardiac Hypertrophy

Ventricular size was significantly increased in animals infused with Ang II (Figure

2-2A). Angiotensin-(1-7) had no significant effect on this increase in ventricle size, but a

trend toward reversal was observed. Ang-(1-7) did, however, significantly prevent the

Ang II-induced increase in individual cardiac myocyte diameter (Figure 2-2B).











170 &Ang II
--Ang II + Ang-(1-7)
1 Sham
160


2 150

0 140

130

120

110 ,
0 1 2 3
Weeks


Figure 2-1. Chronic Ang-(1-7) has no effect on blood pressure increases with chronic
Ang II infusion. Analysis by one-way repeated measures ANOVA reveals a
significant increase in BP with Ang II (P=0.0053), and Ang II + Ang-(1-7)
was not different from Ang II alone (P=0.4579). Data presented as mean +
SE.


17

S16

15
i.
S14

I 13


Ang II Ang II +
Ang-(1-7)


Sham


e B














Ang II Ang II+
Ang-(1-7)


Figure 2-2. Chronic Ang-(1-7) prevents myocyte hypertrophy due to chronic Ang II
infusion. (A) Heart weight, normalized to body weight, is significantly
increased by chronic Ang II. Chronic Ang-(1-7) causes a non-significant
(P=0.09) trend toward reversing this effect. (B) Chronic infusion of Ang II
significantly increases myocyte cross-sectional diameter, and chronic Ang-(1-
7) significantly prevents this effect. (* indicates P<0.05 vs. Sham, and t
indicates P<0.05 vs. Ang II) Data presented as mean SE.


3.4

3.3

, 3.2

3.1*

3.0-

w 2.9-

2.8

2.7


Sham









Cardiac Fibrosis

Interstitial collagen deposition was significantly increased in animals with chronic

Ang II infusion, and this increase was prevented by simultaneous Ang-(1-7) infusion

(Figure 2-3).


Sham Ang II Ang II +
Ang-(1-7)


Sham


Ang II Ang II + Ang-(1-7)


Figure 2-3. Chronic Ang-(1-7) prevents interstitial fibrosis due to chronic Ang II
infusion. (A) Infusion of Ang II significantly increased interstitial fibrosis in
the left ventricle wall. Infusion of Ang-(1-7) significantly prevented this
effect. (* indicates P<0.05 vs Sham, and t indicates P<0.05 vs. Ang II) Data
presented as mean + SE. (B) Representative 250x-magnification images
illustrate changes in myocyte size and interstitial collagen.

Discussion

In this study, chronic Ang II infusion resulted in increased blood pressure,

hypertrophied heart ventricles and cardiac myocytes, and increased interstitial collagen









deposition. Simultaneous infusion of Ang-(1-7) at a nearly equal molar rate prevented

cardiac remodeling, both in terms of hypertrophy and fibrosis, without effect on blood

pressure. These findings are parallel to those observed with ACE2 overexpression during

Ang II infusion (Huentelman et al., 2005).

The finding that chronic Ang-(1-7) provides the same cardioprotection as

overexpression of ACE2 supports the concept that Ang-(1-7) mediates the cardiovascular

actions of ACE2. Further, these findings indicate that Ang-(1-7) is an active hormone

(rather than simply a degradation product of the trophic Ang II peptide).

There are several possible mechanisms by which Ang-(1-7) may inhibit

pathophysiologies induced by Ang II. First, it has been proposed that Ang-(1-7) may

antagonize the AT1 receptor (Gironacci et al. 1999; Caruso-Neves et al., 2000; Ferrario et

al., 2005; Ferrario et al., 2005; Igase et al., 2005). Second, some evidence exists that

Ang-(1-7) selectively activates the AT2 receptor (Santos et al., 2000; De Souza et al.,

2004; Gironacci et al., 2004; Roks et al., 2004), and thereby opposes the effects of AT1

receptor activation. Third, Ang-(1-7) may inhibit ACE (Donoghue et al., 2000), and

therefore inhibit endogenous production of Ang II. Fourth, Ang-(1-7) may act through its

own receptor to effect changes in cellular function directly (Santos et al., 2003; Tallant et

al., 2005).

Several groups have proposed that Ang-(1-7) may bind the AT1 receptor. For

example, Ang-(1-7) stimulates the Na -ATPase, but not the Na+,K+-ATPase activity

present in pig kidney proximal tubules, and this stimulation is attenuated by losartan (a

selective AT1 antagonist), but not PD-123,319 (a selective AT2 antagonist) or D-Ala-

Ang-(1-7) (also known as A779, an Ang-(1-7) receptor binding antagonist) (Caruso-









Neves et al., 2000). Similarly, others have shown that radiolabeled Ang II binds to ATi

receptors in rat renal cortex, and that Ang-(1-7) displaces the Ang II with high affinity

(Gironacci et al., 1999). Clearly it is possible that Ang-(1-7) may bind to the AT1

receptor in target tissues. If Ang-(1-7) did interfere with Ang II binding to AT1 receptors

in the present study, though, one would hypothesize that the Ang-(1-7) would interfere

with pressor effects of chronic Ang II (which are well established to be AT1 dependent).

Indirect pressure data over time (Figure 2-1), however, fail to support this hypothesis.

Therefore, Ang-(1-7) antagonism of AT1 receptors seems unlikely as the cardioprotective

mechanism in the current study.

Activation of the AT2 receptor is another possible mechanism by which Ang-(1-7)

prevents cardiac remodeling in the present study. Ang-(1-7) inhibits the pig renal inner

cortex basolateral Na -ATPase activity in a dose-dependent manner, and this effect is

attenuated by PD-123,319 but not losartan or A779 (De Souza et al., 2004). Ang-(1-7)-

mediated inhibition of the release of norepinephrine in the spontaneously hypertensive rat

is also AT2 dependent (Gironacci et al., 2004), as is the endothelium-dependent, non-

competitive antagonism of Ang II contraction of aortic rings (Roks et al., 2004), and the

osmotic water permeability in isolated toad skin (Santos et al., 2000). It has been shown

that AT2 receptors are cardioprotective (Metcalfe et al., 2004), and these receptors are

known to be expressed in both cardiac myocytes (Matsubara, 1998) and fibroblasts

(Lijnen and Petrov, 1999; Lijnen and Petrov, 2003), although their expression profiles

during adulthood are debated. Therefore, it is possible that AT2-mediated transduction of

Ang-(1-7) signaling could account for the present results.









Another possible mechanism by which Ang-(1-7) may mediate its effects is

through the inhibition of the ACE enzyme. This enzyme is responsible for most of the

endogenous production of Ang II in rats, and is also known to degrade Ang-(1-7). It has

been suggested (Tom et al., 2003) that Ang-(1-7) may work through inhibition of ACE,

and therefore decreased production of Ang II. This inhibition could represent a feedback

loop, with the degradation product of Ang II regulating the production of Ang II.

Although this mechanism seems plausible, it is unlikely that decreased production of Ang

II through ACE inhibition could account for the present results, as Ang II is chronically

infused in the current paradigm. Therefore, it is unlikely that Ang-(1-7) inhibition of Ang

II production through ACE would account for the present results. Inhibition of ACE

could also represent a mechanism of cross-talk with the bradykinin / nitric oxide system,

though, which likely could mediate the observed effects. Active (vasodilatory and

cardioprotective) bradykinin is inactivated and degraded to bradykinin-(1-7) by ACE

(Tom et al., 2003). In fact, more than 50% of the degradation of bradykinin in vivo is

mediated by ACE (Goodman and Gilman, 2001). It is plausible that Ang-(1-7) may, by

inhibiting the destruction of the cardioprotective bradykinin through inhibition of ACE,

elicit the observed cardioprotective actions. One would expect, though, that increased

survival of bradykinin through Ang-(1-7) inhibition of ACE would significantly impact

blood pressure regulation. Therefore, although it is possible that inhibition of ACE may

account for the cardioprotective actions of Ang-(1-7), the observed effects of chronic

Ang-(1-7) on blood pressure do not support this hypothesis.

A few years ago, Santos et al. (2003) established that Ang-(1-7) selectively binds

the G-protein coupled receptor Mas, and that the anti-diuretic actions of Ang-(1-7) in the









mouse kidney are Mas-dependent. Subsequent studies by others have established that

Ang-(1-7) mediates anti-hypertrophic (Tallant et al., 2005) and vasodilator (Lemos et al.,

2005) effects in vitro through the Mas receptor. In light of these findings, it seems

possible that chronic Ang-(1-7) effects on cardiac structure in vivo are mediated through

the Mas receptor.

While the determination that Ang-(1-7) actively opposes the trophic, fibrotic

actions of Ang II is significant, this study does not clarify the mechanism of Ang-(1-7)

action, nor does it determine if the actions of Ang-(1-7) are limited to Ang II-mediated

cardiac remodeling. Therefore, the next study was designed to determine if Ang-(1-7) is

effective in preventing cardiac remodeling in a hypertensive animal model that displays

low circulating Ang II levels.














CHAPTER 3
CHRONIC ANGIOTENSIN-(1-7) PREVENTS CARDIAC FIBROSIS IN THE DOCA-
SALT MODEL OF HYPERTENSION

Note: This chapter has already been published in the American Journal of
Physiology: Heart & Circulatory Physiology. Grobe JL, Mecca AP, Mao H, and
Katovich MJ. Chronic angiotensin-(1-7) prevents cardiac fibrosis in the DOCA-
salt model of hypertension. Am JPhysiol Heart Circ Physiol. [Epub ahead of
print], 2006.

Abstract

Cardiac remodeling is a hallmark hypertension-induced pathophysiology. In the

current study, the role of the angiotensin-(1-7) fragment in modulating cardiac

remodeling was examined. Sprague-Dawley rats underwent uninephrectomy surgery and

were implanted with a deoxycorticosterone acetate pellet (DOCA). DOCA animals had

their drinking water replaced with 0.9% saline solution. A subgroup of DOCA-salt

animals was implanted with osmotic minipumps, which delivered angiotensin-(1-7)

chronically (100 ng/kg/min). Control animals underwent sham surgery and were

maintained on normal drinking water. Blood pressure (BP) was measured weekly using a

tail-cuff method, and after four weeks of treatment, BP responses to graded doses of

angiotensin II were determined by direct carotid artery cannulation. Ventricle size was

measured and cross-sections of the heart ventricles were paraffin embedded and stained

using Masson's Trichrome to measure interstitial and perivascular collagen deposition

and myocyte diameter. DOCA-salt treatment caused significant increases in blood

pressure, cardiac hypertrophy, and myocardial and perivascular fibrosis. Angiotensin-(1-

7) infusion prevented the collagen deposition effects without any effect on blood pressure









or cardiac hypertrophy. These results indicate that angiotensin-(1-7) selectively prevents

cardiac fibrosis independent of blood pressure or cardiac hypertrophy in the DOCA-salt

model of hypertension.

Introduction

Cardiac fibrosis is a major facet of hypertensive cardiac disease, and it interferes

with the normal function and structure of the myocardium (Brilla et al. 1991; Weber and

Brilla 1991; Weber, 2000). Increased deposition of basement membrane collagen is a

hallmark of the remodeling process, and results in an increase in cardiac tissue stiffness.

This remodeling predisposes the patient to an increased risk of adverse cardiac events,

including myocardial ischemia, infarction, arrhythmias and sudden cardiac death (Weber,

2000). Thus, prevention and reversal of cardiac fibrosis are essential in the management

of hypertensive heart disease.

The renin angiotensin aldosterone system (RAAS) has been suggested to

participate in the development of end-organ damage in hypertensive patients (Brunner et

al., 1972; Alderman et al. 1991). Support for this concept comes from clinical trials that

demonstrate treatment of hypertensive patients with either ACE inhibitors (Pfeffer et al.,

1992; The SOLVD Investigators, 1992) or AT1 receptor blockers (Pitt et al., 1997;

Thurmann et al. 1998) provides significant protection from, and even reversal of, end-

organ damage. Animal studies have also demonstrated that ACE inhibitors and ATi

receptor antagonists prevent cardiovascular injury (Jalil et al., 1991; Stier et al., 1991;

Stier et al., 1993) as well as protect against renal (Anderson et al., 1986; Stier et al.,

1991; Stier et al. 1998) and cerebral (Stier et al. 1991; Stier etal., 1993; Stier etal.,

1998) injury.









The use of these antagonists of the RAAS not only reduce the formation and

actions of angiotensin II (Ang II), but also result in a significant elevation of another

fragment of the RAAS, angiotensin-(1-7) (Ang-(1-7)) (Ferrario et al., 2005; Keidar et al.,

2005). This peptide, which can be generated locally in the myocardium (Santos et al.,

1990), has been reported to work antagonistically to Ang II and has been implicated in

protecting against cardiac pathophysiology (Santos et al., 2004). Recently, Loot et al.

(2002) demonstrated that chronic infusion of Ang-(1-7) improved endothelial function

and coronary perfusion, and preserved cardiac function in an animal model of heart

failure. Ang-(1-7) has also been shown to significantly increase cardiac output and stroke

volume in rodents (Ferreira et al., 2002) and to improve contractile function in isolated

perfused rat hearts (Sampaio et al., 2003). Using a fusion protein to generate a transgenic

rat model that overproduces Ang-(1-7), Santos et al. (2004) showed that Ang-(1-7)

reduced the induction of cardiac hypertrophy and the duration of reperfusion arrhythmias,

and improved post-ischemic function in isolated perfused hearts. Thus, Ang-(1-7) may

be an important component of the RAAS that has opposing actions to Ang II, and it may

provide protective actions) in the heart and possibly other end-organs. Angiotensin-(1-

7) also recently has been reported to inhibit the growth of cardiac myocytes in vitro

(Tallant et al., 2005), suggesting that elevated levels of Ang-(1-7) may also protect

against cardiac hypertrophy in hypertension. Interestingly, blockade of Ang-(1-7)

receptors has also been shown to reverse the antihypertensive effects of long term

administration of ACE inhibitors (Iyer et al., 1998). Thus, Ang-(1-7) may play a

significant role in the beneficial effects on the cardiovascular system attributed to some

antihypertensive agents.









Not only has Ang II been implicated in the development of cardiac fibrosis, but so

has the aldosterone component of the RAAS (Funder, 2001). It has been demonstrated in

experimental studies utilizing uninephrectomized rats, that deoxycorticosterone acetate

supplementation (DOCA-salt) treatment results in significant cardiac and renal

remodeling, including intersititial fibrosis and hypertrophy (Dobrzynski, et al, 2000;

Young et al. 1995). The purpose of this study was to determine if chronically elevated

levels of Ang-(1-7) alters the development of hypertension, cardiac hypertrophy and/or

cardiac fibrosis in the DOCA-salt model of hypertension.

Methods

Animals

Male Sprague-Dawley rats weighing between 170 and 250 grams were used for this

study. Animals were housed in a temperature and humidity controlled room in standard

shoebox cages, and maintained on a 12:12 hour light:dark schedule with free access to

food and water. All procedures were approved by the University of Florida Institutional

Animal Care and Use Committee.

Chemicals

Angiotensin-(1-7) was obtained from Bachem Bioscience, Inc.

Deoxycorticosterone acetate was purchased from Sigma Aldrich (St. Louis, MO).

DOCA-salt Model

Animals were anesthetized using an intramuscular injection of a Ketamine,

Xylazine Acepromazine, and (30, 6, and 1 mg/kg, i.m., respectively) before

uninephrectomy. During the surgery, fifteen animals were implanted with a

subcutaneous pellet of deoxycorticosterone acetate (40 mg). Seven of these animals were

additionally implanted with a subcutaneous osmotic pump that delivered Ang-(1-7) (100









ng/kg/min) for the duration of the experiment. After surgery (and for the remainder of

the experiment), drinking water for these animals was replaced with 0.9% saline solution.

Ten animals underwent a sham surgery, and were maintained on normal drinking water.

Four additional sham animals were implanted with only an Ang-(1-7) pump.

Indirect Blood Pressure

Systolic blood pressures were determined weekly by an indirect method utilizing a

tail-cuff and pneumatic pulse sensor as previously reported (Katovich et al., 2001).

Briefly, animals were heated using a 200-watt heat lamp for 3-5 minutes before being

restrained in a heated Plexiglas restrainer to which the animals had been conditioned

prior to the experiment. A pneumatic pulse sensor was then attached to the tail distal to a

pressure cuff under the control of a Programmed Electro-Sphygmomanometer (Narco).

Voltage outputs from the pressure sensor bulb and inflation cuff were recorded and

analyzed electronically using a PowerLab signal transduction unit and associated Chart

software (ADInstruments). At least three separate indirect pressures were averaged for

each animal.

Direct Blood Pressure/Acute Angiotensin II Responses

After four weeks of chronic treatment, animals underwent carotid artery and jugular

cannula implantation surgery as previously reported (Lu et al., 1997). Briefly, animals

were anesthetized with a mixture ofKetamine, Xylazine, and Acepromazine (30, 6, and 1

mg/kg, i.m., respectively). A polyethylene cannula (PE-50, Clay Adams) was introduced

into the carotid artery for direct blood pressure measurements, while a silicone elastomer

cannula (Helix Medical) was introduced into the descending jugular vein for acute

intravenous injections of drug. Both cannulae were filled with heparin saline (40 U/mL,

sigma), and sealed with stylets. Animals were allowed 24 hours to recover before









experiments were performed. Direct blood pressure responses to acute intravenous

injections of Ang II (5 to 320 ng/kg) in awake, freely moving animals were recorded by

connecting the carotid cannula to a liquid pressure transducer, which was interfaced to a

PowerLab (ADInstruments) signal transduction unit. Data were analyzed using the Chart

program that was supplied with the PowerLab system.

Cardiac Remodeling

Cardiac remodeling was determined at the end of the direct blood pressure

experiment by ventricular hypertrophy and cardiac fibrosis after four weeks of respective

treatments. Following the acute Ang II blood pressure experiment, animals were

anesthetized by inhaled halothane, then euthanized by decapitation. Ventricular

hypertrophy was determined by measuring ventricle mass normalized by body mass.

Cross-sections of the ventricles were then embedded in paraffin, sectioned at 4 microns,

and stained for collagen deposition using Masson's Trichrome. Single sections from

each animal were then viewed and photographed with a Moticam 1000 digital camera

(Motic, Richmond, British Columbia, Canada) under 10X (whole heart) and 250X

(perivascular collagen and myocyte diameter) magnifications. The collagen content of

the left ventricle wall was determined by measuring the blue stain density of the left wall

(from the septum in the posterior to the septum in the anterior) and was normalized to the

red stain density of the same portion of the image. The area of blue-stained collagen

surrounding the left anterior descending coronary artery was measured and normalized to

the area of the lumen of the vessel. Myocyte cross-sectional diameter was also measured

to confirm ventricular hypertrophy results. Color density, perivascular collagen and

lumen areas, and myocyte diameter were determined using the ImageJ program from

NIH (Rasband, 1997).










Statistics

Basal direct blood pressure and ventricular hypertrophy (by mass and by myocyte

diameter) were analyzed by two-way ANOVA, while indirect blood pressures and direct

blood pressure responses to graded, acute doses of Ang II were analyzed by two-way

ANOVA with repeated measures. Cardiac fibrosis measurements were compared using

the more stringent nonparametric Kruskal-Wallis ANOVA, followed by post-hoc Mann-

Whitney U tests. Data were considered significantly different with p<0.05.

Results

Indirect Blood Pressure

190
--Sham
180 '- DOCA
-D-DOCA + Ang-(1-7)
) 170

E 160

S150

S140

130

120
0 1 2 3
Weeks of Treatment


Figure 3-1. Chronic Ang-(1-7) has no effect on blood pressure increases with DOCA-salt
treatment. DOCA-salt treatment led to a significant increase in systolic blood
pressure as measured by an indirect tail-cuff method (p=0.006). Chronic
infusion of Ang-(1-7) had no effect on the systolic blood pressure increases
due to DOCA-salt treatment (p=0.22). Data presented as mean +/- SE.

DOCA-salt treatment caused a significant increase (p=0.006) in systolic blood

pressure over the course of the experiment. Ang-(1-7), when infused simultaneously,

failed to prevent these increases (p=0.22) (Figure 3-1). Ang-(1-7), when infused alone,










did not significantly alter blood pressure when compared to sham-implanted animals

(p=0.69, data not shown).

Direct Blood Pressure/Acute Angiotensin II Responses

Direct pressure measurements confirmed that while DOCA-salt treatment

significantly increased pressure (p=0.007), Ang-(1-7) had no effect (p=0.20) on basal

systolic blood pressure (Sham, 129.23.7; DOCA, 160.05.2; DOCA + Ang-(1-7),

171.610.8; Ang-(1-7) alone, 143.8+21.0 mmHg). In addition, DOCA-salt treatment

significantly potentiated (p=0.02), and Ang-(1-7) infusion had no effect (p=0.41), on the

dose-response curves to acute, graded injections of Ang II (Figure 3-2).

60
60 Sham

50 DOCA
I J-DOCA + Ang-(1-7)
E 40

C. 30







-10
c r- N G W CI D N
Acute Dose of Ang II (nglkg)


Figure 3-2. Chronic Ang-(1-7) has no effect on acute pressor responses to Ang II in
animals treated with DOCA-salt. DOCA-salt treatment significantly
potentiated the dose-response curve to acute Ang II (p=0.02). Chronic Ang-
(1-7) had no effect (p=0.41) on the acute responses to Ang II. Data presented
as mean +/- SE.

Cardiac Remodeling

DOCA-salt treatment significantly increased ventricle mass (p=0.02), and Ang-(1-

7) failed to prevent this effect (p=0.35) (Figure 3-3A). These effects were confirmed by










measurement of myocyte diameter, which uncovered a significant increase in size with

DOCA-salt treatment (p<0.0001), but no effect of Ang-(1-7) (p=0.71) (Figure 3-3B).


A B
3.4 A is

S17 *
3.2 "*


E
>3.0 M 15
t 8
i 11 14
46 2.8-
M ; 13

2.6 12
E E (< +
0 0
= a
0o 0
04

Figure 3-3. Chronic Ang-(1-7) has no effect on cardiac hypertrophy due to DOCA-salt
treatment. (A) DOCA-salt treatment significantly increased heart mass
(p=0.02). Chronic Ang-(1-7) had no effect on the hypertrophy due to DOCA-
salt treatment (p=0.35). (B) Cardiac myocyte diameter was also significantly
increased by DOCA-salt treatment (p<0.0001), but chronic Ang-(1-7) had no
effect (p=0.71). Data are presented as mean +/- SE. (* indicates p<0.05 vs.
\/hl /l)

A significant increase in left ventricular wall fibrosis was observed with DOCA-

salt treatment (p=0.04 vs. sham), and chronic Ang-(1-7) treatment significantly prevented

this trend (p=0.04 vs. DOCA) (Figure 3-4). While the increase in ventricular wall

fibrosis is only approximately 4%, this value is most likely diluted by our measurement

of collagen in the entire left ventricular wall at 10X magnification (as opposed to the

common method of measuring selected "representative samples" at high magnification).

Clearly, from Figure 3-4B, analysis of "representative areas" within the left wall would

lead to much larger relative changes.













S104-

tt
I 102
L.
-98







Sham DOCA DOCA + Ang-(1-7)










Figure 3-4. Chronic Ang-(1-7) prevents interstitial fibrosis with DOCA-salt treatment.
(A) DOCA-salt treatment caused a significant increase in collagen deposition
in the left ventricle wall. Chronic Ang-(1-7) significantly prevented this
increase. Data are presented as mean +/- SE. (* indicatesp<0.05 vs. .\ln, f
indicates p< 0.05 vs. DOCA). (B) Representative images of mid-myocardium
at 250X magnification, stained with Masson's Trichrome. Blue color
indicates collagen fibers.

DOCA-salt treatment further resulted in a significant (p=0.0002 vs sham) increase

in perivascular collagen deposition around the left anterior descending coronary artery,

and Ang-(1-7) completely prevented this effect (p=0.0007 vs DOCA) (Figure 3-5).

Discussion

In this study, DOCA-salt treatment resulted in increases in blood pressure, acute

pressor responses to Ang II, cardiac hypertrophy, and mid-myocardial and perivascular

fibrosis. The Ang-(1-7) fragment had no effect on the elevated blood pressure, acute










pressor responses or cardiac hypertrophy, but significantly prevented the cardiac mid-

myocardial and perivascular fibrosis in the DOCA-salt hypertensive rat model.


500 A

.- 400
U)

._ 300

200

100

4) 0
S E ? +




B
Sham DOCA DOCA +Ang 1-7










Figure 3-5. Chronic Ang-(1-7) prevents perivascular fibrosis with DOCA-salt treatment.
(A) DOCA-salt significantly increased perivascular collagen around the left
anterior descending coronary artery. Chronic Ang-(1-7) significantly
prevented this effect. Data are presented as mean +/- SE. (* indicates p<0.05
vs. Nhi /n, f indicates p<0.05 vs. DOCA). (B) Representative images of
perivascular collagen surrounding the left anterior descending coronary artery,
stained with Masson's Trichrome. Blue color indicates collagen fibers.

Deoxycorticosterone treatment in uninephrectomized rats drinking 1.0% NaCl has

previously been shown to cause hypertension, cardiac hypertrophy and interstitial and

perivascular fibrosis (Young et al. 1995; Dobrzynski et al., 2000). These cardiac effects

are mediated via mineralocorticoid receptors (Young and Funder, 2000; Rocha and Stier,









2001; Weber, 2001), and are not observed in animals on a low salt diet (Brilla et al.,

1992). Others have suggested the involvement of AT1 receptors in the fibrosis associated

with DOCA-salt treatment (Kim et al. 1994; Nishikawa, 1998). Elevation of cardiac AT1

receptors associated with DOCA-salt treatment may potentiate the well described fibrotic

effect of Ang II. There is evidence that the trigger for cardiac fibrosis in this animal

model is coronary vasculitus, as the inflammatory cell infiltration occurs within a few

days of DOCA treatment (Young and Funder, 2003).

In a study by Young et al. (1995), the cardiac hypertrophy response to DOCA

treatment was restricted to the left ventricle, whereas the collagen deposition was

indistinguishable between left and right ventricle, thus supporting a humoral, rather than

a hemodynamic, etiology. Further support for a humoral etiology for collagen deposition

comes from Weber's (1994) laboratory, where spironolactone prevented aldosterone-

induced cardiac fibrosis without lowering blood pressure. Additionally, interstitial and

perivascular fibrosis can be produced by mineralocorticoid and salt without substantial

hypertension or cardiac hypertrophy (Young et al., 1995). Young et al. (1995) also

demonstrated that central infusion of a mineralocorticoid receptor antagonist lowered

blood pressure but not the pattern of cardiac fibrosis, further suggesting an independence

of fibrosis and blood pressure. Our results also demonstrate the fibrosis observed in this

model was found in both the right and left ventricle walls (right ventricle data not

shown), and such fibrosis appears to be independent of both blood pressure and

hypertrophy.

The effects of DOCA on cardiac hypertrophy have been postulated to involve

angiotensin II or transforming growth factor 0 (TGFp) (Young et al., 1994; Takeda et al.,









2002). Whether these same mechanisms are responsible for the cardiac fibrosis are not

clear. Young and Funder (2003) demonstrated that animals treated for as few as 8 days

with DOCA/salt treatment demonstrated a significant increase in cardiac perivascular

collagen deposition, with no changes in heart weight, and the collagen formation was

inhibited by a mineralocorticiod receptor antagonist. Further evidence that the cardiac

hypertrophy and fibrosis may be independent include results from Lekgabe et al. (2005),

who demonstrated that cardiac hypertrophy, and the related markers of hypertrophy that

were elevated in the SHR compared to the WKY, were not reversed with chronic relaxin

treatment, whereas the fibrosis and collagen content in the hearts of the SHR were

normalized by relaxin treatment to levels observed in the WKY. In the current study,

Ang-(1-7) also showed differential effects upon hypertrophy and fibrosis in the DOCA-

salt model of hypertension, suggesting different mechanisms may exist for these two

endpoints of cardiac remodeling.

Nehme et al. (2005) reported that both aldosterone and Ang II receptor antagonists

significantly reduced the cardiac fibrosis induced by coronary artery ligation in male

Wistar rats. The effects of the aldosterone receptor antagonist (spironolactone) were

independent of changes in mean blood pressure. This form of experimental myocardial

infarction (MI) in rats has been shown to activate the RAAS (Pinto et al., 1993,

Schunkert et al., 1993). Spironolactone also has been shown to reduce arterial collagen

production, independent of changes in systemic blood pressure in the spontaneously

hypertensive rat (Benetos et al., 1997) and in an L-NAME (nitro-L-arginine methyl ester)

plus Ang II with salt model of hypertension (Rocha et al., 2000). As in the current study,

Nehme et al. (2005) also reported that these treatments did not prevent the cardiac









hypertrophy induced in this MI model. This is further evidence that the mechanisms for

cardiac hypertrophy and fibrosis may be independent.

Generally, the actions of Ang-(1-7) are opposite of those attributed to Ang II

(Santos et al., 2000). Ang-(1-7) has been reported to have intrinsic vasoactive effects in

some studies (Brosnihan et al., 1996; Iyer et al., 1998; Sasaki et al., 2001; Ren et al.,

2002), whereas these effects are absent in other studies (Davie et al., 1999; Wilsdorf et

al., 2001; Zhu et al., 2002; Stegbauer et al., 2003). Bayorh et al. (2002) demonstrated the

hypotensive effects of Ang-(1-7) were not observed in Dahl animals on a low salt diet

and Eatman et al. (2001) noted a gender difference in the vasodilatory action of Ang-(1-

7) in Dahl rats. Neves et al. (2004) recently reported that the vasodilatory effects of Ang-

(1-7) are modulated during the estrus cycle. Thus, the contrasting findings of Ang-(1-7)

on the vasculature may be related to the differences in animal models studied, dose of

Ang-(1-7), vascular bed studied, or the experimental condition (in vitro versus in vivo) in

which the peptide is evaluated. In the current study, at the dose of Ang-(1-7) infused,

there was no effect on the basal blood pressure of normotensive SD animals or those

animals made hypertensive with DOCA-salt treatment. There are situations, such as in

models of endothelial dysfunction (Le Tran et al., 1997), where the vasodilatory effects

of Ang-(1-7) have been shown to be blunted. This may be the reason for a lack of effect

in our hypertensive model. In the current study, no reduction in the pressor response to

acute administration of Ang II was observed. Benter et al. (1995) reported that infusion

of Ang-(1-7) did not alter the pressor responses to Ang II or phenylephrine in WKY

animals; however, it did reduce the response to these agents in the SHR. A higher dose

of Ang-(1-7) was utilized in the Benter study compared to ours. Although there was no









observable effect on blood pressure at the dose of Ang-(1-7) used in the current study,

there was a significant attenuation of cardiac fibrosis.

The mechanism of action of Ang-(1-7), an active peptide fragment of the renin

angiotensin system, is currently controversial. It has been reported to act as an

angiotensin receptor antagonist, an ACE inhibitor, as well as altering prostaglandins and

the bradykinin-nitric oxide system. These mechanisms have been exhaustively reviewed

by Santos et al. (2000) and others. The mechanisms may involve the reported mas

receptor (Tallant et al., 2005) or direct interaction with other angiotensin receptors

(Santos et al., 2000).

There are several mechanisms by which Ang-(1-7) may decrease collagen

deposition in models of hypertension. First, Ang-(1-7) could reduce blood pressure,

which would decrease hemodynamic-dependent pro-fibrotic signaling. Our results would

not support this mechanism, as changes in blood pressures and pressor responses to acute

Ang II were not observed by indirect or direct methods during chronic Ang-(1-7)

treatment (Figures 3-1 and 3-2). Second, Ang-(1-7) could directly inhibit the secretion of

collagen or the activation of the fibroblasts or their differentiation into myofibroblasts.

Third, Ang-(1-7) may increase collagen degradation by activating matrix

metalloproteinases (MMP's), or by inhibiting tissue inhibitors of matrix

metalloproteinases (TIMP's). Finally, Ang-(1-7) may inhibit the actions of various pro-

fibrotic factors such as norepinephrine, angiotensin II, TGFp, and/or endothelin-1, as

Ang-(1-7) infusion has been reported to result in a reduction of cardiac Ang II levels

(Mendes et al., 2005). Future studies will be aimed at dissecting the possible









mechanisms by which Ang-(1-7) reduces cardiac fibrosis in hypertension and to

determine if this anti-fibrotic action also occurs in non-hypertensive fibrotic conditions.

This study also supports the idea that angiotensin-(1-7) has direct physiological

actions, rather than simply being the result of metabolized Ang II. Our in vivo results are

supported by a recent in vitro study (Iwata et al., 2005). In this study, the authors

examined angiotensin fragment binding in cultured adult rat cardiac fibroblasts, and

showed that Ang-(1-7) binds to two separate sites that are unaffected by the AT1 receptor

antagonist valsartan and the AT2 receptor antagonist PD-123,319. Importantly, these

authors showed that within the concentration ranges studied for binding, Ang-(1-7)

inhibited collagen formation as measured by [3H]proline incorporation, and also

decreased mRNA expression of various growth factors including transforming growth

factor 31 (TGFpi), endothelin-1 (ET-1), and leukemia inhibitory factor (LIF).

Pretreatment of the cardiac fibroblasts with Ang-(1-7) inhibited Ang II-induced collagen

protein. Together, the current in vivo study and the in vitro study by Iwata et al. (2005)

strong support the hypothesis that Ang-(1-7) actively regulates cardiac fibrosis. These

findings further indicate the significant role that Ang-(1-7) plays in the heart.

Our results parallel those of Loot et al. (2002), who demonstrated cardioprotective

effects of Ang-(1-7). In the current study, we utilized one fourth of the dose of Ang-(1-7)

that these authors employed. We did not determine the plasma or cardiac levels of Ang-

(1-7) in the current study. It is conceivable that utilization of higher concentrations of

Ang-(1-7) may result in greater cardioprotection, and it may also uncover effects on

hypertrophy.









The results of the current study fit well with the findings of our recent study, in

which systemic overexpression of ACE2 (the most important enzyme involved in

angiotensin-(1-7) production) in angiotensin-infusion hypertensive rats resulted in blood

pressure-independent decreases in cardiac fibrosis (Huentelman et al., 2005). This study

supports the concept that ACE2 mediates many (or most) of its protective effects through

the production of angiotensin-(1-7). Myocardial infarction increases ACE2 expression in

rats and humans (Burrell et al., 2005). Averill et al. (2003) also reported that Ang-(1-7)

immunoreactivity was significantly increased in the ventricular tissue surrounding an area

of myocardial infarction and absent in the remodeled area of infarction. Thus, there is

evidence suggesting that the ACE2 enzyme and the formation of Ang-(1-7) play

significant roles in modifying cardiac remodeling.

Recently, Keider et al. (2005) demonstrated that the mineralocorticoid aldosterone

significantly increased cardiac ACE and decreased ACE2 mRNA and activity levels in

both humans and mice, whereas aldosterone antagonists produced the opposite responses.

This data would suggest that ACE2 levels, and the subsequent generation of Ang-(1-7)

may be the mechanism by which mineralocorticoid receptor blockers improve cardiac

function in myocardial infarct patients (Pitt et al., 2003).

Several investigators (Abraham and Simon, 1994; Simon et al., 1995; Melargno

and Fink, 1996) have demonstrated that chronic infusion of angiotensin II results in the

development of hypertension, cardiac hypertrophy and fibrosis. Functional Ang II

receptors have been documented in cardiac fibroblasts (Crabbos et al., 1994) and Ang II-

stimulated expression and secretion of collagen is completely abolished by AT1 and not

AT2 receptor antagonism in vivo and in vitro (Lijnen and Petrov, 1999, Lijnen et al.,









2001). As Ang-(1-7) is significantly elevated with the use of AT1 antagonists (Ferrario et

al., 2005), it is possible that some of the anti-fibrotic effects attributed to AT1 antagonism

are mediated via actions of Ang-(1-7).

The development of cardiac fibrosis is a major complication of hypertensive

cardiac disease. The increased deposition of basement membrane collagen is a hallmark

of the remodeling process. This remodeling can predispose a patient to increased risk of

adverse cardiac events, and therefore any reduction or reversal of cardiac fibrosis would

facilitate the management of hypertensive heart disease. Our results suggest that Ang-(1-

7) may be a beneficial component of the RAAS that provides this cardioprotection during

hypertension, and any maneuver that would increase cardiac levels of ACE2 and

subsequently Ang-(1-7) may be used as an effective therapeutic intervention in

hypertensive heart disease patients.














CHAPTER 4
PROTECTIVE EFFECTS OF ANGIOTENSIN-(1-7) DURING ISOPROTERENOL-
INDUCED CARDIAC REMODELING

Abstract

P-Adrenergic stimulation of cardiac tissue leads to myocyte hypertrophy and can

result in heart failure. A recent study by Santos et al. showed that transgenic rats

overexpressing an angiotensin-(1-7) (Ang-(1-7)) fusion gene were significantly protected

from cardiac hypertrophy and arrhythmias induced by administration of the 0-adrenergic

receptor agonist isoproterenol. Here, we first characterized the effects of isoproterenol in

male and female rats, then examined the hypothesis that chronic infusion of Ang-(1-7)

would prevent cardiac hypertrophy and fibrosis due to isoproterenol administration.

Intact male, intact female, and ovariectomized female Sprague-Dawley rats between 180-

280 grams were injected with isoproterenol at either 1 or 2 mg/kg daily for fourteen

consecutive days. A subset of the 2 mg/kg isoproterenol-treated male rats were

implanted with osmotic minipumps which chronically delivered Ang-(1-7) (100

ng/kg/min). Hearts were harvested and weighed, and the (2 mg/kg isoproterenol) male

rat hearts were sectioned and stained using Masson's Trichrome. Isoproterenol injections

resulted in large increases in heart size in males at both doses, but had no effect on

cardiac fibrosis. While isoproterenol also induced large increases in heart size in intact

females, this effect was blunted in ovariectomized animals. Chronic infusion of Ang-(1-

7) in the 2 mg/kg male group resulted in significantly attenuated cardiac hypertrophy.

From these studies we conclude both that Ang-(1-7) inhibits the hypertrophic response to









isoproterenol, and that ovarian hormones are permissive to the hypertrophic effects of

isoproterenol. These findings lead us to the hypothesis that ovarian hormones may alter

cardiac tissue sensitivity to the protective Ang-(1-7).

Introduction

High blood pressure is a key risk factor for cardiac remodeling and subsequent

heart failure (Regitz-Zagrosek and Lehmkuhl, 2005; Thom et al., 2006). Others have

previously demonstrated that reductions in arterial pressure can prevent or even reverse

the cardiac remodeling associated with high blood pressure (Diez et al., 2001). To

determine whether the cardioprotective effects of Ang-(1-7) are mediated by decreased

arterial pressure (or limited to hypertensive models), the following study was carried out

using a normotensive model of cardiac remodeling.

Isoproterenol is a pharmacological catecholamine with an N-alkyl substitution,

making it almost entirely selective for P-adrenergic receptors (Jacob, 1996; Goodman and

Gilman, 2001). Administration of isoproterenol results in decreased peripheral resistance

through stimulation of 32 receptors, which leads to a decrease in diastolic pressure.

Although systolic pressure typically is normal or slightly increased, mean arterial

pressure is usually depressed. Stimulation of 31 receptors in the heart results in positive

inotropic and chronotropic effects, thus increasing cardiac output. Stimulation of cardiac

tissue by isoproterenol may lead to sinus tachycardia, palpitations, and more serious

arrhythmias, and large doses may cause myocardial necrosis (Goodman and Gilman,

2001). These properties of isoproterenol have led many investigators to use the

compound to induce cardiac remodeling without increasing blood pressure.

A recent study by Santos et al. (2004) found that increasing circulating Ang-(1-7)

levels protected rats from isoproterenol-induced hypertrophy and associated arrhythmias.









While the data are encouraging, the authors utilized transgenic rats that overexpressed an

Ang-(1-7) fusion protein, which confounds interpretation of the physiological data as the

role of Ang-(1-7) during development is currently unknown. Further, the authors did not

examine the effect of Ang-(1-7) on isoproterenol-induced cardiac fibrosis.

In the present study, daily injections of isoproterenol were used to induce cardiac

remodeling. Several groups of both male and female animals were tested at various

doses of isoproterenol to characterize this normotensive model of cardiac remodeling

before testing the cardioprotective effects of angiotensin-(1-7) in this model.

Methods

Animals

Group A Males with isoproterenol at 1 mg/kg

Fourteen male Sprague-Dawley rats weighing 220-250 grams were divided into

two groups. The first group, with eight animals, was treated with daily injections of 0.9%

saline solution (1 mL/kg, s.c.). The second group, with six animals, was treated with

daily injections of isoproterenol (1 mg/kg, s.c.). Daily injections were performed

between 11:00 AM and 1:00 PM. Both groups were treated for fourteen consecutive

days, and on the fifteenth day all animals were sacrificed without a final injection.

Group B Ovariectomized and intact females with isoproterenol at 1 and 2 mg/kg

Twenty-four female Sprage-Dawley rats weighing 180-210 grams were divided

into two groups of twelve animals each. The first group underwent ovariectomy surgery,

while the second group served as intact controls. Within each group, three subgroups

were formed, including a saline control-injection subgroup and two subgroups that

received daily isoproterenol injections at either 1 mg/kg or 2 mg/kg, s.c. Daily injections









were performed between 11:00 AM and 1:00 PM for fourteen days. On the fifteenth day,

animals were sacrificed without a final injection.

Group C Males with isoproterenol at 2 mg/kg

Seven male Sprague-Dawley rats weighing 240-280 grams were divided into three

groups. Three animals served as saline-injection controls. Two animals received daily

injections of isoproterenol (2 mg/kg, s.c.). The final two animals were implanted with

subcutaneous osmotic minipumps (Alzet, model 2002), which chronically delivered

angiotensin-(1-7) at 100 ng/kg/min. This final group also received daily injections of

isoproterenol (2 mg/kg, s.c.). Again, daily injections were performed between 11:00 AM

and 1:00 PM for fourteen days. On the fifteenth day, animals were sacrificed without a

final injection.

Indirect Blood Pressure

Indirect blood pressures were recorded from animals in group C one day before

pump implantation and isoproterenol injections, and weekly during the isoproterenol

treatment using an indirect tail-cuff method as described in previous experiments.

Briefly, animals were heated using a 200-watt heat lamp for 3-5 minutes before being

restrained in a heated Plexiglas restrainer to which the animals had been conditioned

prior to the experiment. A pneumatic pulse sensor was then attached to the tail distal to a

pressure cuff under the control of a Programmed Electro-Sphygmomanometer (Narco).

Voltage outputs from the pressure sensor bulb and inflation cuff were recorded and

analyzed electronically using a PowerLab signal transduction unit and associated Chart

software (ADInstruments). At least three separate indirect pressures were averaged for

each animal.









Cardiac Remodeling

Following fourteen days of isoproterenol injections with or without chronic

infusion of angiotensin-(1-7), animals were sacrificed by overdose of halothane inhalant.

Hearts were removed by blunt dissection, weighed, and sectioned. Again, as in previous

experiments, cross-sections at the level of the papillary muscles were then fixed in 10%

formalin solution, embedded in paraffin, sectioned at 4 microns, and stained using

Masson's Trichrome. Single sections from each animal were then viewed and

photographed with a Moticam 1000 digital camera (Motic, Richmond, British Columbia,

Canada) with a 10X magnification. The collagen content of the left ventricle wall was

determined by measuring the blue stain density of the left wall (from the septum in the

posterior to the septum in the anterior) and was normalized to the red stain density of the

same portion of the image. The area of blue-stained collagen surrounding the left

anterior descending coronary artery was measured and normalized to the area of the

lumen of the vessel. Color density and perivascular collagen and lumen areas were

determined using the ImageJ program from NIH (Rasband, 1997).

Results

Cardiac Hypertrophy

Male rats injected daily with 1 mg/kg isoproterenol (Group A) displayed marked

increases in cardiac hypertrophy (Figure 4-1). The increase in size was approximately

28%, which is much larger than that observed with the Ang II (5%) or DOCA-salt (14%)

models of hypertension-induced cardiac remodeling.










5.0


4.5

r*
0 4.0


3.5


I 3.0


2.5
Saline Iso

Figure 4-1. Isoproterenol (1 mg/kg/day) induces cardiac hypertrophy in male rats.
Fourteen days ofisoproterenol treatment resulted in a significantly increased
heart size (P<0.0001). Data presented as mean SE.


MSaline
5.0 lDso, 1 mg/kg

Ilso, 2 mglkg
4.5


0 4.0


S3.5


I 3.0


2.5
Intact Ovariectomized


Figure 4-2. Ovariectomy potentiates the isoproterenol-mediated induction of cardiac
hypertrophy in female rats. Daily injections of isoproterenol resulted in
differential effects on cardiac hypertrophy, depending on ovarian hormone
status. Two-way ANOVA reveals a significant effect of isoproterenol
treatment level (P<0.0001), but ovariectomy treatment only resulted in a non-
signficant trend (P=0.0765). The presence of a significant interaction
(P=0.0308), however, indicates some impact of ovary status. Data presented
as mean SE.






51


Female rats injected daily with isoproterenol (Group B) showed differential

hypertrophic responses to isoproterenol injection based on ovarian hormone status

(Figure 4-2). Isoproterenol significantly increased heart size in both surgery groups

(P<0.0001). Ovariectomy appeared to attenuate the response to isoproterenol, and

although two-way analysis of variance reveals only a non-significant trend with regard to

the effect of surgery (P=0.0765), an significant interaction between surgery and

isoproterenol dose was uncovered (P=0.0308).


5.0 A 108 B


S4.5
significantly increased cardiac hypertrophy (P=0.0007), and chronic infusion
S104
0 4.0 U


33.5 LL
M- 100

S3.0


2,5 96
Saline Iso Iso+ Saline Iso Iso+
Ang(1-7) Ang-(1 -7)

Figure 4-3. Chronic Ang-(1-7) prevents the hypertrophic, but not fibrotic effects of
isoproterenol (2 mg/kg/day) in male rats. (A) Isoproterenol resulted in
significantly increased cardiac hypertrophy (P=0.0007), and chronic infusion
of angiotensin-(1-7) at 100 ng/kg/min significantly attenuated this effect
(P=0.0082). (B) Isoproterenol and angiotensin-(1-7) had no effect on cardiac
fibrosis. Data presented as mean + SE.

Male rats injected daily with 2 mg/kg isoproterenol (Group C) exhibited

tremendous cardiac hypertrophy. As this combination of gender and drug dose resulted

in the greatest effect upon cardiac hypertrophy, the effect of angiotensin-(1-7) was

evaluated with this combination. Chronic infusion of angiotensin-(1-7) significantly









attenuated the hypertrophic response to isoproterenol (Figure 4-3A). These hearts were

then processed to determine the effect of isoproterenol and angiotensin-(1-7) on cardiac

fibrosis. Isoproterenol and angiotensin-(1-7) had no significant effects on cardiac fibrosis

(Figure 4-3B).

Blood Pressure

170 ISaline
~Iso
165
15 so + Ang-(1-7)
C 160
I
E 155
150
145
.2 140
-* 135
5 130
CO)
125
120
0 1 2
Weeks

Figure 4-4. Isoproterenol and Ang-(1-7) had no chronic effects on blood pressure in male
rats. (P=0.4207 and P=0.6615 vs. saline, respectively) Data presented as
mean + SE.

Indirect blood pressure was also determined in the male rats with 2 mg/kg

isoproterenol, as this combination of gender and dose resulted in the greatest changes in

cardiac hypertrophy. Isoproterenol had no significant effect on blood pressure, and

neither did chronic infusion of angiotensin-(1-7) (Figure 4-4).

Discussion

Daily injections of isoproterenol resulted in cardiac hypertrophy in both male and

female rats. It was determined that ovariectomy significantly attenuated the effects of









isoproterenol in females, and that males were slightly more sensitive to isoproterenol-

induced cardiac remodeling than intact females at the 2 mg/kg dose. Angiotensin-(1-7)

infusion significantly attenuated the hypertrophic response in these high-dose

isoproterenol male rats. Cardiac fibrosis and blood pressure were unaffected by the 2

mg/kg dose of isoproterenol in male rats. Further, angiotensin-(1-7) had no effect on

either blood pressure or cardiac fibrosis.

While the lack of effect of angiotensin-(1-7) on blood pressure is unsurprising,

given the results of both the Ang II and DOCA-salt models (Chapters 2 and 3,

respectively), the trend toward increased fibrosis in this model (Figure 4-3B) was

unanticipated. These findings appear to conflict with the results in both the Ang II and

DOCA-salt models, as Ang-(1-7) completely prevented interstitial fibrosis in both of

these models, but also give clues as to the site of Ang-(1-7) interference with pro-fibrotic

signaling. Neves et al. (2005) recently determined that mineralocorticoid receptor

activation mediates the pro-fibrotic actions of Ang II. Bos et al. (2005) found that

isoproterenol-induced fibrosis could be inhibited by antagonism of mineralocorticoid

receptors. Together, these findings suggest that the anti-fibrotic effects of Ang-(1-7) may

work though interference with the pro-fibrotic signaling pathways of Ang II and DOCA,

upstream of the convergence with isoproterenol signals.

The effect of Ang-(1-7) upon ventricular hypertrophy was substantial, which is

again in direct opposition to the effects observed in the Ang II and DOCA-salt models

(Chapters 2 and 3, respectively). Others have determined that antagonism of the

mineralocorticoid receptor (Bos et al., 2005) or AT1 receptors (Nakano et al., 1995) in

vivo can prevent isoproterenol-induced cardiac effects, thus clearly demonstrating cross-









talk among these signaling cascades. Ang-(1-7) likely inhibits the hypertrophy process

between the convergence of signals from Ang II or isoproterenol with signals from

mineralocorticoids.

The difference in inhibitory roles for Ang-(1-7) with regard to hypertrophy and

fibrosis in the Ang II, DOCA-salt, and isoproterenol models likely is related to the

differential effects of Ang-(1-7) in the various cardiac cell types. Tallant et al. (2005)

showed that Ang-(1-7) acts in vitro at the cardiac myocyte to modulate hypertrophy, and

Iwata et al. (2005) also showed that Ang-(1-7) acts in vitro at the cardiac fibroblast to

elicit changes in collagen turnover. Possible differences in intracellular signaling

cascades in each cell type, or differences in their in vitro models versus our in vivo

models, may account for the differential effects of Ang-(1-7) on hypertrophy and fibrosis

with mineralocorticoid or 3-adrenergic stimulation. Further studies in vitro will help

elucidate the intracellular signaling cascade(s) of Ang-(1-7) in myocytes and fibrosis,

which should uncover the biochemical cause of the different outcomes.

Differences in cardiac responsiveness by gender have previously been noted, which

prompted the inclusion of intact and ovariectomized female rats in the present study.

Schwertz et al., (1999) showed that isolated female rat hearts have smaller inotropic

responses to isoproterenol administration than male hearts. In the present study, only

small differences in cardiac hypertrophy responses between males and females were

observed with 1 and 2 mg/kg isoproterenol, but ovariectomized females exhibited blunted

responses to isoproterenol. This may indicate a role for ovarian hormones in regulating

cardiac structure.









Previous work by Tallant et al. (2005) uncovered that the anti-hypertrophic actions

of Ang-(1-7) are mediated at the cardiac myocyte through the Mas receptor. Preliminary

in silico analysis of the Mas receptor promoter indicates that there are several possible

estrogen receptor and AP-1 binding sites within the first few kilobases 5' of the

transcription start site, thus suggesting that estrogens may alter expression of this receptor

(data not shown). These findings, along with our present study of cardiac hypertrophy

responses in ovariectomized versus intact animals, lead us to the hypothesis that ovarian

hormones may decrease tissue sensitivity to the cardioprotective angiotensin-(1-7)

hormone.

It has been documented that the Ang-(1-7) receptor (Mas) is found in the

vasculature, and stimulation of this receptor results in a relaxation response (le Tran and

Forster, 1997). Therefore, in the subsequent study we decided to evaluate estrogenic

modulation of vascular reactivity to Ang-(1-7) in rat aorta.














CHAPTER 5
ALTERATIONS IN AORTIC VASCULAR REACTIVITY TO ANGIOTENSIN-(1-7)
IN 17-P-ESTRADIOL-TREATED FEMALE SD RATS

Note: This chapter has already been published in Regulatory Peptides. Grobe, JL
and Katovich MJ. Alterations in aortic vascular reactivity to angiotensin-(1-7) in
17-P-estradiol-treated female SD rats. RegPept 133:62-67, 2006.

Abstract

Estrogen's suggested cardio-protective effects have come into question following

the results of recent clinical trials. Two major components of the renin-angiotensin

system (RAS) that are modulated by estrogen are angiotensin converting enzyme, and the

angiotensin II type 1 receptor. Further research has revealed several new components of

the RAS, including angiotensin converting enzyme 2, its peptide product angiotensin-(1-

7) (Ang-(1-7)), and that peptide's receptor, Mas. These components appear to oppose the

classical effects of the RAS, and may act to buffer the RAS in vivo. Recent work has

shown that during pregnancy, when estradiol levels are elevated, renal and urinary Ang-

(1-7) are greatly increased. This study examined the effects of estradiol on the efficacy of

Ang-(1-7) in the rat aorta. Female Sprague-Dawley rats were ovariectomized and a

subgroup was chronically treated with subcutaneous pellets of estradiol for 3 weeks.

Thoracic aortas were harvested for assessment of in vitro vascular reactivity to Ang-(1-

7). The results demonstrated that increased estradiol exposure attenuated the relaxation

response to Ang-(1-7) in a dose-dependent manner. These findings are in contrast to

recent work showing potentiated responses to Ang-(1-7) in mesenteric arteries from









estrogen-manipulated rats, and may suggest a regional specificity in estradiol-mediated

changes in the RAS.

Introduction

The leading cause of death in women is cardiovascular disease (CVD). Blood

pressure (BP) is higher in men than in premenopausal women at similar ages, but

following menopause, BP increases in women to levels higher than men (Tremollieres et

al., 1999; Harrison-Bernard and Raij, 2000). In the United States, nearly 38% of

postmenopausal women use some form of hormone replacement therapy (HRT) (Keating

et al., 1999). For many years, it has been thought that HRT provided women some level

of protection against CVD, specifically with regard to myocardial infarction (Grodstein

and Stampfer, 1995; Grodstein and Stampfer, 1998; Mendelsohn and Karas, 1999;

Arkhrass et al., 2003).

Recent clinical trials, including the Women's Health Initiative (WHI) and the Heart

and Estrogen-progestin Replacement Study (HERS), have cast some doubt on the

protective effects of estrogens in cardiovascular biology, as these studies failed to show a

decreased risk for stroke in postmenopausal women treated with HRT (Simon et al.,

2001). The WHI was stopped 2 years early due to an increase in risk of heart disease,

strokes, and blood clots (Hulley et al., 2002; Rossouw et al., 2002). Questions remain,

then, as to what specific harmful and/or protective effects estrogens have on the

cardiovascular system.

One endogenous hormonal system that is very important in cardiovascular

regulation and is altered by estrogen is the renin-angiotensin system. Specifically,

estrogen has been shown to stimulate the production of angiotensinogen (Healy et al.,

1992) and the angiotensin II type 2 receptor (AT2R) (Armando et al., 2002), and to









inhibit the production of prorenin (Szilagyi et al., 1995), angiotensin converting enzyme

(ACE) (Turner and Hooper, 2002), neprilysin (Pinto et al., 1999), aminopeptidase N (Seli

et al., 2001), and the angiotensin II type 1 receptor (ATiR) (Nickenig et al., 2000). These

changes in the RAS have the net effect of attenuating the pressor effects of the RAS, but

potentiating the depressor effects. Collectively, these effects of estrogen on components

of the renin-angiotensin system should reduce blood pressure and offer some

cardiovascular protective effects.

Angiotensin-(1-7) (Ang-(1-7)) is an endogenous product of the renin-angiotensin

system. Production of this peptide hormone is achieved by three distinct pathways,

including breakdown of angiotensin II (Ang II) by angiotensin converting enzyme 2

(ACE2) (Donoghue et al., 2000; Tipnis et al., 2000), and breakdown of angiotensin I

(Ang I) both directly by various endopeptidases and also through the intermediate

formation of angiotensin-(1-9) by ACE2 followed by digestion by ACE (Ferrario and

Chappell, 2004). Ang-(1-7) is then degraded by ACE to form an inactive product,

angiotensin-(1-5) (Ferrario and Chappell, 2004).

At least three possible physiological actions of Ang-(1-7) have been suggested.

First, it is possible that Ang-(1-7) acts as a partial agonist or antagonist of the AT1 and/or

AT2 receptors. This is supported by evidence that Ang-(1-7) can block the effects of Ang

II (Brosnihan et al., 1998; Chaves et al., 2000; Machado et al., 2001; Pinheiro et al.,

2004), and that in some tissues Ang-(1-7) can signal through the AT1R (Gironacci et al.,

1998; Caruso-Neves et al., 2000) and the AT2R (Santos et al., 2000; De Souza et al.,

2004; Gironacci et al., 2004; Roks et al., 2004). Second, it is possible that Ang-(1-7) acts

through its own receptor. The Mas receptor has been suggested as one possible Ang-(1-7)









receptor, and various experiments have shown changes in Ang-(1-7) signaling in Mas-

altered tissues and animals (Santos et al., 2003). Third, it has been suggested that Ang-(1-

7) may act as an endogenous ACE inhibitor. This is supported by experiments which

examined the catalytic efficiency of ACE in the presence of Ang-(1-7) (Iyer et al., 1998;

Collister and Hendel, 2003; Tom et al., 2003).

Recent work by Broshinhan and colleagues (Brosnihan et al., 2003; Neves et al.,

2003) has shown that both the production and the efficacy of Ang-(1-7) are altered in

pregnancy. Specifically, during pregnancy, plasma and urinary Ang-(1-7) increase

significantly, and mesenteric artery preparations are much more reactive to Ang-(1-7).

Because circulating estrogen increases during pregnancy, it is conceivable that estrogens

may control production and/or sensitivity to Ang-(1-7).

The current study was undertaken to further examine the effects of estrogen on

vascular reactivity to Ang-(1-7). Aortic rings were used to examine the Ang-(1-7) effects

upon the rat vasculature, as it has been shown that rat aorta relax to Ang-(1-7) in an

endothelium-dependent manner (Le Tran and Forster, 1997). As estrogen significantly

increases in pregnancy, it was hypothesized that estrogen would cause a significant

potentiation of vascular responses to Ang-(1-7).

Methods

Animal Housing and Surgery

Twelve female Sprague-Dawley rats (150-200 g) were housed doubly in shoebox

style cages with free access to standard rat chow and tap water, and were maintained on a

12 : 12 h light: dark cycle, beginning at 7:00 AM. Twenty-one days before the aortic

vascular reactivity experiment, all animals were ovariectomized while under anesthesia

by xylazine/acepromazine/ketamine injectable cocktail. Subsets of animals (four each









group) received estradiol replacement via subcutaneous pellet (0.05 or 25 mg). All

procedures involving animals were approved by the University of Florida Institutional

Animal Care and Use Committee.

Reagents

Subcutaneous estradiol pellets were obtained from IRA (Innovative Research of

America, Sarasota, FL). All chemicals and peptides for aortic vascular reactivity

experiments were obtained from Sigma Aldrich (St. Louis, MO). Estradiol ELISA kit

was obtained from ALPCO, Inc. (Windham, NH).

Aortic Vascular Reactivity Assay

Rats were decapitated and aortas were isolated using blunt dissection. The thoracic

aorta was cleaned of all adherent tissue and two adjacent 3.0 mm ring sections were

obtained from each animal. These rings were then placed onto pressure transducer arms

and submerged in tissue baths containing a modified Krebs solution (118 mM NaC1, 4.7

mM KC1, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25.0 mM NaHCO3, 11.1 mM Dextrose, and

2.5 mM CaC12) with 95% 02 / 5% CO2 compressed gas mixture bubbling through.

Pressure transducers were connected to a PowerLab signal transduction unit

(ADInstruments), and data was continuously recorded on a PC computer, and further

analyzed using the Chart program (ADInstruments). Rings were pretensioned to 4 g over

1 h, as preliminary studies determined this to provide the optimal length-tension ratio

(data not shown). A single dose of potassium chloride was applied (60 mM final

concentration) to determine smooth muscle viability. After maximal contraction was

reached, the tissue baths were purged and fresh modified Krebs solution was applied.

Following relaxation back to the 4 g baseline, phenylephrine (10 7 M final concentration)

was applied. After maximal contraction was reached, graded doses of Ang-(1-7) were









applied (10 10 M through 10 6 M final concentrations), separated by 3 min intervals.

Baths were then purged, and the contractile response to angiotensin II (10 7 M) was

assessed. Finally, relaxation responses to acetylcholine and sodium nitroprusside were

determined in a similar manner to those of Ang-(1-7). Rings were then removed from the

tissue chambers and allowed to dry overnight in a dessicating oven. Dry ring masses were

then measured, and contractile responses to each drug were normalized to the mass of the

dried ring. Relaxation responses were determined by dividing maximal relaxation

responses (tension change from maximal contraction to phenylephrine) by total active

tension (total tension minus 4 g baseline).

Plasma Steroid Measurement

Trunk blood was collected from the animals at decapitation into EDTA-coated

collection tubes and centrifuged for 5 min at 3000 xg in a Triac centrifuge (Becton,

Dickinson). Plasma was removed and placed into cryogenic vials for storage at -20 C

until assayed. To determine plasma estradiol levels, an ELISA kit (ALPCO, sensitivity of

4.6 pg/mL) was utilized following the manufacturer's instructions, in duplicate.

Statistics

Body, uterine and heart masses, plasma estradiol, ring masses and contractile

responses to potassium chloride and angiotensin II were analyzed using one-way

ANOVA with significance set at P<0.05. Relaxation responses to angiotensin-(1-7) were

analyzed using repeated measures ANOVA with significance set at P<0.05. All post-hoc

tests were performed using Fisher's method with significance set at P<0.05.









Results

Estradiol Levels and Effects on Body, Heart and Uterine Growth

Estrogen caused significant dose-related reductions in overall body mass at all

doses, but no significant effects were observed on heart size with any level of estrogen

replacement. Uterine mass was significantly increased in a dose-dependent manner with

estrogen replacement. Plasma estradiol was significantly (P < 0.05) increased over

ovariectomized levels in the 25 mg pellet group (Ovex, 8.90 + 0.34; 0.05 mg pellet,

16.28 3.99; 25 mg pellet, 95818.26 3515.76 pg/mL).

Body Mass (g) Heart (g kl-,) Uterus (g k.,'
Ovariectomized 274.48.1 3.240.14 0.61+0.11
Estrogen, 0.05 mg 198.43.8 3.670.24 2.670.24 *
Estrogen, 25 mg 171.25.5 3.25+0.15 5.360.80 *

Table 5-1. Effects of steroid treatments on body parameters in female Sprague-Dawley
rats. Estrogen exposure caused significant changes in body size and uterine
mass at all doses. No significant changes were observed in heart size with any
treatment. Data presented as means SE. (* indicates P<0.05 versus
ovariectomized).

Aortic Ring Properties

60 mMKCl (g) Phenylephrine (g) Ring mass (g)
Ovariectomized 3.31+0.09 1.960.50 1.350.02
Estrogen, 0.05 mg 3.170.16 1.680.17 1.41+0.11
Estrogen, 25 mg 2.830.48 3.130.43 1.270.07

Table 5-2. Aortic ring properties. Aortas were sectioned into 3 mm length rings for
vascular reactivity assay. Control contractions by potassium chloride (KC1)
and phenylephrine were performed to determine treatment effects on
contractile responses. Following the assay, rings were dried in a dessicator
and then weighed to determine effects of treatment on protein content. Data
presented as means SE.

Vascular smooth muscle contractions to 60 mM potassium chloride and

phenylephrine were similar across treatments, as were ring masses (Table 5-2). Estradiol

treatment, however, resulted in significant alterations of the vascular smooth muscle









response to stimulation of components of the renin-angiotensin system. Aortic rings from

animals treated with estrogen showed an estrogen-dose dependent attenuation of

constrictor response to angiotensin II (Fig. 5-1).

90





O3
& 60









Ovex Estradiol, Estradiol
0.05 mg 25 mg


Fig. 5-1. Maximal contraction to angiotensin II in aortic rings from ovariectomized rats
with estrogen replacement. Aortic rings were exposed to 107 M angiotensin
II, and the maximal contractile response was recorded. These responses were
normalized by dividing each ring's response by its own maximal response to
107 M phenylephrine. (* indicates P<0.05 versus ovariectomized). Data
presented as means + SE.
Aortic Relaxation to Angiotensin-(1-7)

Increasing concentrations of Ang-(1-7) in preconstricted vessels from

ovariectomized rats resulted in a dose-dependent relaxation response that was

significantly greater than matched time controls (data not shown). This response was

similar in animals treated with a low dose of estradiol. Aortic rings harvested from

animals maintained on a pharmacological estradiol treatment were completely

unresponsive to any dilatory effects of Ang-(1-7) (Fig. 5-2), and even appeared to display

a slight contractile response to lower concentrations of Ang-(1-7), although this effect


was not significant. It is interesting to note that the maximum percent-relaxation of rings







64


from ovariectomized animals at each dose is similar in magnitude to those of the

mesenteric arteries from pregnant animals in the work from Neves et al. (2003).


C
0
. -10

C
o -15
0
X
I -20
?


-30 ----Estradiol, 25 mg I
----Estradiol, 0.05 mg
-35 -o-- Ovariectomized
-10 -9 -8 -7 -6
Log Molar Angiotensin 1-7


Fig. 5-2. Estradiol attenuates the aortic relaxation response to acute Ang-(1-7). Increasing
doses of estrogen caused dose-dependent attenuations of vascular reactivity to
angiotensin-(1-7). Repeated measures ANOVA reveals a significant effect of
steroid treatment. Post-hoc analyses reveal no differences between
ovariectomized and the 0.05 mg estradiol treatment, but 25 mg estradiol
replacement caused a significant attenuation of the relaxation response when
compared to either ovariectomized or 0.05 mg estradiol treatments. Data
presented as means + SE.

In a subsequent study, the dependence of aortic relaxation responses to Ang-(1-7)

upon the Mas receptor was determined. At doses of 10 6 M and lower, aortic relaxation

responses in endothelium-intact rings were completely abolished by 105 M D-Ala7-

Angiotensin-(1-7) (A779), an antagonist to the Mas receptor. At 10 5 M, however, Ang-

(1-7) relaxation responses were minimally effected by this receptor antagonist (data not









shown). These observations have been reported previously for rat aortic rings (Le Tran

and Forster, 1997).

Aortic Ring Integrity

Endothelial integrity was examined by exposing rings pre-constricted with 10 7 M

phenylephrine to 10-4 M acetylcholine. Rings from all animals relaxed more than 60%

(with the exception of one animal, which was dropped from all analyses), and no

statistical difference exists between groups (P=0.5219). Smooth muscle integrity and

responsiveness to nitric oxide was also tested by exposing rings pre-constricted with 10 7

M phenylephrine to 105 M sodium nitroprusside. All rings relaxed between 80% and

100%, with no differences between groups (P=0.8397).



Discussion

This study provides some significant insight into the effects of estradiol on

cardiovascular physiology. Although it was determined that estrogen does appear to have

effects on the vasodilatory responses of the aorta to Ang-(1-7), the directions of responses

were unexpected. Estrogen was, in fact, detrimental to the vasodilatory responses to Ang-

(1-7).

Estrogen caused the expected dose-dependent attenuation of body weight, and a

potentiation of uterine mass (Table 5-1). These effects are well documented in the

literature (Harris et al., 2002), as is the dose-dependent attenuation of aortic ring

constrictor responses to Ang II (Cheng and Gruetter, 1992). These effects of estrogen on

the response to Ang II have been attributed to decreased transcription and activity of the

AT1R (Schunkert et al., 1997; Nickenig et al., 1998; Fisher et al., 2002). Other responses

to Ang II, such as the dipsogenic or in vivo pressor response are also attenuated with









elevated levels of estrogen (Jonklaas and Buggy, 1984; Fregly, et al, 1985). It is possible

that Ang-(1-7) works through interactions with the AT1 and/or AT2 receptors (Jaiswal et

al., 1992; Gironacci et al., 1998; Caruso-Neves et al., 2000; Santos et al., 2000; Roks et

al., 2004), and that altered levels of these receptors may account for the changes in

responsiveness to Ang-(1-7) in these aortic rings. Actions of Ang-(1-7) through the ATi

receptor seem less likely, in that the 0.05 mg estradiol pellet group displayed a

significantly decreased constrictor response to Ang II (Figure 5-1), but its relaxation

response to Ang-(1-7) was not different from ovariectomized (Figure 5-2).

Chronic estrogen treatment did not appear to alter the in vitro vascular contractile

response to a depolarizing agent (potassium chloride), or a receptor-mediated

vasoconstrictor phenylephrinee). The lack of effect on vascular ring mass also suggests

no significant effect of steroid treatment on aortic vessel musculature. Likewise, the

similar relaxation responses of the vessels to sodium nitroprusside demonstrate that the

vascular smooth muscle response to nitric oxide was not altered by estrogen treatment.

Maximal responses to acetylcholine suggest that endothelial function was not

compromised with estrogen treatment. Utilization of lower doses of acetylcholine may

have been able to distinguish if the sensitivity to acetylcholine is altered by estrogen

treatment. Other literature reports would suggest that this may be the cause, as estradiol

increases endothelial and calcium-dependent nitric oxide synthase (Saito, et al, 1999;

Morschl et al., 2000). Estradiol, however, does appear to alter the vascular response to

components of the renin-angiotensin system.

Vasodilatory responses to Ang-(1-7) were hypothesized to increase with estrogen

exposure, as the circulating level of estradiol significantly increases along with Ang-(1-7)









levels and sensitivity during pregnancy (Neves et al., 2003). The results of the current

study suggest that in the aorta, increased estrogen appears to have a dose-dependent

inhibitory effect on the vasodilatory response to Ang-(1-7) (Figure 5-2). These results,

obtained from aortic rings, are in contrast to the recent findings ofNeves et al. (2004),

who found that estrogen mediates increased vasodilatory responses to angiotensin-(1-7)

in mesenteric artery preparations of female rats. These differential effects on the aorta

and the mesentery may suggest a regional specificity of the effects of estrogen, which has

been suggested for other estrogen-mediated physiological effects (Zubair et al., 2005).

Further, methodological differences between this study and those of Brosnihan et al. may

contribute to the conflicting results. Brosnihan's group utilizes the peptide endothelin-1 to

preconstrict their vessels before relaxation with Ang-(1-7), whereas we have used

phenylephrine. Additionally, their measurements of vascular reactivity in mesenteric

arteries are performed using a pressurized myograph system, whereas our measurements

are made using an isometric wire myograph system. Finally, our experiments utilized a

pharmacological dose of estradiol, whereas their group has used more physiological

doses.

Differential effects between vascular beds may contribute to the ongoing debate

regarding the cardio-protective effects of estrogen. Further studies in this area, examining

the different mechanisms of action of estrogen in the aorta versus the mesenteric arteries

may help to discover a mesentery-selective estrogen analog that would be able to produce

the cardio-protective effects of estrogen, but not cause the detrimental aorta-specific

effects.









One potential shortfall to this design was the absence of an intact control. This

particular group was not utilized as we anticipated that the normal estrous cycle could

impact the results and confound data interpretation. An intact control could be used in

future studies, provided the stage of estrous cycle was determined prior to harvesting the

vascular tissue. Another confounding effect with the estrogen treatment paradigm

incorporated was that a normally cycling estrogen was replaced with a chronic exposure.

This chronic exposure is of interest, despite its difference from normal physiology, as it

more closely approximates the estrogen dosing paradigm of women on monthly estrogen

pills. Further, this chronic exposure mimics the chronically increased levels of estradiol

observed during pregnancy. The doses of estradiol utilized were recommended by IRA to

mimic levels achieved during pregnancy. At the completion of the physiological

measurements, the results of the estradiol ELISA assay revealed much higher levels than

expected, and future studies will evaluate lower levels of estradiol that more closely

mimic those observed during normal pregnancy in the rat. The fact that such high

estrogen levels were achieved in the current study may suggest that nonspecific effects of

estrogen are mediating the observed attenuation of Ang-(1-7) responses, however the

responses observed to angiotensin II are consistent with the literature (Cheng and

Gruetter, 1992).

In a preliminary study with human coronary artery endothelial cells, we observed

that estrogen treatment significantly reduced the transcription of the Mas receptor mRNA

when examined using realtime RT-PCR. This observation would further support our

current demonstration that estrogen decreased relaxational responses to Ang-(1-7) in rat

aorta. These observations were made in cells at a relatively late passage number, and this









preliminary study will have to be repeated at early passages before a definitive effect of

estrogen on the Mas receptor could be validated.

In conclusion, we have found that angiotensin-(1-7) causes a dose-dependent

relaxation of the female rat aorta, and that this relaxation response is attenuated by 17-3-

estradiol. This aortic relaxation has previously been shown to be endothelium- and Mas

receptor-dependent (Le Tran and Forster, 1997). These results are in contrast to findings

from other studies on the effects of estradiol on the relaxation response to angiotensin-(1-

7), although as these other studies have been performed in mesenteric arteries, a regional

specificity of action of estradiol is suggested. Future studies will examine this regional

(vessel) specificity, the dose-response relationship of estradiol with the Mas receptor,

and the influence of 17-p-estradiol and other estrogenic compounds on Mas receptor

expression.














CHAPTER 6
PRELIMINARY CELL CULTURE EXPERIMENTS

Abstract

Angiotensin-(1-7) (Ang-(1-7)) has been previously demonstrated to provide

protection from cardiac remodeling in vivo. Here, the role of Ang-(1-7) in mediating the

production of collagen by cultured neonatal rat cardiac fibroblasts following stimulation

by both myocyte preconditioned media and by hypoxia-reperfusion injury was examined.

It was determined that Ang-(1-7) inhibits myocyte-derived pro-fibrotic stimuli, possibly

including transforming growth factor-3 (TGFp). Further, it was determined that lentiviral

delivery of the angiotensin converting enzyme 2 gene to fibroblasts prevented hypoxia-

induced collagen and TGFp production. Together, these results indicate protective roles

for ACE2 and Ang-(1-7) in vitro, and may suggest future therapeutic avenues for

myocardial injury.

Introduction

The preceding chapters have explored the beneficial effects of angiotensin-(1-7)

(Ang-(1-7)) in vivo. While these studies provide an integrated-systems verification of the

cardioprotective role of Ang-(1-7), they fail to examine the tissue-level mechanisms) of

these effects.

The two major components of cardiac remodeling, myocyte hypertrophy and

cardiac fibrosis, are primarily mediated by (and between) cardiac myocytes and

fibroblasts. When these cells are cultured independently, cardiac myocyte hypertrophy

can be triggered by treatment with media formerly on cardiac fibroblasts (Iwata et al.,









2005), and collagen production by cardiac fibroblasts can be potentiated by co-culture

with cardiac myocytes (Sarkar et al., 2004). Together, these findings indicate that cross-

talk between cardiac cell types is an important factor in the regulation of normal tissue-

level regulation.

Iwata et al. (2005) recently demonstrated that cultured adult rat cardiac fibroblasts

show a blunted collagen-production response to various stimuli when the culture media is

supplemented with Ang-(1-7). Tallant et al. (2005) also recently demonstrated that Ang-

(1-7) inhibits cardiac myocyte growth in vitro through the Mas receptor. These

investigators, though, did not examine the effect of Ang-(1-7) on myocyte-fibroblast

cross-talk demonstrated by Sarkar et al. (2004). Therefore, the effects of Ang-(1-7) on

myocyte-mediated stimulation of collagen production by cardiac fibroblasts were

examined.

In addition to myocyte-derived pro-fibrotic stimuli, certain pathologies can lead to

aberrant activity of cardiac fibroblasts. One pathophysiological state which leads to

excessive collagen production by cardiac fibroblasts is hypoxia, which can result from

acute or chronic myocardial ischemia (Agocha et al., 1997).

Averill et al. (2003) recently determined that Ang-(1-7) is conspicuously absent in

the infarct zone of surgically-induced myocardial ischemia in Lewis rats, although intact

myocardium surrounding the infarct zone stains with normal levels of the peptide.

Preliminary studies from our lab have also determined that cardiac myocytes have a

moderate basal activity of the angiotensin converting enzyme 2 (ACE2) enzyme, which is

the major source of Ang-(1-7) in the heart (Zisman et al., 2003). Cultured cardiac

fibroblasts, on the other hand, do not have detectable ACE2 activity. Together, these









findings next led us to the hypothesis that the absence of ACE2 in an infarct zone is

permissive for aberrant extracellular matrix production by fibroblasts that are able to

survive in the hypoxic local environment.

Methods

Isolation and Culturing of Neonatal Rat Cardiac Myocytes

Neonatal rat cardiac myocytes were isolated using a commercially available kit

(Worthington Biochemical Corp., Lakewood, NJ). Briefly, hearts from 5-day old rats

were removed by blunt dissection, then digested with trypsin and collagenase. Myocytes

were grown in DMEM/F-12 media supplemented with 1% penicillin/streptomycin and

10% fetal bovine serum (FBS) in a 5% CO2 / 95% air humidified incubator. Cells were

used for experiments within one week.

Isolation and Culturing of Neonatal Rat Cardiac Fibroblasts

Neonatal rat cardiac fibroblasts were isolated using a protocol adapted from Zhang

et al. (2001). Five day old Sprague-Dawley rat pups were deeply anesthetized by

inhalation of halothane. Following heart excision and mincing, heart tissue was digested

for 2 hours with 1% collagenase (Worthington Biochemical Corp, Lakewood, NJ), spun

down at 3000 rpm for 2 min, then digested for 1 hour with 1% trypsin (Worthington

Biochemical Corp, Lakewood, NJ). Cells were then spun down for 2 min at 3000 rpm,

and reconstituted in media and plated in a T-75 flask. Cells were maintained in DMEM

supplemented with 1% penicillin/streptomycin, 50 tg/mL ascorbic acid, and 10% fetal

bovine serum. Cells were split by standard trypsin procedure, and were used within the

first three passages. Cells were seeded at 3000 cells/cm2.









Myocyte-Fibroblast Cross-Talk Paradigm

Cultured cardiac myocytes were grown for three to five days before serum-

starvation for 24 hours. Following this starvation, cells were treated with 10% FBS-

supplemented media with or without angiotensin-(1-7) at 10-7 M for 24 hours. At the end

of this incubation, medias were transferred to confluent cultured fibroblasts, and some

(previously 10% FBS-only) media was supplemented with angiotensin-(1-7) at this time,

as indicated in Figure 6-1A. Collagen was sampled after 12 hours of incubation on the

cardiac fibroblasts.

An additional set of cardiac myocytes was treated in a parallel fashion, but

following the 24 hour incubation with or without angiotensin-(1-7), RNA from these cells

was isolated using a commercially available kit from Ambion. These RNA samples were

then used to measure transcript for transforming growth factor-p 1.

Real-time RT-PCR

Real-time RT-PCR was used to determine levels of transforming growth factor-p 1

mRNA. Primers and probe for the gene, along with all reagents for the reaction and

primers and probe for the loading control (the 18S gene), were obtained from Applied

Biosystems. One-step RT-PCR reactions were carried out using the manufacturer's

instructions in a Prism 7000 thermocycler.

Lentiviral ACE2 Gene Transfer

Lentivirus harboring the gene for ACE2 under transcriptional control of the

elongation factor-la promoter (EF a) was obtained from the Raizada laboratory. This

batch of virus was determined to exhibit an infectious titer of 6.68x109 ifu/mL.

Cultured cardiac fibroblasts were grown to approximately 80% confluence before

infection. The virus was diluted in serum-free media for infection. Residual media was









removed from the fibroblasts, and the minimal volume of virus-containing media (200 [L

for a single well of a 24-well plate) was placed onto the cells. After four hours, 10% FBS

media was added to the cells to bring the media volume up to normal (500 [L total for a

single well of a 24-well plate).

Hypoxia-Reperfusion Paradigm

Upon confluence, cardiac fibroblasts were serum-starved for 48 hours. Medias

were then replaced with 10% FBS media immediately before cells were transferred to a

hypoxia chamber (or a normoxia control chamber). Cells in the hypoxia chamber were

exposed to a continual flow of 95% N2 / 5% CO2 gas for one hour. After one hour, cells

were returned to the normoxia condition for 12 or 24 hour hours before assays for ACE2

activity, collagen or transforming growth factor-p 1 were performed.

ACE2 Activity

ACE2 activity was determined in cardiac fibroblasts as previously described

(Huentelman et al., 2004). Briefly, cells were scraped into a buffer consisting of 1 M

NaC1, 75 mM Tris, 0.5 mM ZnCl2, pH 7.5. Cells were then sonicated and protein content

was determined by a standard Bradford Assay. ACE2 activity was then determined by

kinetic assay using the Fluorogenic Peptide Substrate VI (FPS VI, 7Mca-Y-V-A-D-A-P-

K(Dnp)-OH, R&D Systems, Minneapolis, MN). Samples containing ACE2 enzyme (up

to 50 [l) were incubated with 100 tM FPS VI, 10 tM captopril (to inhibit ACE activity),

and reaction buffer (1 M NaC1, 75 mM Tris, 0.5 mM ZnCl2, pH 7.5) in a final reaction

volume of 100 ul at 37 C. In the assay, ACE2 removes the C-terminal dinitrophenyl

moiety that quenches the inherent fluorescence of the 7-methoxycoumain group resulting

in an increase in fluorescence in the presence of ACE2 activity at excitation and emission

spectra of 328 and 392 nm, respectively.









Soluble Collagen

Soluble collagen was determined using a commercially available Sircol Soluble

Collagen kit (Accurate Chemical) according to manufacturer's instructions.

Transforming Growth Factor- 1

Total transforming growth factor-p 1 protein was determined by ELISA (R&D

Systems) according to manufacturer's instructions.

Results

Cardiac Myocyte-Fibroblast Cross-Talk is Inhibited by Ang-(1-7) at the Myocyte

In this first series of experiments, cardiac myocytes were serum starved for twenty-

four hours before treatment with normal (10% FBS) media with or without Ang-(1-7)

supplementation. After twenty-four hours of incubation, this myocyte-preconditioned

media was transferred to cardiac fibroblasts. A preliminary study determined that

myocyte-preconditioned media caused increases in fibroblast collagen production only if

the myocytes were stimulated with FBS (data not shown). In this experiment

(summarized in Figure 6-1A), Ang-(1-7) supplementation caused a significant decrease

in collagen production by the cardiac fibroblasts if the peptide was allowed to act at the

cardiac myocyte, but had no effect if only the fibroblast was treated with Ang-(1-7)

(Figure 6-1B).

In a subsequent set of cultured cells, myocytes were treated with serum-free, 10%

FBS, or 10% FBS media supplemented with Ang-(1-7). After twenty-four hours of

incubation, cells were scraped from their growth chambers and total RNA was isolated.

One-step real-time RT-PCR measurement of mRNA for TGFpi demonstrated that while

10% FBS results in a significant increase in TGFpi mRNA, Ang-(1-7) co-incubation

significantly attenuates this effect (Figure 6-2).







76


A 10% FBS B 80
Ang-(1-7)
E 75
Myocytes/
(24 hours) 70


u656 *
Ang-(1-7) O
60

Fibroblasts ss. 55
(12 hours) Cc
60
10% FBS 10% FBS+ 10% FBS+
Ang-(1-7) on Ang-(1-7) on
(Collagen Assay) Myocytes Fibroblasts

Figure 6-1. Angiotensin-(1-7) attenuates myocyte-derived pro-fibrotic signaling. (A)
Neonatal rat cardiac myocytes were incubated 24 hours with 10% fetal bovine
serum-supplemented media with or without 10-7 M Ang-(1-7). Medias were
then transferred to neonatal rat cardiac fibroblasts, and a subset of fibroblasts
receiving Ang-(1-7)-free media were spiked with Ang-(1-7) at time of
transfer. (B) Significant attenuation of collagen production (P=0.0320 vs.
10% FBS) was only achieved if Ang-(1-7) was allowed to act at the cardiac
myocyte.

1.10
-6


IJ.
0 1.05

U-

z
W 1.00
E

U-

0.95
Serum-free 10% FBS 10% FBS +
Ang-(1-7)

Figure 6-2. Angiotensin-(1-7) attenuates expression of TGFpi mRNA in cardiac
myocytes. Neonatal rat cardiac myocytes were stimulated for 24 hours with
serum-free, 10% serum, or 10% serum supplemented with 10-5 M Ang-(1-7).
Real-time RT-PCR was used to measure mRNA for the TGFpi cytokine.
Serum significantly increased transcription of TGFpi (P=0.0009), and Ang-(1-
7) significantly attenuated this effect (P=0.0282).









Lentiviral Delivery of EFla-ACE2 Results in Increased ACE2 Activity and
Protection from Hypoxia

Preliminary studies determined that while cultured cardiac myocytes do exhibit a

moderate basal level of ACE2 activity, cultured cardiac fibroblasts have no detectable

basal activity (data not shown). Lentiviral delivery of the murine ACE2 gene under

transcriptional control of the elongation factor- a (EF a) promoter, though, results in a

viral dose-dependent increase in ACE2 activity in cultured cardiac fibroblasts (Figure 6-

3A).


O Low
0 None


3 6 9 12 15
Time (min)


B 40
-J
E 38*
28
E
,-% 36.

O
0
S32
C-)
301
I')
28.


DNormoxia
1Hypoxia


Illrl1
None Low Medium High
Viral dose


S T


Figure 6-3. Lentiviral delivery of ACE2 attenuates the college production response to
hypoxia in cultured cardiac fibroblasts. (A) Lentiviral delivery of the EFla-
ACE2 construct results in a dose-dependent increase in ACE2 activity in
cultured cardiac fibroblasts. (B) Lentiviral delivery of ACE2 attenuates the
collagen production response to hypoxia in cultured cardiac fibroblasts.
Fibroblasts, previously infected with various doses of lentivirus harboring the
EF 1la-ACE2 construct, were exposed to 1 hour of hypoxia, before being
returned to normoxic conditions for 12 hours. Two-way ANOVA indicates
significant effects of hypoxia (P<0.0001) and virus (P<0.0001) on collagen
production, and a significant interaction between these factors (P=0.0111).
The viral dose of "Low" indicates 2.36x105 ifu/cm2, "Medium" indicates
5.9x105 ifu/cm2, and "High" indicates 11.82x105 ifu/cm2.


A 2001 High


150'


M 100.
Q.
(N
O 50'


~mmqg~~










Cardiac fibroblasts exhibit a strong collagen-production response to acute hypoxia

followed by normoxia. Lentiviral delivery of the murine ACE2 gene caused a significant

attenuation of this response to hypoxia (Figure 6-3B).

ACE2 Gene Delivery Attenuates Hypoxia-Reperfusion Induction of Transforming
Growth Factor-pl

In addition to attenuating the collagen-production response to hypoxia, lentiviral

delivery of murine ACE2 significantly attenuated both basal and hypoxia-stimulated

media levels of TGFpi protein (Figure 6-4).

1350o 3 Normoxia

1300 M Hypoxia

E 1250

1200

0) 1150

r 1100
1-
1050

1000
None Low Mid High
Viral dose


Figure 6-4. Lentiviral delivery of ACE2 attenuates hypoxia-induced expression of
transforming growth factor-p31 protein. Two-way ANOVA indicates
significant effects of hypoxia (P=0.0019) and virus (P=0.0002) on TGF-31
protein, but no interaction between these factors (P=0.5405). Viral doses are
the same as above.

Discussion

In this preliminary set of experiments utilizing cultured neonatal rat cardiac

myocytes and fibroblasts, we determined that Ang-(1-7) inhibits myocyte-fibroblast

cross-talk, and that this inhibition may be due to decreased transcription of the myocyte-

derived pro-fibrotic cytokine TGFp.









In addition to examining the effects of Ang-(1-7) on myocyte-fibroblast cross-talk,

the protective effects of ACE2 in fibroblasts treated with hypoxia were also examined.

Here, it was determined that while cultured neonatal rat cardiac fibroblasts show no

detectable basal ACE2 activity, lentiviral delivery of the murine ACE2 gene dose-

dependently increases ACE2 activity. This increased activity prevents hypoxia-

reperfusion induced collagen production by the fibroblasts, and this may be mediated by

decreased TGFp protein. Finally, it was demonstrated that increased ACE2 activity, even

if temporally limited to the onset of hypoxia, is sufficient in cultured fibroblasts to

provide some anti-fibrotic protection.

The determination that ACE2 protects fibroblasts from hypoxia-induced metabolic

changes may lead to a future therapy for myocardial infarction. During the hypoxic

phase of a myocardial infarction, cardiac myocytes die and formerly quiescent fibroblasts

are stimulated by the hypoxic environment to produce large quantities of collagens I and

III, and these cells morphologically change into myofibroblasts (Stenmark et al., 2002).

The formation of myofibroblasts is vital, as this is a necessary step in wound healing.

Death of cardiac myocytes is also important, as these cells represent the endogenous

cardiac source of ACE2 and Ang-(1-7) (Averill et al., 2003; Zisman et al., 2003).

Future studies should be directed at determining the mechanism of ACE2-mediated

protection in cultured fibroblasts. Although Iwata et al. (2005) determined that Ang-(1-

7) can act upon cultured fibroblasts, our cross-talk experiment (Figure 6-1) suggests that

Ang-(1-7) did not have direct effects upon cultured fibroblasts. In light of these

conflicting findings, one can not simply assume that ACE2 mediates its effects in

cultured fibroblasts through Ang-(1-7). In addition to increasing Ang-(1-7) levels, which









may or may not be able to act upon fibroblasts, it is possible that ACE2 may work in this

paradigm solely through degradation of Ang II. Agocha et al. (1997) demonstrated that

during hypoxia, Ang II is a significantly more potent stimulus for cardiac fibroblasts, and

decreased Ang II levels would presumably contribute to decreased stimulation of the

fibroblasts. Lijnen et al. (2006) recently showed that Ang II mediates its actions in

cultured cardiac fibroblasts through NAD(P)H oxidase, and Byrne et al. (2003) published

a suggestive review article regarding the role of reactive oxygen species (which are

produced by NAD(P)H oxidase) in the development and progression of heart failure.

Okada et al. (2005) showed that viral delivery of a gene encoding a soluble TGFpII

receptor (thereby artificially decreasing available TGFp cytokine in the myocardium)

resulted in significant protection from cardiac remodeling, function loss, and apoptosis of

the reparative myofibroblasts. Together with our current findings regarding the effects of

Ang-(1-7) and ACE2 on TGFp, we conclude that Ang-(1-7) and ACE2 may mediate their

cardioprotective effects through inhibition of TGFp, and that loss of myocytes (and

therefore the source of ACE2 and Ang-(1-7)) may permit increased TGFp signaling.

Collectively, these preliminary studies in vitro suggest important roles for both

Ang-(1-7) and ACE2. In particular, it was determined that Ang-(1-7) likely mediates

myocyte-fibroblast cross-talk through actions on the cardiac myocyte, and that it may be

the loss of myocytes (which are the endogenous cardiac source of ACE2, and therefore

Ang-(1-7)) that permits increased TGFp signaling and overactivity of cardiac fibroblasts

in an infarct zone. These findings lead to the conclusion that ACE2, and possibly Ang-

(1-7), may represent an as-yet unexplored therapy for myocardial infarction. Ongoing









studies are currently examining the utility of viral and pharmacological stimulation of

ACE2 in vivo during surgically-induced infarction.

Future studies should also repeat and expand upon the examination of Ang-(1-7)

modulation of myocyte-fibroblast cross-talk, both to confirm these results, and to

determine whether Ang-(1-7) can act directly upon cardiac fibroblasts. Interpretation of

the current findings (Figure 6-1) is hindered by the experimental design, as Ang-(1-7)

was delivered in a single bolus, and therefore the rate of degradation of the peptide in

cultures of cardiac fibroblasts may mask the effects of Ang-(1-7) upon these cells.

Ongoing studies are designed to utilize lentiviral-mediated delivery of the gene for an

Ang-(1-7) fusion protein (Santos et al., 2004) to provide a constant source of the peptide,

and thereby overcome this design limitation.














CHAPTER 7
GENERAL DISCUSSION

Summary and Conclusions

The studies presented here aimed to examine the cardioprotective actions of

angiotensin-(1-7) (Ang-(1-7)). Lentiviral-mediated increases in expression of

angiotensin converting enzyme 2 (ACE2) resulted in significant protection from cardiac

remodeling during chronic infusion of angiotensin II (Ang II) in vivo. Here, it was

demonstrated that chronic infusion of Ang-(1-7) in rats resulted in the same

cardioprotective effects during chronic Ang II infusion, thus suggesting that Ang-(1-7)

mediates the anti-trophic, anti-fibrotic actions of ACE2 on the myocardium. These

findings lead us to several important conclusions. First, this provides evidence that the

predominant mechanism of ACE2 cardioprotection during hypertension is to produce

Ang-(1-7) rather than simply degrade Ang II or metabolize ACE2's other endogenous

ligands, including apelin-13 and -32, some of the kinin metabolites (with the exception of

bradykinin), neurotensin, and opioid peptides such as dynorphin (Vickers et al., 2002).

Second, this establishes Ang-(1-7) as an active cardioprotective hormone. The lack of

effect of Ang-(1-7) on blood pressure is also important, as this indicates a direct effect of

Ang-(1-7) on cardiac tissue, and establishes that any changes in blood pressure associated

with ACE2 overexpression may be a by-product of the degradation of Ang II.

Next, it was determined that the cardioprotective actions of Ang-(1-7) are not

limited to the Ang II-infusion model of hypertension and cardiac remodeling. In the

deoxycorticosterone acetate (DOCA)-salt hypertensive model, it was observed that









chronic infusion of Ang-(1-7) attenuated both interstitial and perivascular fibrosis,

without effect on cardiac hypertrophy. The anti-fibrotic actions of Ang-(1-7) in the

DOCA-salt (mineralocorticoid-dependent) model are potent but independent of effect

upon hypertrophy and blood pressure, which leads to two important conclusions. First,

these findings further support the notion that the signaling mechanisms of cardiac

hypertrophy and fibrosis are independent. Second, Ang-(1-7) must either only oppose the

effects of mineralocorticoids at the cardiac fibroblast, or act at the cardiac myocyte to

alter mineralocorticoid-induced paracrine pro-fibrotic signals but not intracellular (or

autocrine) hypertrophic signals.

Use of the normotensive isoproterenol-injection model of cardiac remodeling

further demonstrated that the cardioprotective effects of Ang-(1-7) are not mediated by

decreased blood pressure. In the isoproterenol model, it was observed that infusion of

Ang-(1-7) prevents cardiac hypertrophy, without effect on fibrosis. These findings, while

in contrast to those of the DOCA-salt model, establish that the effects of Ang-(1-7) are

model-dependent, and that Ang-(1-7) may have diverse effects across the cell types

within cardiac tissue. Protective effects in this model further establish that Ang-(1-7) has

an active role in the myocardium, and that this hormone is not a passive inhibitor of the

RAS. A follow-up study on the effect of ovarian hormones on isoproterenol-induced

cardiac remodeling indicated that ovarian hormones potentiate the hypertrophic response

to isoproterenol injection.

Next, it was determined that estrogen attenuates cardiovascular tissue sensitivity to

Ang-(1-7). Aortic rings from ovariectomized rats showed a dose-dependent relaxation

response to Ang-(1-7), while rings from ovariectomized rats with 17-P-estradiol









replacement showed an estrogen dose-dependent attenuation of this relaxation response.

Other hormones, including progesterone and testosterone were examined, but these

hormones showed little effect on vascular reactivity to Ang-(1-7) (data not shown).

Finally, the roles of ACE2 and Ang-(1-7) in cultured cardiac fibroblasts and

myocytes were examined to begin to dissect their mechanisms) of action in these cell

types. It was determined that Ang-(1-7) inhibits myocyte-derived pro-fibrotic signaling,

which may be mediated by decreased transforming growth factor-3 (TGFp) gene

transcription. Further, it was determined that ACE2 gene delivery to cultured cardiac

fibroblasts, which have no detectable basal ACE2 activity, causes dose-dependent

increases in ACE2 activity and provides protection from hypoxia-induced collagen and

TGFp production. Together with the literature, these findings suggest that within an

infarct zone, where myocytes (the endogenous source of ACE2 in myocardium) are

absent, lack of ACE2 (and possibly Ang-(1-7)) may be permissive for overactivity of

cardiac fibroblasts. This overactivity would result in severe cardiac remodeling, and loss

of function, eventually resulting in heart failure.

Pitfalls and Shortcomings

Few studies are ever perfect in design and execution, and the studies presented here

are no exception. There are several design flaws and shortcomings in these studies,

which should be noted so as not to repeat these errors in future studies.

In chapter 2, chronic infusion of Ang II resulted in increased blood pressure,

cardiac hypertrophy, and interstitial fibrosis. Chronic infusion of Ang-(1-7) had no effect

on the increased blood pressure, but significantly attenuated the hypertrophy and fibrosis.

In this study, a single dose of Ang II, and a single dose of Ang-(1-7) were utilized. The

dose of Ang II (100 ng/kg/min) was selected based on a long series of previous and









preliminary studies within the Katovich lab to provide a moderate increase in blood

pressure and moderate cardiac remodeling over four weeks, and the dose of Ang-(1-7)

was selected to represent a nearly-equimolar dose to that of the Ang II. The four-week

timeframe for the study was selected for practical limitations, namely the maximum time

afforded by the commercially available osmotic minipumps that were used to infuse both

hormones. Timeframes longer than four weeks would require removal of original pumps

and implantation of secondary pumps for each hormone after every four weeks of

treatment, and it was decided that additional anesthesia and subcutaneous "surgery" was

an unnecessary risk for the animals. Presumably, a longer treatment timecourse would

lead to overt heart failure in the Ang II-infused animals, as direct effects on cardiac

remodeling and indirect effects through increased vascular resistance and peripheral

blood pressure would result in gross morphological changes of the myocardium.

An additional problem with this first study was the use of Masson's Trichrome to

stain the extracellular matrix of the myocardium. While this stain provides beautiful

pictures, it is not particularly well suited for the specific analysis of collagen deposition

in the matrix. This treatment results in red-stained myocytes, green-stained endothelial

cells, and blue-stained matrix. To analyze matrix deposition in the heart with two

different dominant stain colors on each slide (red and blue), pictures of the slides must be

analyzed by "splitting" the RGB data into three separate files (using the ImageJ program

from NIH Rasband, 2005). These three individual files represent a pixel-by-pixel

color-saturation matrix for each of the primary (red, green, blue) values. Sections of the

left ventricle wall are then outlined in the red and blue files, and the average color

saturation for red and for blue are then determined for each animal's heart-slice picture.









The blue saturation is then normalized to the red saturation within each animal to control

for batch-to-batch differences in the stain. Together, this process, along with the analysis

of the entire left ventricle wall (as described in chapter 2), artificially compresses the

magnitude of matrix deposition change with treatments, as is evidenced by only an

approximate 5% increase with chronic Ang II infusion (Figure 2-3). This problem is

again reflected in chapters 3 (mineralocorticoid model) and 4 (isoproterenol model). This

same extracellular matrix deposition, if analyzed using a different stain and analyzed

through a "percent-area" method, may result in more impressive numerical changes with

treatments. Regardless of the magnitude of the numerical values, though, the changes

which are visually obvious in the pictures (especially in chapter 3) are significantly

different even when analyzed by stringent non-parametric analysis when analyzed this

way. Heart slices from animals in each treatment group (from at least one future

experiment) should be stained by both Masson's Trichrome and another more specific

stain, such as Sirius Red, to confirm the reliability of Masson's Trichrome and to

determine the relationship between numerical results (in other words, to determine what a

5% difference when Masson's Trichrome is used relates to if Sirius Red had been used).

In chapter 3, where deoxycorticosterone acetate (DOCA) was delivered by

subcutaneous pellet to uninephrectomized animals, similar design flaws existed. Again, a

single dose of DOCA was administered, and a single dose of Ang-(1-7) was utilized.

Once again, preliminary studies in the Katovich lab led to the use of the DOCA dose.

The Ang-(1-7) dose of 100 ng/kg/min was used because of successful results in the Ang

II study (chapter 2). Once again, four weeks was used as the timecourse, as this

represented the time limit imposed by the use of osmotic minipumps. This study was









also plagued by the numerically-unimpressive, yet statistically significant differences in

interstitial extracellular matrix changes with DOCA treatment. Future studies certainly

should be done with either the Ang II or DOCA models to determine the dose-response

relationship of Ang-(1-7) and its anti-remodeling effects.

Chapter 4 examined the effects of chronic Ang-(1-7) infusion on isoproterenol-

induced cardiac remodeling. Again, similar flaws in design were present as were

mentioned above. Several preliminary studies were carried out to determine the

appropriate dose of isoproterenol and timecourse, as the literature is hyper-variable with

regard to the specifics of this model. It was determined that while daily subcutaneous

injections of 1 mg/kg isoproterenol for one week resulted in moderate cardiac

hypertrophy in males, the variability between animals would require a very large sample

size to see statistical differences. Increasing the timecourse of treatment to two weeks

resulted in only slightly larger hypertrophy, and thus it was decided to increase the dose

of isoproterenol to 2 mg/kg daily. This increase in dosage resulted in a much larger

cardiac hypertrophy, but also greatly increased spontaneous deaths of animals.

Preliminary experiments led to the conclusion that in male Sprague-Dawley rats, 2 mg/kg

daily for 14 days was required to see large, reliable changes in cardiac hypertrophy.

Further, to decrease the spontaneous death rate of these animals, it was determined that

the daily injections of isoproterenol should be administered during the middle of the light

cycle of the animals. Animals should also be allowed to recover for at least one day

before the first injection of isoproterenol is administered, as the precipitous acute drop in

blood pressure is potentiated by anesthesia and minipump delivery of blood pressure

lowering compounds.