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Defining the Role of the Angiotensin II Type 2 Receptor in Cardiovascular Disease


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DEFINING THE ROLE OF THE ANGIOT ENSIN II TYPE 2 RECEPTOR IN CARDIOVASCULAR DISEASE By BEVERLY L. METCALFE 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 2004

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Copyright 2004 by Beverly L. Metcalfe

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I dedicate this work to my family and Alaric Falcn for their constant encouragement and support in every endeavor I pursue.

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ACKNOWLEDGMENTS I would first like to thank my mentor, Mohan K. Raizada, for his constant encouragement and invaluable advice. His infectious enthusiasm for scientific research has encouraged me to do things that I never would have thought possible. His advice, both professionally and personally will undoubtedly prove to be invaluable in my future endeavors and I thank him for not only being such a great mentor, but for also being a wonderful role model and friend. In addition, I would like to thank the members of my dissertation committee: Drs. Colin Sumners, Michael Katovich, and Gerry Shaw. They have all provided me with sound advice to advance my projects. These people are much more then committee members as they have been heavily involved in my studies. I appreciate their dedication in shaping me as a better scientist. Id also like to thank my labmates in Dr. Raizadas laboratory. They are always willing to lend a hand, and I have learnt so many new skills and techniques thanks to them. I would like to especially thank Dr. Shereeni Veerasingham for her help and advice concerning both the microarray project and the brain microinjections; Dr. Matthew Huentelman who established the lentiviral vector in our laboratory and with whom I also worked closely on the in vivo project in the SHR; Dr. Carlos Dez-Freire for help with radiotelemetry surgeries; Ms. Jillian Stewart for lentiviral preparation and help with the angiogenesis arrays; and Ms. Jasenka Zubcevic and Mr. Michael Anthony Cometa for indirect BP measurements. iv

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In addition, I would like to thank Ms. Laura Dixon for her help with AT1R and AT2R primer optimization; Mr. Adam Mecca for help with osmotic minipump implantation; Dr. Harm Knot and his laboratory and Randy Harris for their help with echocardiographies; the UF ICBR Microarray Facility, especially Dr. Mick Popp and Ms. Blanca Ostmark for their help with the microarrays; the AMRIS facilities at the McKnight Brain Institute, especially Dr. Glenn Walter, Mr. Xeve Silver, and Ms. Raquel Torres for their help with the MRI imaging and the Paton laboratory for their collaboration on the lentiviral transduction efficiency in the brain. Id also like to thank Dr. Jeffery Harrison for providing AT2R cDNA; Dr. Peter Sayeski for providing multiple cell lines; and Dr. Pushpha Kalra and her former graduate student, Dr. Erin Rhinehart, for teaching me how to do brain microinjections. v

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TABLE OF CONTENTS Page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES.............................................................................................................ix LIST OF FIGURES.............................................................................................................x ABSTRACT......................................................................................................................xii CHAPTER 1 INTRODUCTION........................................................................................................1 Impact and Importance.................................................................................................1 Hypertension.................................................................................................................1 Classic Circulating Renin-Angiotensin System............................................................3 Angiotensin Fragments..........................................................................................4 Tissue Renin Angiotensin System................................................................................5 Angiotensin II Receptors..............................................................................................6 Angiotensin II Type 1 Receptor............................................................................6 Structural and molecular function..................................................................6 Signaling.........................................................................................................7 Physiological effects......................................................................................8 Angiotensin II Type 2 Receptor............................................................................9 Structural and Molecular function..................................................................9 Signaling.......................................................................................................11 Physiological effects....................................................................................13 Targeting the RAS for the Treatment of CV Diseases...............................................15 RAS as a Gene Therapy Target..................................................................................16 Ideal Viral Vector.......................................................................................................16 Non-viral vectors.................................................................................................17 Viral Vectors.......................................................................................................18 Adenovirus...................................................................................................19 Adeno-Associated Virus..............................................................................20 Retrovirus/Lentivirus...................................................................................21 Aims and Rational......................................................................................................23 Aim 1: Characterize the Lenti-AT2R Virus In Vitro..........................................24 vi

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Aim 2: Identify Genes Involved in Angiotensin II Type 2 Receptor-Mediated Inhibition of Endothelial Cell Migration by Expression Profiling..................25 Aim 3: Determine the Effect of AT2R Overexpression on CV Pathophysiologies............................................................................................25 Aim 4: Determine the Dipsogenic Responses Following Angiotensin II Type 2 Receptor Gene Transfer into the Paraventricular Nucleus..............................25 2 IN VITRO CHARACTERIZATION OF LENTI-AT2R...........................................26 Introduction.................................................................................................................26 Materials and Methods...............................................................................................27 Lentiviral Constructs and Preparation.................................................................27 Cell Culture.........................................................................................................31 RNA Isolation and Quantification.......................................................................31 Ligand Binding Assay.........................................................................................32 Protein Isolation and Determination....................................................................32 Detection of Activated MAPK............................................................................33 Statistical Analysis..............................................................................................34 Results.........................................................................................................................34 Overexpression of the Receptor by Lenti-AT2R.................................................34 Characterization of AT2R Transgene Function..................................................36 Discussion...................................................................................................................38 3 IDENTIFYING GENES INVOLVED IN ANGIOTENSIN II TYPE 2 RECEPTOR-MEDIATED SIGNALING PATHWAYS BY EXPRESSION PROFILING............41 Introduction.................................................................................................................41 Materials and Methods...............................................................................................42 Cell Culture, AT2R Transduction, and Treatments.............................................42 Real-Time RT-PCR.............................................................................................44 AT2R Binding Assay..........................................................................................45 Microarray Analysis............................................................................................45 Microarray Analysis Controls and Data Analysis...............................................46 Migration Assay..................................................................................................48 Angiogenesis Protein Array................................................................................49 Statistics...............................................................................................................49 Results.........................................................................................................................50 Characterization of AT2R Transduction of HCAEC..........................................50 Expression Profiling of AT2R-Transduced HCAEC..........................................50 Gene Validation...................................................................................................53 The Role of the AT2R in Migration and Angiogenesis......................................59 Discussion...................................................................................................................60 4 PREVENTING CARDIAC PATHOPHYSIOLOGIES BY ANGIOTENSIN II TYPE 2 RECEPTOR GENE TRANSFER............................................................................70 Introduction.................................................................................................................70 vii

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Materials and Methods...............................................................................................71 Animals and Lentiviral Delivery.........................................................................71 Viral Production and Transgene and AT1R Expression Measurements.............72 Physiological Measurements...............................................................................73 Statistics...............................................................................................................75 Results.........................................................................................................................75 In vivo Gene Transfer..........................................................................................75 Pathophysiology in the SHR...............................................................................78 Pathophysiology in the Angiotensin II-Infusion Model......................................80 Discussion...................................................................................................................85 5 DETERMINING THE DIPSOGENIC RESPONSES FOLLOWING ANGIOTENSIN II TYPE 2 RECEPTOR GENE TRANSFER INTO THE PARAVENTRICULAR NUCLEUS..........................................................................89 Introduction.................................................................................................................89 Methods......................................................................................................................92 Lentiviral Vector Production, Concentration and Titers.....................................92 Animal Care.........................................................................................................92 Lentiviral Vector Delivery into Brain Nuclei......................................................92 Physiological Measurements...............................................................................93 Results.........................................................................................................................94 Lentiviral Vector-Mediated Transduction into the PVN.....................................94 Physiological Effects of the AT2R in the PVN...................................................95 Discussion...................................................................................................................97 6 OVERALL DISCUSSION AND CONCLUSIONS................................................104 AT2R Prevents Cardiac Pathophysiologies..............................................................104 Role of the AT2R in Ubiquitination.........................................................................105 AT2R Effects on Migration and Angiogenesis........................................................106 AT2R-Mediated Effects on the AT1R......................................................................106 Ligand-Independent Activity of the AT2R...............................................................107 Role of the AT2R in the PVN...................................................................................107 Perspectives..............................................................................................................108 LIST OF REFERENCES.................................................................................................109 BIOGRAPHICAL SKETCH...........................................................................................123 viii

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LIST OF TABLES Table Page 1-1 Advantages and disadvantages of gene transfer techniques.....................................18 3-1 AT2R decreases gene expression without CGP42112A stimulation.......................53 3-2 List of genes whose expression was significantly increased with AT2R expression independent of ligand...............................................................................................54 3-3 List of genes whose expression was significantly altered in the AT2R-transduced cells stimulated with CGP42112A...........................................................................57 ix

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LIST OF FIGURES Figure Page 1-1 If left untreated, hypertension can lead to a number of devastating diseases............2 1-2 Circulating renin-angiotensin system.........................................................................4 1-3 Signal transduction cascades of the AT1R.................................................................8 1-4 Known signaling mechanisms of the AT2R............................................................12 1-5 Lentiviral vector system...........................................................................................23 2-1 Lentiviral vectors......................................................................................................28 2-2 AT2R overexpression in CHO-AT1R cells.............................................................35 2-3 Time course of AngII-induced Erk42/44 activation in CHO-AT1R cells...............36 2-4 AT2R transduction prevents AngII-mediated increases in phosphorylated Erk42/44...................................................................................................................37 2-5 AT2R-mediated effects of Erk42/44 activity cannot be reversed by PD123,319....37 3-1 Timeline for the HCAEC used in the microarray experiments................................43 3-2 Outline of the microarray protocol as described in detail in the Materials and Methods section........................................................................................................47 3-3 Lentiviral transduction in HCAEC...........................................................................51 3-4 Scatter plots of the microarray data..........................................................................52 3-5 Gene validation of the microarray analysis..............................................................60 3-6 AT2R prevents HCAEC migration..........................................................................61 3-7 AT2R effects are independent of the typical regulators of angiogenesis represented on Panomics Angiogenesis Array...........................................................................62 3-8 Graphical representation of the pathway where the AT2R-regulated genes could exert its actions to inhibit endothelial cell migration and angiogenesis...................66 x

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xi 4-1 In vivo lentiviral transduction efficiency..................................................................76 4-2 Lentiviral transduction efficiency in the heart.........................................................77 4-3 AT2R and AT1R expression in isolated cardiomyocytes........................................77 4-4 Effect of AT2R transduction on CV pathologies in the SHR..................................79 4-5 Blood pressure response to AngII infusion..............................................................80 4-6 Role of the AT2R in the CV pathol ogies associated with AngII infusion...............82 4-7 MRI analysis indicates AT2R tran sduction prevents ca rdiac hypertrophy..............83 4-8 Effect of the AT2R and A ngII on other CV physiologies........................................84 4-9 Effect of the AT2R on the pathology of the heart following AngII infusion for 4 weeks........................................................................................................................85 5-1 Lentiviral transduction into the PVN.......................................................................94 5-2 Role of the AT2R in the PVN on basal BP, HR, and activity..................................95 5-3 Effect of icv injecti on of AngII in animals overe xpressing the AT2R in the PVN..........................................................................................................................96 5-4 Effect of the AT2R in the PVN on basal activity and activity following icv injection of AngII.....................................................................................................98 5-5 Basal effects of AT2R overexpression in the PVN..................................................98 5-6 Role of the AT2R in the PVN on dehydration-induced water intake......................99 5-7 Effects of the AT2R in the PVN on subcutaneous injection of AngII.....................99

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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 DEFINING THE ROLE OF THE ANGIOTENSIN II TYPE 2 RECEPTOR IN CARDIOVASCULAR DISEASE By Beverly L. Metcalfe May, 2004 Chair: Mohan K. Raizada Major Department: Physiology and Functional Genomics Despite recent advances in understanding the renin angiotensin system, the role of the angiotensin II type 2 receptor (AT2R) in cardiovascular diseases remains elusive. Given that the AT2R has been implicated to play a role in embryonic development, traditional transgenic animal models may not be an effective means for studying the role of the AT2R. To overcome the inherent problems of compensatory gene expression with transgenic animals, we developed a lentiviral vector system in which overexpression of the AT2R could be accomplished after embryonic development. By injecting lentiviral vector either directly into the heart or into specific brain regions, we were able to study both the peripheral and the central actions of this receptor. In addition, this same vector was used to characterize the AT2R in vitro in cells that are typically difficult to transduce. These studies indicate that the AT2R plays a cardioprotective role in several models of disease. Overexpression of the AT2R in the heart prevents the development of xii

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cardiac hypertrophy and heart failure in both a genetic model of hypertension and one with AngII-induction. In addition, the AT2R prevents migration in human coronary artery endothelial cells. This effect could prevent angiogenesis and its induction of atherosclerosis. Additionally, microarray analysis in these cells indicated a number of genes whose regulation may play a role in this effect. Finally, it appears that the AT2R in the paraventricular nucleus causes a decrease in water intake, both basally and in response to either dehydration or AngII. These effects could play a role to decrease circulatory volume, cardiac output, and blood pressure. All of which could prevent other cardiovascular abnormalities. These studies indicate that delivery of the AT2R by lentiviral vectors may provide a novel therapeutic option in the prevention of cardiovascular diseases. xiii

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CHAPTER 1 INTRODUCTION Impact and Importance Cardiovascular (CV) disease is composed of many diseases of the heart and circulation, including high blood pressure (HBP), heart disease, stroke, and atherosclerosis. It is the number-one killer of both women and men in the United States. One of every five people has some form of CV disease, with an average of one death every 33 seconds. In fact, CV disease kills more people each year than the next 5 leading causes of death combined. In addition to the large physical impact on society, it also has a very large economic impact, with an estimated cost of over $350 billion being spent in the United States in the year 2003. In the next section, I present an introduction to hypertension and its devastating effects on the cardiovascular system. We then examine the renin-angiotensin system (RAS) and its role in hypertension and CV disease, comparing and contrasting the tissue versus circulating RAS. Next, I introduce the major receptors of the RAS, and what is known about their role in CV disease. Finally, we investigate the use of gene therapy as a way to study the RAS and CV disease, as well as its use as a novel therapeutic option. Hypertension More than 50 million Americans have hypertension, which is defined as having a diastolic pressure 90 mmHg and a systolic pressure 140 mmHg. Hypertension is a multifactorial disease in which the cause of disease in nearly 95% of the patients is unknown. While hypertension itself is asymptomatic, if left untreated it can lead to other 1

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2 CV diseases (Figure 1-1). Chronic elevation of BP can lead to increased workload of the heart that can eventually lead to cardiac hypertrophy and heart failure, as well as myocardial ischemia and infarction. Hypertension also causes damage to the arteries. This damage leads to endothelial dysfunction and atherosclerosis, which can ultimately cause stroke, myocardial ischemia and infarction. Damaged vessels can also lead to renal failure and retinopathy. HypertensionCardiac WorkloadArterial DamageCardiac HypertrophyEndothelial Cell DysfunctionAtherosclerosisHeart FailureMyocardial Ischemia and InfarctionStrokeRenal FailureRetinopathy Figure 1-1: If left untreated, hypertension can lead to a number of devastating diseases. Even with increased education of the general population as to the risk factors of hypertension and CV disease and improved therapies, the incidence of CV disease in the U.S. remains high. In fact, of all the people with high BP, 31.6% are unaware that they have the disease, 14.8% are not on medication for the disease, 27.4% are on medication and have it under control, while 26.2% are on medication but do not have the disease under control. These statistics indicate that the current therapies are ineffective. There are several explanations for this: (1) despite increased education, a high percentage of the people still do not understand the risk factors for CV disease, (2) because many of the diseases do not produce any symptoms, diagnosis and treatment are delayed, (3) the high

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3 number of side effects causes compliance issues with patients needing to be on medication, and (4) a lack of understanding of the systems involved in the disorders inhibits the ability of physicians to properly treat and manage the disease. Therefore, a better understanding of the systems involved in hypertension and the development of CV disease and alternative therapeutics for these diseases must be further investigated. Classic Circulating Renin-Angiotensin System The RAS has been shown to play a major role in hypertension, cardiac hypertrophy, and electrolyte balance.1,2 Research involving antagonists, antisense gene delivery, and genetic polymorphisms of multiple components of the RAS has been shown to have effects on the cardiovascular (CV) system. Due to the relative importance of the RAS in CV disease, it continues to be under intense study for future treatments. Discovery of the RAS began in 1898 when Tigerstedt and Bergman found a pressor compound in renal extracts that they named renin.3,4 Almost 40 years later, two independent research groups described another pressor substance that would later become known as angiotensin.3,4 After the discovery of angiotensin, it took nearly another decade to delineate the cascade that has become the classic circulating renin angiotensin system. In this system, angiotensinogen (AOGEN), which is largely produced in the liver, is converted to the decapeptide angiotensin I (AngI) by the proteolytic enzyme renin that is produced mainly in the kidney. Angiotensin I is then cleaved by a second proteolytic enzyme produced in the lungs, angiotensin converting enzyme (ACE), to give the physiologically-active hormone angiotensin II (AngII). AngII elicits distinct actions by binding to either the angiotensin II type 1 receptor (AT1R) or the angiotensin II type 2

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4 receptor (AT2R). In general, AngII binding to the AT1R is associated with the development of cardiovascular pathophysiologies, while binding to the AT2R is thought to counteract the AT1R and elicit cardioprotective effects (Figure 1-2). A more detailed examination of these receptors is given later in the text, ANG IANG 1-9 ANG(1-7)ACE2ACE2ACEANG II ACE2ACE2AT1RAT2RAT1-7R AOGEN ANG IIIAng(2-8)ANG IVAng(3-8) AT4R? Anti-AT1RVasodilationNa+ ReabsorptionHypertrophyMemory and LearningVasoconstrictionNa+ & H2O ReabsorptionSympathetic OutputGrowth and ProliferationVasopressin ReleaseAnti-AT1RVasodilationAnti-ProliferationPro-ApoptoticTissue Development and RepairVasodilationAnti-ProliferationVasopressin ReleaseDiuresisNatriuresis ACE Renin Figure 1-2: Circulating renin-angiotensin system. Angiotensin Fragments While the classical RAS pathway leading to the formation of AngII has stood the test of time, recent studies indicate an important role for new receptors, enzymes, and angiotensin fragments in the RAS (Figure 1-2). It has been shown that aminopeptidase A can break down the octapeptide AngII to yield the angiotensin fragment angiotensin(2-8) or AngIII. AngIII can then be further degraded by additional aminopeptidases to yield angiotensin(3-8) or AngIV. While the roles of AngIII and AngIV are not well defined,

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5 there is evidence that both fragments have cardiovascular functions. While a specific receptor for AngIII has not been identified, it has been shown to have a similar affinity for the AT1R in the brain to elicit arginine vasopressin release and an increase in BP.5 AngIV has been shown to play an important role in the regulation of blood flow, cardiac hypertrophy, sodium reabsorption, and learning and memory.6 In addition to these angiotensin fragments, the recently identified carboxypeptidase ACE2 was to elicit another angiotensin fragment has been shown to have cardiovascular effects. ACE2 has been shown to breakdown AngII to Ang(1-7) and AngI to Ang(1-9), which is further degraded to Ang(1-7) by ACE.7 Ang(1-7) has been shown to play a role in vasodilation, antiproliferation, and sodium and water reabsorption.8,9 Tissue Renin Angiotensin System Recent evidence indicates both circulating and tissue production of all of the components of the RAS. This local RAS was first described almost 20 years ago to explain the blood pressure-independent effects of ACE and AT1R inhibitors on cardiovascular diseases. This local RAS has now been shown in many tissues including those of cardiovascular importance, such as the brain, heart, adrenal, vasculature, and kidney.10 The heart provides an interesting example of a local RAS with differing levels of its components. Angiotensinogen, ACE, and the AT1R are abundantly expressed in the heart, while renin and the AT2R are only moderately expressed. Even though the expression of renin is low, the heart has alternative enzymes such as cathepsin G, chymase, and tonin that have been shown to convert AngI to AngII.10 In addition, the AT2R is not highly expressed in the heart under normal conditions, but expression levels

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6 increase during heart failure and fibrosis.11 This local production of AngII has been shown in transgenic animals to play an important role in the development of cardiac hypertrophy and fibrosis.12 Therefore, in studying CV disease, one must consider its effects on hypertension; and also its effects on end organ damage, independent of elevated BP. Angiotensin II Receptors Evidence for AngII receptor subtypes was found in 1989, when two independent groups provided pharmacological evidence for the existence of 2 receptor subtypes; the AT1R and the AT2R.13 Later studies identified the AT1R as mediating the known effects of AngII at that time, while the role of the AT2R still remains elusive. Angiotensin II Type 1 Receptor Structural and molecular function Numerous studies on the molecular characteristics of the AT1R began after its successful cloning in 1991.14,15 The AT1R is a seven-transmembrane domain receptor, with 359 amino acids, and a molecular weight of 41 kDa. In humans, the AT1R has only one subtype that has 5 exons and exists on chromosome 3.16 In contrast to humans, rodents have 2 subtypes of the AT1R (AT1A and AT1B). The AT1A and AT1B receptors are 96% homologous; but they are highly different in their non-coding region, indicating differential regulation of these receptors. The AT1A receptor is located on chromosome 17, and has 4 exons; while AT1B receptor is situated on chromosome 2, with only 3 exons.16 While both receptors are expressed in CV tissues, the AT1A receptor subtype predominates in most tissues.

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7 Signaling The AT1R couples to multiple signaling cascades, leading to diverse biological actions. These signaling events are dependent on both time of activation and cell type. They have been shown to have effects though G protein-dependent (Gq/11, Gi, G12/13) and independent coupling (JAK, Src, FAK; Figure 1-3). One of the well-characterized signaling pathways of the AT1R is G protein (Gq/11, G12/13) activation of phospholipase C (PLC), diacylglycerol (DAG), and inositol triphosphate (IP3) that leads to Ca2+ mobilization and activation of the Ca2+ dependent kinases, protein kinase C (PKC) and Ca2+/calmodulin-dependent protein kinase II (CAMKII).2,17 Activation of Gi, however, leads to the inhibition of cAMP which inhibits the activation of PKC.18 In addition, the AT1R activates a number of small G proteins, which signals to Raf to activate mitogen-activated protein kinases (MAPK, ERK42, ERK44). These activated MAPKs are then transported to the nucleus, where they increase the expression of early-response genes (c-fos, c-jun, c-myc).2 The AT1R also activates a number of nonreceptor tyrosine kinases (Src, JAK, FAK) independent of G proteins (Figure 1-3). Activation of Src leads to Ca2+ mobilization through a PLC and IP3 pathway; and also regulates gene transcription through Ras, Raf, and MAPK. Janus kinase (JAK) activates signal transducers and activators of transcription (STAT), which translocates to the nucleus to regulate early growth-response genes. The AT1R-induced autophosphorlyation of focal adhesion kinase (FAK) leads to its translocation and phosphorlyation of paxillin and talin, which may regulate cell morphology and movement.19

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8 Physiological effects One of the most important physiological effects of AngII mediated by the AT1R is its regulation of BP. AngII binding to the AT1R can cause transient increases in BP. AT1RG ProteinsTyrosine KinaseGq, G12/13GiRasIP3RafDAGCa2+MAPKPKCCAMKIIcAMP PLC SrcJAKFAKRasRafMAPK IP3Ca2+PKCCAMKII PLC STAT PaxillinTalin ContractionDifferentiationProliferationVesicle Transport TranscriptionRegulation ContractionDifferentiationProliferationVesicle Transport TranscriptionRegulation MobilityMorphology Figure 1-3: Signal transduction cascades of the AT1R. This can occur either directly, through the vasoconstriction of vascular smooth muscle cells (VSMC) and vessels; or indirectly, through increases in vasopressin release and sympathetic nerve activity. Blood pressure is also regulated chronically through the regulation of renal sodium and water reabsorption. Again, the AT1R plays a direct and indirect role in this effect. Angiotensin II can act directly by binding to the AT1R in the kidney, or indirectly by AT1R-mediated increases in aldosterone and thirst. In addition to its hemodynamic actions, activation of the AT1R has also been associated with cell

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9 growth and proliferation that could lead to increases in vascular and cardiac hypertrophy.20 Angiotensin II Type 2 Receptor Since its discovery, the actions of the AT2R have remained an enigma. Its binding properties, signaling, and even its physiological effects have all remained elusive. Structural and Molecular function The AT2R is a seven-transmembrane domain protein. It is located on the X chromosome; and has three exons, with the entire coding region being in the 3rd exon. It has 363 amino acids, with a molecular mass between 60 to 140 kDa depending on the amount of glycosylation on five possible sites. The AT2R only has 34% homology to the AT1R, with the 3rd intracellular loop having the lowest amount of homology. The distribution of the AT2R is quite different from that of the AT1R. Unlike the AT1R, which is highly expressed in adults, the AT2R is abundantly expressed during embryonic development. Its expression dramatically decreases after birth, but can increase after vascular and cardiac injury, wound healing, and renal failure.13 These expression patterns indicate that the AT2R may play a role in development, growth, and/or remodeling.19 Thus studies using transgenic or knockout animals that contain genetic alterations during embryonic development may develop inadvertent compensatory mechanisms that may not reflect the true role of the AT2R. In addition, it has been shown that the AT2R expression can be regulated by various intracellular and extracellular factors. For example, a number of factors such as AngII, insulin growth factor, basic fibroblast growth factor, and transforming growth

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10 factor beta 1, as well as estrogen and sodium have been shown to increase the AT2R expression; while cAMP and aldosterone have been shown to decrease its expression.13,21 Recent studies indicate that the AT2R can act in a constitutively active manner. Studies from Karniks group showed that the amount of the AT2R expression determined the level of apoptosis; independent of AngII in cultured fibroblasts, epithelial cells, and vascular smooth muscle cells.22 In addition, modifying the side chain of AngII drastically affected the AT1R effects, but had only modest effects on the AT2R.23 This idea is further supported by studies that replaced the 3rd intracellular loop of the AT2R with that of the AT1R, and resulted in an increase in c-fos, independent of AngII.24 Even though the AT2R has been found to have ligand-independent activities, studies mutating the AT2R have revealed important sites for both AngII binding to the AT2R and signaling of the AT2R. A series of studies from the Pulakat laboratory showed that amino acids Lys215, Asp287, Arg182, and His273 are important for AngII binding to the AT2R.25-28 In addition, they were able to identify areas of the receptor important for signaling, by replacing the 3rd intracellular loop or the C-terminus of the AT2R with that of the AT1R. In these studies, they have shown that the 3rd intracellular loop is important in inhibiting AT1R-mediated increases in IP3 and in inhibiting cell growth; while the C-terminus plays a role in AngII binding and inhibition of cell growth.29-31 These ideas are in direct contradiction of each other, and future studies are critically needed to identify when the AT2R is constitutively active or not. If the AT2R needs an agonist to elicit its effects, then one can control its effects with agonists or antagonists; however, if the AT2R is constitutively active, then one would have to regulate the AT2R expression in order to control its effects.

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11 Signaling The AT2R was initially thought to be a clearance receptor, because there was no consistent evidence linking the AT2R to any of the well-known intracellular signaling pathways. To date, the signaling cascades of the AT2R remain to be well defined, but it appears that it modulates a number of different signaling mechanisms, depending on the tissue/cell type (Figure 1-4). The AT2R has been shown to increase a number of protein phosphatases. AT2R coupling to the G protein Gi has been shown to increase both mitogen-activated protein kinase phosphatase 1 (MKP-1) and serine threonine phosphatase 2A (PP2A) activity; while the SH2 domain containing phosphatase 1 (SHP-1) has been shown to be activated independent of any G protein signaling. In addition, a recent study showed that in transfected COS-7 cells, the AT2R can increase SHP-1 when the AT2R couples to Gs independent of the and subunits.32 These increases in protein-phosphatase activity play a role to inhibit growth-promoting factors such as MAPK (Erk42/44). In a neuronal cell line, however, the AT2R plays a role in neurite outgrowth by signaling through Rap1 and B-Raf to increase ERK1/ERK2 activity.33 Protein phosphatase 2A (PP2A) has also been shown to inhibit Ca2+ channels in a neuroblastoma cell line, while increasing Kv current in cultured neurons, which could lead to hyperpolarization of the cell.34,35 In addition to the actions of the protein phosphatases, the AT2R has been shown to modulate several lipid-signaling pathways. Stimulation of the AT2R can lead to an increase in phospholipase A2 (PLA2) activity and arachidonic acid (AA) release.13 This leads to modification of eicosanoids to increase potassium currents. The AT2R has also been shown to play a role in the induction of apoptosis, and several pathways have been

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12 implemented in this role. AT2R-mediated increases in MKP-1 can inactivate the anti-apoptotic signal Bcl-2 while increasing the pro-apoptotic signal Bax. In addition, it has been shown that the AT2R can increase ceramides to induce apoptosis. Finally, the AT2R has also been shown to increase bradykinin (BK), nitric oxide (NO), and cyclic GMP levels that may play a role in vasodilation and natriuresis.36,37 From these studies it is evident that the AT2R can couple to a number of various signaling molecules; however, there are some discrepancies among these studies. These differences can most likely be accounted for by the differences in cell/tissue type. Therefore, more studies are needed to fully understand the signaling pathways of the AT2R in each of these cell types. AT2R GiGsRapCeramideSHP1MKPPLA2PP2ABcl-2BaxERK1/2 Apoptosis Gene TranscriptionAAEicosanoidsIKICaERK1/2 Hyperpolar-ization Gene Transcription RafERK1/2 NeuriteOutgrowthNO Apoptosis cGMP BK VasodilationNatriuresis Figure 1-4: Known signaling mechanisms of the AT2R. Recent studies have further advanced the understanding of the AT2R. The AT2R was initially thought not to be internalized because its binding efficiency did not change upon AngII activation, and it did not contain a nuclear localization signal. Recently,

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13 however, Inagamis group has shown that upon AngII stimulation, the promyelocytic zinc finger protein (PLZF) co-localizes and then internalizes the AT2R. This internalization can then act in the nucleus to activate a number of factors to increase protein synthesis.38 In addition, it has been shown that the AT2R can form a heterodimer with the AT1R to directly inhibit its actions independent of the AT2R signaling.39 Finally, the AT2R can play a role to decrease the expression of the AT1R in certain cell types.40 Physiological effects Since the role of the AT2R is not well understood in many aspects, it should come as no surprise that the physiological role of the AT2R is elusive as well. Among these is the role of the AT2R in the heart. The AT2R knockout animals indicate that the AT2R plays a role in the development of cardiac hypertrophy induced by both pressure overload and AngII-infusion.41,42 Another AT2R knockout experiment, however, indicates that the AT2R plays a protective role in the heart. In that study, the investigators showed that the AT2R deficiency exacerbated death rates and heart failure after myocardial infarction.43 Transgenic animals overexpressing the AT2R specifically in the heart show conflicting results. Results from one study show that AT2R overexpression does not affect the development of high BP and cardiac hypertrophy after AngII-infusion,44 but does play a role in the prevention of perivascular fibrosis.45 However, a separate study indicates that ventricular-specific overexpression of the AT2R is involved in the development of dilated cardiomyopathy and heart failure.46 Studies using AT2R-specific antagonists, however, indicate that the AT2R prevents or reverses a number of cardiac pathophysiologies. A study conducted by Mukawa et al, showed that simultaneous

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14 administration of AT1R and AT2R antagonists negated the antihypertrophic effects of the AT1R blocker alone.47 While another study demonstrated that the AT2R blockade did not affect cardiac hypertrophy but did regulate a number of growth-promoting factors in an AngII-infusion model.48 In addition, it has been shown that inhibition of the AT2R in hypertrophied hearts amplifies LV growth response to AngII.49 These inherent controversies on the cardiac role of the AT2R may be due to intrinsic problems associated with different strains of transgenic and knockout mice, developmental abnormalities associated with overexpressing or knocking out the AT2R in embryonic development, and/or stability issues with the AT2R-specific antagonist. The physiological role of the AT2R in the brain is not very well understood. Expression of the AT2R is concentrated in the areas involved in learning and control of motor activity and sensory areas.50 The AT2R is thought to play a role in brain development and cognitive function, as it was recently found that patients with X-linked mental retardation have mutations in the AT2R.51 Studies using simultaneous injection of AngII and the AT2R antagonist indicate that the AT2R in the brain increases drinking while decreasing BP.52 In addition, the AT2R antagonists have been shown to potentiate AngII-induced vasopressin release by the AT1R.53 Finally, studies using the AT2R knockout animals indicate that the AT2R plays a role in regulating pain threshold and exploratory behavior while having no effect on learning behavior.54,55 In addition to these physiological actions in the heart and the brain, the AT2R has also been found to play a role in differentiation and in the inhibition of endothelial cell migration and tube formation as well as cell growth and proliferation.56-59 The AT2R has

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15 been shown to have vasodilatory effects and to reduce pressor responses to AngII.55,60 Finally, the AT2R has been shown to play a role in regulating apoptosis, which may indicate a role for the AT2R in development and/or tissue remodeling and repair.22,61 Targeting the RAS for the Treatment of CV Diseases The RAS has been shown to be an integral part of the development of many CV diseases. In addition, pharmacological evidence indicates that the RAS is an important target for the treatment CV diseases. Both AT1R antagonists and ACE inhibitors have been used to successfully treat CV diseases such as hypertension, heart failure, myocardial infarction, and stroke.62 Despite these advances, the incidence of CV diseases remains on the rise. This has led many to believe that the current pharmacotherapies have reached a plateau, and novel approaches for the treatment of CV disease must be explored. Gene therapy is a rapidly emerging field for the study of genetic diseases. There are many advantages of using gene therapy over traditional pharmacological drugs, making it useful in studying CV diseases. (1) Viral vectors can be designed to either overexpress a deficient or dysfunctional gene, or to reduce the expression levels of an overactive gene by antisense technology, ribozymes, or double-stranded RNA inhibition. (2) Transduction with a viral vector can elicit long-term effects. Therefore, daily regimens can be eliminated. (3) Viral vectors can be directed to specific target tissues, which could reduce unwanted side effects. (4) Viral vectors can be designed to be regulated by a system such as doxycycline that can effectively toggle between an on and off expression level that can be regulated dose-dependently.

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16 RAS as a Gene Therapy Target The RAS has been intensely studied for its potential use as a gene therapy target. The RAS has been targeted for several reasons: While the complexity of our understanding of the RAS has increased recently, the role of the RAS in CV disease is fairly well understood The circulating RAS affects multiple targets, which is ideal for systemic delivery Pharmacological treatment blocking the RAS reverses many CV pathologies. Most of the studies targeting the RAS have focused on three major targets: angiotensinogen, the AT1R, and ACE. Studies performed using antisense oligonucleotides, targeting angiotensinogen, provided proof of concept for targeting the RAS. These initial studies showed a transient decrease in BP.63 Further studies using recombinant adeno-associated virus (rAAV) to decrease the levels of angiotensinogen showed an attenuation of both hypertension and cardiac hypertrophy.64 Retroviral delivery of antisense targeting ACE showed similar results with an observed decrease in BP.65 Cardioprotective effects have also been observed with retroviral delivery of antisense targeting the AT1R. Studies by Dr. Mohan Raizadas laboratory have characterized the effects of AT1R antisense (AT1R-AS) on the CV system. When a single injection of the retroviral AT1R-AS is delivered into the heart of 5-day old SHR animals, a decrease in BP, cardiac hypertrophy, and perivascular and interstitial fibrosis, as well as improved vessel reactivity is observed.66-69 In addition, AT1R-AS treatment prevented increases in BP induced by AngII-infusion, L-NAME, and fructose.70-72 Ideal Viral Vector For gene therapy to be effective, the best possible vector must be chosen to fit the need. For example, if you are studying restenosis, which is a short-term, secondary effect

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17 of balloon angioplasty, then you would choose a vector system that can efficiently transduce endothelial and vascular smooth muscle cells, and only elicits its effects for a short period of time. Someone studying a long-term disease such as hypertension or heart failure, however, would want a viral vector that can transduce cardiac and vascular tissues, and whose effects are stable and long-lasting. Currently, a number of both nonviral and viral delivery systems are used, each having their own advantages and disadvantages that must be considered when determining which system to use for a particular disease. In the next sections, we examine these delivery systems, their advantages and disadvantages, and a few examples of successful studies using these techniques (Table 1). Non-viral vectors Nonviral gene delivery techniques do not rely on a viral system to effectively transduce cells of interest. Instead they utilize the ionic properties of the cell membrane in order to enter the cell. Essentially, cationic or lipogenic vehicles are used to aid delivery into the cells, but naked, noncomplexed DNA can also be used. Advantages of these nonviral delivery systems are that it is safe, and because there are no viral particles, it elicits no immune response. The disadvantages, however, are that it is less efficient than most viral methods, and it does not integrate into the genome; therefore, the expression is only transient. Several studies highlight the success of using nonviral gene delivery methods. Dr. Phillips group showed decreases in hypertension by antisense oligonucleotides directed towards the AT1R, angiotensinogen, ACE, or -adrenergic receptor.73 Another series of studies using naked plasmid delivery are now in clinical trials. These studies show that

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18 delivery of vascular endothelial growth factor (VEGF) improved collateral angiogenesis, which could lead to prevention of myocardial infarction and angina.74,75 Viral Vectors A number of viral vectors have been developed for gene therapy. Most of these viruses depend on binding to a specific membrane receptor of the host cell for cellular uptake. Once inside the nucleus, the virus can then either replicate on its own, or integrate into the host genome. Each viral vector has its own unique properties that make them ideal for particular situations (Table 1). Table 1-1: Advantages and disadvantages of gene transfer techniques. Advantages Disadvantages Nonviral Safe Less efficient Low immune response Short-term expression Easily produced Adenovirus Infect dividing and non-dividing cells Short-term expression Large capacity High immune response No insertional mutagenesis Easily produced AAV Long-term expression Limited capacity Infect dividing and non-dividing cells Delayed expression Little immune response Hard to produce Safe Retrovirus Long-term expression Insertional mutagenesis Large capacity Only infects dividing cells Little immune response Easily produced Lentivirus Long-term expression Insertional mutagenesis Large capacity Safe Infect dividing and non-dividing cells Little immune response Easily produced

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19 Adenovirus Recombinant adenoviruses are double-stranded DNA viruses. They are currently among the most-often-used viral vectors for gene transfer into cardiovascular tissue. Adenoviruses can efficiently infect both dividing and nondividing cells, with high efficiency. In addition, their insert size can handle 7 to 8 kb and they can be easily produced. The adenovirus does not integrate into the genome; therefore, there is little risk for insertional mutagenesis. This lack of integration, however, limits its time of expression to approximately 1 month. In addition, the adenovirus elicits a strong immune response that decreases its expression, and prevents the use of repeated administrations. Recently, a new generation of adenovirus has been introduced. This generation of adenovirus has fewer endogenous viral proteins; therefore, a helper virus is required for viral production. This helper-dependent adenovirus (hdAd) has a larger capacity for transgene, can elicit longer transgene expression (up to 9 months), and has a reduced immune response.76 This hdAd is a promising gene therapy vector, since it has resolved many of the disadvantages of earlier generations of adenoviral vectors. There are, however, still limitations. The hdAd still requires a specific receptor on the cell surface for infection. Therefore, its transduction efficiency is dependent on the cells expressing the adenoviral receptor. Secondly, the production of the virus is more difficult, because it requires a helper virus. Finally, safety issues may still be a concern because of low levels of contaminating helper virus in the hdAd preps. Despite its high immunogenicity and its short-term expression, the adenovirus has been a successful gene therapy vector for the cardiovascular system. Studies expressing fibroblast growth factor and apolipoprotein A-I show improvement in myocardial

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20 perfusion and a decrease in atherosclerosis, respectively.77,78 A number of studies have used the adenoviral vector to overexpress superoxide dismutase. These studies show that superoxide dismutase expression can improve endothelial dysfunction, decrease arterial pressure, and attenuate myocardial ischemia reperfusion injury.79-81 Adeno-Associated Virus Adeno-associated virus (AAV) is a single-stranded linear DNA virus. While wild-type AAV is known to specifically integrate into chromosome 19, the recombinant virus used for gene therapy has lost this characteristic. Instead, it is thought to exist in an episomal manner. A recent study, however, indicated that a new generation of recombinant AAV (rAAV) can integrate site-specifically in muscle cells.82 Despite being largely episomal, the rAAV elicits long-term expression. Its other advantages are that it can infect both dividing and non-dividing cells and elicits minimal immune response. There are some disadvantages of rAAV, however. First, it has a limited size capacity of only 4.4kb. In addition, even though there are many serotypes used with rAAV, many humans contain antibodies for these serotypes. These antibodies may act to neutralize the virus before eliciting its effects. Additionally, the rAAV takes weeks to months before expression can be observed; therefore, immediate effects cannot be seen. Finally, viral replication is dependent on helper virus to initiate amplification and viral production; so the production of rAAV is difficult and laborious. Studies in Dr. Dzaus group have shown that delivery of the heme-oxygenase 1 (HO-1) gene before myocardial injury protects the heart from myocardial infarction by as much as 75%.83 In addition, rAAV has been shown to be useful in decreasing

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21 hypertension by AT1R antisense delivery and improving muscular dystrophy and its associated cardiomyopathy by sarcoglycan delivery.84,85 Retrovirus/Lentivirus Retroviruses are single-stranded RNA viruses that can stably integrate into the host genome, allowing for long-term expression of the transgene. Because it integrates into the host genome, however, retroviruses can cause insertional mutagenesis, thus affecting normal gene expression. Additional advantages of retroviruses include its low immune response, large transgene capacity and its optimization for efficient viral production. Their major limitation is that they can only infect dividing cells, thus decreasing their efficiency and somewhat limiting their usefulness to ex vivo gene transfer in which target cells are removed, transduced, and then reintroduced. Recent advances have identified a new retroviral gene therapy vector based on the human immunodefiency virus (HIV), named lentivirus. After removal of 5 of the unnecessary wild type (wt) proteins, this virus has been shown to be an efficient vector for gene therapy. Lentivirus has several significant advantages over the previously-mentioned gene therapy vectors. (1) Viral particles are produced with the necessary wt viral proteins being produced in trans; therefore, none of the wt viral proteins are packaged in the lentivirus. This increases the safety of this viral system as well as decreasing the immune response. (2) The viral coat of the lentivirus can be made with a variety of glycoproteins. This allows one to pseudotype the lentivirus to optimize its transduction efficiency to target tissues. (3) Since the lentivirus does not require the cell cycle for transduction, unlike other retroviruses, it can infect both dividing and non-dividing cells. (4) It has a large capacity for transgene of up to 18kb. (5) Recent studies

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22 have developed methods for efficient large-scale production of the virus.86 (6) The lentivirus also integrates into the genome with efficient transgene expression levels being achieved within 12-24 hours and lasting the life of the animal. A disadvantage of integration into the genome is insertional mutagenesis, however, a recent report may have identified a way to fix from this problem. These investigators showed that fusing the HIV-1 integrase with a sequence-specific DNA binding protein is an efficient approach for directing integration into a predetermined DNA site.87 The major drawback of lentiviral vectors is that it is based on the human pathogen HIV-1 and therefore safety concerns remain. To address these problems, 5 of the 9 viral genes of wild type HIV-1 have been deleted in the lentiviral vectors. For additional safety, the majority of the remaining wt genes (gag, pol, rev, tat) are produced in trans (pHP vector; Figure 1-5). In addition, the wt envelope gene (env) has been replaced with a pseudotyped vesicular stomatitus virus glycoprotein (VSVG; Figure 1-5). With these modifications, only the necessary packaging signal, psi (), and the long terminal repeats (LTR) which is necessary for transcription initiation are packaged with the transgene of interest (pTYF vector; Figure 1-5). Our group has had much success using the retroviral vector. We have previously shown that AT1R antisense delivered by an intracardiac injection of retroviral vector prevents the development of hypertension and its associated pathologies in the SHR.66-72,88 The lentivirus has also been shown to be effective against pathologies associated with hypertension as lentiviral delivery of angiostatin reduces retinopathy.89

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23 Aims and Rational Gene therapy to overexpress the AT2R could prove to be a novel therapeutic approach for the treatment of CV diseases. It has been indicated that unopposed stimulation of the AT2R may contribute to the effectiveness of AT1R antagonists. In fact, it has been shown that AT2R antagonists negate the antihypertrophic affect of AT1R antagonists.47 In addition, studies have shown that the AT2R can inhibit AT1R actions.39 These studies indicate that overexpression of the AT2R in cardiovascularly-relevent tissues would prevent the effects of the AT1R and may prevent the development of or even cause the reversal of CV diseases. Transgene FRRELTRLTR EF1SV40-PolyA VSVG EF1SV40-PolyA VSVG EF1 VSVG EF1pTYFVector (Transducing Vector)VSVG Vector (Envelope Vector) Gag CMV Pol Tat Rev SV40-PolyApHPVector (Packaging Vector) Figure 1-5: Lentiviral vector system. Three viral vectors are used to produce lentivirus in order to minimize the introduction of wild type HIV-1 proteins. In addition to these advantages, AT2R gene therapy could also provide novel insights into the role of this receptor in CV diseases. Based on the current literature, it is quite evident that the physiological role of the AT2R remains elusive. There are

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24 conflicting studies involving many aspects of the AT2R from its activation to its signaling to its physiological effects. While some of these inconsistent results can be accounted for by differences in cell lines and animals models, innovative approaches must be investigated to determine the true role of this receptor. Since the AT2R has been shown to play a role in and is highly expressed during embryonic development, transgenic and knockout animals may not be an accurate way of determining the role of the AT2R. Overexpression or absence of the AT2R during this critical developmental stage may cause inadvertent compensatory mechanisms in these animals, thus not truly reflecting the effects of the AT2R. In addition, the AT2R is only expressed at low levels in the adults making studies using AT2R-specific antagonists inaccurate. To further add to this problem, the current AT2R antagonists are expensive and only short-lived; therefore, physiological measurements are hard to accurately record and long-term studies in vitro and in vivo are nearly impossible. Successful gene therapy for the prevention of hypertension and cardiac hypertrophy has previously been established in our laboratory by decreasing the levels of the AT1R using a retroviral vector. Recent work in our laboratory has established the lentiviral vector for the transduction of cardiovascularly-relevent tissues. This viral vector is ideal for several reasons: (1) it can carry a large transgene, (2) it can be produced easily and reproducibly, (3) it elicits long-term expression, (4) it produces little immune response, and finally (5) it transduces both dividing and non-dividing cell types. Based on this information, the specific aims of my project are outlined below: Aim 1: Characterize the Lenti-AT2R Virus In Vitro. Infection Efficiency and Expression. Functionality.

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25 Aim 2: Identify Genes Involved in Angiotensin II Type 2 Receptor-Mediated Inhibition of Endothelial Cell Migration by Expression Profiling. Perform Microarray Analysis to Determine Novel Gene Profiles Following AT2R Overexpression in Human Coronary Artery Endothelial Cells. Associate Novel Gene Expression Profile with a Physiological Function. Aim 3: Determine the Effect of AT2R Overexpression on CV Pathophysiologies. Determine the Affects of the AT2R Overexpression in the Spontaneously Hypertensive Rat. Provide Further Proof of Concept of the Effects of the AT2R Overexpression in an AngII-Infused Model of CV Disease. Aim 4: Determine the Dipsogenic Responses Following Angiotensin II Type 2 Receptor Gene Transfer into the Paraventricular Nucleus. Determine the Affect of the AT2R Overexpression in the Paraventricular Nucleus.

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CHAPTER 2 IN VITRO CHARACTERIZATION OF LENTI-AT2R Introduction The physiological role of the angiotensin II type 2 receptor (AT2R) remains elusive. Since the AT2R has been implicated to play a role in embryonic development, genetic abnormalities associated with development in transgenic and knockout animals may limit the effectiveness in studying this receptor. Thus, gene transfer of AT2R following embryonic development offers a novel way to investigate this receptor and its role in the CV system. The use of lentiviral vector has many appealing traits in studying the physiological role of the AT2R in CV diseases. (1) It can accommodate large transgenes. This flexibility can allow one to overexpress the transgene of interest along with a marker gene such as the neomyocin resistance gene (NeoR), an alkaline phosphatase gene, or even green fluorescent protein (GFP). (2) The lentivirus can infect both dividing and non-dividing cells. This is particularly appealing when studying the CV system because both the myocytes of the heart and the neurons of the brain do not divide. (3) Modifications in the wild type HIV-1 virus made while creating this gene therapy vector have increased its safety and allows the virus to infect cells in vivo with little to no immune response. (4) Finally, the lentiviral vector has been shown to efficiently transduce cardiovascularly-relevant tissues.86 A single injection of 2.5x107 titer units (TU) into the left ventricular cavity of the heart leads to efficient transduction of the heart, liver, lung and kidney.86 26

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27 Before we can begin physiological studies, we must ensure that our AT2R transgene has been successfully cloned and is functional. In addition, each viral construct should be tested to ensure that it can infect and overexpress the AT2R transgene. Thus the objective of this study was 2-fold: (1) determine if infections of cells with the previously-cloned Lenti-AT2R construct leads to AT2R overexpression; and (2) examine if the AT2R overexpression is functional by examining the transgenes ability to regulate the activation of mitogen-activated protein kinase (MAPK), Erk42/44. Previous studies have shown antagonistic actions of the AT1R and AT2R in the regulation of Erk42/44. Studies have shown that stimulation of the AT1R leads to activation of MAPK through Ras and Raf, indicating a role for this receptor in cell growth and differentiation.90 Activation of the AT2R, however, has been shown to decrease MAPK activities.90,91 In fact, a study performed by Dr. Colin Sumners group showed that in neurons derived from neonatal rat hypothalamus and brainstem, the AT1R increased and the AT2R decreased MAPK activity.90 However, since the cells used were mixed cultures, the investigators were not able to determine whether these effects were occurring in the same neuron or in neurons containing either the AT1R or the AT2R. Materials and Methods Lentiviral Constructs and Preparation A series of lentiviral constructs (pTYF) used throughout this study, was created as previously described86,92 (Figure 2-1). Expression vectors created to express either GFP (Lenti-GFP) or human placental alkaline phosphatase (Lenti-PLAP) were used to determine the transduction efficiency of the virus both in vitro and in vivo. Additional vectors were created to bicistronically overexpress, through the use of an internal

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28 ribosome entry site (IRES), both the AT2R and neomyocin resistance gene (NeoR; Lenti-AT2R-I-Neo) genes or the AT2R and PLAP (Lenti-AT2R-I-PLAP). Finally, control vectors were used, which only contained the bicistronic element and either the NeoR (Lenti-I-NeoR) or PLAP (Lenti-I-PLAP) genes. GFP FRRELTRLTR PLAP FRRELTRLTRExpression Vectors PLAP FRRELTRLTR AT2R NeoR FRRELTRLTR AT2RExperimental VectorsControl Vectors: PLAP FRRELTRLTR NeoR FRRELTRLTRLenti-I-PLAPEF1 EF1Lenti-I-NeoREF1 Lenti-AT2R-I-PLAPLenti-AT2R-I-NeoR EF1EF1 Lenti-PLAPLenti-GFP IRES IRES IRES IRESEF1 Figure 2-1: Lentiviral vectors. A series of lentiviral vectors were previously constructed in our laboratory. For the experiments described in this dissertation, there were 2 expression vectors used to characterize lentiviral transduction that contain either GFP (Lenti-GFP) or PLAP (Lenti-PLAP), 2 experimental plasmids which bicistronically express both the AT2R and either NeoR (Lenti-AT2R-I-NeoR) or PLAP (Lenti-AT2R-I-PLAP) genes, and finally, 2 control plasmids that contain all the elements of the experimental plasmids minus the AT2R (Lenti-I-NeoR, Lenti-I-PLAP).

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29 Lentivirus was produced, concentrated, and titered according to established protocols.86 293FT cells (Invitrogen) maintained in Dulbeccos Modified Eagles Medium (DMEM; Gibco) containing 10% FBS and antibiotics were plated at a density of 1x107 cells/75-cm2 flask (T-75). The next day, the cells were transfected with a mixture of 400L of serum-free DMEM, 7g pHP, 3.5 g of the desired pTYF plasmid, 2.8 g of VSVG plasmid and 0.6 g of Tat plasmid with the use of Superfect (Gibco). After the complexes were mixed and allowed to form for 10 minutes, 5 mL of DMEM + FBS + antibiotics (growth media) were mixed with the complexes and added to the cells. The complexes remained on the cells for 4-5 hours at 37 C after which time the media on the cells are changed to 5 mL growth media. The first collection of virus was harvested ~30 hours post-transfection and an additional 5 mL of growth media is added to the transfected cells. The media from this first collection is spun down at 2000 x g for 10 minutes at 4 C and then filtered through a 0.45 micron low-protein-binding (PES) membrane (Nalgene). This first collection is then divided and centrifuged through a Centricon-80 ultrafiltration column (Millipore) for 1 hour at 2000 x g at 4 C according to manufacturers protocols. This first collection of virus is added to or spiked into the final collection of virus. Approximately 45 hours post-transfection, the second virus collection is performed. Again, this collection is centrifuged at 2000 x g for 10 min and filtered through a Nalgene membrane, after which, the first collection is added to this virus. A cushion of 220 l of 60% iodixanol is added to the bottom of 4 conical-bottom tubes (Beckman), and 30 mL of the media containing the virus is gently added to the tube. The virus samples are then centrifuged for 2.5 hours at 50000 x g at 4 C in a Beckman SW-28 swinging bucket

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30 rotor. Following this spin, the media above the iodixanol interface is removed. The residual media containing the virus and iodixanol are then carefully mixed and collected into a 3mL conical bottom tube (Beckman). This sample is then spun for 22-24 hours at 6100 x g at 4 C using a Beckman SW-50.1 rotor to separate the virus from the iodixanol. The supernatant is then removed and discarded. The remaining pellet containing the virus is resuspended in 30 L of phosphate buffered saline (PBS) or artificial cerebral spinal fluid ( -csf) overnight at 4 C. Finally, the virus is gently mixed, aliquoted and stored at -80 C until used. Lentiviral vectors were titered using a p24-Antigen Assay (Beckman). First, 1L of virus was diluted 1:100 in the provided lysis buffer and allowed to lyse at 37 C for 1 hour. Following this incubation, the virus is serially diluted to a final concentration, which is 10-7 and 10-8 from the original. The diluted samples (200 L) were added to the wells containing the p24-antibody and incubated at 37 C for 1 hour. The samples were then washed a total of 6 times with the provided wash buffer. Next, 200 L of the biotinlyated reagent was added to the samples and incubated at 37 C for 1 hour. After an additional series of washes, streptavidin conjugated to horseradish peroxidase (200 L) was added and incubated for 30 minutes at 37 C. Another series of washes were performed on the samples, and tetramethlbenzidine was added to the samples and incubated at 37 C for 30 minutes. Finally, 50 L of hydrogen sulfate was added to the sample and read on a microplate reader at an absorbance of 450 nm. Titers were then calculated by comparing the absorbance to the provided standard curve and the original dilution factor.

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31 Cell Culture Chinese hamster ovary cells transfected to overexpress the AT1R (CHO-AT1R) was a kind gift from Dr. Peter Sayeski (University of Florida). Cells were grown in Hams F-12 media (Cellgro) supplemented with 10% fetal bovine serum (Hams complete; Cellgro). Once the cells had reached confluency, they were subcultured and ~25,000 cells/cm2 were plated in 6-well dishes. The following day, the cells were incubated with the appropriate lentiviral vector (Lenti-AT2-I-PLAP or Lenti-I-PLAP) at a multiplicity of infection (MOI) of 1 in the presence of 8 g/L polybrene (Sigma) in the Hams complete media for ~15 hours. Following this period, the medium was replaced with fresh Hams complete media and allowed to grow for an additional 3 days prior to use in the experiments. RNA Isolation and Quantification Real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to quantitate the mRNA levels of the AT2R. Total RNA was isolated from the CHO cells using Ambions RNaqueous-4-PCR kit (Ambion) according to the manufacturers protocol. Two-step RT-PCR was used to quantitate the receptor. First, a reverse transcription reaction was performed where the total RNA is converted to cDNA using TaqMan reverse transcription reagents (Applied Biosystems). This was followed by the PCR reaction using the TaqMan Universal PCR Master Mix and an ABI Prism 7000 HT Detection System (Applied Biosystems). Again, all reactions performed were done according to manufacturers protocols. The primers and probe that were used were as follows: AT2R (forward): 5-CCGCATTTAACTGCTCACACA-3; (reverse): 5-ATCATGTAGTAGAGAACAGGAATTGCTT-3; (probe): 5-FAMCCGGCAGATAAGCAT-MGBNFQ-3. Relative quantitation was performed

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32 using ribosomal rRNA (18S) as an endogenous control for the comparative method described in Applied Biosystems User Bulletin #2. No reverse transcriptase and no template controls were used for each sample to ensure that there was no contaminating amplification. Ligand Binding Assay Ligand-specific binding of 125I-SI-AngII to the AT2R was performed as previously described.92 Briefly, cells were washed with Dulbeccos phosphate-buffered saline (PBS; Cellgro), followed by a 30-minute incubation with the reaction mixture containing 0.1-10 nmol/L 125I-SI-AngII (Washington State University), 0.5% bovine serum albumin (Sigma), and 1 M of the AT1R-specific antagonist, losartan (Los; Merck) prepared to a final volume of 0.4 mL in PBS. Additionally, 1 M of the AT2R-specific antagonist, PD123,319 (PD; Sigma) was added to the binding reaction mixtures. Following this incubation, the cells were washed with ice-cold PBS to remove the unbound ligand; the cells were then dissolved and collected in 0.1 N NaOH (Sigma) before being read on a Beckman 5500 counter. AT2R-specific binding was calculated by subtracting the binding of the non-specific reactions (Los + PD) from the total binding (Los only). These values were then normalized to the protein content of each reaction as determined by the method of Lowry et al.93 Protein Isolation and Determination Total cell lysates were isolated from CHO cells and used to determine the levels of activated MAPK by measuring the levels of phosphorylated Erk42/44 via Western blot analysis. CHO cells infected with either the control virus (Lenti-I-PLAP) or the experimental virus (Lenti-AT2R-I-PLAP) were plated at a concentration of 140,000 cells

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33 in a 35 mm dish. They were grown in Hams complete media to confluency, changing the media every 2 days. Once confluent, the media was replaced with serum-free Hams media. The cultures were incubated with the indicated drugs at the following concentrations: 100 nM AngII, 1 M Los, or 1 M PD. In a time-course, cells were incubated with 100nM AngII from 0-20 minutes, to determine the amount of time need to reach maximum activation of Erk42/44. At the end of the incubations, the cells were washed with ice-cold PBS and the proteins were isolated using ice-cold lysis buffer composed of 1% NP40, 10% glycerol, 150 mM NaCl, 20 mM Tris-HCl (pH 7.4), and protease inhibitor cocktail (125 mM PMSF, 2.5 mg/mL aprotinin, 2.5 mg/mL leupeptin, 2.5 mg/mL antipain, 2.5 mg/mL chymostatin; Sigma). Cell lysates were scraped using a rubber policeman and collected in a microcentrifuge tube. The samples were then sonicated 3 times for 5 seconds each on ice and centrifuged at 14,000 rpm for 10 minutes at 4 C. The supernatant was then saved, and protein levels were determined using the manufacturers protocol for BioRads Bradford-based protein assay (BioRad). Detection of Activated MAPK Samples (20g) were separated on Ready-Made 10% Tris-HCl gels (Bio-Rad), and proteins were transferred to a nitrocellulose membrane using the Bio-Rad Mini-Protean system. Protein detection of phosphorylated Erk42/44 was performed as suggested by the manufacturers protocol (Promega). Briefly, nitrocellulose membranes were washed for 5 minutes in TBS, followed by an overnight incubation at 4 C in blocking buffer composed of 3% BSA in TBS. The membrane was then incubated for 2 hours at room temperature in Anti-ACTIVE-MAPK (Promega) primary antibody diluted 1:2500 in TBS + 0.1% Tween (TBST) plus 0.1% BSA. The membrane was then washed 3 times, 15 minutes each in TBST and then incubated for 1 hour at room temperature in the horse

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34 radish peroxidase conjugated anti-rabbit secondary antibody (Promega) diluted 1:5000 in TBST + 5% milk. This was followed by 3 15 minute washes in TBST. Finally, the bands were visualized using a Western Lightning chemiluminescence (DuPont). Following this, the membranes were stripped of antibody and a second Western blot was performed on the same membrane to detect paxillin, a control protein used to normalize the samples. Membranes were stripped for 18 minutes at 57 C using a buffer consisting of 60 mM Tris (pH 6.8), 2% SDS, 0.7% -Mercaptoethanol. Next, the membranes were washed 4 times for 5 minutes each in TBST and blocked for 1 hour at room temperature in a solution containing 5% milk in TBST. The membrane was then incubated for 1 hour at room temperature with anti-paxillin (Promega) primary antibody diluted 1:10,000 in the 5% milk/TBST solution. This was followed by 3-5 minute washes in TBST and a 45 minute incubation at room temperature with the secondary anti-mouse antibody (Promega) diluted 1:5000 in the 5% milk/TBST solution. Finally, the membranes were washed 3 times for 5 minutes each in TBST and bands were visualized using chemiluminescence. All bands for active MAPK and paxillin were quantitated using BioRads GS-710 densitometer. Statistical Analysis Results were analyzed with a students t-test when only 2 samples were being examined. All other experiments were analyzed using a one-way ANOVA. Results are indicated as mean +/standard error with a p value of 0.05 being considered significant. Results Overexpression of the Receptor by Lenti-AT2R CHO-AT1R cells were infected with either the Lenti-AT2R-I-PLAP (AT2R) or Lenti-I-PLAP (Cntrl) at a MOI of one. Real-time RT-PCR analysis revealed a significant

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35 increase in the levels of the AT2R mRNA in those CHO-AT1R cells transduced with the AT2R (Figure 2-2A). This increase was associated with an increase in AT2R binding. CHO-AT1R cells do not contain any endogenous AT2R. When tranduced with the Lenti-AT2R-I-PLAP, however, the total binding activity for the AT2R was 4.85 pmol/mg protein with a Kd of 0.82 nM (Figure 2-2B). These results indicate that the lentiviral vector can efficiently deliver the AT2R in vitro and that these transduced cells exhibit binding characteristics typically associated with the AT2R. 0500100015002000250030003500CntrlAT2RArbitrary Units (Normalized to 18S)* Saturation Curve012345020004000Free 125I-SI-AII (pmol/L)Bound 125I-SI-AII (pmol/mg) ScatchardGraph00.0020.0040.0060.0080.0124Bound(pmol/mg)Bound/Free (pm/mg/pm/L)0 6 Kd= 0.82nMBmax = 4.85 pmol/mgAB Figure 2-2: AT2R overexpression in CHO-AT1R cells. Lenti-AT2R-I-PLAP (AT2R) or Lenti-I-PLAP (Cntrl) was used to transduce CHO-AT1R cells at a MOI of 1. A) Three days post-infection, the cells were analyzed for AT2R mRNA by real-time RT-PCR (n=3). B) In addition, AT2R-specific binding was analyzed by ligand binding. (* = p<0.05).

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36 Characterization of AT2R Transgene Function CHO-AT1R cells were first used to determine the effect of AngII on MAPK activation in these cells. Cells were incubated with AngII or AngII + Los. Western blot analysis for phosphorylated Erk42/44 (activated MAPK) revealed little endogenous MAPK activity. Incubation with AngII caused a time-dependent increase in MAPK activation with maximum activation being achieved in 10 minutes (Figure 2-3). This activation was reduced when Los was added to the cells at all time points (Figure 2-3) while Los alone had no effect on MAPK activity (Figure 2-3). Based on these results, subsequent experiments were carried out at the 10 minute timepoint. Untrt5min10 min20min5min10 min20minLosAngII(1uM)AngII + Los(1uM each) Erk42Erk44 Figure 2-3: Time course of AngII-induced Erk42/44 activation in CHO-AT1R cells. CHO-AT1R cells were tested to determine if AngII induced ERK42/44 activity through the AT1R. The cells were either left untreated or treated for 5, 10, or 20 minutes with either 1 M AngII, 1 M AngII + 1 M Los or 20 minutes with Los. Following these incubations, proteins were isolated and analyzed for phosphorylated Erk42/44 (Active MAPK) by Western blot analysis. Next, we determined if AT2R transduction in the CHO-AT1R cells would inhibit AT1R-mediated activation of MAPK. CHO-AT1R cells transduced with Lenti-I-PLAP (Cntrl) showed a 65-fold increase in Erk42 activity following AngII stimulation (Figure 2-4). In the CHO cells transduced with Lenti-AT2R-I-PLAP (AT2R), however, stimulation of MAPK with AngII resulted in only a 7-fold increase in activity (Figure 2-4). This effect, however, was not reversed by PD (Figure 2-5) nor altered by Los (Figure 2-5).

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37 Erk42 00.511.522.533.54CntrlCntrl+ AIIAT2RAT2R + AIIOD/mm2Norm to Paxillin PaxillinErk42Erk44* p=0.08p=0.09 Figure 2-4: AT2R transduction prevents AngII-mediated increases in phosphorylated Erk42/44. CHO-AT1R cells were either transduced with Lenti-I-PLAP (Cntrl) or Lenti-AT2R-I-PLAP (AT2R). Three days post-transduction, the cells were treated with AngII for 10 minutes, proteins were isolated, and phosphorylated Erk42/44 was examined by Western blot analysis. (Top: Representative Western blot showing Erk42/44 activity; Bottom: Quantitation of Erk42 expression. n = 2/group; = p<0.05 compare to Cntrl). PaxillinErk42Erk44UntrtAngIIUntrtAngIIAngII+LosAngII+PDLosPDAT2R Cntrl Figure 2-5: AT2R-mediated effects of Erk42/44 activity cannot be reversed by PD123,319. CHO-AT1R cells transduced with either Lenti-I-PLAP (Cntrl) or Lenti-AT2R-I-PLAP (AT2R) were treated for ten minutes with the indicated drugs (untreated (Untrt) or 1 M AngII, 1 M Los or 1 M PD) and analyzed for Erk42/44 activity by Western blot analysis.

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38 Discussion The results presented in this chapter demonstrate that (1) infection of CHO-AT1R cells with Lenti-AT2R results in an increase in the expression of the AT2R transgene, (2) the AT2R is functional, (3) the transduced AT2R may be acting ligand independently. Previous studies overexpressing the AT2R in vitro were unable to associate the AT2R with a functional consequence.94,95 Our study is one of the first demonstrations of an AT2R transgene functionally coupling to its signaling cascade. The advantage of this study over the others is that the Lenti-AT2R was able to transduce and overexpress the AT2R at high levels without selection, thus allowing us to study the effects of AT2R overexpression as early as 3 days post-infection. Our data indicate that the AT2R inhibits AngII-mediated increases in MAPK. This is consistent with previous data. These studies have shown that the AT2R can directly bind to the AT1R to inhibit the actions of the AT1R.39 In addition, it has been shown that the AT1R and AT2R have opposing actions on MAPK in the same primary cultured cells. However, the researchers could not conclusively state that these opposing actions were occurring in the same cell and whether or not the receptors were having a direct effect on each other.90 In similar cultures, however, the same group was able to show that the AT1R and AT2R have opposing actions on potassium current.96 Finally, it has been shown in chromaffin cells that the AT2R can negatively regulate the AT1R signaling pathways through the regulation of cGMP.97 Based on our findings and those of others, it will be interesting to the mechanism of these actions, whether it is through a steric hindrance of the AT2R directly binding to the AT1R, the regulation of cGMP,

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39 AT2R-mediated dephosphorylation by activation of phosphatases, or by some other unknown mechanism. The antagonistic effect we observed in these studies could not be due to non-specific effects, whereby stimulation of the AT1R by AngII is decreased because of the abundance of AT2Rs to compete for binding sites? There are two explanations that indicate that it is a signaling effect rather than just a binding effect. First, the Kd for both the AT1R and AT2R is less than 1 nM. In these studies we are using 100 nM of AngII to stimulate the cells. Therefore, there is an abundance of AngII available to bind to the AT1R. Secondly, the affects we are seeing cannot be reversed by an AT2R antagonist. These results indicate that AT2R overexpression decreases the ability of AngII to stimulate MAPK activity presumably through the AT1R. These results also indicate that the AT2R appears to be acting in a ligand-independent manner. This conclusion is supported by several factors. (1) We were unable to reverse the actions of the AT2R on MAPK activity by the addition of an AT2R specific antagonist. We believe that this is due to an inability to reverse the actions and not a timing effect of the addition of the antagonist because the blockade by PD was performed in two different ways (addition with AngII or addition 1 hour before AngII) with similar results each time. (2) Previous studies have also indicated ligand-independent roles of the AT2R.22,24 These studies indicate that the level of AT2R expression determines whether or not the AT2R is acting through a ligand-independent manner or not. These studies are fascinating in that they provide us with a lentiviral vector which can transduce a functional AT2R. However, they also raise some important questions related to the mechanism by which a ligand-independent AT2R attenuates AT1R

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40 mediated actions. Does the AT2R directly interact with the AT1R as a heterodimer to prevent the actions of AngII on this receptor? Or is the AT2R mediating its effects downstream through the inhibition of signaling mechanisms? If so, what are those signaling cascades? Finally, does the AT2R have a mechanism that increases the internalization of the AT1R? In spite of these questions, our observation is significant in that we were able to demonstrate that the Lenti-AT2R can transduce and overexpress a functional receptor. Future studies examining the mechanisms by which the AT2R mediates its ligand-independent activity and its effects on the AT1R will be fascinating.

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CHAPTER 3 IDENTIFYING GENES INVOLVED IN ANGIOTENSIN II TYPE 2 RECEPTOR-MEDIATED SIGNALING PATHWAYS BY EXPRESSION PROFILING Introduction The AT2R has been implicated to play a role in the development and prevention of different pathophysiologies. In the cardiovascular system, however, the physiological role of the AT2R remains elusive. A number of studies indicate that the AT2R plays a cardioprotective role to induce vasodilation as well as prevent the development of cardiac hypertrophy and heart failure.36,98 There are, however, just as many studies indicating that the AT2R actually plays a role in the development of some of these CV pathophysiologies. For example, the AT2R has been shown to be necessary for the development of cardiac hypertrophy, heart failure, and even cardiomyopathies.41,42,45,99,100 In addition, separate studies have shown that (1) the AT2R prevents or reduces cell migration and angiogenesis56 and (2) blockade of the AT2R reduces angiogenesis.101 These studies illustrate the discrepancies associated with the physiological role of the AT2R. Because of these differing roles of the AT2R in the CV system, a better understanding of the cellular processes and signal transduction cascades of the AT2R may provide some insight as to how and when the AT2R acts in a protective versus a detrimental manner. One way to globally assess the cellular response to the AT2R is through the use of microarrays. Microarray analysis is a growing technology based on a chip containing thousands of genes to simultaneously and quantitatively analyze genetic 41

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42 profiles in one experiment. These methods have been applied for both clinical and basic science research. Clinically, microarrays analysis is an emerging field to determine disease mechanisms and to identify novel candidates for therapeutic interventions. Basic scientists use these techniques to assess the transcriptional affects and transduction cascades associated with specific genetic and pharmacological interventions. Endothelial cells line blood vessels in circulation and thus play an important role in CV regulation. Endothelial dysfunction can lead to atherosclerosis, ultimately leading to stroke and myocardial ischemia and infarction. In addition, the endothelium has been shown to play an important role in angiogenesis. In fact, endothelial cell activation is the first process to take place in both physiological and pathological angiogenesis. Through the use of microarray analysis and the vast knowledge of the role of the endothelium in CV disease, the goal of this aim was two-fold. (1) Elucidate novel signaling pathways of the AT2R using microarray technology. This high throughput method, will allow us to identify multiple signaling pathways and genetic profiles associated with the AT2R. (2) Determine if any of these genes segregate with a functional aspect associated with endothelial cells that is modulated by the AT2R. Materials and Methods Cell Culture, AT2R Transduction, and Treatments Frozen vials of human coronary artery endothelial cells (HCAEC) of passage 3 were obtained from Clonetics. Cells were grown in EGM2-MV growth medium (Clonetics). The protocol is outline in Figure 1-1. Briefly, the cells were thawed, plated, and allowed to grow, changing the medium every 2 days. Once they reached confluency (approximately 7 days), the cells were passaged at a concentration of 2500 cells/cm2 in 4-100mm dishes. The following day, the cells were transduced with either Lenti-GFP

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43 (MOI 10), Lenti-I-Neo (MOI 1) or Lenti-AT2R-I-Neo (MOI 1) as described in Chapter 2. The medium containing lentivirus was removed from the cells the next day and allowed to grow for an additional 2 more days before selection began. At this time (day 11), the growth medium on the cells was changed to EGM2-MV plus 800 g/l geneticin (G418). The cells remained on G418 selection for 2 weeks, providing fresh medium and G418 every 2 days. After one week of selection, the cells were passaged at a concentration of 2500 cells/cm2. At the end of the 2 weeks of selection, the cells were confluent. At this time the cells were passaged for the microarray experiment at 2500 cells/cm2 and grown for an additional 5 days, changing the medium every 2 days. PlateHCAECPassage 3 Subculture HCAEC 1.4105Cells Passage 4 LentiviralTransduction MOI <1.0 Selection Subculture TransducedCells 1.4105Cells Passage 5 Treat Cells Isolate RNA for Microarray Cell Growth and Maintenance13334Time (Days)7 811281823 LigandBinding Isolate RNA for Real-Time Figure 3-1: Timeline for the HCAEC used in the microarray experiments. At this time, the cells were treated for 24 hours with either the AT2R-specific agonist, CGP42112A (Sigma), or viral resuspension buffer as a control (untreated). Following this incubation, total RNA was isolated from the cells for microarray experiments and additional plates were used for binding and expression analysis. There were essentially 4 groups that were compared by microarray analysis: (1) Lenti-I-Neo (Control) transduced cells that were left untreated, (2) Control transduced cells treated

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44 with CGP42112A, (3) Lenti-AT2R-I-Neo transduced cells that were left untreated, and (4) Lenti-AT2R-I-Neo transduced cells that were treated with CGP42112A. Real-Time RT-PCR Total RNA was isolated from cells using Ambions RNaqueous-4-PCR kit using the manufacturers protocol, and real-time RT-PCR was performed for the AT2R as described in Chapter 2. Real-time RT-PCR for gene validation was performed using primers and probes from Applied Biosystems Assays-on-Demand (catalog numbers: Ubiquitin Thiolesterase, Hs00188233_m1; RGS-7, Hs00175619_m1; IGFBP-3, Hs00426287_m1). For all these experiments, two-step real-time RT-PCR protocols were used. RNA (1ug) was converted to cDNA using Applied Biosystems TaqMan reverse transcription reagents in a total volume of 50 L. Following the conversion to cDNA, a PCR reaction was set-up using TaqMan Universal PCR Master Mix. For the AT2R, 1 ng of cDNA, 0.9 M of each primer and 0.5 M of probe was used. For the Assays-on-Demand reactions, 20 ng of cDNA was used, and 12.5 L of the primers and probe mixture was added to each 25 L reaction. The PCR plate was set-up according to the manufacturers suggestions and was run at 50 C for 2 minutes, 95 C for 10 minutes, and then 40 cycles of 95 C for 15 seconds and 60 C for 1 minute. In all of the experiments, cDNA was diluted to 100 pg for the AT2R and 20 pg for the Assays-on-Demand experiments to be used to quantitate ribosomal 18S as an endogenous control (Applied Biosystems). Each sample had a reaction set up that did not contain any reverse transcriptase as a control for genomic DNA contamination. In addition, another control that did not contain any RNA was used to measure non-specific amplification. Relative quantitation of gene expression was determined using the comparative CT method. The average threshold cycle (CT) of each sample was related to the CT of its endogenous

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45 control, 18S. The samples were then compared to the lowest expressing sample (calibrator) as described in Applied Biosystems User Bulletin #2. AT2R Binding Assay Ligand-binding assays for the AT2R were performed to determine the Bmax and Kd for the transduced AT2R as previously described in Chapter 2. Microarray Analysis Total RNA was isolated, DNase-inactivated and concentrated from HCAEC using Ambions RNAqueous-4PCR according the manufacturers protocols. For these protocols, 4-100 mm dishes were used for each group. The initial lysis was performed with 500 L of lysis buffer added to each dish. The RNA was eluted from each filter cartridge in 2 aliquots of 80 L and 40 L of elution buffer. The RNA was DNase-inactivated for 30 minutes and concentrated to a volume of 35 L. The RNA was then prepared for hybridization to the human U133A microarray chip using the protocols outlined in GeneChip Expression Analysis Overview (Affymetrix) and highlighted in Figure 3-2. Total RNA isolated from the treated HCAEC was assessed for quality and concentration using both a spectrometer and a bioanalyzer. The RNA was used to synthesize double-stranded cDNA using the SuperScript Choice System (Invitrogen). First strand cDNA was synthesized using the HPLC purified T7-(dT)24 primer; 5 GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24 3. The primer is first hybridized to the RNA by mixing 8g of RNA with 100 pmol T7-(dT)24 primer for 10minutes at 70 C. Following this incubation, 10 mM DTT and 500 M of dNTP mix was added to the reaction and the temperature was adjusted to 42 C for 2 minutes. The Superscript reverse transcriptase is then added to the reaction and the first-strand of cDNA is synthesized at 42 C for 1 hour. The second strand of cDNA is

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46 synthesized by adding a mixture of second-strand reaction buffer, 200 M of each dNTP, 10 units of DNA ligase, 40 units of DNA polymerase I and 2 units of RNase H to the first strand cDNA reaction and incubating at 16 C for 2 hours. Following this reaction, 10 units of T4 DNA polymerase is added and incubated for an additional 5 minutes. Finally, 10 L of 0.5 M EDTA is added to stop the reaction. The double-stranded cDNA was then cleaned-up using Affymetrix Gene Chip Sample Cleanup Module according to the manufacturers protocol. Following this cleanup procedure, biotinylated cRNA was created in an in vitro transcription reaction using the ENZO BioArray High Yield RNA Transcript Labeling Kit (Affymetrix) and cleaned up using the GeneChip Sample Cleanup Kit (Affymetrix) according to the manufacturers protocol. To produce the biotinylated cRNA, 10 L of the cleaned-up cDNA was added to a reaction containing 1X each of the provided reaction buffers, biotin-labeled ribonucleotides, DTT, RNase inhibitor mix and T7 RNA polymerase and incubated at 37 C for 4-5 hours. Once cleaned-up, the cRNA was quantified spectrophotometrically and fragmented in a fragmentation buffer (200 mM Tris-acetate, pH 8.1, 500 mM KOAc, 150 mM MgOAc) and incubated at 94 C for 35 minutes. The fragmented cRNA (20ug) was then hybridized to the microarray chip for 16 hours at 45 C. This is followed by a series of washing and staining with streptavidin phyoerythrin conjugate performed by an Affymetrix fluidics station. Once the chips are stained, they are scanned by a GeneArray Scanner (Affymetrix) at an excitation wavelength of 488 nm. Microarray Analysis Controls and Data Analysis There are a number of controls that are used to assess quality of each chip and enhance the ability to compare between multiple microarray chips. Comparisons of

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47 probe sets which hybridized to the 5 and 3 ends of housekeeping genes such as GAPDH and actin are used to assess the quality of cRNA hybridized to each microarray chip. Validation of select genes usingReal-time RT-PCRTransducedHCAEC Total RNA cDNA cDNA Scanning & VisualizationRTIVT & fragmentationHybridization Untreated or Treat CGP42112A for 24hrs.Gene Expression Profiling B B Biotinylated cRNA fragments B B B BHuman Chip U133AMultiple Probe SetsRepresent ~33,000 Well Substantiated Human Genes Figure 3-2: Outline of the microarray protocol as described in detail in the Materials and Methods section. Hybridization efficiency is monitored by control samples that are spiked into the hybridization cocktail. Probe Profiler (Corimbia, Inc) is used to standardize each array. First, automatic artifact detection, saturation correction, and outlier detection and removal are performed. Probe Profiler also scales the mean array intensity of each chip to a target intensity, called global normalization, before comparisons between chips are made. Following these procedures, an expression score (e-score) is calculated which reflects the expression level of each gene. Any gene with an e-score less than 25 in all four treatment groups is considered absent in the experiment and is removed from any further analysis.

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48 The gene profiles were analyzed for statistical significance of the e-scores by 2-way ANOVA using custom written software from the UF ICBR Microarray Core Facility. Only those significantly changing genes (p<0.05) were investigated further. Gene Spring (Silicon Genetics) gene expression analysis software was used to graphically represent and clarify the significance of the differentially expressed genes. In addition, gene ontology was determined through the use of custom written software from the UF ICBR Microarray Core Facility. Migration Assay Transwell cell migration assays were performed using a 48-well Boyden chemotaxis apparatus (Neuroprobe) as previously described.56 Briefly, 8m membranes were coated in 100 g/mL type1 collagen (Sigma) diluted in 20mM acetic acid overnight. The next day the membranes were rinsed in PBS and placed over the lower chambers of the apparatus which contained EBM-2 medium supplemented with 0.1% BSA (experimental medium) and either 10 ng/mL vascular endothelial growth factor (VEGF) or suspension buffer as a control (no VEGF). HCAEC transduced with either Lenti-I-Neo (Cntrl) or Lenti-AT2R-I-Neo (AT2R) was trypsinized, counted, and 5x103 cells were added to each well of the upper chamber. In addition, the cells in the upper chamber were either not treated or treated with 1 M PD or 10 nM CGP42112A. The filled apparatus was incubated at 37 C at 5% CO2 for 3 hours. Following this incubation, the non-migrated cells were removed, and the membrane was fixed in methanol and stained with Diff-Quick (Fischer Scientific) according to the manufacturers protocols. The number of migrated cells was counted in 5 randomly-chosen fields under 20x

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49 magnification. Experiments were performed a total of 3 times, all with similar results. Figure 3-6 is a representative of one experiment. Angiogenesis Protein Array A TranSignal Angiogenesis Antibody Array (Panomic) was used according to manufacturers protocol. Using 1mL of RIPA buffer (1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mg/mL PMSF, 20 L/mL sprotinin, 100 mM sodium orthovanadate made in PBS), proteins were isolated from Cntrl and AT2R-transduced HCAEC either not treated or treated with 10nM of the AT2R specific agonist, CGP42112A. Proteins were then quantitated using the Bradford method described in Chapter 2. The angiogenesis protein array was incubated in Blocking Buffer for 2 hours at room temperature. Following this, the membranes were rinsed 2x in Wash Buffer II and 1 mg of protein was added to each membrane. The membranes were incubated with the proteins for 2 hours at room temperature, washed 3 times for 5 minutes each in Wash Buffer I and washed once for 5 minutes in Wash Buffer II. Following this, the Biotin-conjugated Angiogenesis Antibody Mix was added to the membranes and incubated for 3 hours at room temperature. The membranes were washed again with Wash Buffer I and II as described above. Then the Strepavidin-HRP Conjugate was added to each membrane and incubated for 60 minutes at room temperature. The membranes were washed a final time in Wash Buffer I and II as described above and incubated in the Detection Buffer for 5 minutes. The proteins were visualized using x-ray film. Statistics Microarray data was analyzed by 2-way ANOVA as described above. All other experiments were analyzed by 1-way ANOVA. Results are indicated as mean +/standard error with statistical significance being set at the 95% confidence level.

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50 Results Characterization of AT2R Transduction of HCAEC The ability of the lentiviral vector to efficiently transduce HCAEC was investigated. Gene transfer into endothelial cells has historically been difficult to accomplish, and the Lenti-GFP expression plasmid was first used to determine if the lentivirus could transduce this cell type. Cells transduced at an MOI of 10 efficiently infected the majority of the HCAEC at 48 hours post-transduction, as evidenced by the fluorescent green cells (Figure 3-3A). HCAEC were transduced with either Lenti-I-Neo (Cntrl) or Lenti-AT2R-I-Neo (AT2R) at an MOI of 1. The AT2R-transduced cells showed a 12-fold increase in mRNA of the AT2R over the Cntrl-transduced cells (Figure 3-3B). This increase in AT2R mRNA was also associated with an increase in AT2R-specific binding. Saturation and scatchard binding analysis revealed total AT2R binding of 11.5 pmol/mg of protein with a Kd of 1.25 nM (Figure 3-3C). These results indicate that the lentivirus can efficiently transduce HCAEC to overexpress the AT2R. Expression Profiling of AT2R-Transduced HCAEC Microarray analysis was used to determine the novel signaling effects of AT2R overexpression and AT2R activation in HCAEC. Four experimental groups (n=2 microarray chips per group) were used in the following experiments: (1) Lenti-I-Neo transduced cells with no treatment (Cntrl-Untrt); (2) Lenti-I-Neo transduced cells treated with 10 nM CGP42112A (Cntrl-CGP); (3) Lenti-I-AT2R-transduced cells left untreated (AT2R-Untrt); (4) Lenti-AT2R-transduced cells treated with 10 nM CGP42112A (AT2R-CGP).

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51 UninfectedpTYF-GFP 0246810121416CntrlAT2RArbitrary Units*AT2R mRNA Scatchard Plot2468100510Bound (pmol/mg)Bound/Free (pm/mg)/(pm/L)x 10-30 Scatchard Plot2468100510Bound (pmol/mg)Bound/Free (pm/mg)/(pm/L)x 10-30 Saturation Curve0246810120246125I-SI-AII (cpm x 105)125I-SI-AII Bound (pmol/mg) 8 Saturation Curve0246810120246125I-SI-AII (cpm x 105)125I-SI-AII Bound (pmol/mg) 8 Kd = 1.25nMBmax = 11.5 pmol/mgABC Figure 3-3: Lentiviral transduction in HCAEC. Lenti-GFP was used to determine the transduction efficiency of the lentivirus. A) HCAEC were transduced at a MOI of 10 and analyzed for green fluorescence 2 days post transduction. HCAEC were transduced with Lenti-I-NeoR (Cntrl) or Lenti-AT2R-I-NeoR (AT2R). B) Following selection with G418, RNA was isolated from the cells and examined for AT2R mRNA by real-time RT-PCR (n = 3; = p<0.05). C) HCAEC transduced with the AT2R was also examined for AT2R ligand binding. Scatchard and Saturation binding curves performed, as described in the Materials and Methods, revealed a Bmax of 11.5 pmol/mg of protein and a Kd of 1.25 nM. Of the 33,000 genes represented on the microarray chip, 5,224 of them were differentially expressed following AT2R overexpression. In addition, 1,235 genes were differentially expressed with treatment of 10 nM CGP42112A for 24 hours. Scatter plotswere used to graphically represent those genes which are differentially expressed following overexpression of the AT2R (Figure 3-4A). Treatment of the AT2R

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52 transduced cells with CGP42112A produced fewer changes in gene expression compared to only overexpressing the AT2R (Figure 3-4B). 0.11101001000 0.010.11101001000 CntrlAT2R 0.11101001000 0.010.11101001000 CntrlAT2R AT2R-Untrt AT2R-CGPAB UbiquitinThiolesterase Fibrillin2IGFBP3 RGS-7ILK 16 PAK4 HMG-CoAReductase Ubiquitin-Specific Protease 4Decorin Figure 3-4: Scatter plots of the microarray data. HCAEC were transduced with either Lenti-I-NeoR (Cntrl) or Lenti-AT2R-I-NeoR (AT2R) and either left untreated (Untrt) or treated for 24 hours with the AT2R specific agonist, CGP42112A (CGP) for microarray analysis as outlined in Figure 3-1. A) Microarray analysis revealed 5,224 genes that were significantly differentially expressed between the Cntrl and AT2R groups and are represented as a scatter plot. Each dot represents a gene that had a significantly-altered profile between these two groups (p<0.05). B) Comparison of the number and expression of genes significantly differentially expressed with just AT2R expression (green) and those that are stimulated by CGP42112A (red). Again, each dot represents a significantly-changing gene (B; p<0.05). To clarify our gene list into a manageable number of genes to analyze, genes that changed more than 50% and those which are of particular interest are listed in Tables 4-1 through 4-3. Genes of particular interest are genes which were defined by gene ontology analysis as having a role in cell adhesion, mobility, and/or migration. This selection criteria was based on our results presented in the following sections. Table 3-1 lists those genes which are down-regulated by the expression of the AT2R. Many of these genes played a role in the regulation of cell adhesion, mobility and/or migration. Table 3-2

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53 indicates those genes which are upregulated by AT2R transduction. Finally, Table 3-3 lists the genes which are differentially expressed in the AT2R-transduced cells following treatment with CGP42112A. The results of AT2R transduction and treatment in Table 3-3 are compared to the combined control cells left untreated and the control cells treated with CGP42112A. Each table list the genes accession number, common name, average e-score for each group, and the percentage change. Genes highlighted in yellow are of particular interest and are described more in the discussion. Table3-1: AT2R decreases gene expression without CGP42112A stimula tion. Access #Gene TitleAvg AT2Avg Neo% Chan g eNM_004181 Ubiquitin thiolesterase96.08531.18-81.91NM 001999 Fibrillin 250.15206.83-75.75NM_000598 Insulin-like growth factor binding protein 338.73141.28-72.59U71300 Small nuclear RNA activating com p lex30.2864.68-53.19NM 005132 Rec8 p 36.1374.35-51.41M90391Interleukin 1640.1050.78-21.024867555 RCPAK-4165.55204.68-19.12NM_000859 HMG-CoA reductase105.38120.43-12.50 Microarray data was analyzed for significantly differentially expressed genes by 2-way ANOVA. This table represents those genes that were either decreased at least 50%, or identified by gene ontology studies as having a role in cell adhesion, mobility or cell migration. The accession number (Access #), common gene name/title, e-scores for both AT2R (Avg AT2) and Cntrl (Avg Neo), and percent change (% Change) are given. Genes highlighted in yellow are described further in the text. Gene Validation Real-time RT-PCR was used to validate those genes, which were significantly differentially expressed from the microarray analysis. Selection of the genes to be quantitated was based upon their extent of regulation and its proposed role in the CV

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54 system. The genes that were validated were greatly regulated by AT2R transduction and literature searches revealed that they also played an important regulatory role in the CV system. Table 3-2: List of genes whose expression was significantly increased with AT2R expression independent of ligand. Access # Gene Title Avg AT2 Avg Neo % Change BC000977 Aminolevulinate 127.78 85.03 50.28 AF015524 Chemokine (C-C motif) receptor-like 2 26.63 17.65 50.85 NM_004125 Guanine nucleotide binding protein 10 424.75 280.58 51.39 NM_005028 Phosphatidylinositol-4-phosphate 5-kinase 22.95 15.15 51.49 AL042733 BRCA1 associated protein 52.43 34.35 52.62 J03620 Dihydrolipoamide dehydrogenase 367.15 239.93 53.03 NM_000995 Ribosomal protein L34 2017.63 1315.50 53.37 M96651 Interleukin 5 receptor 34.30 22.25 54.16 NM_014059 RGC32 protein 347.48 224.53 54.76 NM_014135 PRO0641 protein 26.83 17.30 55.06 NM_016216 Debranching enzyme homolog 1 29.63 19.10 55.10 BC002666 Guanylate binding protein 1 159.30 102.63 55.23 BF000239 Chromatin assembly factor 1 25.15 16.18 55.49 NM_001655 Archain 1 528.75 336.78 57.00 D43968 Runt-related transcription factor 1 24.23 15.43 57.05 BG532690 Integrin, alpha 4 25.53 16.05 59.03 BF593908 TATA element modulatory factor 1 45.23 28.33 59.66

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55 Table 3-2: Continued. Access # Gene Title Avg AT2 Avg Neo % Change AI761771 Chromodomain helicase DNA binding protein 4 26.48 16.58 59.73 NM_004202 Thymosin, beta 4 139.63 86.90 60.67 NM_000557 Growth differentiation factor 5 26.45 16.38 61.53 M12423 T cell receptor alpha locus 28.05 17.33 61.90 NM_002886 RAP2B 29.70 18.30 62.30 NM_003668 MAPK-activated protein kinase 5 33.55 20.63 62.67 NM_002028 Farnesyltransferase 25.63 15.63 64.00 AI989512 HIV-1 Rev binding protein 104.80 63.73 64.46 NM_002601 Phosphodiesterase 6D 51.03 30.95 64.86 AL574096 Tissue factor pathway inhibitor 2 25.00 15.15 65.02 AF055994 PPAR binding protein 27.45 16.58 65.61 U20760 Calcium-sensing receptor 25.38 15.08 68.33 D42045 DNA cross-link repair 1A 28.18 16.73 68.46 BC000103 NCK adaptor protein 2 129.63 76.43 69.61 AI005066 Arginine vasopressin receptor 1A 41.75 24.48 70.58 AF074717 RAD1 homolog 25.80 15.08 71.14 M12959 T cell receptor alpha locus 43.48 25.20 72.52 BG260658 CS box-containing WD protein 94.65 54.50 73.67 AF249671 NK3 transcription factor homolog A 52.43 30.05 74.46 NM_024430 Pro-ser-thr phosphatase interacting protein 2 76.93 43.68 76.13

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56 Table 3-2: Continued. Access # Gene Title Avg AT2 Avg Neo % Change NM_004500 Heterogeneous nuclear ribonucleoprotein C 712.83 402.63 77.04 NM_015930 Transient receptor potential cation channel 129.48 70.65 83.26 NM_016277 RAB23 76.00 40.83 86.16 X75940 SMA3 31.43 16.73 87.89 NM_004381 cAMP responsive element binding protein-like 1 33.05 17.53 88.59 NM_025019 Tubulin, alpha 4 56.75 29.68 91.24 NM_002924 Regulator of Gprotein signalling 7 40.53 19.78 104.93 NM_003430 Zinc finger protein 91 35.93 16.83 113.52 AF047190 Sarcosine dehydrogenase 41.25 18.73 120.29 NM_030786 Intermediate filament protein syncoilin 35.35 15.45 128.80 NM_000439 Proprotein convertase subtilisin/kexin type 1 182.78 65.63 178.51 BE875592 Vesicle docking protein p115 49.90 17.45 185.96 NM_005526 Heat shock transcription factor 1 48.68 15.63 211.52 This table represents those genes that were increased significantly by 50% or more as described in Table 3-1. Validation of both ubiquitin thiolesterase and regulator of G-protein signaling 7 (RGS-7) showed a significant difference in the same direction as the microarray had predicted (Figure 3-5; 92% decrease for ubiquitin thiolesterase; 94% increase in RGS-7). In addition, insulin growth factor binding protein 3 showed the same trend as the microarray data of a 60% decrease with AT2R expression although it only reached the 94% confidence level (Figure 3-5). These results indicate that the microarray data is

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57 valid and some of the observed changes seen by microarray analysis can also be observed through real-time RT-PCR. Table 3-3: List of genes whose expression was significantly altered in the AT2R-transduced cells stimulated with CGP42112A. Accession # Gene Title AT2 Untrt AT2 CGP Avg Neo % Change AT2 Untrt % Change AT2 CGP NM_003128 Spectrin, beta 105.60 58.20 66.15 59.64 -12.02 BC005354 Ribosomal protein 61.00 25.40 27.08 125.30 -6.19 BC000603 Ribosomal protein L38 361.25 160.05 236.33 52.86 -32.28 NM_014292 Chromobox homolog 6 84.30 83.65 55.38 52.23 51.06 NM_003363 Ubiquitin specific protease 4 20.75 35.95 22.25 -6.74 61.57 NM_003893 LIM domain binding 1 18.55 22.80 15.08 23.05 51.24 NM_003430 Zinc finger protein 91 49.05 22.80 16.83 191.53 35.51 NM_014212 Homeo box C11 38.40 27.85 65.85 -41.69 -57.71 NM_005732 RAD50 homolog 38.55 48.90 30.10 28.07 62.46 AW025108 Topoisomerase I 28.40 22.85 18.00 57.78 26.94 AI281593 Decorin 29.00 24.55 50.98 -43.11 -51.84 NM_016073 Transmembrane 6 superfamily member 1 73.45 51.80 48.00 53.02 7.92 U35139 Necdin homolog 48.70 59.20 105.48 -53.83 -43.87 D42045 DNA cross-link repair 1A 34.85 21.50 16.73 108.37 28.55 AF130102 Retinoic acid repressible protein 53.60 28.45 34.28 56.38 -16.99 U71300 Small nuclear RNA activating complex 27.05 33.50 64.68 -58.18 -48.20 BC001259 Adaptor-related protein complex 4 18.35 21.10 37.88 -51.55 -44.29

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58 Table 3-3: Continued. Accession # Gene Title AT2 Untrt AT2 CGP Avg Neo % Change AT2 Untrt % Change AT2 CGP AF061193 Ectodermal dysplasia 1 26.15 20.60 15.65 67.09 31.63 M95489 Follicle stimulating hormone receptor 17.85 23.60 15.08 18.41 56.55 AF258449 Estrogen receptor 1 19.10 27.50 45.03 -57.58 -38.92 AF120491 Potassium voltage-gated channel 17.85 23.15 15.08 18.41 53.57 BE737027 Ribosomal protein L27a 119.95 62.70 62.63 91.54 0.12 NM_015322 Fem-1 homolog 59.10 49.30 37.63 57.08 31.03 NM_003668 MAPK-activated protein kinase 5 29.25 37.85 20.63 41.82 83.52 BF680255 Ribosomal protein S11 48.00 20.80 30.58 56.99 -31.97 BF593727 Ras homolog 42.75 22.65 24.30 75.93 -6.79 AA748649 YY1 transcription factor 48.20 27.50 27.53 75.11 -0.09 BE312027 Ribosomal protein L27 151.40 85.45 89.58 69.02 -4.61 L07335 SRY-box 2 34.25 20.75 47.73 -28.23 -56.52 BE857772 Ribosomal protein L37a 170.70 96.10 83.90 103.46 14.54 H71805 Myeloid cell leukemia sequence 1 48.30 24.45 31.05 55.56 -21.26 H71805 Myeloid cell leukemia sequence 1 30.65 20.60 17.38 76.40 18.56 BE877796 Collagen, type VIII, alpha 1 218.55 172.80 131.13 66.67 31.78 W87901 Small nuclear ribonucleoprotein polypeptide E 77.85 47.55 47.33 64.50 0.48 AA215854 Integrin, beta 1 187.50 88.65 100.50 86.57 -11.79

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59 Table 3-3: Continued. Accession # Gene Title AT2 Untrt AT2 CGP Avg Neo % Change AT2 Untrt % Change AT2 CGP NM_019034 Ras homolog gene family 17.85 22.80 15.08 18.41 51.24 NM_014168 HSPC133 protein 36.05 20.60 21.50 67.67 -4.19 BF970427 UDP-glucose ceramide glucosyltransferase 170.15 129.05 107.50 58.28 20.05 AI189609 RAB2 81.90 41.95 39.93 105.13 5.07 J02761 Surfactant 55.20 44.45 96.70 -42.92 -54.03 Microarray data was analyzed for significantly differentially-expressed genes by 2-way ANOVA. This table represents those genes that were increased significantly by 50% or more. The accession number (Access #), common gene name/title, e-scores for both AT2R transduced cells left untreated (AT2 Untrt), AT2R transduced cells treated with CGP42112A (AT2 CGP), and Cntrl (Avg Neo), and percent change of AT2R Untrt versus Cntrl (% Change AT2 Untrt) and AT2R CGP versus Cntrl (% Change AT2 CGP) is given. Genes highlighted in yellow indicate gene which are described further in the text. The Role of the AT2R in Migration and Angiogenesis The second goal of this aim was to determine if AT2R overexpression in the HCAEC leads to a functional or physiological effects that can then be related to the changes observed in the microarray analysis. We chose to look at cell migration, since endothelial cells play a key role in the initiation of migration and thus angiogenesis. Both Cntrl and AT2R-transduced cells were analyzed for its ability to migrate in the absence and presence of AT2R stimulation (10 nM CGP42112A) with angiogenesis activation (VEGF) or in the presence and absence of VEGF (Figure 3-6). AT2R-overexpressing cells that were not stimulated with CGP42112A showed a significant decrease in migration in both the presence and absence of VEGF (Figure 3-6). This effect was not altered by activation of the AT2R with CGP42112A (Figure 3-6). In addition, the AT2R-specific antagonist PD123,319 did not reverse these effects (Figure 3-6). These results

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60 indicate that AT2R transduction inhibits endothelial cell migration independent of both VEGF and AT2R activation, again indicating that the AT2R elicits its actions independent of ligand. RGS-7051015202530CntrlAT2RArbitrary Units* Ubiquitin Thiolesterase048121620CntrlAT2RArbitrary Units* IGFBP301234CntrlAT2RArbitrary Units p = 0.06ABC Figure 3-5: Gene validation of the microarray analysis. Gene validation by real-time RT-PCR was performed on RNA isolated from HCAEC either transduced with Lenti-I-NeoR (Cntrl) or Lenti-AT2R-I-NeoR (AT2R). Reaction set-ups are as described in the Materials and Methods using pre-designed Assays-on-Demand primers and probe for A) ubiquitin thiolesterase, B) regulator of G-protein signaling-7 (RGS-7), and C) insulin growth factor binding protein 3 (IGFBP3). (n = 6, = p<0.05 vs. Cntrl). In addition to the actions of the AT2R on cell migration, we also wanted to determine if the AT2R had any effects on the cytokines whose dysregulation is typically associated with the activation of angiogenesis. A protein array specifically designed to detect 19 different angiogenesis-specific cytokines was used. AT2R overexpression with and without activation with CGP42112A in HCAEC did not appear to have any effect on these specific cytokines (Figure 3-7). Therefore, the AT2R effects on migration are independent of these specific cytokines. Instead these actions of the AT2R may involve a novel mechanism. Discussion These results indicate that the AT2R regulates genes that may play a role in inflammation, protein regulation, cell migration, and extracellular matrix interactions.

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61 Many of these effects can be seen without stimulation of the AT2R, once again indicating that the AT2R has some constitutive activity. In addition, we were able to show that in these cells, the AT2R decreases cell migration, with several of the genetic profiles correlating to this functional aspect. Finally, we were able to show that the effects of the AT2R on migration and angiogenesis may be independent of the typically-regulated cytokines, and may be eliciting actions through an unknown pathway. 0123456UntrtVEGFVEGF + CGPVEGF + PD +CGPMigrated Cells/Field(Avg5 fields at 20x) Cntrl AT2R*** P=0.07 Figure 3-6: AT2R prevents HCAEC migration. HCAEC were transduced with either Lenti-I-NeoR (Cntrl) or Lenti-AT2R-I-NeoR (AT2R). Cell migration was determined using a 48-well Boyden chamber. Cells were left uninduced (Untrt) or induced with 10 ng/mL VEGF in the lower chamber. In addition, the HCAEC were left unstimulated or stimulated with 10 nM CGP42112A (CGP) or 1 M PD123,319 (PD) in the upper chamber. (n = 4-6/group, = p<0.05 vs. Cntrl). A number of genes were found to be differentially regulated by the AT2R in the HCAEC. In the following sections, I will highlight a few of the more relevant and interesting genes found in this study. Two genes that were found to be differentially regulated by the AT2R (ubiquitin thiolesterase and ubiquitin specific protease 4) are involved in the ubiquitination pathway. Ubiquitination is a process by which proteins are modified post-translationally

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62 to mark the protein for degradation. Three steps are involved in this process. First, the protein is identified for degradation. Signals for this include genetic programming, Cntrl-UntrtCntrl-CGPAT2R-UntrtAT2R-CGPPosAngG-CSFHGFLeptinVEGFPos IL-1aIL-1bIL-6IL-8PIGFPosFGFaFGFbTNFaTGFaNegPosIFNgIL-12IP-10TIMP-1TIMP-2 AB Figure 3-7: AT2R effects are independent of the typical regulators of angiogenesis represented on Panomics Angiogenesis Array. A) Proteins represented are as indicated in the table (Ang = Angiostatin, G-CSF = Granulocyte Colony Stimulating Factor, HGF = Hepatocyte Growth Factor, VEGF = Vascular Endothelial Growth Factor, IL = Interleukin, PIGF = Placental Growth Factor, FGF = Fibroblast Growth Factor, TNF = Tumor Necrosis Factor, TGF = Transforming Growth Factor, Neg = Negative Control, Pos = Positive Control, IFN = Interferon, IP = Interferon Inducible Protein, TIMP = Tissue Inhibitors of Metalloproteinases). Each sample is represented in duplicate on the membrane. B) HCAEC were transduced with Lenti-I-NeoR (Cntrl) or Lenti-AT2R-I-NeoR (AT2R) and either left untreated or treated for 24 hours with CGP42112A (CGP). Following these incubations, protein was isolated and bound to the membrane as described in the Materials and Methods.

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63 phosphorylation or protein damage. Next, the protein is marked for degradation by using a series of enzymes to attach ubiquitin. Finally, the marked protein is delivered to the proteasome where it is degraded and the ubiquitin recycled. Ubiquitination is, however, a reversible process by means of the deubiquitinating enzymes (DUB) that are either ubiquitin C-terminal hydrolases or ubiquitin-specific proteases. In this study, we found that the AT2R decreases an enzyme responsible for marking proteins for degradation (ubiquitin thiolesterase), while increasing the DUB, ubiquitin-specific protease 4. Together, these results indicate that the AT2R decreases ubiquitination. This is especially interesting because a previous study has shown that there is an increase in ubiquitination in both dilated cardiomyopathy and ischemia of the heart.102 These results raise an interesting question; can AT2R overexpression in the coronary artery endothelial cells prevent ubiquitination to such an extent as to prevent these pathophysiologies? Another interesting gene shown to be differentially-regulated by AT2R overexpression was the regulator of G-protein signaling-7 (RGS-7). The regulators of G-protein signaling are a class of proteins that accelerate intrinsic GTP hydrolysis of activated G-proteins, Gi and Gq, to inactivate these signals. In addition, RGS-7 has specifically been shown to reduce Ca2+ mobilization in CHO cells.103 We observed an increase in RGS-7 protein expression with the overexpression of the AT2R. Because the AT2R has been shown to directly effect the signaling of the AT1R and the AT1R has signal transduction cascades to regulate Ca2+ mobilization through both Gi and Gq, we speculate that the AT2R can reduce AT1R-mediated increases in Ca2+ mobilization through this RGS-7 protein.

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64 In addition to these genes, a functional aspect of the AT2R in the HCAEC was identified that can be related to a physiological/pathological state. We observed that AT2R overexpression inhibits endothelial cell migration. Because endothelial cell migration is one of the first steps of angiogenesis, this leads us to speculate that AT2R overexpression would also inhibit angiogenesis. Angiogenesis is the formation of new blood vessels from pre-existing capillaries. Endothelial cells (EC) have been shown to play an important role in angiogenesis. Upon activation by factors such as an increase in immune response or ischemia, EC penetrate new areas of the body by degrading the extracellular matrix (ECM). The EC then proliferate and migrate by forming new attachments with the ECM at the leading edge and detach at the trailing edge. Finally, these sprouting EC roll up to form a new blood vessel. The regulation of angiogenesis is associated with many different and non-related diseases. In diseases such as ischemia, new blood vessel formation provides essential nutrients to improve the pathological state. This indicates that an increase in angiogenesis would be beneficial to patients with myocardial ischemia and infarction as well as stroke. In contrast, in other diseases such as cancer, atherosclerosis, rheumatoid arthritis, and retinopathy, excessive angiogenesis may be contributing to the pathology. In these diseases, the new blood vessels either provide nutrients to promote additional cell growth, or they promote an increase in inflammation to cause additional damage. Because of this dual role for angiogenesis in CVD, it would be interesting to determine the overall effect of AT2R overexpression on these pathophysiologies. Is the AT2R cardioprotective in the sense that it prevents further development of atherosclerosis through the inhibition of angiogenesis? Or is it detrimental to the heart because it prevents the formation of new blood vessels to provide

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65 nutrients to ischemic tissue? Finally, since angiogenesis prevents the development of atherosclerosis which ultimately leads to ischemia of the heart, will new blood vessels ever need to be formed to feed the ischemic tissue? In addition, we were able to associate AT2Rs prevention of cell migration with the observed changes in gene expression seen in our microarray analysis. A number of differentially-expressed genes with AT2R expression can be associated with the regulation of cell migration and angiogenesis. The majority of these genes exert their effects by either (1) inhibiting the initiation of angiogenesis or migration by decreasing the immune response, (2) inhibiting the proteases from degrading the ECM needed to initiate migration, or (3) decreasing the ability of the EC to migrate by reducing their adhesion to the ECM (Figure 3-8). Microarray analysis indicates that the AT2R causes a decrease in 3-hydroxy-3-methylglutaryl CoEnzyme A Reductase (HMG-CoA). HMG-CoA is one of the key enzymes in the production of cholesterol. Numerous studies have shown that inhibitors of HMG-CoA, called statins, effectively decrease migration, prevent the development of atherosclerosis and improve overall endothelial function.104,105 These inhibitors are thought to accomplish this by reducing the amount of circulating LDL and/or reducing the inflammatory response elicited by LDL production.106 The AT2R in the HCAEC could be mimicking these statins and eliciting its affects through the inhibition of HMG-CoA. Since these statins have been shown to play a role in inhibiting inflammatory responses, it is feasible that the AT2R inhibits migration and angiogenesis, which inhibits inflammatory responses to prevent the development of atherosclerosis and thus stroke and myocardial ischemia. This line of thought would indicate that the AT2R-mediated

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66 decrease in cell migration and thus angiogenesis is playing a cardioprotective role to prevent atherosclerosis and myocardial ischemia. In addition, the AT2R was shown to decrease interleukin 16. Interleukin 16 is a T-cell specific chemoattractant factor, which plays a major role in trafficking immune cells. In addition, it has been shown to initiate migration in dendritic cells.107,108 Thus, the AT2R may be inhibiting cell migration by decreasing its initiation induced by the immune system, specifically through the inhibition of interleukin 16 and HMG-CoA. EC Migration & AngiogenesisAT2R ResponseSignal for AngiogenesisEC Breaks Down ECMEC Migration and Proliferation through ECM contactsEC Proliferation and Tube FormationNew Vessel Formation Interleukin 16HMG-CoAReductaseFibrillin2DecorinPAK-4 IGFBP3 Figure 3-8: Graphical representation of the pathway where the AT2R-regulated genes could exert its actions to inhibit endothelial cell migration and angiogenesis. Another way the AT2R may inhibit cell migration and angiogenesis is through the prevention of protease-mediated breakdown of the ECM. One of the first steps in cell migration and angiogenesis is matrix metalloproteinases (MMP) breakdown the ECM, allowing room for the endothelial cells to grow and migrate to form new vessels. Insulin growth-factor binding protein 3 (IGFBP-3) has been shown to be a substrate for some of these MMPs.109 Our microarray and real-time RT-PCR studies indicate that the AT2R

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67 reduces the expression of IGFBP-3. This would theoretically decrease the amount of substrate for the MMP to elicit its actions, thus preventing cell migration and angiogenesis. In addition to these affect on migration initiation, a number of genes indicate that the AT2R may be preventing migration through its effects on cell adhesion to the ECM. Fibrillin-2 is an extracellular matrix glycoprotein that provides an adhesive substrate for migrating cells.110 In our studies, we see that AT2R expression decreases the expression of fibrillin 2. Theoretically, this reduction would then reduce cell adhesion and thus prevent cell migration. Decorin, which is a proteoglycan associated with tissue development and assembly, has previously been shown to play a role in angiogenesis. It has been shown that endothelial cells have increased expression of decorin during angiogenesis.111,112 In addition, in an in vitro model of angiogenesis, Schonherr et. al. was able to show that adenoviral-mediated overexpression of decorin is sufficient to induce angiogenesis.113 In our studies, we see a decrease in expression of decorin with AT2R overexpression, indicating that the AT2R may be inhibiting cell migration and thus angiogenesis through the regulation of decorin. Finally, p21-activated kinase 4 (PAK-4) was shown to decrease with AT2R expression. PAK-4 is a serine/threonine kinase which plays a role in regulating cytoskeletal organization through Rac and Cdc-42. This organization promotes the formation of focal complexes needed for migration.114 In addition, it has been shown that a dominant negative PAK inhibits cell migration.114 Our results are consistent with this finding and indicate that AT2R-mediated decreases in PAK-4 may prevent the regulation

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68 of the cytoskeleton needed for the formation of focal adhesions through a Rac and Cdc-42 pathway. All of these genes indicate that the AT2R regulates pathways that would be beneficial to normal CV function, whether it be through the prevention of cardiomyopathies, or through decreasing AT1R-mediated increases in calcium mobilization, or through the inhibition of atherosclerosis and myocardial ischemia though the regulation of migration and angiogenesis. It will be interesting to see how all of these factors may influence each other to prevent the development of these and other pathophysiologies. These studies indicate once again that the AT2R may be acting in a ligand independent manner. There were a greater number of genes differentially expressed by overexpressing the AT2R and the AT2R-mediated inhibition of endothelial cell migration both indicates a constitutive role of this receptor. Future studies will need to address whether this constitutive activity is due to the overexpression of the receptor or if endogenous receptors also elicits its effects independent of ligand. Our migration assay indicated that the AT2R inhibited cell migration independent of VEGF activation. Previous studies have shown that the AT2R inhibits VEGF-induced cell migration in a different human coronary artery endothelial cell line.56 In fact, many cells do not migrate without VEGF induction. We were shown able to show, however, with our protein array analysis that our HCAEC express endogenous VEGF. This indicates that the cells did not require additional VEGF to induce migration because of basal VEGF expression.

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69 Our angiogenesis protein array revealed that the AT2R did not regulate the activators and inhibitors typically associated with the initiation of angiogenesis. This indicates that the AT2R is inhibiting this function independent of these cytokines. The AT2R may have novel mechanism by which it signals to inhibit angiogenesis. Our microarray data confirms this idea. We described several differentially-expressed genes which may inhibit migration and angiogenesis by factors not represented on the protein array. In addition, we performed these experiments on crude protein isolated from the cells. Since many of the factors represented on the protein array are secreted, it would be interesting to investigate changes in these factors in the media from these cell.

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CHAPTER 4 PREVENTING CARDIAC PATHOPHYSIOLOGIES BY ANGIOTENSIN II TYPE 2 RECEPTOR GENE TRANSFER Introduction The conventional concept of cardiac hypertrophy (CH) is that it is an adaptive response of the heart to sustained increases in BP to preserve cardiac function. In addition, recent evidence indicates that other non-hemodynamic factors such as the tissue RAS may also play a role in its development.88,115,116 This sustained and uncontrolled growth ultimately leads to diminished cardiac performance and cardiac pathophysiologies such as heart failure. Numerous studies have shown that the AT1R plays a role in the development of cardiac hypertrophy. Both AT1R-antisense gene therapy and AT1R antagonists have resulted in prevention in cardiac hypertrophy and its associated pathophysiologies.88,115,117-119 Although the mechanism by which AT1R antagonists prevent cardiac pathophysiologies is still speculative, it has been suggested that unopposed stimulation of the AT2R may contribute to their effectiveness. In fact, a study by Mukawa et al showed that simultaneous administration of an AT2R antagonist with an AT1R antagonist negated the antihypertrophic effects of the AT1R blocker alone.47 In addition, studies performed in cultured cardiomyocytes and hypertrophied hearts indicate a role for the AT2R in the prevention of cardiac pathophysiologies.49,120,121 Despite all of the support for the role of the AT2R in the prevention or regression of CH, the role of the AT2R in cardiac pathophysiologies remains controversial. Studies 70

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71 performed in transgenic and knockout animals indicate a different role for the AT2R. Inagamis group has shown that the absence of the AT2R in knockout mice prevents the induction of CH by both AngII-infusion and pressure overload.41,42 In contrast to these studies, other transgenic studies showed no effects of the AT2R on cardiac hypertrophy.45,99,100 These conflicting observations for the role of the AT2R in cardiac pathophysiologies may be due to inherent problems associated with the experimental design. Since the AT2R has been found to play a role in embryonic development, altering the expression levels of the AT2R, as in the development of transgenic and knockout animals, may result in improper CV development. In addition, AT2R antagonists are expensive and only short-lived. Therefore, long-term extensive studies are hard to accomplish. To alleviate from these problems, our laboratory has established an efficient method of lentiviral vector-mediated gene delivery following the natural development of the CV system. In the present study, we use this system to determine the effects of AT2R gene transfer on cardiac pathophysiologies such as CH, HF, and high BP. Materials and Methods Animals and Lentiviral Delivery All animals used in these studies were purchased from Charles River Laboratories. Initial studies used to determine the transduction efficiency of 1.5x108 TU and the AngII-infusion studies used Sprague Dawley (SD) rats, while spontaneously hypertensive rats (SHR) were used for the other experiments. Offspring from timed-pregnant mothers were removed at 5-days of age, lightly anesthetized with methoxyflurane (Schering

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72 Plough), and a bolus of 30-60 L of either viral resuspension buffer, 5x109 TU/mL of Lenti-AT2R-I-Neo or Lenti-PLAP was injected directly into the left ventricular chamber as previously described.86,88,122 Following this intracardiac delivery, the pups were lightly coated in peanut oil and returned to their respective mothers until weaning. All animal procedures were conducted with our Institutional Animal Care and Use Committee (IACUC) approval and adhered to the guidelines for the care and use of laboratory animals. Viral Production and Transgene and AT1R Expression Measurements Lenti-PLAP or Lenti-AT2R-I-Neo production and titration was performed as described in Chapter 2. Histochemical staining was performed on the animals transduced with Lenti-PLAP as previously described.86 The animals were perfused with cold PBS followed by perfusion with 4% paraformaldehyde (PFA; Sigma). The tissues were collected and allowed to post-fix in 4% PFA for an additional 2 hours at 4 C. Following this incubation, the organs were rinsed in PBS 4 times and heated to 72 C for 3 hours to inactivate any endogenous alkaline phosphatase activity. Next, the tissues were allowed to cool to room temperature. Then they were incubated in a pre-incubation BCIP solution (100 mM Tris, 100 mM sodium chloride, 50 mM magnesium chloride, and 0.5 mM levamisole, pH 9.5) for 1 hour at room temperature. Finally, the tissues were incubated in BCIP solution containing 100 mM Tris, 100 mM sodium chloride, 50 mM magnesium chloride, 0.5 mM levamisole, 1 mg/mL nitro blue tetrazolium, and 0.1 mg/mL 5-bromo-4-chloro-3-iodolyl-phosphate, pH 9.5 for 2 hours at room temperature, followed by an overnight incubation at 4 C. The following day, the tissues

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73 were rinsed in PBS + 50 mM EDTA and whole mount pictures were taken through a dissecting microscope. Following the pictures, the tissues were cryoprotected in 20% sucrose overnight at 4 C and frozen in Tissue-Tek freezing medium. Sections at 30 microns were taken and stained for PLAP and/or DAPI. Total RNA was isolated from the cardiomyocytes of the transduced animals using Qiagens RNeasy Fibrous Tissue Mini Kit. Real-time RT-PCR for both the AT1R and the AT2R was performed as described in Chapter 2. Primers and probe used for the AT1R were as follows: (forward): 5-CCATCGTCCACCCAATGAAG-3; (reverse): 5-GTGACTTTGGCCACCAGCAT-3; (probe): 5-FAMCTCGCCTTCGCCGCAMGBNFQ-3. Physiological Measurements Indirect blood pressure (BP) was monitored by the tail-cuff method as previously described.88 Direct BP measurements were monitored using radiotelemetry devices (Data Sciences, Inc.) according to the manufacturers protocols. Briefly, the animals were anesthetized with isoflurane. The abdominal cavity was exposed and the cannula of the radiotelemetry device was inserted into the abdominal aorta. Once the cannula was secured into the aorta, the radiotelemetry device was sutured into the abdominal wall and the wound was closed. Heart weight to body weight ratios were taken as previously described.88 The animals were weighed prior to euthanasia; the chest cavity was then opened and the heart carefully extracted. The whole heart was rinsed in PBS or Krebs solution, blotted dry and weighed. Measurements are presented as the heart weight (mg) divided by the body weight (g).

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74 Echocardiographies were used to monitor wall thickness and ejection fraction of the hearts. The rats were lightly anesthetized with isoflurane and echocardiographies were performed using a Hewlett Packard Sonos Model 5500 with a 12-MHz transducer. Parasternal long and short-axis images and end-diastolic diameter, end-systolic diameter, ejection fraction and wall thickness were obtained by standard echocardiographic measurements. The magnetic resonance imaging (MRI) of the in vivo rat cardiac cycle was performed at the University of Florida, McKnight Brain Institutes Advanced Magnetic Resonance Imaging and Spectroscopy Facility. All animals were imaged on a 4.7T Oxford Magnet using a Bruker Avance console and Para vision software. The rats were anesthetized by reflexive inhalation of 1.5-2% isoflorane and 1L/min oxygen and monitored using the Small Animal Instrument (SAI) monitoring and gating system for respiration rate and cardiac triggering. The heart was centered in a custom-built 3.5-5cm receive-only quadrature saddle surface coil (each element of the coil is a 3.5 x 3.5 cm rectangle) on a plexiglass cradle and tuned loaded to resonant frequency. The rat and receive coil were inserted into an 8.8 cm in diameter transmit-only quadrature volume coil. Following pilot images, dorsal and sagittal images were acquired using a cardiac gated gradient echo (GEFI_TOMO) sequence with the following parameters: FOV 7.0 x 3.0cm, matrix 256 x 128, TR=12 msec, TE=2.2 msec, 4 AVG, slice thickness 1.5 mm, and a total of 14 frames. These parameters resulted in a resolution of 273 x 234 microns in-plane. Based on the sagittal and dorsal views, transverse images were prescribed and were collected with the GEFI_TOMO sequence with the following parameters: FOV 4.0 x 3.0cm, matrix 256 x 128, TR=12 msec, TE=2.3 msec, 4 AVG, slice thickness

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75 2.0 mm, and a total of 14 frames to capture the entire cardiac cycle. These parameters resulted in a resolution of 156 x 234 microns. Pathology of the heart was analyzed by Massons Trichrome staining through the UF Molecular Pathology Core Facilities. Portions of the heart were fixed in 4% paraformaldehyde or PLP solution (2% paraformaldehyde, 75 mM lysine, 37 mM sodium phosphate, and 10 mM sodium periodate), sectioned and stained. Statistics Results are presented as mean +/SE. All data was analyzed by ANOVA using the student-Newman-Keuls method for all pairwise multiple comparisons. Values of p<0.05 were considered statistically significant. The number of animals per group is indicated in the figure legends. Results In vivo Gene Transfer Delivery of 1.5x108 TU of the expression vector, Lenti-PLAP, into the heart of 5-day old SD animals resulted in efficient transduction of CV-relevant tissues measured at 21 days of age. The lentiviral vector highly transduced the liver, adrenal and heart (Figure 4-1A, E, I and Figure 4-2B-D) compared to its controls (Figure 4-1B, F, J and Figure 4-2A), while it only moderately transduced the lungs and the kidneys (Figure 4-1C,G,K) compared to its controls (Figure 4-1D,H,L). Thin sections of the adrenal shows that the lentiviral vector can transduce both the medulla and the cortex (Figure 4-1I,J). Thin sections of the kidney did not reveal any preference for viral transduction (Figure 4-1K,L), and thin sections of the heart indicated that the lentivirus can efficiently transduce cardiomyocytes (Figure 4-2D).

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76 The effect of Lenti-AT2R-I-Neo on the expression of both the AT1R and AT2R was also examined. Real-time RT-PCR was performed on isolated cardiomyocytes from the hearts of 31-week-old SHR injected into the heart at 5 days of age with either LentiLiverAdrenalLungKidneyPLAPCntrlPLAPCntrl ABCEFDGHIJKLmc Figure 4-1: In vivo lentiviral transduction efficiency. 1.5x108 lentiviral particles of Lenti-PLAP (PLAP) or viral resuspension buffer (Cntrl) were injected into the cavity of the left ventricle at 5-days of age and viral transduction efficiency was examined by PLAP immunochemistry as described in the materials and methods at 21 days of age. Examination of the whole tissue revealed transduction of the A) liver, C) lung, E) adrenal, and G) kidney. Cntrl animals revealed little to no PLAP staining (B,D,F,H) in the whole tissues. I) Thin sections of the adrenal showed efficient transduction in the cortex (c) and the medulla (m) with J) no staining present in either area in the Cntrl animals. Thin sections of the K) PLAP kidney and the L) Cntrl kidney reveal diverse staining within the kidney. AT2R-I-Neo (AT2R) or viral resuspension buffer (Cntrl). Using AT2R-specific primers and probe, there was negligible expression of the AT2R in the Cntrl animals, while the AT2R animals had significantly higher AT2R mRNA levels (Figure 4-3A). In

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77 contrast to this, the AT1R was abundantly expressed in all samples and its expression was not altered by AT2R transduction (Figure 4-3B). A BCD Cntrl PLAP Figure 4-2: Lentiviral transduction efficiency in the heart. B-D) Lenti-PLAP (PLAP) or A) viral resuspension buffer (Cntrl) was delivered and examined as described in Figure 4-1. Whole hearts stained for PLAP in A) Cntrl and B) Lenti-PLAP. C) A section of the Lenti-PLAP transduced heart, looking into the ventricles. D) A thin section of the Lenti-PLAP transduced heart reveals cardiomyocyte morphology. A 0123456CntrlAT2RNRTArbitrary Units 0510152025CntrlAT2RNRTArbitrary Units B*AT2RAT1R Figure 4-3: AT2R and AT1R expression in isolated cardiomyocytes. Viral resuspension buffer (Cntrl) or Lenti-AT2R-I-Neo (AT2R) was delivered into the heart of 5-day old SHR. At 31 weeks of age, the SHR were sacrificed and cardiomyocytes were isolated. Total RNA was collected and examined for A) AT2R and B) AT1R mRNA levels by real-time RT-PCR. No reverse transcriptase (NRT) was used as a negative control. (n = 4/group; = p<0.05 vs. the other two groups).

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78 Pathophysiology in the SHR SHR animals were administered with either Lenti-AT2R-I-Neo (AT2R) or viral resuspension buffer (Cntrl) at 5 days of age. Previous studies in our laboratory indicated no differences between the HW/BW of the animals injected with a control lentiviral vector versus viral resuspension buffer (data not shown). At 12, 18, and 22 weeks of age, echocardiographies (ECHO) were performed to characterize the effects of AT2R transduction on the heart. At 12 weeks of age, the control ECHO revealed that none of the groups had begun to develop cardiac hypertrophy as the wall thickness (WT) of both the Cntrl and AT2R SHR (Cntrl 1.41+/-0.03 mm; AT2R 1.36+/-0.03 mm) animals were comparable to age-matched normotensive WKY animals (Figure 4-4A; WKY 1.42+/-0.03 mm). At 18 and 22 weeks, however, we saw a significant increase in WT of the Cntrl SHR (18 wks 1.78+/-0.03 mm; 22 wks 2.0+/-0.11 mm) animals but not of the AT2R-treated SHR (18 wks 1.53+/-0.03 mm; 22 wks 1.54+/-0.09 mm) or the age-matched WKY (Figure 4-4A; 18 wks 1.52+/-0.09 mm; 22 wks 1.52+/-0.09 mm). In fact the Cntrl-treated SHR had a significantly larger WT compared to both the WKY and the AT2R-treated SHR (Figure 4-4A). These results were supported by HW/BW which revealed that the AT2R-treated SHR (3.7+/-0.02 mg/g) had a significantly lower ratio than the Cntrl-treated SHR animals (Figure 4-4B; 4.0+/-0.1 mg/g). In contrast to these results, there were no differences observed in the ejection fraction or the BP of the Cntrl and AT2R-treated SHR (Figure 4-4C,D). Comparison of the BP with age-matched WKY animals, however, revealed that both the Cntrl and AT2R-treated SHR animals had elevated BP (Figure 4-4D) at both 18 (Cntrl 184 +/-5 mmHg; AT2R 184+/-6 mmHg) and 22 weeks (Cntrl 187+/-13 mmHg; AT2R 193+/-14 mmHg). These data indicate that

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79 AT2R transduction of cardiomyocytes alters cardiac hypertrophy without influencing high BP. ABHW/BW (mg/g)CtrlAT2RC* 2.533.544.5 00.511.522.5CtrlCtrlAT2RAT2RWKYWKY12 weeks18 weeksWall Thickness (mm)CtrlAT2RWKY22 weeks* 68707274767880EF (%)CtrlCtrlAT2RAT2R12 weeks18 weeksCtrlAT2R22 weeks 050100150200250Indirect BP (mmHg)CtrlAT2RWKY18 weeksCtrlAT2RWKY22 weeks**D Figure 4-4: Effect of AT2R transduction on CV pathologies in the SHR. Five-day-old SHR animals were injected into the heart with either viral resuspension buffer (Cntrl) or Lenti-AT2R-I-Neo (AT2R). Age matched WKY animals were used as a control where possible. At 12, 18, and 22 weeks of age, the animals were subjected to ECHOs (n = 4 Cntrl and 4 AT2R) to measure both A) wall thickness and C) ejection fraction (EF). D) At 18 and 22 weeks indirect blood pressures were measure by the tail cuff method (n = 5 Cntrl and 3 AT2R). B) Finally, at the end of the study (31 weeks) the animals were sacrificed and heart weight to body weight ratios were determined (n = 4/group). ( = p<0.05 compared to 12 weeks. = p<0.05 compared to the other groups in that timepoint).

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80 Pathophysiology in the Angiotensin II-Infusion Model SD animals were injected at 5 days of age with either viral resuspension buffer (Cntrl) or Lenti-AT2R-I-Neo (AT2R). BP was monitored both before AngII-infusion (Pre) or following AngII-infusion either indirectly by the tail-cuff method or directly through the use of radiotelemetry devices. Following the implantation of osmotic mini-pumps to continuously deliver either saline or 200 ng/kg/min AngII, the BP was monitored at 1 and 2 weeks. By 2 weeks, the BP was significantly increased by ~70 mmHg compared to the Pre values in both the Cntrl and the AT2R-treated animals infused with AngII. In addition, both groups treated with AngII had a significantly higher BP then those treated with saline (Cntrl Saline 94+/-3 mmHg; Cntrl AngII 172+/-5 mmHg; AT2R AngII 160+/-8 mmHg; Figure 4-5). 6080100120140160180200Pre1 week2 weeksBP (mmHg) Cntrl-Saline Cntrl-AngII AT2R-AngII ** Figure 4-5: Blood pressure response to AngII infusion. SD animals injected into the heart at 5-days of age with either viral resuspension buffer (Cntrl) or Lenti-AT2R-I-Neo (AT2R). BP was taken at 8 weeks of age (Pre) and 1 week and 2 weeks following the start of AngII infusion. (n = 3-4/group; = p<0.05 vs. Pre; = p<0.05 vs. Cntrl-Saline).

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81 ECHOs were taken both before (Pre) and 2 weeks after minipump implantation. After 2 weeks of AngII infusion, it appears that the AT2R-treated animals infused with AngII have a reduced wall thickness compared to the Cntrl-treated animals infused with AngII (Cntrl AngII 2.2+/-0.3 mm; AT2R AngII 1.9+/-0.2 mm; Figure 4-6A). This is better represented when the change in wall thickness from before infusion and 2 weeks post infusion is presented (Figure 4-6B). This was confirmed by studies using MRI. These studies showed that AT2R-transduced animals had a reduced wall thickness following 2 weeks of AngII-infusion (Cntrl AngII 2.1+/-0.06 mm; AT2R AngII 1.8+/-0.05 mm; Figure 4-7A-B,D). In fact, the WT of the AT2R transduced animals was similar to that of saline-infused controls (Cntrl Saline 1.7+/-0.04 mm; Figure 4-7B-D). In addition to these changes in WT, the change in ejection fraction was significantly improved with the AT2R-treated animals infused with AngII (Cntrl AngII -28+/-6%; AT2R AngII -3+/-6%; Figure 4-6C). Finally, at the end of the experiments, HW/BW was determined. Again we see a similar trend with the SHR experiment in that the AT2R-treated animals exhibited a decrease in HW/BW (Cntrl AngII 10.9+/-1.1 mg/g; AT2R AngII 9.3+/-0.4 mg/g; Figure 4-6D). Metabolic parameters, heart rate, and activity were also monitored in these animals. Two weeks following the implantation of the osmotic mini-pumps infusing either saline or 200 ng/kg/min AngII, the animals metabolic parameters were observed. Following 2 weeks of AngII infusion, both the Cntrl and the AT2R-treated animals showed a significant increase in water intake (Figure 4-8A) and urine output (Figure 4-8B) while significantly decreasing the body weight (Figure 4-8C) compared to the saline-infused Cntrl animals. There were, however, no differences in these parameters observed

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82 between the Cntrl and the AT2R-treated animals infused with AngII. In contrast to these effects, there were no effects of AngII infusion or AT2R-treatment on food intake (Figure 4-8D), fecal output (Figure 4-8E), activity (Figure 4-8F) or heart rate (Figure 4-8G). 0.40.50.60.70.80.911.11.21.3Cntrl-SalineCntrl-AIIAT2R-AIIChange in Wall Thickness (mm) 00.050.10.150.20.250.3Pre2 weeksWall Thickness (mm) Cntrl-Saline Cntrl-AngII AT2R-AngII ABCD 4681012Cntrl-SalineCntrl-AIIAT2R AIIHW/BW (mg/g)* 4681012Cntrl-SalineCntrl-AIIAT2R AIIHW/BW (mg/g)* -30-25-20-15-10-50Cntrl-SalineCntrl-AIIAT2R-AIIChange in Ejection Fraction (%)* p=0.55 -30-25-20-15-10-50Cntrl-SalineCntrl-AIIAT2R-AIIChange in Ejection Fraction (%)* p=0.55 Figure 4-6: Role of the AT2R in the CV pathologies associated with AngII infusion. The hearts of SD animals injected with either viral resuspension buffer (Cntrl) or Lenti-AT2R-I-Neo (AT2R) were monitored by ECHOs both before and 2 weeks following the start of a constant infusion of either Saline or 200 ng/kg/min AngII. A) ECHOs revealed trends towards changes in wall thickness. B) This is highlighted when the change in wall thickness from Pre to 2 weeks post infusion is represented. C) In addition, ECHOs reveal that the AT2R-treated AngII infused animals had a significant prevention of the observed reduction in ejection fraction of the Cntrl animals infused with AngII. D) The effects on CH is supported by the observed differences in HW/BW taken at the end of the experiment after 4 weeks of AngII infusion. (n = 3-4/group; = p<0.05 vs Cntrl-AngII).

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83 Cntrl-SalineCntrl-AngIIAT2-AngII 0.17cm 0.17cm 0.21cm 0.21cm 0.18cm 0.18cm 00.050.10.150.20.25 Cntrl-SalineCntrl-AIIAT2R-AIIWT (cm)**ABCD Figure 4-7: MRI analysis indicates AT2R transduction prevents cardiac hypertrophy. SD animals injected with either Lenti-AT2R-I-Neo (AT2R) or viral resuspension buffer (Cntrl) were subjected to a constant infusion of 200 ng/kg/min of AngII infusion. Following 2 weeks of infusion, MRI images were taken. A) Cntrl animals infused with AngII elicited an increased wall thickness compared to both the B) AT2R treated animals infused with AngII and the C) Cntrl animals infused with saline. D) A graphical representation for all the animals is also presented (n = 3/group; = p<0.05 vs. Cntrl-AngII). Finally, at the end of the experiment, the pathology of the heart was examined. After the animals were sacrificed after 4 weeks of infusion, the hearts were fixed and stained with Massons trichrome. Analysis of the sections indicate that the AT2R-treated animals infused with AngII have decreased myocardial fibrosis (Figure 4-9A) compared to the Cntrl-treated animals infused with AngII (Figure 4-9B). In contrast, the

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84 AT2R-treated animals appeared to have increased perivascular fibrosis (Figure 4-9C) compared to the Cntrl animals (Figure 4-9D). 050100150200250300350Cntrl-SalineCntrl-AngIIAT2R-AngIIWater Intake (ml/kg) 050100150200250Cntrl-SalineCntrl-AngIIAT2R-AngIIUrine Output (ml/kg) 05101520253035Cntrl-SalineCntrl-AngIIAT2R-AngIIFecal Output (mg/kg) 010203040506070Cntrl-SalineCntrl-AngIIAT2R-AngIIIFood Intake (g/kg) 00.10.20.30.40.50.6Cntrl-SalineCntrl-AngIIAT2R-AngIIBW (kg) 00.511.522.533.544.5CntrllSalineCntrll-AngIIIAT2R-AIIActivity (Counts/min) 0100200300400Cntrl-SalineCntrl-AngIIAT2R-AngIIHeart Rate (Beats/min)Pre2 wksPre2 wksPre2 wksABCDEFG p=0.197 p=0.637****** Figure 4-8: Effect of the AT2R and AngII on other CV physiologies. SD animals treated as described in Figure 4-6 were used to monitor A) water intake; B) urine output; C) body weight; D) food intake; E) fecal output through the use of metabolic cages and F) activity and G) heart rate through radiotransmittors.

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85 ControlAT2RMassons TrichromeStaining ABCDPerivascularFibrosisMyocardial Fibrosis Figure 4-9: Effect of the AT2R on the pathology of the heart following AngII infusion for 4 weeks. SD animals treated as described in Figure 4-6 were analyzed for pathologies of the heart at the end of the experiment, following the full 4-week infusion of AngII. Hearts were isolated from the animals, post-fixed, and stained with Massons Trichrome. A,B) Analysis of the tissue indicates that the AT2R prevents the development of myocardial fibrosis within the tissue of the heart while C,D) increasing perivascular fibrosis. (Pictures represent 1 animal from each group. Similar results were seen in the other animals; n = 3-4/group). Discussion These results show that we were able to overcome the inherent problems with transgenic and knockout animals through the use of lentiviral vector-mediated gene transfer injected into the ventricular space of 5-day-old animals. Using such techniques we were able to show: (1) 1.5x108 TU of lentiviral vector was able to transduce CV relevant tissues. (2) This transduction was able to overexpress the AT2R without effecting the expression of the AT1R. (3) AT2R overexpression lead to beneficial effects on cardiac hypertrophy (CH) and ejection fraction (EF). (4) These effects on CH and EF appear to be regulated by the local/tissue RAS because the AT2R overexpression has no

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86 effect on BP regulation. (5) It appears that only ~40% transduction of the cardiomyocytes is efficient to produce these significant effects. These studies show a complete inhibition of cardiac hypertrophy despite only ~40% transduction of the heart. This result indicates that the AT2R may be acting in a autocrine/paracrine manner to propagate its effects. In addition, recent evidence indicates that new cardiomyocytes can form from stem-like cells, which could play a role in the development of CH.123,124 Therefore, transduction of a few cells can propagate to elicit larger effects. Previous studies have shown an increase in AT2R expression in response to CH,125 yet the present results do not show an increase in AT2R expression in the control SHR despite the development of CH. There are several explanations for this observation. In this study we only examined the AT2R expression levels in isolated cardiomyocytes, while the previous studies looked at AT2R binding in the whole heart. Therefore, the observed increases in AT2R expression could be occurring either at a post-transcriptional level or in cell types other than cardiomyocytes, such as endothelial cells and/or fibroblasts. Evidence indicates that all of the components of the RAS exist in the heart. This tissue RAS appears to regulate normal cardiac functions. ACE inhibitors, AT1R antagonists and AT1R-antisense have shown a reduction in CV pathologies independent of changes in arterial pressures.88,126,127 This study indicates that AT2R expression in the heart is exerting its effects on CH and EF independent of BP. This can be explained several ways. First, the lentiviral transduction of the vasculature is not high enough to

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87 elicit any effects on BP. Secondly, the AT2R in the heart is exerting its effects through the tissue RAS rather than the systemic RAS. Many previous studies using AT2R antagonists and transgenic and knockout animals showed conflicting data as to the role of the AT2R in cardiac function. We believe that our gene transfer model has an advantage over these more traditional methods since the genetic manipulations do not occur until after the cardiac development has occurred. This raises some questions as to whether or not the AT2R overexpression prevents the development of CH or simply delays it. In addition, it would be interesting to determine if AT2R overexpression can reverse the development of CH. Massons trichrome staining of the AngII-infused hearts indicates that the AT2R may be playing opposing roles to prevent myocardial fibrosis while increasing perivascular fibrosis. The significance of this observation is yet to be determined. It is possible that the AT2R is playing opposing roles in different cell types. It is also possible that the prevention in myocardial fibrosis is an AT2R effect while the perivascular fibrosis is an inflammatory response to lentiviral delivery. Lentiviral controls were not used in the present studies because previous studies did not show any differences in HW/BW and BP between lentiviral controls or viral resuspension buffer. These controls may be needed, however, to address whether the observed effects on perivascular fibrosis is an effect of the AT2R or lentiviral transduction. We have shown that the AT2R prevents the development of cardiac pathologies. We do not know, however, the mechanisms by which the AT2R elicits these effects. Is it though the prevention of AngII binding to the AT1R? Are the effects we observed through AT2R overexpression in the heart, or from AT2R overexpression in other tissues

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88 such as the adrenal gland? Future studies need to examine the mechanism of these AT2R effects as well as establish cardiac-specific overexpression of the receptor.

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CHAPTER 5 DETERMINING THE DIPSOGENIC RESPONSES FOLLOWING ANGIOTENSIN II TYPE 2 RECEPTOR GENE TRANSFER INTO THE PARAVENTRICULAR NUCLEUS Introduction The studies presented thus far have focused on the role of the AT2R in the periphery. The AT2R, however, is also expressed in the brain and has been implicated to play an important role in the central regulation of CV disease. In the adult rat brain, the AT2R is largely distributed in areas involved in sensory, motor, and visual control. These areas include the inferior olive, locus coeruleus, medial geniculate nucleus, mediodorsal thalamic nuclei, and superior colliculus.13,50,128 In addition, the AT2R has been found to be expressed in the medial amygdala, lateral septum, and ventral septum.13,50,128 These areas are involved in the regulation of drinking and salt intake. Finally, while the AT1R predominates in the paraventricular nucleus (PVN), evidence has shown that the excitatory action of AngII can be blocked in the PVN with AT2R-specific antagonists. The central role of the AT2R has been heavily investigated ever since its discovery, but yet it still remains elusive. Early studies established that the AT2R played a distinct role on the membrane ionic currents in cultured neurons. One study found that the AT2R plays a role in the AngII-mediated increases in net outward current (Ino) of the neurons,129 indicating a role for these receptors in the repolarization of the cell. Later, it was established that the AT2R increased potassium current (IK) in cultured neurons.34,130,131 89

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90 Studies performed in vivo indicated a role for the AT2R in BP, vasopressin (AVP) and drinking responses to AngII. These studies found that the AT2R antagonist alone did not have much effect on intracerebroventricular (icv) injection of AngII; however, the AT1R antagonists were more effective at preventing the pressor response to AngII when delivered with the AT2R antagonist.132 This result suggests that AngII increases BP via the AT2R. On the other hand, a study performed by Hogarty et al. indicated that the AT2R played a role to increase AVP secretion and drinking in response to AngII but did not play a role in the pressor response.133 More recent studies have confirmed and clarified the drinking response mediated by the AT2R. These studies have shown that the AT2R is involved in dehydration-induced drinking134 and AngII-induced drinking in high sodium chloride-fed rats but had no effect in animals fed a normal sodium chloride diet.135 A recent study using knockout mice confirmed some of these earlier results on drinking. These investigations showed that the AT1R and AT2R worked synergistically in the regulation of water to increase water intake in response to AngII. In contrast, the AT1R and AT2R were shown to play an antagonistic role in the regulation of BP induced by AngII, with the AT2R decreasing BP in response to AngII.52 As with many of the other studies with the AT2R, we see evidence for opposing roles of this receptor. There could be several explanations for these controversies. (1) The expression levels of the AT2R are not high enough to see significant changes with AT2R antagonists. In addition, the high levels of the AT1R in the brain may be masking the effects of the AT2R in response to AngII. (2) The global effect of AngII delivered icv may have opposing actions on the AT2R in different brain nuclei. These actions may

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91 cancel each other out, leading to an absence of effect. (3) Once again, studies using transgenic and knockout mice may have developmental abnormalities which restrict the ability of determining the true role of the AT2R by using these animal models. The purpose of this project was to establish a method of gene delivery in which we could overexpress the AT2R in specific brain nuclei to better understand its function. We believe that if we express more of this receptor in a particular brain region, we may be able to overcome some of the inherent problems discussed above and gain some insight into the physiological role of the AT2R in the brain. Once established, these protocols can then be used to not only clarify the central role of the AT2R but also the specific role of the AT2R in particular brain nuclei. For this study, the role of the AT2R in the paraventricular nucleus (PVN) was investigated. The PVN is known to be an important regulator of pressor responses, vasopressin release, and dipsogenic actions in response to AngII.136-138 In addition, the AT2R has been shown to be expressed in the PVN, and recent evidence indicates that angiotensinergic projections into the PVN may regulate dipsogenic responses through the AT2R.136 Thus the objective of this study was 3-fold: (1) establish coordinates and lentiviral transduction efficiency in the PVN; (2) determine the effects of AT2R overexpression in the PVN on basal BP and BP responses to AngII; and (3) establish dipsogenic responses to basal AT2R overexpression and AT2R-mediated responses to dehydration and AngII in the PVN.

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92 Methods Lentiviral Vector Production, Concentration and Titers The lentiviral vector was constructed, produced, concentrated and titered as described in Chapter 2. Lenti-GFP or Lenti-PLAP was used to determine the infection efficiency into specific brain nuclei (Figure 5-1). Lenti-AT2R-I-PLAP and Lenti-I-PLAP was used for the PVN studies. Animal Care Male Wistar (275 g), Sprague Dawley (180-220 g), or Spontaneously Hypertensive (180-220 g) rats purchased from Charles River were used in these studies. All coordinates were tested for accuracy using 25% India Ink (Fischer Scientific) in each animal strain. Lentiviral Vector Delivery into Brain Nuclei Coordinates and transduction effiency of the lentiviral vector into the PVN were previously established in Wistar rats in collaboration with Dr. Julian Patons laboratory as previously described.86 SD and SHR animals, however, were optimized in our laboratory for bilateral injections into the PVN. Animals were anesthetized with isoflurane and placed in a stereotaxic frame. Following a midline incision, the skull was cleaned and checked with an alignment tool (David Kopf Instruments) to ensure that the skull is flat. Next, the skull was marked for the PVN (bregma -1.6 mm, lateral +/0.5 mm) and right lateral cerebroventricle (bregma -1.3 mm, lateral 1.5 mm) coordinates with a fine marker. Holes were then carefully drilled at these marks. A 5 L Hamilton syringe (88000) filled with viral vector (1x109 TU/mL concentration) was used in a Kopf microinjection unit to reestablish the PVN coordinates (bregma -1.6 mm, lateral +/-0.5 mm). The needle was inserted into the right hemisphere -7.3 mm from the dura and

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93 1L was slowly injected at a rate <0.1 L/min. Following the injection, the needle remained in the brain for an additional 10 minutes to ensure that the virus remained at the injection site. The same delivery procedure was then repeated on the left hemisphere. Following this bilateral delivery into the PVN, the holes were sealed with bone wax and a 22 gauge stainless steel guide cannula was placed into the lateral ventricle as previously described.139 Following the surgery, the animals were injected with the analgesic, banamine (1.1 mg/kg body weight intramuscularly) and allowed to recover for 1 week. Physiological Measurements One week after the brain surgery, radiotelemetry devices were implanted into the abdominal aorta to measure direct blood pressure (BP), heart rate (HR), and activity as described in Chapter 3. These parameters were measured from 1-3 weeks following radiotelemetry surgery. During this time of measurement, the response to AngII was also measured. AngII (30 ng) was injected into the lateral ventricle cannula using a 5 L Hamilton syringe (88000). In addition, AngII was delivered subcutaneously at a concentration of 100 g/kg to determine the effects on water intake. Water intake, urine output, food intake, and fecal output were all monitored through the use of a metabolic cage approximately 4 weeks following the radiotelemetry surgery. Animals were placed into a metabolic cage and allowed to acclimate to the new environment for 2-3 days. Once acclimated, the animals food, water, spilled water, urine, and feces were weighed daily. For dehydration studies, the animals water was removed for 20 hours. After this time, everything was weighed and water was given back to the animals. Following reintroduction to the water, the water bottles and spilled water were weighed at 30 minutes, 1 hour, 2 hours and 8 hours to monitor their water intake. Water intake was

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94 monitored in a similar manner to determine the effect of subcutaneous injection of saline or AngII. Following a subcutaneous injection, the animals water bottles and spilled water was monitored at 30 minutes, 1 hour and 2 hours. Results Lentiviral Vector-Mediated Transduction into the PVN Lentiviral vectors carrying either GFP or PLAP were injected into the PVN (Figure 5-1). Transverse cross sections of the brain reveal that the lentiviral vector transduced a high proportion of the cells in the PVN. In fact, the GFP-positive cells (Figure 5-1A) revealed a predominant neuronal phenotype of the transduced cells. In addition, the lentiviral vector was able to be bilaterally introduced in the PVN (Figure 5-1B). AB Figure 5-1: Lentiviral transduction into the PVN. A) Lenti-GFP was injected into the PVN of Wistar rats as previously described.86 Viral transduction is indicated by the green fluorescence present in these brain regions. B) Lenti-PLAP was used to transduce the PVN bilaterally in SD animals using the coordinates that our lab has established as described in the Materials and Methods. (A. Adapted from Coleman, Huentelman, et al. Phys. Genomics. 2003).

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95 Physiological Effects of the AT2R in the PVN The animals overexpressing the AT2R in the PVN were first analyzed for changes in basal BP, HR and activity. Basal BP, HR and activity was monitored weekly 2-6 weeks post-transduction. AT2R overexpression in the PVN did not appear to have any effects on these basal parameters (Figure 5-2). 110120130140150CntrlAT2RBP (mmHg) p=0.698A 200250300350400450500Heart RateCntrlAT2R 0123456CntrlAT2RActivity p=0.357BC Figure 5-2: Role of the AT2R in the PVN on basal BP, HR, and activity. A) Basal BP, B) HR, and C) activity were measured by radiotelemetry devices from 2-6 weeks post-transduction of either Lenti-I-PLAP (Cntrl) or Lenti-AT2R-I-PLAP (AT2R). Data is presented as the averages of measurements taken each minute for ~ 4 daylight hours. (n = 2 Cntrl and 3-4 AT2R). In addition, the effect of AngII icv injection on BP, HR, and activity was also analyzed in these animals. Both Cntrl and AT2R-treated animals showed similar BP and HR responses to icv AngII injection (Figure 5-3A-D). Figures 5-3A and C shows the profile of a randomly selected animal in each group, while Figures 5-3B and D show the collective response to AngII for 1 hour prior to and 1 hour post AngII delivery. Finally, AT2R overexpression in the PVN showed a 45% decrease in activity following AngII delivery (Figure 5-4). This effect was not observed in the Cntrl animals (Figure 5-4). Despite seeing no effects on BP and HR, the effect of AT2R transduction in the PVN on metabolic parameters were examined. A recent report indicates that the AT2R

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96 in the PVN increases water intake in response to medial septal stimulation.138 Approximately six weeks following delivery of Lenti-AT2R-I-PLAP (AT2R) or Lenti-I-PLAP (Cntrl) into the PVN, basal effects of AT2R transduction on metabolic parameters were measured. These studies revealed that the AT2R increases body weight by ~15% (Figure 5-5A) while decreasing basal water intake by 55% (Figure 5-5B) and urine output by 76% (Figure 5-5C). AT2R overexpression in the PVN, however, did not seem to affect food intake or fecal output (Figures 5-5D-E). Time (minutes)Time (minutes) 200250300350400450500BasalAngIIHeart RateCntrlAT2RCntrlAT2R 100120140160180200220BasalAngIIBP (mmHg)CntrlAT2RCntrlAT2RCABD 050100150200250-60-300306090BP (mmHg) Control AT2R 120 050100150200250-60-300306090BP (mmHg) Control AT2R Control AT2R 120 0100200300400500-60-300306090Heart Rate(Beats/min) Control AT2R Control AT2R 120 Figure 5-3: Effect of icv injection of AngII in animals overexpressing the AT2R in the PVN. Basal responses and the response of icv injection of AngII on BP and HR was monitored by radiotelemetric devices in SHR injected with either Lenti-I-PLAP (Cntrl) or Lenti-AT2R-I-PLAP (AT2R). Profiles of the response of icv injection of AngII are given for A) BP and C) HR. Average responses 1 hour before injection (Basal) and 1 hour post delivery (AngII) are also given for the B) BP and D) HR responses. (n = 1-2 Cntrl and 3-4 AT2R).

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97 Finally, the effects of dehydration and subcutaneous injection of AngII on water intake was examined in these animals overexpressing the AT2R in the PVN. Following 20 hours of dehydration, the Cntrl animals drank ~40% more than the AT2R-treated animals (Figure 5-6). This effect was seen at all the time points examined: 30 minutes, 1 hour, 2 hours and 8 hours (Figure 5-6). Similar trends were also seen in response to subcutaneous injection of AngII (Figure 5-7). Following subcutaneous injection of AngII (100 g/kg) the Cntrl animals drank ~45% more than the AT2R animals, while a volume control of saline showed no differences between the animals (Figure 5-7). Discussion These results indicate that the AT2R in the PVN affects basal and AngII and dehydration-induced water intake but not BP responses. In addition, the animals transduced with the AT2R in the PVN appeared to have an increase in BW and a decrease in urine output. Finally, these results also indicate that the AT2R may also play a role to decrease the activity of the animals in response to icv injection of AngII. While these observed trends are exciting, they may contradict previous studies. Camargo et al. has shown that the AT2R in the PVN increases water and sodium input, urine and sodium excretion, and BP in response to AngII injection into the medial septal area.138 In addition, other studies using AT2R antagonists and knockout animals indicate that the AT2R plays a role to increase water intake.133-136 There are many explanations for the differences between these and our studies. (1) Several of the previous studies used AT2R antagonists. Many antagonists have non-selective properties. In addition, AngII has a higher potency in the brain than in the periphery. Coupled together, these properties of AT2R antagonists could cause some nonspecific or secondary effect

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98 0123456CntrlAT2RActivityBasalAngIIBasalAngII* P=0.357 Figure 5-4: Effect of the AT2R in the PVN on basal activity and activity following icv injection of AngII. Activity was measured by radiotelemetric devices in SHR previously injected with either Lenti-I-PLAP (Cntrl) or Lenti-AT2R-I-PLAP (AT2R) into the PVN as described above both before (Basal) and after icv AngII delivery (AngII). (n = 2 Cntrl, 4 AT2R; = p<0.05 vs. Basal). 020406080100120140CntrlAT2RWater Intake (mL/kg) p=0.08 01020304050607080CntrlAT2RUrine Output (mL/kg) p=0.08 00.050.10.150.20.250.30.35CntrlAT2RBody Weight (kg) 05101520253035CntrlAT2RFecal Output (g/kg) p=0.08 0102030405060CntrlAT2RFood Intake (g/kg)EDCAB Figure 5-5: Basal effects of AT2R overexpression in the PVN. Lenti-I-PLAP (Cntrl) or Lenti-AT2R-I-PLAP (AT2R) was injected into the PVN as described in the materials and methods. Following transduction, basal effects on A) body weight, B) water intake, C) urine output, D) food intake, E) fecal output and F) blood pressure were examined for up to 8 weeks following viral delivery (n = 2 Cntrl and 3-4 AT2R; statistical results are given).

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99 02040608010012030 min.1 hr.2 hrs.8 hrs.Water Intake (mL/kg)CntrlAT2RCntrlAT2RCntrlAT2RCntrlAT2R Figure 5-6: Role of the AT2R in the PVN on dehydration-induced water intake. Animals transduced with either Lenti-I-PLAP (Cntrl) or Lenti-AT2R-I-PLAP (AT2R) were dehydrated for 20 hours. Following this time, water was reintroduced to the animals and water intake was monitored at 30 min, 1h, 2 h, and 8 h. (n = 2 Cntrl and 3 AT2R; = p<0.05 compared to the Cntrl group). 0510152025303530 min.60 min.120 min.Water Intake (mL/kg)Cntrl-SalineAT2R-SalineCntrl-AngIIAT2R-AngIICntrl-SalineAT2R-SalineCntrl-AngIIAT2R-AngIICntrl-AngIIAT2R-AngII Figure 5-7: Effects of the AT2R in the PVN on subcutaneous injection of AngII. Animals transduced with Lenti-I-PLAP (Cntrl) or Lenti-AT2R-I-PLAP (AT2R) were first subcutaneously injected with saline and monitored for water intake at 30 minutes and 1 hour. This was followed by a subcutaneous injection of 100 g/kg AngII and water intake was measured at 30 minutes, 1 hour and 2 hours. (n = 2 Cntrl and 3 AT2R).

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100 unrelated to the AT2R. (2) Previous studies have relied on the inhibition of low levels of the AT2R, while our experiments are a result of AT2R overexpression. (3) The effect of the AT2R cannot be accurately determined by icv injection of the AT2R antagonist. It is feasible that the AT2R exerts different effects in different brain regions. Therefore, our system which allows one to determine the role of the AT2R in specific brain regions may eliminate background effects which hamper ones ability to see the true role of the AT2R. (4) We have discussed in Chapter 3 the possibility of compensatory mechanisms in AT2R-knockout animals. These compensatory mechanisms may also play a role in the development of the brain. Therefore, studies using these animals may not reflect the role of the AT2R in the brain. (5) Finally, the trends we have observed in response to AngII were by subcutaneous delivery. Therefore, we do not know where AngII is exerting its action. Essentially, we may be signaling to the PVN in a manner independent of the medial septal area. We are most likely signaling to the PVN from the subfornical organ (SFO) since that is a region of the brain where the blood brain barrier does not exist. Future studies will provide more animals so the effect of icv injection of AngII can be determined. This data is tantalizing because it demonstrates for the first time our ability to separate BP and dipsogenic responses to AngII in the PVN. AngII stimulation in the brain elicits a number of effects. Parvocellular neurons from the PVN regulate sympathetic activity and BP. In addition, AngII receptors in the PVN also regulate vasopression release and water intake. These results indicate that our injection technique is able to separate the dipsogenic responses from the BP responses. This may be useful to try to understand the mechanism by which AngII receptors in the brain regulate water

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101 intake. Future studies will need to try to determine the mechanism by which we were able to separate these functions. Is our injection protocol targeting a certain neuronal cell type which only effects water intake? If so, what type of neurons are they? If we inject deeper into the PVN will we see affects on BP? And finally are we eliciting any effects on vasopressin release? Future studies will also need to address the mechanism by which the AT2R regulates BW. In this study, we observed a trend towards an increase in BW despite having similar food intake, basal activities and BP. A balance between energy intake (food intake) and energy expenditure regulates BW. Since we do not see any changes in food intake, this leads us to speculate that the AT2R effect on BW is through the regulation of energy expenditure. There are three main categories for energy expenditure; (1) expended energy for basic cellular and physiologic functions, (2) energy used in physical activity, and (3) adaptive thermogenesis or energy expended in response to external stresses such as cold or the amount of food ingested. Therefore, future studies need to study the role of the AT2R on physical activity through the use of a rodent wheel and on stress-induced thermogenesis. The results presented here raise some interesting questions about how the AT2R may be exerting its actions in the PVN. Are the observed effects a direct AT2R action, or is it acting through the inhibition of the AT1R? In rodents, the AT1R has been shown to have two subtypes, the AT1AR and the AT1BR. It has recently been shown that the AT1BR elicits the drinking effects in response to AngII while AT1AR elicits the pressor responses in the brain.140 So, if the AT2R is eliciting its actions through the inhibition of

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102 the AT1R, is it specifically inhibiting the AT1BR? If these responses are a direct action of the AT2R, what are the signaling pathways by which it is eliciting its effects? These results indicate that the established method of AT2R delivery by the lentiviral vector is feasible and plausible. Previous studies performed in collaboration with Dr. Paton (Bristol University) and other members of our laboratory, have shown that the lentiviral vector can efficiently transduce specific brain nuclei, specifically the NTS and the PVN.86 These studies established proof of principle that the lentiviral vector can be introduced to specific brain regions and efficiently transduce neurons. In addition, it appears that these injections remain localized and the pseudotype and promoter of the lentivirus actually appears to prefer neuronal cell types. We have taken these protocols and have adapted them to our own system, where we are now able to efficiently overexpress the AT2R through the use of these lentiviral vector systems. In addition, we have developed a method in which we can simultaneously microinject into specific brain nuclei and insert an icv cannula. This allows us to study both the basal and the drug-induced effects of AT2R overexpression in the specific brain regions. In this case, we cannulated the lateral ventricle, but similar methods can be used to deliver drugs into other brain regions. We believe that our system will prove to be more versatile and will allow us to delineate the mechanisms of the actions of the AT2R in the brain. For example, it is feasible that following our physiological measurements in vivo, one can perform electrophysiological measurements on brain slices from the same animals to establish the physiological effects at the cellular level. In fact, because our system is set up to bicistronically express the AT2R and an expression gene such as GFP, one can ensure

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103 that they are only taking recordings from AT2R overexpressing cells because those cells will also be fluorescent. To enhance this even further, recent experiments have established techniques in which one can use in vivo retrograde tracing techniques followed by electrophysiological examination in brain slices.141 This would allow one to understand the role of the AT2R in a specific type of neuron, such as autonomic neurons, projecting to a known location, such as RVLM, IML, or NTS. In addition to the advantage of using these methods to understand the role of the AT2R in BP and drinking responses to AngII, they could be expanded to further the understanding of the function of the AT2R in behavior, global ischemia and stroke. Previous studies have shown that the expression of the AT2R in adult animals is in areas associated with sensory, motor, and visual control,128,142 yet the physiological role of the AT2R in these processes has not been identified. Therefore, one could direct lentivirus containing antisense or RNAi to specifically decrease the expression of the AT2R in these particular brain regions to determine its physiological effects. In addition, it has been shown that there is an increase in AT2R expression levels following global ischemia and/or stroke, which may play a role in tissue repair following injury.143,144 It would be interesting to see if overexpression of the AT2R in these typically affected areas could prevent the damage associated with these pathologies.

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CHAPTER 6 OVERALL DISCUSSION AND CONCLUSIONS All of the results presented here indicate that the AT2R is playing a cardioprotective role. (1) Lentiviral-mediated AT2R overexpression in the heart prevents the development of cardiac hypertrophy and heart failure in two models of hypertension. (2) Gene profiling suggests that in endothelial cells, the AT2R may play a role to prevent cardiomyopathies and ischemia of the heart by decreasing ubiquitination. (3) AT2R expression in EC also inhibits migration and possibly angiogenesis, which could prevent the development of atherosclerosis, cardiac ischemia and myocardial infarction. (4) The AT2R inhibits AT1R-mediated signal transduction cascades. (5) The AT2R may be exerting its effects in a ligand-independent manner. (6) Finally, the AT2R appears to counteract the AT1R in the PVN to decrease basal water intake and water intake in response to dehydration and subcutaneous AngII delivery. AT2R Prevents Cardiac Pathophysiologies The studies presented in Chapter 4 provide strong evidence that the AT2R prevents cardiac pathophysiologies in both the SHR and with AngII-infusion. It would be interesting to determine the mechanism by which the AT2R is exerting its actions in these animal models. In addition, previous studies have shown that the lentiviral vector can efficiently transduce the heart in adult animals.145,146 It would be interesting to apply these surgical procedures to determine if the AT2R can reverse as well as prevent these pathological conditions of the heart. Alternative methods of determining this can also be investigated. There are a number of inducible promoters, such as the tetracycline 104

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105 systems, which could essentially allow one to turn-on AT2R expression in the adult animal. Additionally, Cre-Lox systems could also be used in association with gene transfer or even transgenic animals. In this system, the expression can be repressed until after the pathology has ensued. Then one can remove the inhibition, to gain AT2R overexpression. Future studies also need to determine if the effects we are seeing is a direct effect of the AT2R in the heart. Our method of delivery transduces many other organs other than the heart. It would be interesting to use cardiomyocyte-specific promoters to determine if the effects we are seeing are a direct effect of the cardiomyocytes or through some other tissue. Role of the AT2R in Ubiquitination Microarray analysis revealed a role of the AT2R in the down-regulation of ubiquitination. While there is only a small amount of literature describing a role for ubiquitination in CV diseases, there is some evidence that there is an increase in ubiquitination in patients with cardiomyopathies and ischemia of the heart. Although the mechanism of this overexpression is still speculative, the authors believe that excessive proteolysis may contribute to these cardiac pathophysiologies.102 Taking these results together with our gene profiling effects of the AT2R, it will be interesting to see if AT2R transduction in the endothelial cells can prevent cardiac pathophysiologies though a decrease in ubiquitination. In addition, it would be interesting to see if ubiquitination also plays a role in the development of cardiac hypertrophy and heart failure. If so, are our observed effects of the AT2R in the SHR and AngII-infused animals through the inhibition of ubiquitination? Future in vivo studies should be performed with these ideas in mind.

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106 AT2R Effects on Migration and Angiogenesis We have shown that the AT2R prevents endothelial cell migration, and propose a number of genes identified by microarray analysis which could play a role in the inhibition of migration and/or angiogenesis. Migration is the key initiator of angiogenesis, thus its regulation may play a profound role in the regulation of angiogenesis. Future studies need to determine if the AT2R also prevents angiogenesis. This can be performed by determining the effect of the AT2R on endothelial tube formation. Angiogenesis has been implicated in a number of disease states including atherosclerosis and myocardial ischemia. The role of angiogenesis in these pathological states is opposing. Angiogenesis plays a role in the development of the pathological state of atherosclerosis, while during myocardial ischemia angiogenesis is beneficial in providing nutrients to the tissues. It will be interesting to see how the AT2R plays a role in these two conditions. Is the AT2R cardioprotective in the sense that it prevents the development of these ischemic conditions through the inhibition of atherosclerosis? AT2R-Mediated Effects on the AT1R The results shown here, illustrate that the AT2R may be functioning to inhibit two different AT1R-mediated signaling cascades, either by inhibiting AT1R-mediated increases in MAPK activity or by inhibiting AT1R G-proteins through RGS-7. While both of these findings are preliminary, it will be fascinating to determine the mechanism by which the AT2R is eliciting its actions on the AT1R. Is it through downstream signaling effects of the AT2R? The overexpression of RGS-7 suggests that it may be acting in this manner. Or is the AT2R affecting the AT1R through some direct, steric hindrance or even by just sequestering the circulating levels of AngII? In addition, it

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107 would be interesting to see what cell types elicit which actions. Does the AT2R completely inhibit all AT1R signaling or is it cell-type dependent? Does similar affects occur in vivo? Is it possible that the affects of the AT2R in the SHR and AngII-infusion animals or even in the PVN injected animals an effect of AT2R-mediated inhibition of the AT1R? Ligand-Independent Activity of the AT2R In addition, I have shown that the AT2R may have some ligand-independent activity. In the CHO cells, the effects of AT2R overexpression on MAPK activity could not be reversed by the AT2R-specific antagonist. In the microarray experiment, we saw the majority of the changes in gene expression occurring without stimulation. This is an important issue to address because it is hypothesized that unopposed stimulation of the AT2R contributes to the effectiveness of AT1R antagonists. This raises some interesting questions. Does the AT2R always act in a constitutive manner? Or only when it is highly expressed? Does the AT2R really contribute to the effectiveness of AT1R antagonists? If so, is it though AngII stimulation of the AT2R or just though sequestration of AngII binding to the AT1R? This raises some interesting questions about our in vivo experiments as well. Does the AT2R have a direct effect on cardiac hypertrophy, heart failure and water intake? Or are the actions of the AT2R elicited through sequestration of AngII binding to the AT1R? In addition, it will be interesting to determine the levels of ligand-independent activity. Is there any activation of the AT2R through AngII or is there even some other unknown agonist for this receptor? Role of the AT2R in the PVN While the results of the role of the AT2R in the PVN are preliminary, they do provide some interesting trends. It appears that the AT2R is acting to reduce basal water

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108 intake and water intake in response to dehydration and subcutaneous injection of AngII. Further studies are needed to confirm these studies and to determine the mechanism by which the AT2R is eliciting these actions. Are these effects through the inhibition of the AT1R or some other mechanism? Could the AT2R be regulating the osmoreceptors in the hypothalamus to elicit its effects? What neuronal cell types are our injection protocols infecting to exert these actions? In addition, since the PVN is known to play a role in vasopressin release, it would be interesting to see the effect of AT2R overexpression on the levels of vasopressin release. Perspectives A better understanding of the role of the AT2R in CV disease may be influential in the future therapeutics of this disease. The data presented here provides evidence that the AT2R plays a role to prevent cardiac hypertrophy, heart failure, and migration. These finding along with the findings with the microarray analysis and PVN injections can be applied to even more CV diseases such as angiogenesis, atherosclerosis, cardiomyopathy and myocardial ischemia. It will be important to determine if our systems of AT2R gene transfer can also reverse some of these CV pathophysiologies. If this can be achieved, viral delivery of the AT2R may prove to have the potential to be a novel therapy to treat multiple pathologies of the CV system

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BIOGRAPHICAL SKETCH Beverly Lynn Metcalfe was born on August 29, 1977 and grew up in the small town of Ottoville, Ohio. Beverly first discovered her joy for science in junior high and high school. In fact, she went to the state science fair in both the 7th and 8th grade. She decided to pursue her science career at Bowling Green State University, in Bowling Green, Ohio. During her junior year, she was introduced to basic scientific research. Under the guidance of Dr. Nara Gavini, she worked on a project to characterize some of the genes involved in nitrogen fixation. To further expand her research interests, Beverly applied for a summer undergraduate program at the Medical College of Ohio, in Toledo, Ohio. In this program, working with Dr. James Trempe, she was first introduced to the field of gene therapy. This field excited her, and prompted her to apply to graduate school at the University of Florida (Gainesville, FL), where a lot of top-notch gene therapy research occurs. In 1999, Beverly began her graduate career in the Interdisciplinary Program in Biomedical Research at the University of Florida. Upon exploring many other fields of research in biochemistry and molecular biology, she decided on a laboratory in the Department of Physiology and Functional Genomics that uses gene therapy to study cardiovascular disease. Under the supervision of Dr. Mohan K. Raizada, she has been studying the role of the angiotensin II type 2 receptor in cardiovascular function. 123


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DEFINING THE ROLE OF THE ANGIOTENSIN II TYPE 2 RECEPTOR IN
CARDIOVASCULAR DISEASE















By

BEVERLY L. METCALFE


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


2004

































Copyright 2004

by

Beverly L. Metcalfe

































I dedicate this work to my family and Alaric Falc6n for their constant encouragement and
support in every endeavor I pursue.















ACKNOWLEDGMENTS

I would first like to thank my mentor, Mohan K. Raizada, for his constant

encouragement and invaluable advice. His infectious enthusiasm for scientific research

has encouraged me to do things that I never would have thought possible. His advice,

both professionally and personally will undoubtedly prove to be invaluable in my future

endeavors and I thank him for not only being such a great mentor, but for also being a

wonderful role model and friend.

In addition, I would like to thank the members of my dissertation committee:

Drs. Colin Sumners, Michael Katovich, and Gerry Shaw. They have all provided me

with sound advice to advance my projects. These people are much more then committee

members as they have been heavily involved in my studies. I appreciate their dedication

in shaping me as a better scientist.

I'd also like to thank my labmates in Dr. Raizada's laboratory. They are always

willing to lend a hand, and I have learnt so many new skills and techniques thanks to

them. I would like to especially thank Dr. Shereeni Veerasingham for her help and

advice concerning both the microarray project and the brain microinjections;

Dr. Matthew Huentelman who established the lentiviral vector in our laboratory and with

whom I also worked closely on the in vivo project in the SHR; Dr. Carlos Diez-Freire for

help with radiotelemetry surgeries; Ms. Jillian Stewart for lentiviral preparation and help

with the angiogenesis arrays; and Ms. Jasenka Zubcevic and Mr. Michael Anthony

Cometa for indirect BP measurements.









In addition, I would like to thank Ms. Laura Dixon for her help with AT1R and

AT2R primer optimization; Mr. Adam Mecca for help with osmotic minipump

implantation; Dr. Harm Knot and his laboratory and Randy Harris for their help with

echocardiographies; the UF ICBR Microarray Facility, especially Dr. Mick Popp and Ms.

Blanca Ostmark for their help with the microarrays; the AMRIS facilities at the

McKnight Brain Institute, especially Dr. Glenn Walter, Mr. Xeve Silver, and Ms. Raquel

Torres for their help with the MRI imaging and the Paton laboratory for their

collaboration on the lentiviral transduction efficiency in the brain. I'd also like to thank

Dr. Jeffery Harrison for providing AT2R cDNA; Dr. Peter Sayeski for providing multiple

cell lines; and Dr. Pushpha Kalra and her former graduate student, Dr. Erin Rhinehart, for

teaching me how to do brain microinjections.
















TABLE OF CONTENTS

Page

ACKNOW LEDGM ENTS ........................................ iv

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

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

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

CHAPTER

1 INTRODUCTION ................... .................. .............. .... ......... .......

Im pact and Im portance .............................................. ............... .... ...............
H hypertension ............... ...... .......... ................... ....................
Classic Circulating Renin-Angiotensin System......................................................3
A ngioten sin F ragm ents.............................................................. ............... 4
Tissue Renin A ngiotensin System ........................................................................ .... 5
A ngiotensin II R eceptors .................................................. ...............6
Angiotensin II Type 1 Receptor .............................. ...............6
Structural and molecular function ..........................................................6
Signaling...............................................7
Physiological effects ............................... ............ ..............8
Angiotensin II Type 2 Receptor .............................. ...............9
Structural and M olecular function............................ ...............9
Signaling................................................... ...............11
Physiological effects ........................... ....................... 13
Targeting the RAS for the Treatment of CV Diseases ..........................................15
R A S as a G ene Therapy Target ........................................................................ ...... 16
Ideal Viral Vector .............. ................... ............. .16
N on-viral vectors .................. ........... .. ........ ........... .. ........ 17
Viral Vectors ............................................. .........18
Adenovirus ...................................... ............................ .. ......... 19
A deno-A associated V irus ........................................................ 20
Retrovirus/Lentivirus .................................. ........ .........21
Aims and Rational ........................................ ....... ......... 23
Aim 1: Characterize the Lenti-AT2R Virus In Vitro .................. .......24









Aim 2: Identify Genes Involved in Angiotensin II Type 2 Receptor-Mediated
Inhibition of Endothelial Cell Migration by Expression Profiling. ..............25
Aim 3: Determine the Effect of AT2R Overexpression on CV
Pathophysiologies. .............. ............. ... .. .........25
Aim 4: Determine the Dipsogenic Responses Following Angiotensin II Type 2
Receptor Gene Transfer into the Paraventricular Nucleus. .............................25

2 IN VITRO CHARACTERIZATION OF LENTI-AT2R ........................................26

Introduction ................... ............................................................... 26
M materials and M ethods .................................................. ............... 27
Lentiviral Constructs and Preparation ........................................................27
Cell Culture ......................... ... ............... ........ .3 1
RNA Isolation and Quantification......................... ............... 31
L igand B finding A ssay ........................................................... 32
Protein Isolation and Determination...................... ................32
D election of A activated M A PK ..........................................................................33
Statistical Analysis ...................... .......... ..... ........ 34
Results.......... ............... .. ... ...... ................... ........34
Overexpression of the Receptor by Lenti-AT2R.................. ..............34
Characterization of AT2R Transgene Function ..................................36
D discussion .................. ..........................................................38

3 IDENTIFYING GENES INVOLVED IN ANGIOTENSIN II TYPE 2 RECEPTOR-
MEDIATED SIGNALING PATHWAYS BY EXPRESSION PROFILING............41

Introduction ...................................... ......... ........... .41
Materials and Methods .......................................42..... ........42
Cell Culture, AT2R Transduction, and Treatments...............................42
Real-Time RT-PCR .......... ........ .......... .......... ...............44
A T2R B finding A ssay ................................................. ............... 45
M icroarray Analysis ............. .... ..... ................................ 45
Microarray Analysis Controls and Data Analysis.................. .................46
M migration Assay ............................................... ........ 48
A ngiogenesis Protein A rray ........................................ ................. 49
Statistics ...................................... ............................... ......... 49
R results ..................................... ............... .... ........ ......... .. ..................... 50
Characterization of AT2R Transduction of HCAEC ............... ...............50
Expression Profiling of AT2R-Transduced HCAEC .................................50
Gene Validation................................ .. .....................53
The Role of the AT2R in Migration and Angiogenesis ..............................59
Discussion ...................................... ................... .....................60

4 PREVENTING CARDIAC PATHOPHYSIOLOGIES BY ANGIOTENSIN II TYPE
2 RECEPTOR GENE TRAN SFER .................................................................... 70

Introduction...................................... ................................. ........ 70









M materials and M methods ............................................................7 1
A nim als and Lentiviral D delivery .................... .......................................... 71
Viral Production and Transgene and AT1R Expression Measurements ............72
Physiological Measurements ....................................... 73
Statistics ...................................... ............................... ......... 75
R results ............... ............... ................................................ 75
In vivo G ene Transfer ................................. ........................................75
Pathophysiology in the SHR .............................. ...............78
Pathophysiology in the Angiotensin II-Infusion M odel ......................................80
D discussion .................. .................................... .......................85

5 DETERMINING THE DIPSOGENIC RESPONSES FOLLOWING
ANGIOTENSIN II TYPE 2 RECEPTOR GENE TRANSFER INTO THE
PARAVENTRICULAR NUCLEUS ....................................................... 89

Introduction ...................... .... ...... ............ .89
M methods ................. ................ ... .. ..... ........ ........ 92
Lentiviral Vector Production, Concentration and Titers ...................................92
Animal Care.................................... .. ...............92
Lentiviral Vector Delivery into Brain Nuclei.......... .......................92
Physiological Measurements ....................................... 93
R esults................ ................ .. ....... ... ................... ... ..... ...............94
Lentiviral Vector-Mediated Transduction into the PVN...............................94
Physiological Effects of the AT2R in the PVN .................................................95
Discussion ............................ ......... ......... .97

6 OVERALL DISCUSSION AND CONCLUSIONS .........................................104

AT2R Prevents Cardiac Pathophysiologies.....................................104
Role of the AT2R in Ubiquitination ...................... ........................................ 105
AT2R Effects on Migration and Angiogenesis ................ ................. ...........106
AT2R-M ediated Effects on the AT1R................................ .... ............... 106
Ligand-Independent Activity of the AT2R............... .............. ....... .................107
R ole of the A T2R in the PV N ......................................................................... ...... 107
Perspectives ....................................................... 108

LIST OF REFEREN CES ..................................... ................... .....109

B IO G R A PH IC A L SK E T C H ...................................................................................... 123











viii
















LIST OF TABLES


Table Page

1-1 Advantages and disadvantages of gene transfer techniques..................................18

3-1 AT2R decreases gene expression without CGP42112A stimulation.....................53

3-2 List of genes whose expression was significantly increased with AT2R expression
independent of ligand. ................. ...................... ......... ...54

3-3 List of genes whose expression was significantly altered in the AT2R-transduced
cells stim ulated w ith CG P42112A .................................................................. 57
















LIST OF FIGURES


Figure Page

1-1 If left untreated, hypertension can lead to a number of devastating diseases. ............2

1-2 Circulating renin-angiotensin system. ................. .......................................... .4

1-3 Signal transduction cascades of the AT1R..........................................................8

1-4 Known signaling mechanisms of the AT2R. ................ .................. ............12

1-5 L entiviral v ector sy stem ..................................................................................... 23

2-1 Lentiviral vectors................ ..... ................. 28

2-2 AT2R overexpression in CHO-AT1R cells. ............. ................ ........ ....... 35

2-3 Time course of AngII-induced Erk42/44 activation in CHO-AT1R cells. ...........36

2-4 AT2R transduction prevents AngII-mediated increases in phosphorylated
Erk42/44. ........................... ........................37

2-5 AT2R-mediated effects of Erk42/44 activity cannot be reversed by PD123,319....37

3-1 Timeline for the HCAEC used in the microarray experiments.............................43

3-2 Outline of the microarray protocol as described in detail in the Materials and
Methods section..................... .............. .......... 47

3-3 Lentiviral transduction in HCAEC................................... ........ 51

3-4 Scatter plots of the microarray data........................ ..... ........................52

3-5 Gene validation of the microarray analysis.................... ........ ......60

3-6 AT2R prevents HCAEC migration.. .......................................... 61

3-7 AT2R effects are independent of the typical regulators of angiogenesis represented
on Panomic's Angiogenesis Array. ................................ ............... 62

3-8 Graphical representation of the pathway where the AT2R-regulated genes could
exert its actions to inhibit endothelial cell migration and angiogenesis...................66









4-1 In vivo lentiviral transduction efficiency................................................................76

4-2 Lentiviral transduction efficiency in the heart.. ..........................................77

4-3 AT2R and AT1R expression in isolated cardiomyocytes. ....................................77

4-4 Effect of AT2R transduction on CV pathologies in the SHR. ..............................79

4-5 Blood pressure response to AngII infusion.. ............ ........................80

4-6 Role of the AT2R in the CV pathologies associated with AngII infusion...............82

4-7 MRI analysis indicates AT2R transduction prevents cardiac hypertrophy.............83

4-8 Effect of the AT2R and AngII on other CV physiologies...............................84

4-9 Effect of the AT2R on the pathology of the heart following AngII infusion for 4
w eeks...............................................................85

5-1 Lentiviral transduction into the PVN .. ..................................................................94

5-2 Role of the AT2R in the PVN on basal BP, HR, and activity.............................95

5-3 Effect of icy injection of AngII in animals overexpressing the AT2R in the
PVN ..............................................................96

5-4 Effect of the AT2R in the PVN on basal activity and activity following icy
injection of A ngII.. ................... ................... ................... ................. ..98

5-5 Basal effects of AT2R overexpression in the PVN...............................................98

5-6 Role of the AT2R in the PVN on dehydration-induced water intake ....................99

5-7 Effects of the AT2R in the PVN on subcutaneous injection of AngII.....................99
















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

DEFINING THE ROLE OF THE ANGIOTENSIN II TYPE 2 RECEPTOR IN
CARDIOVASCULAR DISEASE

By

Beverly L. Metcalfe

May, 2004

Chair: Mohan K. Raizada
Major Department: Physiology and Functional Genomics

Despite recent advances in understanding the renin angiotensin system, the role of

the angiotensin II type 2 receptor (AT2R) in cardiovascular diseases remains elusive.

Given that the AT2R has been implicated to play a role in embryonic development,

traditional transgenic animal models may not be an effective means for studying the role

of the AT2R. To overcome the inherent problems of compensatory gene expression with

transgenic animals, we developed a lentiviral vector system in which overexpression of

the AT2R could be accomplished after embryonic development. By injecting lentiviral

vector either directly into the heart or into specific brain regions, we were able to study

both the peripheral and the central actions of this receptor. In addition, this same vector

was used to characterize the AT2R in vitro in cells that are typically difficult to

transduce.

These studies indicate that the AT2R plays a cardioprotective role in several

models of disease. Overexpression of the AT2R in the heart prevents the development of









cardiac hypertrophy and heart failure in both a genetic model of hypertension and one

with AngII-induction. In addition, the AT2R prevents migration in human coronary

artery endothelial cells. This effect could prevent angiogenesis and its induction of

atherosclerosis. Additionally, microarray analysis in these cells indicated a number of

genes whose regulation may play a role in this effect. Finally, it appears that the AT2R

in the paraventricular nucleus causes a decrease in water intake, both basally and in

response to either dehydration or AngII. These effects could play a role to decrease

circulatory volume, cardiac output, and blood pressure. All of which could prevent other

cardiovascular abnormalities. These studies indicate that delivery of the AT2R by

lentiviral vectors may provide a novel therapeutic option in the prevention of

cardiovascular diseases.














CHAPTER 1
INTRODUCTION

Impact and Importance

Cardiovascular (CV) disease is composed of many diseases of the heart and

circulation, including high blood pressure (HBP), heart disease, stroke, and

atherosclerosis. It is the number-one killer of both women and men in the United States.

One of every five people has some form of CV disease, with an average of one death

every 33 seconds. In fact, CV disease kills more people each year than the next 5 leading

causes of death combined. In addition to the large physical impact on society, it also has

a very large economic impact, with an estimated cost of over $350 billion being spent in

the United States in the year 2003.

In the next section, I present an introduction to hypertension and its devastating

effects on the cardiovascular system. We then examine the renin-angiotensin system

(RAS) and its role in hypertension and CV disease, comparing and contrasting the tissue

versus circulating RAS. Next, I introduce the major receptors of the RAS, and what is

known about their role in CV disease. Finally, we investigate the use of gene therapy as

a way to study the RAS and CV disease, as well as its use as a novel therapeutic option.

Hypertension

More than 50 million Americans have hypertension, which is defined as having a

diastolic pressure > 90 mmHg and a systolic pressure > 140 mmHg. Hypertension is a

multifactorial disease in which the cause of disease in nearly 95% of the patients is

unknown. While hypertension itself is asymptomatic, if left untreated it can lead to other










CV diseases (Figure 1-1). Chronic elevation of BP can lead to increased workload of the

heart that can eventually lead to cardiac hypertrophy and heart failure, as well as

myocardial ischemia and infarction. Hypertension also causes damage to the arteries.

This damage leads to endothelial dysfunction and atherosclerosis, which can ultimately

cause stroke, myocardial ischemia and infarction. Damaged vessels can also lead to renal

failure and retinopathy.

Hypertension


SCardiac Workload Arterial Damage


Endothelial Cell
Cdiac Dysfunction
Cardiac
Hypertrophy
Atherosclerosis


Heart Failure Myocardial Ischemia Stroke Renal Failure Retinopathy
and Infarction

Figure 1-1: If left untreated, hypertension can lead to a number of devastating diseases.

Even with increased education of the general population as to the risk factors of

hypertension and CV disease and improved therapies, the incidence of CV disease in the

U.S. remains high. In fact, of all the people with high BP, 31.6% are unaware that they

have the disease, 14.8% are not on medication for the disease, 27.4% are on medication

and have it under control, while 26.2% are on medication but do not have the disease

under control. These statistics indicate that the current therapies are ineffective. There

are several explanations for this: (1) despite increased education, a high percentage of

the people still do not understand the risk factors for CV disease, (2) because many of the

diseases do not produce any symptoms, diagnosis and treatment are delayed, (3) the high









number of side effects causes compliance issues with patients needing to be on

medication, and (4) a lack of understanding of the systems involved in the disorders

inhibits the ability of physicians to properly treat and manage the disease. Therefore, a

better understanding of the systems involved in hypertension and the development of CV

disease and alternative therapeutics for these diseases must be further investigated.

Classic Circulating Renin-Angiotensin System

The RAS has been shown to play a major role in hypertension, cardiac


hypertrophy, and electrolyte balance.1,2 Research involving antagonists, antisense gene

delivery, and genetic polymorphisms of multiple components of the RAS has been shown

to have effects on the cardiovascular (CV) system. Due to the relative importance of the

RAS in CV disease, it continues to be under intense study for future treatments.

Discovery of the RAS began in 1898 when Tigerstedt and Bergman found a pressor

3,4
compound in renal extracts that they named renin.34 Almost 40 years later, two

independent research groups described another pressor substance that would later become

3,4
known as angiotensin. After the discovery of angiotensin, it took nearly another

decade to delineate the cascade that has become the classic circulating renin angiotensin

system.

In this system, angiotensinogen (AOGEN), which is largely produced in the liver,

is converted to the decapeptide angiotensin I (AngI) by the proteolytic enzyme renin that

is produced mainly in the kidney. Angiotensin I is then cleaved by a second proteolytic

enzyme produced in the lungs, angiotensin converting enzyme (ACE), to give the

physiologically-active hormone angiotensin II (AngII). AngII elicits distinct actions by

binding to either the angiotensin II type 1 receptor (AT1R) or the angiotensin II type 2









receptor (AT2R). In general, AngII binding to the AT1R is associated with the

development of cardiovascular pathophysiologies, while binding to the AT2R is thought

to counteract the AT1R and elicit cardioprotective effects (Figure 1-2). A more detailed

examination of these receptors is given later in the text,

AOGEN

Renin I

ACE2
ANG I ANG 1-9

ACEI ACE

ANG IV ANG III ACE2
Ang(3-8) Ang(2-8) ANG II ANG(1-7)

AT4R? AT1R AT2R AT17R

I 1 1I
Anti-AT1 R Vasoconstriction Anti-AT1 R Vasodilation
Vasodilation Na+ & H20 Anti-Proliferation
1 Na+ Reabsorption Reabsorption Vasodilation Vaso ressin
1Hypertrophy Sympathetic Output Anti-Proliferation R is
Memory and Pro-Apoptotic iuresis
Learning Growth and Natriuresis
Proliferation Tissue
Vasoressin Development and
Re ease Repair

Figure 1-2: Circulating renin-angiotensin system.

Angiotensin Fragments

While the classical RAS pathway leading to the formation of AngII has stood the

test of time, recent studies indicate an important role for new receptors, enzymes, and

angiotensin fragments in the RAS (Figure 1-2). It has been shown that aminopeptidase A

can break down the octapeptide AngII to yield the angiotensin fragment angiotensin(2-8)

or AngIII. AngIII can then be further degraded by additional aminopeptidases to yield

angiotensin(3-8) or AngIV. While the roles of AngIII and AngIV are not well defined,









there is evidence that both fragments have cardiovascular functions. While a specific

receptor for AngIII has not been identified, it has been shown to have a similar affinity

5
for the AT1R in the brain to elicit arginine vasopressin release and an increase in BP.

AngIV has been shown to play an important role in the regulation of blood flow, cardiac

6
hypertrophy, sodium reabsorption, and learning and memory. In addition to these

angiotensin fragments, the recently identified carboxypeptidase ACE2 was to elicit

another angiotensin fragment has been shown to have cardiovascular effects. ACE2 has

been shown to breakdown AngII to Ang(1-7) and AngI to Ang(1-9), which is further

7
degraded to Ang(1-7) by ACE. Ang(1-7) has been shown to play a role in vasodilation,

antiproliferation, and sodium and water reabsorption.8,9

Tissue Renin Angiotensin System

Recent evidence indicates both circulating and tissue production of all of the

components of the RAS. This local RAS was first described almost 20 years ago to

explain the blood pressure-independent effects of ACE and AT1R inhibitors on

cardiovascular diseases. This local RAS has now been shown in many tissues including

those of cardiovascular importance, such as the brain, heart, adrenal, vasculature, and

kidney.

The heart provides an interesting example of a local RAS with differing levels of

its components. Angiotensinogen, ACE, and the AT1R are abundantly expressed in the

heart, while renin and the AT2R are only moderately expressed. Even though the

expression of renin is low, the heart has alternative enzymes such as cathepsin G,

chymase, and tonin that have been shown to convert AngI to AngII. In addition, the

AT2R is not highly expressed in the heart under normal conditions, but expression levels










increase during heart failure and fibrosis. This local production of AngII has been

shown in transgenic animals to play an important role in the development of cardiac

12
hypertrophy and fibrosis. Therefore, in studying CV disease, one must consider its

effects on hypertension; and also its effects on end organ damage, independent of

elevated BP.

Angiotensin II Receptors

Evidence for AngII receptor subtypes was found in 1989, when two independent

groups provided pharmacological evidence for the existence of 2 receptor subtypes; the

13
AT1R and the AT2R. Later studies identified the AT1R as mediating the known effects

of AngII at that time, while the role of the AT2R still remains elusive.

Angiotensin II Type 1 Receptor

Structural and molecular function

Numerous studies on the molecular characteristics of the AT1R began after its

14,15
successful cloning in 1991. The AT1R is a seven-transmembrane domain receptor,

with 359 amino acids, and a molecular weight of 41 kDa. In humans, the AT1R has only

16
one subtype that has 5 exons and exists on chromosome 3. In contrast to humans,

rodents have 2 subtypes of the AT1R (ATIA and AT1B). The ATIA and AT1B

receptors are 96% homologous; but they are highly different in their non-coding region,

indicating differential regulation of these receptors. The ATIA receptor is located on

chromosome 17, and has 4 exons; while ATIB receptor is situated on chromosome 2,

16
with only 3 exons. While both receptors are expressed in CV tissues, the AT1A

receptor subtype predominates in most tissues.










Signaling

The AT1R couples to multiple signaling cascades, leading to diverse biological

actions. These signaling events are dependent on both time of activation and cell type.

They have been shown to have effects though G protein-dependent (Gq/1 1, Gi, G12/13)

and independent coupling (JAK, Src, FAK; Figure 1-3). One of the well-characterized

signaling pathways of the AT1R is G protein (Gq/1 1, G12/13) activation of

phospholipase C (PLC), diacylglycerol (DAG), and inositol triphosphate (IP3) that leads

to Ca2+ mobilization and activation of the Ca2+ dependent kinases, protein kinase C

2,17
(PKC) and Ca2+/calmodulin-dependent protein kinase II (CAMKII). Activation of Gi,

18
however, leads to the inhibition of cAMP which inhibits the activation of PKC. In

addition, the AT1R activates a number of small G proteins, which signals to Raf to

activate mitogen-activated protein kinases (MAPK, ERK42, ERK44). These activated

MAPKs are then transported to the nucleus, where they increase the expression of

2
early-response genes (c-fos, c-jun, c-myc).

The AT1R also activates a number of nonreceptor tyrosine kinases (Src, JAK,

FAK) independent of G proteins (Figure 1-3). Activation of Src leads to Ca2+

mobilization through a PLC and IP3 pathway; and also regulates gene transcription

through Ras, Raf, and MAPK. Janus kinase (JAK) activates signal transducers and

activators of transcription (STAT), which translocates to the nucleus to regulate early

growth-response genes. The AT1R-induced autophosphorlyation of focal adhesion

kinase (FAK) leads to its translocation and phosphorlyation of paxillin and talin, which

19
may regulate cell morphology and movement.









Physiological effects

One of the most important physiological effects of AngII mediated by the AT1R is

its regulation of BP. AngII binding to the AT1R can cause transient increases in BP.

AT1R


G Proteins Tyrosine Kinase


Gq, G12/13 Gi Ras Src JAK FAK

PLC + Raf
cAMP I PLC Ras
DAG IP3 MAPK STAT Paxillin
IP3 Raf Talin
Ca2+ 1
Ca2+ MAPK

PKC PKC
CAMKII CAMKII

Contraction Transcription Contraction Transcription Mobility
Differentiation Regulation Differentiation Regulation Morphology
Proliferation Proliferation
Vesicle Transport Vesicle Transport

Figure 1-3: Signal transduction cascades of the AT1R.

This can occur either directly, through the vasoconstriction of vascular smooth muscle

cells (VSMC) and vessels; or indirectly, through increases in vasopressin release and

sympathetic nerve activity. Blood pressure is also regulated chronically through the

regulation of renal sodium and water reabsorption. Again, the AT1R plays a direct and

indirect role in this effect. Angiotensin II can act directly by binding to the AT1R in the

kidney, or indirectly by ATIR-mediated increases in aldosterone and thirst. In addition

to its hemodynamic actions, activation of the AT1R has also been associated with cell









growth and proliferation that could lead to increases in vascular and cardiac

20
hypertrophy.

Angiotensin II Type 2 Receptor

Since its discovery, the actions of the AT2R have remained an enigma. Its binding

properties, signaling, and even its physiological effects have all remained elusive.

Structural and Molecular function

The AT2R is a seven-transmembrane domain protein. It is located on the X

chromosome; and has three exons, with the entire coding region being in the 3rd exon. It

has 363 amino acids, with a molecular mass between 60 to 140 kDa depending on the

amount of glycosylation on five possible sites. The AT2R only has 34% homology to the

AT1R, with the 3rd intracellular loop having the lowest amount of homology.

The distribution of the AT2R is quite different from that of the AT1R. Unlike the

AT1R, which is highly expressed in adults, the AT2R is abundantly expressed during

embryonic development. Its expression dramatically decreases after birth, but can

13
increase after vascular and cardiac injury, wound healing, and renal failure. These

expression patterns indicate that the AT2R may play a role in development, growth,

19
and/or remodeling. Thus studies using transgenic or knockout animals that contain

genetic alterations during embryonic development may develop inadvertent

compensatory mechanisms that may not reflect the true role of the AT2R.

In addition, it has been shown that the AT2R expression can be regulated by

various intracellular and extracellular factors. For example, a number of factors such as

AngII, insulin growth factor, basic fibroblast growth factor, and transforming growth










factor beta 1, as well as estrogen and sodium have been shown to increase the AT2R

13,21
expression; while cAMP and aldosterone have been shown to decrease its expression.

Recent studies indicate that the AT2R can act in a constitutively active manner.

Studies from Kamik's group showed that the amount of the AT2R expression determined

the level of apoptosis; independent of AngII in cultured fibroblasts, epithelial cells, and

22
vascular smooth muscle cells. In addition, modifying the side chain of AngII

23
drastically affected the AT1R effects, but had only modest effects on the AT2R. This

idea is further supported by studies that replaced the 3rd intracellular loop of the AT2R

24
with that of the AT1R, and resulted in an increase in c-fos, independent of AngII.

Even though the AT2R has been found to have ligand-independent activities,

studies mutating the AT2R have revealed important sites for both AngII binding to the

AT2R and signaling of the AT2R. A series of studies from the Pulakat laboratory

showed that amino acids Lys215, Asp287, Argl82, and His273 are important for AngII

25-28
binding to the AT2R. In addition, they were able to identify areas of the receptor

important for signaling, by replacing the 3rd intracellular loop or the C-terminus of the

AT2R with that of the AT1R. In these studies, they have shown that the 3rd intracellular

loop is important in inhibiting ATIR-mediated increases in IP3 and in inhibiting cell

growth; while the C-terminus plays a role in AngII binding and inhibition of cell

29-31
growth. These ideas are in direct contradiction of each other, and future studies are

critically needed to identify when the AT2R is constitutively active or not. If the AT2R

needs an agonist to elicit its effects, then one can control its effects with agonists or

antagonists; however, if the AT2R is constitutively active, then one would have to

regulate the AT2R expression in order to control its effects.









Signaling

The AT2R was initially thought to be a clearance receptor, because there was no

consistent evidence linking the AT2R to any of the well-known intracellular signaling

pathways. To date, the signaling cascades of the AT2R remain to be well defined, but it

appears that it modulates a number of different signaling mechanisms, depending on the

tissue/cell type (Figure 1-4). The AT2R has been shown to increase a number of protein

phosphatases. AT2R coupling to the G protein Gi has been shown to increase both

mitogen-activated protein kinase phosphatase 1 (MKP-1) and serine threonine

phosphatase 2A (PP2A) activity; while the SH2 domain containing phosphatase 1

(SHP-1) has been shown to be activated independent of any G protein signaling. In

addition, a recent study showed that in transfected COS-7 cells, the AT2R can increase

32
SHP-1 when the AT2R couples to Gas independent of the 0 and y subunits. These

increases in protein-phosphatase activity play a role to inhibit growth-promoting factors

such as MAPK (Erk42/44). In a neuronal cell line, however, the AT2R plays a role in

neurite outgrowth by signaling through Rapl and B-Raf to increase ERK1/ERK2

33
activity. Protein phosphatase 2A (PP2A) has also been shown to inhibit Ca2+ channels

in a neuroblastoma cell line, while increasing K, current in cultured neurons, which could

34,35
lead to hyperpolarization of the cell.34

In addition to the actions of the protein phosphatases, the AT2R has been shown to

modulate several lipid-signaling pathways. Stimulation of the AT2R can lead to an

13
increase in phospholipase A2 (PLA2) activity and arachidonic acid (AA) release. This

leads to modification of eicosanoids to increase potassium currents. The AT2R has also

been shown to play a role in the induction of apoptosis, and several pathways have been









implemented in this role. AT2R-mediated increases in MIKP-1 can inactivate the anti-

apoptotic signal Bcl-2 while increasing the pro-apoptotic signal Bax. In addition, it has

been shown that the AT2R can increase ceramides to induce apoptosis. Finally, the

AT2R has also been shown to increase bradykinin (BK), nitric oxide (NO), and cyclic
36,37
GMP levels that may play a role in vasodilation and natriuresis. From these studies it

is evident that the AT2R can couple to a number of various signaling molecules;

however, there are some discrepancies among these studies. These differences can most

likely be accounted for by the differences in cell/tissue type. Therefore, more studies are

needed to fully understand the signaling pathways of the AT2R in each of these cell

types.


AT2R


3i SHP1- Gs Rap Ceramide BK-* NO


MKP PLA2 PP2A Raf cGMP

BcI-2 Bax ERK1/2 AA
I 1 EK ERK1/I
I 1 Eicosanoids ERK1/2 ERK1/2
Apoptosis 1K C. Apoptosis
Gene 11 Vasodilation
Transcription ii Natriuresis
Hyperpolar- Neurite
ization ,, Outgrowth
I Gene
Transcription

Figure 1-4: Known signaling mechanisms of the AT2R.

Recent studies have further advanced the understanding of the AT2R. The AT2R

was initially thought not to be internalized because its binding efficiency did not change

upon AngII activation, and it did not contain a nuclear localization signal. Recently,


I










however, Inagami's group has shown that upon AngII stimulation, the promyelocytic

zinc finger protein (PLZF) co-localizes and then internalizes the AT2R. This

internalization can then act in the nucleus to activate a number of factors to increase

38
protein synthesis. In addition, it has been shown that the AT2R can form a heterodimer

39
with the AT1R to directly inhibit its actions independent of the AT2R signaling.

Finally, the AT2R can play a role to decrease the expression of the AT1R in certain cell

40
types.

Physiological effects

Since the role of the AT2R is not well understood in many aspects, it should come

as no surprise that the physiological role of the AT2R is elusive as well. Among these is

the role of the AT2R in the heart. The AT2R knockout animals indicate that the AT2R

plays a role in the development of cardiac hypertrophy induced by both pressure overload

41,42
and AngII-infusion. Another AT2R knockout experiment, however, indicates that the

AT2R plays a protective role in the heart. In that study, the investigators showed that the

43
AT2R deficiency exacerbated death rates and heart failure after myocardial infarction.

Transgenic animals overexpressing the AT2R specifically in the heart show conflicting

results. Results from one study show that AT2R overexpression does not affect the

44
development of high BP and cardiac hypertrophy after AngII-infusion, but does play a

45
role in the prevention of perivascular fibrosis. However, a separate study indicates that

ventricular-specific overexpression of the AT2R is involved in the development of

46
dilated cardiomyopathy and heart failure. Studies using AT2R-specific antagonists,

however, indicate that the AT2R prevents or reverses a number of cardiac

pathophysiologies. A study conducted by Mukawa et al, showed that simultaneous










administration of ATIR and AT2R antagonists negated the antihypertrophic effects of the

47
AT1R blocker alone. While another study demonstrated that the AT2R blockade did

not affect cardiac hypertrophy but did regulate a number of growth-promoting factors in

48
an AngII-infusion model. In addition, it has been shown that inhibition of the AT2R in

49
hypertrophied hearts amplifies LV growth response to AngII. These inherent

controversies on the cardiac role of the AT2R may be due to intrinsic problems

associated with different strains of transgenic and knockout mice, developmental

abnormalities associated with overexpressing or knocking out the AT2R in embryonic

development, and/or stability issues with the AT2R-specific antagonist.

The physiological role of the AT2R in the brain is not very well understood.

Expression of the AT2R is concentrated in the areas involved in learning and control of

50
motor activity and sensory areas. The AT2R is thought to play a role in brain

development and cognitive function, as it was recently found that patients with X-linked

51
mental retardation have mutations in the AT2R. Studies using simultaneous injection

of AngII and the AT2R antagonist indicate that the AT2R in the brain increases drinking

52
while decreasing BP. In addition, the AT2R antagonists have been shown to potentiate

53
AngII-induced vasopressin release by the AT1R. Finally, studies using the AT2R

knockout animals indicate that the AT2R plays a role in regulating pain threshold and

exploratory behavior while having no effect on learning behavior.

In addition to these physiological actions in the heart and the brain, the AT2R has

also been found to play a role in differentiation and in the inhibition of endothelial cell

56-59
migration and tube formation as well as cell growth and proliferation. The AT2R has










been shown to have vasodilatory effects and to reduce pressor responses to AngII.55

Finally, the AT2R has been shown to play a role in regulating apoptosis, which may

22,61
indicate a role for the AT2R in development and/or tissue remodeling and repair.

Targeting the RAS for the Treatment of CV Diseases

The RAS has been shown to be an integral part of the development of many CV

diseases. In addition, pharmacological evidence indicates that the RAS is an important

target for the treatment CV diseases. Both AT1R antagonists and ACE inhibitors have

been used to successfully treat CV diseases such as hypertension, heart failure,

62
myocardial infarction, and stroke. Despite these advances, the incidence of CV

diseases remains on the rise. This has led many to believe that the current

pharmacotherapies have reached a plateau, and novel approaches for the treatment of CV

disease must be explored.

Gene therapy is a rapidly emerging field for the study of genetic diseases. There

are many advantages of using gene therapy over traditional pharmacological drugs,

making it useful in studying CV diseases. (1) Viral vectors can be designed to either

overexpress a deficient or dysfunctional gene, or to reduce the expression levels of an

overactive gene by antisense technology, ribozymes, or double-stranded RNA inhibition.

(2) Transduction with a viral vector can elicit long-term effects. Therefore, daily

regimens can be eliminated. (3) Viral vectors can be directed to specific target tissues,

which could reduce unwanted side effects. (4) Viral vectors can be designed to be

regulated by a system such as doxycycline that can effectively toggle between an on and

off expression level that can be regulated dose-dependently.










RAS as a Gene Therapy Target

The RAS has been intensely studied for its potential use as a gene therapy target.

The RAS has been targeted for several reasons:

* While the complexity of our understanding of the RAS has increased recently, the
role of the RAS in CV disease is fairly well understood

* The circulating RAS affects multiple targets, which is ideal for systemic delivery

* Pharmacological treatment blocking the RAS reverses many CV pathologies.

Most of the studies targeting the RAS have focused on three major targets:

angiotensinogen, the AT1R, and ACE. Studies performed using antisense

oligonucleotides, targeting angiotensinogen, provided proof of concept for targeting the

63
RAS. These initial studies showed a transient decrease in BP. Further studies using

recombinant adeno-associated virus (rAAV) to decrease the levels of angiotensinogen

64
showed an attenuation of both hypertension and cardiac hypertrophy. Retroviral

delivery of antisense targeting ACE showed similar results with an observed decrease in

65
BP. Cardioprotective effects have also been observed with retroviral delivery of

antisense targeting the AT1R. Studies by Dr. Mohan Raizada's laboratory have

characterized the effects of AT1R antisense (AT1R-AS) on the CV system. When a

single injection of the retroviral AT1R-AS is delivered into the heart of 5-day old SHR

animals, a decrease in BP, cardiac hypertrophy, and perivascular and interstitial fibrosis,

66-69
as well as improved vessel reactivity is observed. In addition, AT1R-AS treatment

70-72
prevented increases in BP induced by AngII-infusion, L-NAME, and fructose.

Ideal Viral Vector

For gene therapy to be effective, the best possible vector must be chosen to fit the

need. For example, if you are studying restenosis, which is a short-term, secondary effect









of balloon angioplasty, then you would choose a vector system that can efficiently

transduce endothelial and vascular smooth muscle cells, and only elicits its effects for a

short period of time. Someone studying a long-term disease such as hypertension or

heart failure, however, would want a viral vector that can transduce cardiac and vascular

tissues, and whose effects are stable and long-lasting. Currently, a number of both

nonviral and viral delivery systems are used, each having their own advantages and

disadvantages that must be considered when determining which system to use for a

particular disease. In the next sections, we examine these delivery systems, their

advantages and disadvantages, and a few examples of successful studies using these

techniques (Table 1).

Non-viral vectors

Nonviral gene delivery techniques do not rely on a viral system to effectively

transduce cells of interest. Instead they utilize the ionic properties of the cell membrane

in order to enter the cell. Essentially, cationic or lipogenic vehicles are used to aid

delivery into the cells, but "naked," noncomplexed DNA can also be used.

Advantages of these nonviral delivery systems are that it is safe, and because there

are no viral particles, it elicits no immune response. The disadvantages, however, are that

it is less efficient than most viral methods, and it does not integrate into the genome;

therefore, the expression is only transient.

Several studies highlight the success of using nonviral gene delivery methods. Dr.

Phillip's group showed decreases in hypertension by antisense oligonucleotides directed

73
towards the AT1R, angiotensinogen, ACE, or 0-adrenergic receptor. Another series of

studies using naked plasmid delivery are now in clinical trials. These studies show that









delivery of vascular endothelial growth factor (VEGF) improved collateral angiogenesis,

which could lead to prevention of myocardial infarction and angina.74'75

Viral Vectors

A number of viral vectors have been developed for gene therapy. Most of these

viruses depend on binding to a specific membrane receptor of the host cell for cellular

uptake. Once inside the nucleus, the virus can then either replicate on its own, or

integrate into the host genome. Each viral vector has its own unique properties that make

them ideal for particular situations (Table 1).

Table 1-1: Advantages and disadvantages of gene transfer techniques.
Advantages Disadvantages
Nonviral Safe Less efficient
Low immune response Short-term expression
Easily produced
Infect dividing and non-dividing
Adenovirus cells Short-term expression
Large capacity High immune response
No insertional mutagenesis
Easily produced
AAV Long-term expression Limited capacity
Infect dividing and non-dividing
cells Delayed expression
Little immune response Hard to produce
Safe
Retrovirus Long-term expression Insertional mutagenesis
Large capacity Only infects dividing cells
Little immune response
Easily produced
Lentivirus Long-term expression Insertional mutagenesis
Large capacity Safe
Infect dividing and non-dividing
cells
Little immune response
Easily produced









Adenovirus

Recombinant adenoviruses are double-stranded DNA viruses. They are currently

among the most-often-used viral vectors for gene transfer into cardiovascular tissue.

Adenoviruses can efficiently infect both dividing and nondividing cells, with high

efficiency. In addition, their insert size can handle 7 to 8 kb and they can be easily

produced. The adenovirus does not integrate into the genome; therefore, there is little

risk for insertional mutagenesis. This lack of integration, however, limits its time of

expression to approximately 1 month. In addition, the adenovirus elicits a strong immune

response that decreases its expression, and prevents the use of repeated administrations.

Recently, a new generation of adenovirus has been introduced. This generation of

adenovirus has fewer endogenous viral proteins; therefore, a helper virus is required for

viral production. This helper-dependent adenovirus (hdAd) has a larger capacity for

transgene, can elicit longer transgene expression (up to 9 months), and has a reduced

76
immune response. This hdAd is a promising gene therapy vector, since it has resolved

many of the disadvantages of earlier generations of adenoviral vectors. There are,

however, still limitations. The hdAd still requires a specific receptor on the cell surface

for infection. Therefore, its transduction efficiency is dependent on the cells expressing

the adenoviral receptor. Secondly, the production of the virus is more difficult, because it

requires a helper virus. Finally, safety issues may still be a concern because of low levels

of contaminating helper virus in the hdAd preps.

Despite its high immunogenicity and its short-term expression, the adenovirus has

been a successful gene therapy vector for the cardiovascular system. Studies expressing

fibroblast growth factor and apolipoprotein A-I show improvement in myocardial









77,78
perfusion and a decrease in atherosclerosis, respectively. A number of studies have

used the adenoviral vector to overexpress superoxide dismutase. These studies show that

superoxide dismutase expression can improve endothelial dysfunction, decrease arterial

79-81
pressure, and attenuate myocardial ischemia reperfusion injury.

Adeno-Associated Virus

Adeno-associated virus (AAV) is a single-stranded linear DNA virus. While wild-

type AAV is known to specifically integrate into chromosome 19, the recombinant virus

used for gene therapy has lost this characteristic. Instead, it is thought to exist in an

episomal manner. A recent study, however, indicated that a new generation of

82
recombinant AAV (rAAV) can integrate site-specifically in muscle cells. Despite being

largely episomal, the rAAV elicits long-term expression. Its other advantages are that it

can infect both dividing and non-dividing cells and elicits minimal immune response.

There are some disadvantages of rAAV, however. First, it has a limited size capacity of

only 4.4kb. In addition, even though there are many serotypes used with rAAV, many

humans contain antibodies for these serotypes. These antibodies may act to neutralize the

virus before eliciting its effects. Additionally, the rAAV takes weeks to months before

expression can be observed; therefore, immediate effects cannot be seen. Finally, viral

replication is dependent on helper virus to initiate amplification and viral production; so

the production of rAAV is difficult and laborious.

Studies in Dr. Dzau's group have shown that delivery of the heme-oxygenase 1

(HO-1) gene before myocardial injury protects the heart from myocardial infarction by as

83
much as 75%. In addition, rAAV has been shown to be useful in decreasing









hypertension by AT1R antisense delivery and improving muscular dystrophy and its

84,85
associated cardiomyopathy by 6-sarcoglycan delivery.

Retrovirus/Lentivirus

Retroviruses are single-stranded RNA viruses that can stably integrate into the host

genome, allowing for long-term expression of the transgene. Because it integrates into

the host genome, however, retroviruses can cause insertional mutagenesis, thus affecting

normal gene expression. Additional advantages of retroviruses include its low immune

response, large transgene capacity and its optimization for efficient viral production.

Their major limitation is that they can only infect dividing cells, thus decreasing their

efficiency and somewhat limiting their usefulness to ex vivo gene transfer in which target

cells are removed, transduced, and then reintroduced.

Recent advances have identified a new retroviral gene therapy vector based on the

human immunodefiency virus (HIV), named lentivirus. After removal of 5 of the

unnecessary wild type (wt) proteins, this virus has been shown to be an efficient vector

for gene therapy. Lentivirus has several significant advantages over the previously-

mentioned gene therapy vectors. (1) Viral particles are produced with the necessary wt

viral proteins being produced in trans; therefore, none of the wt viral proteins are

packaged in the lentivirus. This increases the safety of this viral system as well as

decreasing the immune response. (2) The viral coat of the lentivirus can be made with a

variety of glycoproteins. This allows one to pseudotype the lentivirus to optimize its

transduction efficiency to target tissues. (3) Since the lentivirus does not require the cell

cycle for transduction, unlike other retroviruses, it can infect both dividing and non-

dividing cells. (4) It has a large capacity for transgene of up to 18kb. (5) Recent studies









86
have developed methods for efficient large-scale production of the virus. (6) The

lentivirus also integrates into the genome with efficient transgene expression levels being

achieved within 12-24 hours and lasting the life of the animal. A disadvantage of

integration into the genome is insertional mutagenesis, however, a recent report may have

identified a way to fix from this problem. These investigators showed that fusing the

HIV-1 integrase with a sequence-specific DNA binding protein is an efficient approach

87
for directing integration into a predetermined DNA site.

The major drawback of lentiviral vectors is that it is based on the human pathogen

HIV-1 and therefore safety concerns remain. To address these problems, 5 of the 9 viral

genes of wild type HIV-1 have been deleted in the lentiviral vectors. For additional

safety, the majority of the remaining wt genes (gag, pol, rev, tat) are produced in trans

(pHP vector; Figure 1-5). In addition, the wt envelope gene (env) has been replaced with

a pseudotyped vesicular stomatitus virus glycoprotein (VSVG; Figure 1-5). With these

modifications, only the necessary packaging signal, psi (xy), and the long terminal repeats

(LTR) which is necessary for transcription initiation are packaged with the transgene of

interest (pTYF vector; Figure 1-5).

Our group has had much success using the retroviral vector. We have previously

shown that AT1R antisense delivered by an intracardiac injection of retroviral vector

prevents the development of hypertension and its associated pathologies in the

SHR.66-72,88 The lentivirus has also been shown to be effective against pathologies

89
associated with hypertension as lentiviral delivery of angiostatin reduces retinopathy.









Aims and Rational

Gene therapy to overexpress the AT2R could prove to be a novel therapeutic

approach for the treatment of CV diseases. It has been indicated that unopposed

stimulation of the AT2R may contribute to the effectiveness of ATIR antagonists. In

fact, it has been shown that AT2R antagonists negate the antihypertrophic affect of AT1R
47 39
antagonists. In addition, studies have shown that the AT2R can inhibit AT1R actions.

These studies indicate that overexpression of the AT2R in cardiovascularly-relevent

tissues would prevent the effects of the AT1R and may prevent the development of or

even cause the reversal of CV diseases.

pHP Vector (Packaging Vector)


mSV4-PolyA




pTYF Vector (Transducing Vector)
LTR LTR
-RE = EFla Transgene

l


VSVG Vector (Envelope Vector)

EF1a t[


SV40-PolyA


Figure 1-5: Lentiviral vector system. Three viral vectors are used to produce lentivirus
in order to minimize the introduction of wild type HIV-1 proteins.

In addition to these advantages, AT2R gene therapy could also provide novel

insights into the role of this receptor in CV diseases. Based on the current literature, it is

quite evident that the physiological role of the AT2R remains elusive. There are









conflicting studies involving many aspects of the AT2R from its activation to its

signaling to its physiological effects. While some of these inconsistent results can be

accounted for by differences in cell lines and animals models, innovative approaches

must be investigated to determine the true role of this receptor. Since the AT2R has been

shown to play a role in and is highly expressed during embryonic development,

transgenic and knockout animals may not be an accurate way of determining the role of

the AT2R. Overexpression or absence of the AT2R during this critical developmental

stage may cause inadvertent compensatory mechanisms in these animals, thus not truly

reflecting the effects of the AT2R. In addition, the AT2R is only expressed at low levels

in the adults making studies using AT2R-specific antagonists inaccurate. To further add

to this problem, the current AT2R antagonists are expensive and only short-lived;

therefore, physiological measurements are hard to accurately record and long-term

studies in vitro and in vivo are nearly impossible.

Successful gene therapy for the prevention of hypertension and cardiac hypertrophy

has previously been established in our laboratory by decreasing the levels of the AT1R

using a retroviral vector. Recent work in our laboratory has established the lentiviral

vector for the transduction of cardiovascularly-relevent tissues. This viral vector is ideal

for several reasons: (1) it can carry a large transgene, (2) it can be produced easily and

reproducibly, (3) it elicits long-term expression, (4) it produces little immune response,

and finally (5) it transduces both dividing and non-dividing cell types.

Based on this information, the specific aims of my project are outlined below:

Aim 1: Characterize the Lenti-AT2R Virus In Vitro.

* Infection Efficiency and Expression.

* Functionality.









Aim 2: Identify Genes Involved in Angiotensin II Type 2 Receptor-Mediated
Inhibition of Endothelial Cell Migration by Expression Profiling.

* Perform Microarray Analysis to Determine Novel Gene Profiles Following AT2R
Overexpression in Human Coronary Artery Endothelial Cells.

* Associate Novel Gene Expression Profile with a Physiological Function.

Aim 3: Determine the Effect of AT2R Overexpression on CV Pathophysiologies.

* Determine the Affects of the AT2R Overexpression in the Spontaneously
Hypertensive Rat.

* Provide Further Proof of Concept of the Effects of the AT2R Overexpression in an
AngII-Infused Model of CV Disease.

Aim 4: Determine the Dipsogenic Responses Following Angiotensin II Type 2
Receptor Gene Transfer into the Paraventricular Nucleus.

* Determine the Affect of the AT2R Overexpression in the Paraventricular Nucleus.














CHAPTER 2
IN VITRO CHARACTERIZATION OF LENTI-AT2R

Introduction

The physiological role of the angiotensin II type 2 receptor (AT2R) remains

elusive. Since the AT2R has been implicated to play a role in embryonic development,

genetic abnormalities associated with development in transgenic and knockout animals

may limit the effectiveness in studying this receptor. Thus, gene transfer of AT2R

following embryonic development offers a novel way to investigate this receptor and its

role in the CV system.

The use of lentiviral vector has many appealing traits in studying the physiological

role of the AT2R in CV diseases. (1) It can accommodate large transgenes. This

flexibility can allow one to overexpress the transgene of interest along with a marker

gene such as the neomyocin resistance gene (NeoR), an alkaline phosphatase gene, or

even green fluorescent protein (GFP). (2) The lentivirus can infect both dividing and

non-dividing cells. This is particularly appealing when studying the CV system because

both the myocytes of the heart and the neurons of the brain do not divide. (3)

Modifications in the wild type HIV-1 virus made while creating this gene therapy vector

have increased its safety and allows the virus to infect cells in vivo with little to no

immune response. (4) Finally, the lentiviral vector has been shown to efficiently

86 7
transduce cardiovascularly-relevant tissues. A single injection of 2.5x10 titer units

(TU) into the left ventricular cavity of the heart leads to efficient transduction of the

86
heart, liver, lung and kidney.









Before we can begin physiological studies, we must ensure that our AT2R

transgene has been successfully cloned and is functional. In addition, each viral construct

should be tested to ensure that it can infect and overexpress the AT2R transgene. Thus

the objective of this study was 2-fold: (1) determine if infections of cells with the

previously-cloned Lenti-AT2R construct leads to AT2R overexpression; and (2) examine

if the AT2R overexpression is functional by examining the transgene's ability to regulate

the activation of mitogen-activated protein kinase (MAPK), Erk42/44.

Previous studies have shown antagonistic actions of the AT1R and AT2R in the

regulation of Erk42/44. Studies have shown that stimulation of the AT1R leads to

activation of MAPK through Ras and Raf, indicating a role for this receptor in cell

90
growth and differentiation. Activation of the AT2R, however, has been shown to

decrease MAPK activities.9091 In fact, a study performed by Dr. Colin Sumners group

showed that in neurons derived from neonatal rat hypothalamus and brainstem, the AT1R

90
increased and the AT2R decreased MAPK activity. However, since the cells used were

mixed cultures, the investigators were not able to determine whether these effects were

occurring in the same neuron or in neurons containing either the AT1R or the AT2R.

Materials and Methods

Lentiviral Constructs and Preparation

A series of lentiviral constructs (pTYF) used throughout this study, was created as

86,92
previously described (Figure 2-1). Expression vectors created to express either GFP

(Lenti-GFP) or human placental alkaline phosphatase (Lenti-PLAP) were used to

determine the transduction efficiency of the virus both in vitro and in vivo. Additional

vectors were created to bicistronically overexpress, through the use of an internal











ribosome entry site (IRES), both the AT2R and neomyocin resistance gene (NeoR; Lenti-

AT2R-I-Neo) genes or the AT2R and PLAP (Lenti-AT2R-I-PLAP). Finally, control


vectors were used, which only contained the bicistronic element and either the NeoR


(Lenti-I-NeoR) or PLAP (Lenti-I-PLAP) genes.


Expression Vectors
Lenti-GFP
LTR


Lenti-PLAP
LTR



Experimental Vectors


Lenti-AT2R-I-NeoR
LTR
RRE

'If"
\V
Lenti-AT2R-I-PLAP
LTR
RRE


I -


IRES

AT2R U


AT2R


Control Vectors:
Lenti-I-NeoR
LTR


T -A I


LTR
-U


IRES


Lenti-I-PLAP

RRE


LTR
-U


Figure 2-1: Lentiviral vectors. A series of lentiviral vectors were previously constructed
in our laboratory. For the experiments described in this dissertation, there
were 2 expression vectors used to characterize lentiviral transduction that
contain either GFP (Lenti-GFP) or PLAP (Lenti-PLAP), 2 experimental
plasmids which bicistronically express both the AT2R and either NeoR
(Lenti-AT2R-I-NeoR) or PLAP (Lenti-AT2R-I-PLAP) genes, and finally, 2
control plasmids that contain all the elements of the experimental plasmids
minus the AT2R (Lenti-I-NeoR, Lenti-I-PLAP).


L


1


E11f l


PLAP









Lentivirus was produced, concentrated, and titered according to established

86
protocols. 293FT cells (Invitrogen) maintained in Dulbeccos' Modified Eagle's

Medium (DMEM; Gibco) containing 10% FBS and antibiotics were plated at a density of

1x107 cells/75-cm2 flask (T-75). The next day, the cells were transfected with a mixture

of 400[tL of serum-free DMEM, 7[tg pHP, 3.5 utg of the desired pTYF plasmid, 2.8 utg of

VSVG plasmid and 0.6 [tg of Tat plasmid with the use of Superfect (Gibco). After the

complexes were mixed and allowed to form for 10 minutes, 5 mL of DMEM + FBS +

antibiotics (growth media) were mixed with the complexes and added to the cells. The

complexes remained on the cells for 4-5 hours at 370 C after which time the media on the

cells are changed to 5 mL growth media.

The first collection of virus was harvested -30 hours post-transfection and an

additional 5 mL of growth media is added to the transfected cells. The media from this

first collection is spun down at 2000 x g for 10 minutes at 40 C and then filtered through

a 0.45 micron low-protein-binding (PES) membrane (Nalgene). This first collection is

then divided and centrifuged through a Centricon-80 ultrafiltration column (Millipore) for

1 hour at 2000 x g at 40 C according to manufacturer's protocols. This first collection of

virus is added to or "spiked" into the final collection of virus.

Approximately 45 hours post-transfection, the second virus collection is performed.

Again, this collection is centrifuged at 2000 x g for 10 min and filtered through a Nalgene

membrane, after which, the first collection is added to this virus. A cushion of 220 ul of

60% iodixanol is added to the bottom of 4 conical-bottom tubes (Beckman), and 30 mL

of the media containing the virus is gently added to the tube. The virus samples are then

centrifuged for 2.5 hours at 50000 x g at 40 C in a Beckman SW-28 swinging bucket









rotor. Following this spin, the media above the iodixanol interface is removed. The

residual media containing the virus and iodixanol are then carefully mixed and collected

into a 3mL conical bottom tube (Beckman). This sample is then spun for 22-24 hours at

6100 x g at 40 C using a Beckman SW-50.1 rotor to separate the virus from the iodixanol.

The supernatant is then removed and discarded. The remaining pellet containing the

virus is resuspended in 30 [tL of phosphate buffered saline (PBS) or artificial cerebral

spinal fluid (a -csf) overnight at 40 C. Finally, the virus is gently mixed, aliquoted and

stored at -800 C until used.

Lentiviral vectors were titered using a p24-Antigen Assay (Beckman). First, 1 pL

of virus was diluted 1:100 in the provided lysis buffer and allowed to lyse at 370 C for 1

hour. Following this incubation, the virus is serially diluted to a final concentration,

which is 10-7 and 10-8 from the original. The diluted samples (200 [tL) were added to the

wells containing the p24-antibody and incubated at 370 C for 1 hour. The samples were

then washed a total of 6 times with the provided wash buffer. Next, 200 [tL of the

biotinlyated reagent was added to the samples and incubated at 370 C for 1 hour. After

an additional series of washes, streptavidin conjugated to horseradish peroxidase (200

[tL) was added and incubated for 30 minutes at 370 C. Another series of washes were

performed on the samples, and tetramethlbenzidine was added to the samples and

incubated at 370 C for 30 minutes. Finally, 50 [tL of hydrogen sulfate was added to the

sample and read on a microplate reader at an absorbance of 450 nm. Titers were then

calculated by comparing the absorbance to the provided standard curve and the original

dilution factor.









Cell Culture

Chinese hamster ovary cells transfected to overexpress the AT1R (CHO-AT1R)

was a kind gift from Dr. Peter Sayeski (University of Florida). Cells were grown in

Ham's F-12 media (Cellgro) supplemented with 10% fetal bovine serum (Ham's

complete; Cellgro). Once the cells had reached confluency, they were subcultured and

-25,000 cells/cm2 were plated in 6-well dishes. The following day, the cells were

incubated with the appropriate lentiviral vector (Lenti-AT2-I-PLAP or Lenti-I-PLAP) at a

multiplicity of infection (MOI) of 1 in the presence of 8 tg/[tL polybrene (Sigma) in the

Ham's complete media for -15 hours. Following this period, the medium was replaced

with fresh Ham's complete media and allowed to grow for an additional 3 days prior to

use in the experiments.

RNA Isolation and Quantification

Real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to

quantitate the mRNA levels of the AT2R. Total RNA was isolated from the CHO cells

using Ambion's RNaqueous-4-PCR kit (Ambion) according to the manufacturer's

protocol. Two-step RT-PCR was used to quantitate the receptor. First, a reverse

transcription reaction was performed where the total RNA is converted to cDNA using

TaqMan reverse transcription reagents (Applied Biosystems). This was followed by the

PCR reaction using the TaqMan Universal PCR Master Mix and an ABI Prism 7000 HT

Detection System (Applied Biosystems). Again, all reactions performed were done

according to manufacturer's protocols. The primers and probe that were used were as

follows: AT2R (forward): 5'-CCGCATTTAACTGCTCACACA-3'; (reverse): 5'-

ATCATGTAGTAGAGAACAGGAATTGCTT-3'; (probe): 5'-

FAMCCGGCAGATAAGCAT-MGBNFQ-3'. Relative quantitation was performed









using ribosomal rRNA (18S) as an endogenous control for the comparative method

described in Applied Biosystems User Bulletin #2. No reverse transcriptase and no

template controls were used for each sample to ensure that there was no contaminating

amplification.

Ligand Binding Assay

Ligand-specific binding of 125I-SI-AngII to the AT2R was performed as previously

92
described. Briefly, cells were washed with Dulbecco's phosphate-buffered saline (PBS;

Cellgro), followed by a 30-minute incubation with the reaction mixture containing 0.1-10

nmol/L 125I-SI-AnglI (Washington State University), 0.5% bovine serum albumin

(Sigma), and 1 [tM of the ATIR-specific antagonist, losartan (Los; Merck) prepared to a

final volume of 0.4 mL in PBS. Additionally, 1 [tM of the AT2R-specific antagonist,

PD123,319 (PD; Sigma) was added to the binding reaction mixtures. Following this

incubation, the cells were washed with ice-cold PBS to remove the unbound ligand; the

cells were then dissolved and collected in 0.1 N NaOH (Sigma) before being read on a

Beckman 5500 y counter. AT2R-specific binding was calculated by subtracting the

binding of the non-specific reactions (Los + PD) from the total binding (Los only).

These values were then normalized to the protein content of each reaction as determined

93
by the method of Lowry et al.

Protein Isolation and Determination

Total cell lysates were isolated from CHO cells and used to determine the levels of

activated MAPK by measuring the levels of phosphorylated Erk42/44 via Western blot

analysis. CHO cells infected with either the control virus (Lenti-I-PLAP) or the

experimental virus (Lenti-AT2R-I-PLAP) were plated at a concentration of 140,000 cells









in a 35 mm dish. They were grown in Ham's complete media to confluency, changing

the media every 2 days. Once confluent, the media was replaced with serum-free Ham's

media. The cultures were incubated with the indicated drugs at the following

concentrations: 100 nM AngII, 1 pM Los, or 1 [tM PD. In a time-course, cells were

incubated with 100nM AngII from 0-20 minutes, to determine the amount of time need to

reach maximum activation of Erk42/44. At the end of the incubations, the cells were

washed with ice-cold PBS and the proteins were isolated using ice-cold lysis buffer

composed of 1% NP40, 10% glycerol, 150 mM NaCl, 20 mM Tris-HCI (pH 7.4), and

protease inhibitor cocktail (125 mM PMSF, 2.5 mg/mL aprotinin, 2.5 mg/mL leupeptin,

2.5 mg/mL antipain, 2.5 mg/mL chymostatin; Sigma). Cell lysates were scraped using a

rubber policeman and collected in a microcentrifuge tube. The samples were then

sonicated 3 times for 5 seconds each on ice and centrifuged at 14,000 rpm for 10 minutes

at 40 C. The supernatant was then saved, and protein levels were determined using the

manufacturer's protocol for BioRad's Bradford-based protein assay (BioRad).

Detection of Activated MAPK

Samples (20[tg) were separated on Ready-Made 10% Tris-HCI gels (Bio-Rad), and

proteins were transferred to a nitrocellulose membrane using the Bio-Rad Mini-Protean

system. Protein detection of phosphorylated Erk42/44 was performed as suggested by

the manufacturer's protocol (Promega). Briefly, nitrocellulose membranes were washed

for 5 minutes in TBS, followed by an overnight incubation at 40 C in blocking buffer

composed of 3% BSA in TBS. The membrane was then incubated for 2 hours at room

temperature in Anti-ACTIVE-MAPK (Promega) primary antibody diluted 1:2500 in TBS

+ 0.1% Tween (TBST) plus 0.1% BSA. The membrane was then washed 3 times, 15

minutes each in TBST and then incubated for 1 hour at room temperature in the horse-









radish peroxidase conjugated anti-rabbit secondary antibody (Promega) diluted 1:5000 in

TBST + 5% milk. This was followed by 3 15 minute washes in TBST. Finally, the

bands were visualized using a Western Lightning chemiluminescence (DuPont).

Following this, the membranes were stripped of antibody and a second Western

blot was performed on the same membrane to detect paxillin, a control protein used to

normalize the samples. Membranes were stripped for 18 minutes at 570 C using a buffer

consisting of 60 mM Tris (pH 6.8), 2% SDS, 0.7% j-Mercaptoethanol. Next, the

membranes were washed 4 times for 5 minutes each in TBST and blocked for 1 hour at

room temperature in a solution containing 5% milk in TBST. The membrane was then

incubated for 1 hour at room temperature with anti-paxillin (Promega) primary antibody

diluted 1:10,000 in the 5% milk/TBST solution. This was followed by 3-5 minute

washes in TBST and a 45 minute incubation at room temperature with the secondary anti-

mouse antibody (Promega) diluted 1:5000 in the 5% milk/TBST solution. Finally, the

membranes were washed 3 times for 5 minutes each in TBST and bands were visualized

using chemiluminescence. All bands for active MAPK and paxillin were quantitated

using BioRad's GS-710 densitometer.

Statistical Analysis

Results were analyzed with a student's t-test when only 2 samples were being

examined. All other experiments were analyzed using a one-way ANOVA. Results are

indicated as mean +/- standard error with a p value of 0.05 being considered significant.

Results

Overexpression of the Receptor by Lenti-AT2R

CHO-AT1R cells were infected with either the Lenti-AT2R-I-PLAP (AT2R) or

Lenti-I-PLAP (Cntrl) at a MOI of one. Real-time RT-PCR analysis revealed a significant










increase in the levels of the AT2R mRNA in those CHO-AT1R cells transduced with the

AT2R (Figure 2-2A). This increase was associated with an increase in AT2R binding.

CHO-AT1R cells do not contain any endogenous AT2R. When tranduced with the Lenti-

AT2R-I-PLAP, however, the total binding activity for the AT2R was 4.85 pmol/mg

protein with a Kd of 0.82 nM (Figure 2-2B). These results indicate that the lentiviral

vector can efficiently deliver the AT2R in vitro and that these transduced cells exhibit

binding characteristics typically associated with the AT2R.

A


3500
S3000
2500
11 2000

-2 E 1500
z 1000
500
0


Cntrl


AT2R


Saturation Curve


0.01

^-0.008
-j

L CL0.006

| 0.004

0.002

0


Scatchard Graph


0 2000 4000 0 2 4 6
Free 1251-SI-AII Bound
(pmol/L) Kd = 0.82nM (pmol/mg)
Bmax = 4.85 pmollmg


Figure 2-2: AT2R overexpression in CHO-AT1R cells. Lenti-AT2R-I-PLAP (AT2R) or
Lenti-I-PLAP (Cntrl) was used to transduce CHO-AT1R cells at a MOI of 1.
A) Three days post-infection, the cells were analyzed for AT2R mRNA by
real-time RT-PCR (n=3). B) In addition, AT2R-specific binding was
analyzed by ligand binding. (* = p<0.05).


B





-0
yE
C o .
0
M









Characterization of AT2R Transgene Function

CHO-AT1R cells were first used to determine the effect of AngII on MAPK

activation in these cells. Cells were incubated with AngII or AngII + Los. Western blot

analysis for phosphorylated Erk42/44 (activated MAPK) revealed little endogenous

MAPK activity. Incubation with AngII caused a time-dependent increase in MAPK

activation with maximum activation being achieved in 10 minutes (Figure 2-3). This

activation was reduced when Los was added to the cells at all time points (Figure 2-3)

while Los alone had no effect on MAPK activity (Figure 2-3). Based on these results,

subsequent experiments were carried out at the 10 minute timepoint.


Erk 44 A
Erk 42 -w

Untrt 5min 10 min 20min 5min 10 min 20min Los
Angll Angll + Los
(luM) (luM each)

Figure 2-3: Time course of AngII-induced Erk42/44 activation in CHO-AT1R cells.
CHO-AT1R cells were tested to determine if AngII induced ERK42/44
activity through the AT1R. The cells were either left untreated or treated for
5, 10, or 20 minutes with either 1 pM AngII, 1 [tM AngII + 1 [tM Los or 20
minutes with Los. Following these incubations, proteins were isolated and
analyzed for phosphorylated Erk42/44 (Active MAPK) by Western blot
analysis.

Next, we determined if AT2R transduction in the CHO-AT1R cells would inhibit

ATIR-mediated activation of MAPK. CHO-AT1R cells transduced with Lenti-I-PLAP

(Cntrl) showed a 65-fold increase in Erk42 activity following AngII stimulation (Figure

2-4). In the CHO cells transduced with Lenti-AT2R-I-PLAP (AT2R), however,

stimulation of MAPK with AngII resulted in only a 7-fold increase in activity (Figure 2-

4). This effect, however, was not reversed by PD (Figure 2-5) nor altered by Los (Figure

2-5).










Erk 44 -
Erk 42

Paxillin

Erk 42


a


p=0.09
p-0.08


Figure 2-4: AT2R transduction prevents AngII-mediated increases in phosphorylated
Erk42/44. CHO-AT1R cells were either transduced with Lenti-I-PLAP
(Cntrl) or Lenti-AT2R-I-PLAP (AT2R). Three days post-transduction, the
cells were treated with AngII for 10 minutes, proteins were isolated, and
phosphorylated Erk42/44 was examined by Western blot analysis. (Top:
Representative Western blot showing Erk42/44 activity; Bottom: Quantitation
of Erk42 expression. n = 2/group; = p<0.05 compare to Cntrl).


Erk 44 00M
Erk 42

Paxillin

Untrt Angll

Cntrl


a w


Untrt Angll Angll Angll Los PD
+Los +PD
AT2R


Figure 2-5: AT2R-mediated effects of Erk42/44 activity cannot be reversed by
PD123,319. CHO-AT1R cells transduced with either Lenti-I-PLAP (Cntrl) or
Lenti-AT2R-I-PLAP (AT2R) were treated for ten minutes with the indicated
drugs (untreated (Untrt) or 1 kM AngII, 1 kM Los or 1 kM PD) and analyzed
for Erk42/44 activity by Western blot analysis.


4-
c
E3.5 -
S3-
2 2.5 -
2-
o
Z 1.5 -
C
E 1
S0.5-
0 0-


Cntrl Cntrl
+ All


AT2R AT2R
+ All










Discussion

The results presented in this chapter demonstrate that (1) infection of CHO-AT1R

cells with Lenti-AT2R results in an increase in the expression of the AT2R transgene, (2)

the AT2R is functional, (3) the transduced AT2R may be acting ligand independently.

Previous studies overexpressing the AT2R in vitro were unable to associate the

94,95
AT2R with a functional consequence. Our study is one of the first demonstrations of

an AT2R transgene functionally coupling to its signaling cascade. The advantage of this

study over the others is that the Lenti-AT2R was able to transduce and overexpress the

AT2R at high levels without selection, thus allowing us to study the effects of AT2R

overexpression as early as 3 days post-infection.

Our data indicate that the AT2R inhibits AngII-mediated increases in MAPK. This

is consistent with previous data. These studies have shown that the AT2R can directly

39
bind to the AT1R to inhibit the actions of the AT1R. In addition, it has been shown that

the AT1R and AT2R have opposing actions on MAPK in the same primary cultured

cells. However, the researchers could not conclusively state that these opposing actions

were occurring in the same cell and whether or not the receptors were having a direct

90
effect on each other. In similar cultures, however, the same group was able to show

96
that the AT1R and AT2R have opposing actions on potassium current. Finally, it has

been shown in chromaffin cells that the AT2R can negatively regulate the AT1R

97
signaling pathways through the regulation of cGMP. Based on our findings and those

of others, it will be interesting to the mechanism of these actions, whether it is through a

steric hindrance of the AT2R directly binding to the AT1R, the regulation of cGMP,









AT2R-mediated dephosphorylation by activation of phosphatases, or by some other

unknown mechanism.

The antagonistic effect we observed in these studies could not be due to non-

specific effects, whereby stimulation of the AT1R by AngII is decreased because of the

abundance of AT2Rs to compete for binding sites? There are two explanations that

indicate that it is a signaling effect rather than just a binding effect. First, the Kd for both

the AT1R and AT2R is less than 1 nM. In these studies we are using 100 nM of AngII to

stimulate the cells. Therefore, there is an abundance of AngII available to bind to the

AT1R. Secondly, the affects we are seeing cannot be reversed by an AT2R antagonist.

These results indicate that AT2R overexpression decreases the ability of AngII to

stimulate MAPK activity presumably through the AT1R. These results also indicate

that the AT2R appears to be acting in a ligand-independent manner. This conclusion is

supported by several factors. (1) We were unable to reverse the actions of the AT2R on

MAPK activity by the addition of an AT2R specific antagonist. We believe that this is

due to an inability to reverse the actions and not a timing effect of the addition of the

antagonist because the blockade by PD was performed in two different ways (addition

with AngII or addition 1 hour before AngII) with similar results each time. (2) Previous

22,24
studies have also indicated ligand-independent roles of the AT2R. These studies

indicate that the level of AT2R expression determines whether or not the AT2R is acting

through a ligand-independent manner or not.

These studies are fascinating in that they provide us with a lentiviral vector which

can transduce a functional AT2R. However, they also raise some important questions

related to the mechanism by which a ligand-independent AT2R attenuates AT1R-









mediated actions. Does the AT2R directly interact with the AT1R as a heterodimer to

prevent the actions of AngII on this receptor? Or is the AT2R mediating its effects

downstream through the inhibition of signaling mechanisms? If so, what are those

signaling cascades? Finally, does the AT2R have a mechanism that increases the

internalization of the AT1R? In spite of these questions, our observation is significant in

that we were able to demonstrate that the Lenti-AT2R can transduce and overexpress a

functional receptor. Future studies examining the mechanisms by which the AT2R

mediates its ligand-independent activity and its effects on the AT1R will be fascinating.















CHAPTER 3
IDENTIFYING GENES INVOLVED IN ANGIOTENSIN II TYPE 2 RECEPTOR-
MEDIATED SIGNALING PATHWAYS BY EXPRESSION PROFILING

Introduction

The AT2R has been implicated to play a role in the development and prevention

of different pathophysiologies. In the cardiovascular system, however, the physiological

role of the AT2R remains elusive. A number of studies indicate that the AT2R plays a

cardioprotective role to induce vasodilation as well as prevent the development of cardiac

36 ,98
hypertrophy and heart failure. There are, however, just as many studies indicating

that the AT2R actually plays a role in the development of some of these CV

pathophysiologies. For example, the AT2R has been shown to be necessary for the

development of cardiac hypertrophy, heart failure, and even cardiomyopathies.41,42,45,99,100

In addition, separate studies have shown that (1) the AT2R prevents or reduces cell

56 101
migration and angiogenesis and (2) blockade of the AT2R reduces angiogenesis.

These studies illustrate the discrepancies associated with the physiological role of the

AT2R.

Because of these differing roles of the AT2R in the CV system, a better

understanding of the cellular processes and signal transduction cascades of the AT2R

may provide some insight as to how and when the AT2R acts in a protective versus a

detrimental manner. One way to globally assess the cellular response to the AT2R is

through the use of microarrays. Microarray analysis is a growing technology based on a

chip containing thousands of genes to simultaneously and quantitatively analyze genetic









profiles in one experiment. These methods have been applied for both clinical and basic

science research. Clinically, microarrays analysis is an emerging field to determine

disease mechanisms and to identify novel candidates for therapeutic interventions. Basic

scientists use these techniques to assess the transcriptional affects and transduction

cascades associated with specific genetic and pharmacological interventions.

Endothelial cells line blood vessels in circulation and thus play an important role in

CV regulation. Endothelial dysfunction can lead to atherosclerosis, ultimately leading to

stroke and myocardial ischemia and infarction. In addition, the endothelium has been

shown to play an important role in angiogenesis. In fact, endothelial cell activation is the

first process to take place in both physiological and pathological angiogenesis.

Through the use of microarray analysis and the vast knowledge of the role of the

endothelium in CV disease, the goal of this aim was two-fold. (1) Elucidate novel

signaling pathways of the AT2R using microarray technology. This high throughput

method, will allow us to identify multiple signaling pathways and genetic profiles

associated with the AT2R. (2) Determine if any of these genes segregate with a

functional aspect associated with endothelial cells that is modulated by the AT2R.

Materials and Methods

Cell Culture, AT2R Transduction, and Treatments

Frozen vials of human coronary artery endothelial cells (HCAEC) of passage 3

were obtained from Clonetics. Cells were grown in EGM2-MV growth medium

(Clonetics). The protocol is outline in Figure 1-1. Briefly, the cells were thawed, plated,

and allowed to grow, changing the medium every 2 days. Once they reached confluency

(approximately 7 days), the cells were passage at a concentration of 2500 cells/cm2 in

4-100mm dishes. The following day, the cells were transduced with either Lenti-GFP










(MOI 10), Lenti-I-Neo (MOI 1) or Lenti-AT2R-I-Neo (MOI 1) as described in Chapter 2.

The medium containing lentivirus was removed from the cells the next day and allowed

to grow for an additional 2 more days before selection began. At this time (day 11), the

growth medium on the cells was changed to EGM2-MV plus 800 tg/ul geneticin (G418).

The cells remained on G418 selection for 2 weeks, providing fresh medium and G418

every 2 days. After one week of selection, the cells were passage at a concentration of

2500 cells/cm2. At the end of the 2 weeks of selection, the cells were confluent. At this

time the cells were passage for the microarray experiment at 2500 cells/cm2 and grown

for an additional 5 days, changing the medium every 2 days.

Lentiviral
Transduction
MOl <1.0 Subculture
Transduced
Plate Cells Isolate RNA
HCAEC 1.4x105 Cells for Real-Time
Passage 3 Selection Passage 5
Isolate RNA
for
Time 1 7 8 11 18 23 28 33 34 Microarray
(Days)
I Cell Growth
Subculture and Treat Ligand
HCAEC Maintenance Cells Binding
1.4x105 Cells
Passage 4

Figure 3-1: Timeline for the HCAEC used in the microarray experiments.

At this time, the cells were treated for 24 hours with either the AT2R-specific

agonist, CGP42112A (Sigma), or viral resuspension buffer as a control (untreated).

Following this incubation, total RNA was isolated from the cells for microarray

experiments and additional plates were used for binding and expression analysis. There

were essentially 4 groups that were compared by microarray analysis: (1) Lenti-I-Neo

(Control) transduced cells that were left untreated, (2) Control transduced cells treated









with CGP42112A, (3) Lenti-AT2R-I-Neo transduced cells that were left untreated, and

(4) Lenti-AT2R-I-Neo transduced cells that were treated with CGP42112A.

Real-Time RT-PCR

Total RNA was isolated from cells using Ambion's RNaqueous-4-PCR kit using

the manufacturer's protocol, and real-time RT-PCR was performed for the AT2R as

described in Chapter 2. Real-time RT-PCR for gene validation was performed using

primers and probes from Applied Biosystems Assays-on-Demand (catalog numbers:

Ubiquitin Thiolesterase, Hs00188233 ml; RGS-7, Hs00175619_ml; IGFBP-3,

Hs00426287_ml). For all these experiments, two-step real-time RT-PCR protocols were

used. RNA (lug) was converted to cDNA using Applied Biosystems TaqMan reverse

transcription reagents in a total volume of 50 [tL. Following the conversion to cDNA, a

PCR reaction was set-up using TaqMan Universal PCR Master Mix. For the AT2R, 1 ng

of cDNA, 0.9 [tM of each primer and 0.5 [tM of probe was used. For the Assays-on-

Demand reactions, 20 ng of cDNA was used, and 12.5 pL of the primers and probe

mixture was added to each 25 [tL reaction. The PCR plate was set-up according to the

manufacturer's suggestions and was run at 500 C for 2 minutes, 950 C for 10 minutes,

and then 40 cycles of 950 C for 15 seconds and 600 C for 1 minute. In all of the

experiments, cDNA was diluted to 100 pg for the AT2R and 20 pg for the Assays-on-

Demand experiments to be used to quantitate ribosomal 18S as an endogenous control

(Applied Biosystems). Each sample had a reaction set up that did not contain any reverse

transcriptase as a control for genomic DNA contamination. In addition, another control

that did not contain any RNA was used to measure non-specific amplification. Relative

quantitation of gene expression was determined using the comparative CT method. The

average threshold cycle (CT) of each sample was related to the CT of its endogenous









control, 18S. The samples were then compared to the lowest expressing sample

calibratorr) as described in Applied Biosystems User Bulletin #2.

AT2R Binding Assay

Ligand-binding assays for the AT2R were performed to determine the Bmax and

Kd for the transduced AT2R as previously described in Chapter 2.

Microarray Analysis

Total RNA was isolated, DNase-inactivated and concentrated from HCAEC using

Ambion's RNAqueousTM-4PCR according the manufacturer's protocols. For these

protocols, 4-100 mm dishes were used for each group. The initial lysis was performed

with 500 [tL of lysis buffer added to each dish. The RNA was eluted from each filter

cartridge in 2 aliquots of 80 [tL and 40 [tL of elution buffer. The RNA was DNase-

inactivated for 30 minutes and concentrated to a volume of 35 [tL. The RNA was then

prepared for hybridization to the human U133A microarray chip using the protocols

outlined in GeneChip Expression Analysis Overview (Affymetrix) and highlighted in

Figure 3-2. Total RNA isolated from the treated HCAEC was assessed for quality and

concentration using both a spectrometer and a bioanalyzer. The RNA was used to

synthesize double-stranded cDNA using the SuperScript Choice System (Invitrogen).

First strand cDNA was synthesized using the HPLC purified T7-(dT)24 primer; 5' -

GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24 3'. The

primer is first hybridized to the RNA by mixing 8utg of RNA with 100 pmol T7-(dT)24

primer for minutes at 700 C. Following this incubation, 10 mM DTT and 500 [tM of

dNTP mix was added to the reaction and the temperature was adjusted to 420 C for 2

minutes. The Superscript reverse transcriptase is then added to the reaction and the first-

strand of cDNA is synthesized at 420 C for 1 hour. The second strand of cDNA is









synthesized by adding a mixture of second-strand reaction buffer, 200 [iM of each dNTP,

10 units of DNA ligase, 40 units of DNA polymerase I and 2 units of RNase H to the first

strand cDNA reaction and incubating at 160 C for 2 hours. Following this reaction, 10

units of T4 DNA polymerase is added and incubated for an additional 5 minutes. Finally,

10 [tL of 0.5 M EDTA is added to stop the reaction. The double-stranded cDNA was

then cleaned-up using Affymetrix Gene Chip Sample Cleanup Module according to the

manufacturer's protocol. Following this cleanup procedure, biotinylated cRNA was

created in an in vitro transcription reaction using the ENZO BioArrayTM High YieldTM

RNA Transcript Labeling Kit (Affymetrix) and cleaned up using the GeneChip Sample

Cleanup Kit (Affymetrix) according to the manufacturer's protocol. To produce the

biotinylated cRNA, 10 [tL of the cleaned-up cDNA was added to a reaction containing

IX each of the provided reaction buffers, biotin-labeled ribonucleotides, DTT, RNase

inhibitor mix and T7 RNA polymerase and incubated at 370 C for 4-5 hours. Once

cleaned-up, the cRNA was quantified spectrophotometrically and fragmented in a

fragmentation buffer (200 mM Tris-acetate, pH 8.1, 500 mM KOAc, 150 mM MgOAc)

and incubated at 940 C for 35 minutes. The fragmented cRNA (20ug) was then

hybridized to the microarray chip for 16 hours at 450 C. This is followed by a series of

washing and staining with streptavidin phyoerythrin conjugate performed by an

Affymetrix fluidics station. Once the chips are stained, they are scanned by a GeneArray

Scanner (Affymetrix) at an excitation wavelength of 488 nm.

Microarray Analysis Controls and Data Analysis

There are a number of controls that are used to assess quality of each chip and

enhance the ability to compare between multiple microarray chips. Comparisons of









probe sets which hybridized to the 5' and 3' ends of housekeeping genes such as GAPDH

and actin are used to assess the quality of cRNA hybridized to each microarray chip.

Transduced HCAEC
Untreated or Treat
CGP42112A for 24hrs. Gene Expression Profiling

I ^ \J'WJ X MI cDNA
Total RNA RT
IVT &
'I 4 fragmentation

Validation of Biotinylated cRNA
select genes using
Real-time RT-PCR fragments
I Hybridization






I

Human Chip U133A
Multiple Probe Sets Scanning &
Represent -33,000 Well Visualization
Substantiated Human Genes

Figure 3-2: Outline of the microarray protocol as described in detail in the Materials and
Methods section.

Hybridization efficiency is monitored by control samples that are spiked into the

hybridization cocktail. Probe Profiler (Corimbia, Inc) is used to standardize each array.

First, automatic artifact detection, saturation correction, and outlier detection and removal

are performed. Probe Profiler also scales the mean array intensity of each chip to a target

intensity, called global normalization, before comparisons between chips are made.

Following these procedures, an expression score (e-score) is calculated which reflects the

expression level of each gene. Any gene with an e-score less than 25 in all four treatment

groups is considered absent in the experiment and is removed from any further analysis.









The gene profiles were analyzed for statistical significance of the e-scores by 2-way

ANOVA using custom written software from the UF ICBR Microarray Core Facility.

Only those significantly changing genes (p<0.05) were investigated further. Gene Spring

(Silicon Genetics) gene expression analysis software was used to graphically represent

and clarify the significance of the differentially expressed genes. In addition, gene

ontology was determined through the use of custom written software from the UF ICBR

Microarray Core Facility.

Migration Assay

Transwell cell migration assays were performed using a 48-well Boyden

56
chemotaxis apparatus (Neuroprobe) as previously described. Briefly, 8[tm membranes

were coated in 100 [tg/mL typel collagen (Sigma) diluted in 20mM acetic acid overnight.

The next day the membranes were rinsed in PBS and placed over the lower chambers of

the apparatus which contained EBM-2 medium supplemented with 0.1% BSA

(experimental medium) and either 10 ng/mL vascular endothelial growth factor (VEGF)

or suspension buffer as a control (no VEGF). HCAEC transduced with either Lenti-I-

Neo (Cntrl) or Lenti-AT2R-I-Neo (AT2R) was trypsinized, counted, and 5x103 cells were

added to each well of the upper chamber. In addition, the cells in the upper chamber

were either not treated or treated with 1 pM PD or 10 nM CGP42112A. The filled

apparatus was incubated at 370 C at 5% CO2 for 3 hours. Following this incubation, the

non-migrated cells were removed, and the membrane was fixed in methanol and stained

with Diff-Quick (Fischer Scientific) according to the manufacturer's protocols. The

number of migrated cells was counted in 5 randomly-chosen fields under 20x









magnification. Experiments were performed a total of 3 times, all with similar results.

Figure 3-6 is a representative of one experiment.

Angiogenesis Protein Array

A TranSignalTM Angiogenesis Antibody Array (Panomic) was used according to

manufacturer's protocol. Using ImL of RIPA buffer (1% NP40, 0.5% sodium

deoxycholate, 0.1% SDS, 10 mg/mL PMSF, 20 [tL/mL sprotinin, 100 mM sodium

orthovanadate made in PBS), proteins were isolated from Cntrl and AT2R-transduced

HCAEC either not treated or treated with 10nM of the AT2R specific agonist,

CGP42112A. Proteins were then quantitated using the Bradford method described in

Chapter 2. The angiogenesis protein array was incubated in Blocking Buffer for 2 hours

at room temperature. Following this, the membranes were rinsed 2x in Wash Buffer II

and 1 mg of protein was added to each membrane. The membranes were incubated with

the proteins for 2 hours at room temperature, washed 3 times for 5 minutes each in Wash

Buffer I and washed once for 5 minutes in Wash Buffer II. Following this, the Biotin-

conjugated Angiogenesis Antibody Mix was added to the membranes and incubated for 3

hours at room temperature. The membranes were washed again with Wash Buffer I and

II as described above. Then the Strepavidin-HRP Conjugate was added to each

membrane and incubated for 60 minutes at room temperature. The membranes were

washed a final time in Wash Buffer I and II as described above and incubated in the

Detection Buffer for 5 minutes. The proteins were visualized using x-ray film.

Statistics

Microarray data was analyzed by 2-way ANOVA as described above. All other

experiments were analyzed by 1-way ANOVA. Results are indicated as mean +/-

standard error with statistical significance being set at the 95% confidence level.









Results

Characterization of AT2R Transduction of HCAEC

The ability of the lentiviral vector to efficiently transduce HCAEC was

investigated. Gene transfer into endothelial cells has historically been difficult to

accomplish, and the Lenti-GFP expression plasmid was first used to determine if the

lentivirus could transduce this cell type. Cells transduced at an MOI of 10 efficiently

infected the majority of the HCAEC at 48 hours post-transduction, as evidenced by the

fluorescent green cells (Figure 3-3A).

HCAEC were transduced with either Lenti-I-Neo (Cntrl) or Lenti-AT2R-I-Neo

(AT2R) at an MOI of 1. The AT2R-transduced cells showed a 12-fold increase in

mRNA of the AT2R over the Cntrl-transduced cells (Figure 3-3B). This increase in

AT2R mRNA was also associated with an increase in AT2R-specific binding. Saturation

and scatchard binding analysis revealed total AT2R binding of 11.5 pmol/mg of protein

with a Kd of 1.25 nM (Figure 3-3C). These results indicate that the lentivirus can

efficiently transduce HCAEC to overexpress the AT2R.

Expression Profiling of AT2R-Transduced HCAEC

Microarray analysis was used to determine the novel signaling effects of AT2R

overexpression and AT2R activation in HCAEC. Four experimental groups (n=2

microarray chips per group) were used in the following experiments: (1) Lenti-I-Neo

transduced cells with no treatment (Cntrl-Untrt); (2) Lenti-I-Neo transduced cells treated

with 10 nM CGP42112A (Cntrl-CGP); (3) Lenti-I-AT2R-transduced cells left untreated

(AT2R-Untrt); (4) Lenti-AT2R-transduced cells treated with 10 nM CGP42112A

(AT2R-CGP).











DTYF-GFP


16-
S14-
12


6-
47
2
Z
0-


Scatchard Plot


AT2R mRNA

T


Cntrl AT2R

Saturation Curve


5
Bound (pmol/mg)


2 4 6 8
1251-SI-All (cpm x 105)


Kd = 1.25nM
Bmax = 11.5 pmol/mg


Figure 3-3: Lentiviral transduction in HCAEC. Lenti-GFP was used to determine the
transduction efficiency of the lentivirus. A) HCAEC were transduced at a
MOI of 10 and analyzed for green fluorescence 2 days post transduction.
HCAEC were transduced with Lenti-I-NeoR (Cntrl) or Lenti-AT2R-I-NeoR
(AT2R). B) Following selection with G418, RNA was isolated from the cells
and examined for AT2R mRNA by real-time RT-PCR (n = 3; = p<0.05). C)
HCAEC transduced with the AT2R was also examined for AT2R ligand
binding. Scatchard and Saturation binding curves performed, as described in
the Materials and Methods, revealed a Bmax of 11.5 pmol/mg of protein and a
Kd of 1.25 nM.

Of the 33,000 genes represented on the microarray chip, 5,224 of them were

differentially expressed following AT2R overexpression. In addition, 1,235 genes were

differentially expressed with treatment of 10 nM CGP42112A for 24 hours. Scatter

plotswere used to graphically represent those genes which are differentially expressed

following overexpression of the AT2R (Figure 3-4A). Treatment of the AT2R-


A
Uninfected










transduced cells with CGP42112A produced fewer changes in gene expression compared

to only overexpressing the AT2R (Figure 3-4B).

A HMG-CoA B
Reductase
10007 PAKR4 1000-
Ubiquitin-Specific
Protease 4 0
1 Ubiquitin 100
Thiolesterase
Ci 10- Fibrillin 2 3 10-
-- IGFBP3
< ILK 16 Of Q\
1 CN 1 Decorin


0.11 0.1-

0.01 --- -I -I --- 0.01 ---- *-*- I
0.1 1 10 100 1000 0.1 1 10 100 1000
Cntrl Cntrl
AT2R-Untrt

Figure 3-4: Scatter plots of the microarray data. HCAEC were transduced with either
Lenti-I-NeoR (Cntrl) or Lenti-AT2R-I-NeoR (AT2R) and either left untreated
(Untrt) or treated for 24 hours with the AT2R specific agonist, CGP42112A
(CGP) for microarray analysis as outlined in Figure 3-1. A) Microarray
analysis revealed 5,224 genes that were significantly differentially expressed
between the Cntrl and AT2R groups and are represented as a scatter plot.
Each dot represents a gene that had a significantly-altered profile between
these two groups (p<0.05). B) Comparison of the number and expression of
genes significantly differentially expressed with just AT2R expression (green)
and those that are stimulated by CGP42112A (red). Again, each dot
represents a significantly-changing gene (B; p<0.05).

To clarify our gene list into a manageable number of genes to analyze, genes that

changed more than 50% and those which are of particular interest are listed in Tables 4-1

through 4-3. Genes of particular interest are genes which were defined by gene ontology

analysis as having a role in cell adhesion, mobility, and/or migration. This selection

criteria was based on our results presented in the following sections. Table 3-1 lists those

genes which are down-regulated by the expression of the AT2R. Many of these genes

played a role in the regulation of cell adhesion, mobility and/or migration. Table 3-2









indicates those genes which are upregulated by AT2R transduction. Finally, Table 3-3

lists the genes which are differentially expressed in the AT2R-transduced cells following

treatment with CGP42112A. The results of AT2R transduction and treatment in Table 3-

3 are compared to the combined control cells left untreated and the control cells treated

with CGP42112A. Each table list the gene's accession number, common name, average

e-score for each group, and the percentage change. Genes highlighted in yellow are of

particular interest and are described more in the discussion.

Table3-l1: AT2R decreases gene expression without CGP42112A stimulation.
Avg Avg %
Access # Gene Title AT2 Neo Change
NM 004181 Ubiquitin
thiolesterase 96.08 531.18 -81.91
NM 001999 Fibrillin 2 50.15 206.83 -75.75
NM 000598 Insulin-like
growth factor
binding protein
3 38.73 141.28 -72.59
U71300 Small nuclear
RNA activating
complex 30.28 64.68 -53.19
NM 005132 Rec8p 36.13 74.35 -51.41
M90391 Interleukin 16 40.10 50.78 -21.02
4867555 RC PAK-4 165.55 204.68 -19.12
HMG-CoA
NM 000859 reductase 105.38 120.43 -12.50
Microarray data was analyzed for significantly differentially expressed genes by 2-way
ANOVA. This table represents those genes that were either decreased at least 50%, or
identified by gene ontology studies as having a role in cell adhesion, mobility or cell
migration. The accession number (Access #), common gene name/title, e-scores for both
AT2R (Avg AT2) and Cntrl (Avg Neo), and percent change (% Change) are given.
Genes highlighted in yellow are described further in the text.

Gene Validation

Real-time RT-PCR was used to validate those genes, which were significantly

differentially expressed from the microarray analysis. Selection of the genes to be

quantitated was based upon their extent of regulation and its proposed role in the CV









system. The genes that were validated were greatly regulated by AT2R transduction and

literature searches revealed that they also played an important regulatory role in the CV

system.

Table 3-2: List of genes whose expression was significantly increased with AT2R
expression independent of ligand.
Avg Avg %
Access # Gene Title AT2 Neo Change
BC000977 Aminolevulinate 127.78 85.03 50.28
AF015524 Chemokine (C-C
motif) receptor-like 2 26.63 17.65 50.85
NM 004125 Guanine nucleotide
binding protein 10 424.75 280.58 51.39
NM 005028 Phosphatidylinositol-
4-phosphate 5-kinase 22.95 15.15 51.49
AL042733 BRCA1 associated
protein 52.43 34.35 52.62
J03620 Dihydrolipoamide
dehydrogenase 367.15 239.93 53.03
Ribosomal protein
NM 000995 L34 2017.63 1315.50 53.37
M96651 Interleukin 5 receptor 34.30 22.25 54.16
NM 014059 RGC32 protein 347.48 224.53 54.76
NM 014135 PR00641 protein 26.83 17.30 55.06
NM 016216 Debranching enzyme
homolog 1 29.63 19.10 55.10
BC002666 Guanylate binding
protein 1 159.30 102.63 55.23
BF000239 Chromatin assembly
factor 1 25.15 16.18 55.49
NM 001655 Archain 1 528.75 336.78 57.00
D43968 Runt-related
transcription factor 1 24.23 15.43 57.05
BG532690 Integrin, alpha 4 25.53 16.05 59.03
BF593908 TATA element
modulatory factor 1 45.23 28.33 59.66









Table 3-2: Continued.
Avg Avg %
Access # Gene Title AT2 Neo Change
AI761771 Chromodomain
helicase DNA
binding protein 4 26.48 16.58 59.73
NM 004202 Thymosin, beta 4 139.63 86.90 60.67
NM 000557 Growth
differentiation factor
5 26.45 16.38 61.53
M12423 T cell receptor alpha
locus 28.05 17.33 61.90
NM 002886 RAP2B 29.70 18.30 62.30
NM_003668 MAPK-activated
protein kinase 5 33.55 20.63 62.67
NM 002028 Farnesyltransferase 25.63 15.63 64.00
AI989512 HIV-1 Rev binding
protein 104.80 63.73 64.46
NM 002601 Phosphodiesterase 6D 51.03 30.95 64.86
AL574096 Tissue factor pathway
inhibitor 2 25.00 15.15 65.02
AF055994 PPAR binding protein 27.45 16.58 65.61
U20760 Calcium-sensing
receptor 25.38 15.08 68.33
D42045 DNA cross-link
repair lA 28.18 16.73 68.46
BC000103 NCK adaptor protein
2 129.63 76.43 69.61
A1005066 Arginine vasopressin
receptor 1A 41.75 24.48 70.58
AF074717 RAD1 homolog 25.80 15.08 71.14
M12959 T cell receptor alpha
locus 43.48 25.20 72.52
BG260658 CS box-containing
WD protein 94.65 54.50 73.67
AF249671 NK3 transcription
factor homolog A 52.43 30.05 74.46
NM 024430 Pro-ser-thr
phosphatase
interacting protein 2 76.93 43.68 76.13









Table 3-2: Continued.
Avg Avg %
Access # Gene Title AT2 Neo Change
NM 004500 Heterogeneous
nuclear
ribonucleoprotein C 712.83 402.63 77.04
NM 015930 Transient receptor
potential cation
channel 129.48 70.65 83.26
NM 016277 RAB23 76.00 40.83 86.16
X75940
SMA3 31.43 16.73 87.89
NM 004381 ccAMP responsive
element binding
protein-like 1 33.05 17.53 88.59
NM 025019 Tubulin, alpha 4 56.75 29.68 91.24
NM 002924 Regulator of G-
protein signalling 7 40.53 19.78 104.93
NM 003430 Zinc finger protein 91 35.93 16.83 113.52
AF047190 Sarcosine
dehydrogenase 41.25 18.73 120.29
NM_030786 Intermediate filament
protein syncoilin 35.35 15.45 128.80
NM_000439 Proprotein convertase
subtilisin/kexin type 1 182.78 65.63 178.51
BE875592 Vesicle docking
protein p115 49.90 17.45 185.96
NM 005526 Heat shock
__transcription factor 1 48.68 15.63 211.52
This table represents those genes that were increased significantly by 50% or more as
described in Table 3-1.

Validation of both ubiquitin thiolesterase and regulator of G-protein signaling 7

(RGS-7) showed a significant difference in the same direction as the microarray had

predicted (Figure 3-5; 92% decrease for ubiquitin thiolesterase; 94% increase in RGS-7).

In addition, insulin growth factor binding protein 3 showed the same trend as the

microarray data of a 60% decrease with AT2R expression although it only reached the

94% confidence level (Figure 3-5). These results indicate that the microarray data is









valid and some of the observed changes seen by microarray analysis can also be observed

through real-time RT-PCR.


Table 3-3:


List of genes whose expression was significantly altered in the AT2R-
transd uced cells stimulated with CGP42 1A.


% %
Change Change
AT2 AT2 Avg AT2 AT2
Accession # Gene Title Untrt CGP Neo Untrt CGP
NM 003128 Spectrin, beta 105.60 58.20 66.15 59.64 -12.02
BC005354 Ribosomal
protein 61.00 25.40 27.08 125.30 -6.19
BC000603 Ribosomal
protein L38 361.25 160.05 236.33 52.86 -32.28
NM 014292 Chromobox
homolog 6 84.30 83.65 55.38 52.23 51.06
NM 003363 Ubiquitin specific
protease 4 20.75 35.95 22.25 -6.74 61.57
NM 003893 LIM domain
binding 1 18.55 22.80 15.08 23.05 51.24
NM 003430 Zinc finger
protein 91 49.05 22.80 16.83 191.53 35.51
NM 014212 Homeobox C11 38.40 27.85 65.85 -41.69 -57.71
NM 005732 RAD50 homolog 38.55 48.90 30.10 28.07 62.46
AW025108 Topoisomerase I 28.40 22.85 18.00 57.78 26.94
AI281593 Decorin 29.00 24.55 50.98 -43.11 -51.84
NM0 16073 Transmembrane 6
superfamily
member 1 73.45 51.80 48.00 53.02 7.92
U35139 Necdin homolog 48.70 59.20 105.48 -53.83 -43.87
D42045 DNA cross-link
repair lA 34.85 21.50 16.73 108.37 28.55
AF130102 Retinoic acid
repressible
protein 53.60 28.45 34.28 56.38 -16.99
U71300 Small nuclear
RNA activating
complex 27.05 33.50 64.68 -58.18 -48.20
BC001259 Adaptor-related
protein complex
4 18.35 21.10 37.88 -51.55 -44.29









Table 3-3: Continued.
% %
Change Change
AT2 AT2 Avg AT2 AT2
Accession # Gene Title Untrt CGP Neo Untrt CGP
AF061193 Ectodermal
dysplasia 1 26.15 20.60 15.65 67.09 31.63
M95489 Follicle
stimulating
hormone receptor 17.85 23.60 15.08 18.41 56.55
AF258449 Estrogen receptor
1 19.10 27.50 45.03 -57.58 -38.92
AF120491 Potassium
voltage-gated
channel 17.85 23.15 15.08 18.41 53.57
BE737027 Ribosomal
protein L27a 119.95 62.70 62.63 91.54 0.12
NM 015322 Fem-1 homolog 59.10 49.30 37.63 57.08 31.03
NM 003668 MAPK-activated
protein kinase 5 29.25 37.85 20.63 41.82 83.52
BF680255 Ribosomal
protein S11 48.00 20.80 30.58 56.99 -31.97
BF593727 Ras homolog 42.75 22.65 24.30 75.93 -6.79
AA748649 YY1 transcription
factor 48.20 27.50 27.53 75.11 -0.09
BE312027 Ribosomal
protein L27 151.40 85.45 89.58 69.02 -4.61
L07335 SRY-box 2 34.25 20.75 47.73 -28.23 -56.52
BE857772 Ribosomal
protein L37a 170.70 96.10 83.90 103.46 14.54
H71805 Myeloid cell
leukemia
sequence 1 48.30 24.45 31.05 55.56 -21.26
H71805 Myeloid cell
leukemia
sequence 1 30.65 20.60 17.38 76.40 18.56
BE877796 Collagen, type
VIII, alpha 1 218.55 172.80 131.13 66.67 31.78
W87901- Small nuclear
ribonucleoprotein
polypeptide E 77.85 47.55 47.33 64.50 0.48
AA215854 Integrin, beta 1 187.50 88.65 100.50 86.57 -11.79





Microarray data was analyzed for significantly differentially-expressed genes by 2-way
ANOVA. This table represents those genes that were increased significantly by 50% or
more. The accession number (Access #), common gene name/title, e-scores for both
AT2R transduced cells left untreated (AT2 Untrt), AT2R transduced cells treated with
CGP42112A (AT2 CGP), and Cntrl (Avg Neo), and percent change of AT2R Untrt
versus Cntrl (% Change AT2 Untrt) and AT2R CGP versus Cntrl (% Change AT2 CGP)
is given. Genes highlighted in yellow indicate gene which are described further in the
text.

The Role of the AT2R in Migration and Angiogenesis

The second goal of this aim was to determine if AT2R overexpression in the

HCAEC leads to a functional or physiological effects that can then be related to the

changes observed in the microarray analysis. We chose to look at cell migration, since

endothelial cells play a key role in the initiation of migration and thus angiogenesis. Both

Cntrl and AT2R-transduced cells were analyzed for its ability to migrate in the absence

and presence of AT2R stimulation (10 nM CGP42112A) with angiogenesis activation

(VEGF) or in the presence and absence of VEGF (Figure 3-6). AT2R-overexpressing

cells that were not stimulated with CGP42112A showed a significant decrease in

migration in both the presence and absence of VEGF (Figure 3-6). This effect was not

altered by activation of the AT2R with CGP42112A (Figure 3-6). In addition, the AT2R-

specific antagonist PD123,319 did not reverse these effects (Figure 3-6). These results


Table 3-3: Continued


% %
Change Change
AT2 AT2 Avg AT2 AT2
Accession # Gene Title Untrt CGP Neo Untrt CGP
NM 019034 Ras homolog gene
family 17.85 22.80 15.08 18.41 51.24
NM_014168 HSPC133 protein 36.05 20.60 21.50 67.67 -4.19
BF970427 UDP-glucose
ceramide
glucosyltransferase 170.15 129.05 107.50 58.28 20.05
A1189609 RAB2 81.90 41.95 39.93 105.13 5.07
J02761 Surfactant 55.20 44.45 96.70 -42.92 -54.03










indicate that AT2R transduction inhibits endothelial cell migration independent of both

VEGF and AT2R activation, again indicating that the AT2R elicits its actions

independent of ligand.

A Ubiquitin B RGS-7 C IGFBP3
Thiolesterase 3.0 p=0.06
4-
1625



02 0 0 3
-o 2


Cntrl AT2R Cntrl AT2R Cntrl AT2R

Figure 3-5: Gene validation of the microarray analysis. Gene validation by real-time
RT-PCR was performed on RNA isolated from HCAEC either transduced
with Lenti-I-NeoR (Cntrl) or Lenti-AT2R-I-NeoR (AT2R). Reaction set-ups
are as described in the Materials and Methods using pre-designed Assays-on-
Demand primers and probe for A) ubiquitin thiolesterase, B) regulator of G-
protein signaling-7 (RGS-7), and C) insulin growth factor binding protein 3
(IGFBP3). (n = 6, = p<0.05 vs. Cntrl).

In addition to the actions of the AT2R on cell migration, we also wanted to

determine if the AT2R had any effects on the cytokines whose dysregulation is typically

associated with the activation of angiogenesis. A protein array specifically designed to

detect 19 different angiogenesis-specific cytokines was used. AT2R overexpression with

and without activation with CGP42112A in HCAEC did not appear to have any effect on

these specific cytokines (Figure 3-7). Therefore, the AT2R effects on migration are

independent of these specific cytokines. Instead these actions of the AT2R may involve a

novel mechanism.

Discussion

These results indicate that the AT2R regulates genes that may play a role in

inflammation, protein regulation, cell migration, and extracellular matrix interactions.









Many of these effects can be seen without stimulation of the AT2R, once again indicating

that the AT2R has some constitutive activity. In addition, we were able to show that in

these cells, the AT2R decreases cell migration, with several of the genetic profiles

correlating to this functional aspect. Finally, we were able to show that the effects of the

AT2R on migration and angiogenesis may be independent of the typically-regulated

cytokines, and may be eliciting actions through an unknown pathway.

6- *Cntrl
x- 5- *AT2R P=0.07
4-







Untrt VEGF VEGF + VEGF + PD +
CGP CGP

Figure 3-6: AT2R prevents HCAEC migration. HCAEC were transduced with either
Lenti-I-NeoR (Cntrl) or Lenti-AT2R-I-NeoR (AT2R). Cell migration was
determined using a 48-well Boyden chamber. Cells were left uninduced
(Untrt) or induced with 10 ng/mL VEGF in the lower chamber. In addition,
the HCAEC were left unstimulated or stimulated with 10 nM CGP42112A
(CGP) or 1 pM PD123,319 (PD) in the upper chamber. (n = 4-6/group,
= p<0.05 vs. Cntrl).

A number of genes were found to be differentially regulated by the AT2R in the

HCAEC. In the following sections, I will highlight a few of the more relevant and

interesting genes found in this study.

Two genes that were found to be differentially regulated by the AT2R (ubiquitin

thiolesterase and ubiquitin specific protease 4) are involved in the ubiquitination

pathway. Ubiquitination is a process by which proteins are modified post-translationally









to mark the protein for degradation. Three steps are involved in this process. First, the

protein is identified for degradation. Signals for this include genetic programming,


VEGF
Y W


PIGF


Neg


Cntrl-Untrt


TIMP-2
a + ama


Cntrl-CGP


AT2R-Untrt


AT2R-CGP


Figure 3-7: AT2R effects are independent of the typical regulators of angiogenesis
represented on Panomic's Angiogenesis Array. A) Proteins represented are as
indicated in the table (Ang = Angiostatin, G-CSF = Granulocyte Colony
Stimulating Factor, HGF = Hepatocyte Growth Factor, VEGF = Vascular
Endothelial Growth Factor, IL = Interleukin, PIGF = Placental Growth Factor,
FGF = Fibroblast Growth Factor, TNF = Tumor Necrosis Factor, TGF =
Transforming Growth Factor, Neg = Negative Control, Pos = Positive
Control, IFN = Interferon, IP = Interferon Inducible Protein, TIMP = Tissue
Inhibitors of Metalloproteinases). Each sample is represented in duplicate on
the membrane. B) HCAEC were transduced with Lenti-I-NeoR (Cntrl) or
Lenti-AT2R-I-NeoR (AT2R) and either left untreated or treated for 24 hours
with CGP42112A (CGP). Following these incubations, protein was isolated
and bound to the membrane as described in the Materials and Methods.


Pos Pos Pos Pos
Ang IL-la FGFa IFNg
G-CSF IL-1b FGFb IL-12
HGF IL-6 TNFa IP-10
Leptin IL-8 TGFa TIMP-1









phosphorylation or protein damage. Next, the protein is marked for degradation by using

a series of enzymes to attach ubiquitin. Finally, the marked protein is delivered to the

proteasome where it is degraded and the ubiquitin recycled. Ubiquitination is, however, a

reversible process by means of the deubiquitinating enzymes (DUB) that are either

ubiquitin C-terminal hydrolases or ubiquitin-specific proteases. In this study, we found

that the AT2R decreases an enzyme responsible for marking proteins for degradation

(ubiquitin thiolesterase), while increasing the DUB, ubiquitin-specific protease 4.

Together, these results indicate that the AT2R decreases ubiquitination. This is

especially interesting because a previous study has shown that there is an increase in

102
ubiquitination in both dilated cardiomyopathy and ischemia of the heart. These results

raise an interesting question; can AT2R overexpression in the coronary artery endothelial

cells prevent ubiquitination to such an extent as to prevent these pathophysiologies?

Another interesting gene shown to be differentially-regulated by AT2R

overexpression was the regulator of G-protein signaling-7 (RGS-7). The regulators of G-

protein signaling are a class of proteins that accelerate intrinsic GTP hydrolysis of

activated G-proteins, Gai and Gaq, to inactivate these signals. In addition, RGS-7 has

103
specifically been shown to reduce Ca2+ mobilization in CHO cells. We observed an

increase in RGS-7 protein expression with the overexpression of the AT2R. Because the

AT2R has been shown to directly effect the signaling of the AT1R and the AT1R has

signal transduction cascades to regulate Ca2+ mobilization through both Gai and Gaq, we

speculate that the AT2R can reduce AT1R-mediated increases in Ca2+ mobilization

through this RGS-7 protein.









In addition to these genes, a functional aspect of the AT2R in the HCAEC was

identified that can be related to a physiological/pathological state. We observed that

AT2R overexpression inhibits endothelial cell migration. Because endothelial cell

migration is one of the first steps of angiogenesis, this leads us to speculate that AT2R

overexpression would also inhibit angiogenesis. Angiogenesis is the formation of new

blood vessels from pre-existing capillaries. Endothelial cells (EC) have been shown to

play an important role in angiogenesis. Upon activation by factors such as an increase in

immune response or ischemia, EC penetrate new areas of the body by degrading the

extracellular matrix (ECM). The EC then proliferate and migrate by forming new

attachments with the ECM at the leading edge and detach at the trailing edge. Finally,

these sprouting EC roll up to form a new blood vessel. The regulation of angiogenesis is

associated with many different and non-related diseases. In diseases such as ischemia,

new blood vessel formation provides essential nutrients to improve the pathological state.

This indicates that an increase in angiogenesis would be beneficial to patients with

myocardial ischemia and infarction as well as stroke. In contrast, in other diseases such

as cancer, atherosclerosis, rheumatoid arthritis, and retinopathy, excessive angiogenesis

may be contributing to the pathology. In these diseases, the new blood vessels either

provide nutrients to promote additional cell growth, or they promote an increase in

inflammation to cause additional damage. Because of this dual role for angiogenesis in

CVD, it would be interesting to determine the overall effect of AT2R overexpression on

these pathophysiologies. Is the AT2R cardioprotective in the sense that it prevents

further development of atherosclerosis through the inhibition of angiogenesis? Or is it

detrimental to the heart because it prevents the formation of new blood vessels to provide









nutrients to ischemic tissue? Finally, since angiogenesis prevents the development of

atherosclerosis which ultimately leads to ischemia of the heart, will new blood vessels

ever need to be formed to feed the ischemic tissue?

In addition, we were able to associate AT2R's prevention of cell migration with the

observed changes in gene expression seen in our microarray analysis. A number of

differentially-expressed genes with AT2R expression can be associated with the

regulation of cell migration and angiogenesis. The majority of these genes exert their

effects by either (1) inhibiting the initiation of angiogenesis or migration by decreasing

the immune response, (2) inhibiting the proteases from degrading the ECM needed to

initiate migration, or (3) decreasing the ability of the EC to migrate by reducing their

adhesion to the ECM (Figure 3-8).

Microarray analysis indicates that the AT2R causes a decrease in 3-hydroxy-3-

methylglutaryl CoEnzyme A Reductase (HMG-CoA). HMG-CoA is one of the key

enzymes in the production of cholesterol. Numerous studies have shown that inhibitors

of HMG-CoA, called stations, effectively decrease migration, prevent the development of

104,105
atherosclerosis and improve overall endothelial function. These inhibitors are

thought to accomplish this by reducing the amount of circulating LDL and/or reducing

106
the inflammatory response elicited by LDL production. The AT2R in the HCAEC

could be mimicking these stations and eliciting its affects through the inhibition of HMG-

CoA. Since these stations have been shown to play a role in inhibiting inflammatory

responses, it is feasible that the AT2R inhibits migration and angiogenesis, which inhibits

inflammatory responses to prevent the development of atherosclerosis and thus stroke

and myocardial ischemia. This line of thought would indicate that the AT2R-mediated









decrease in cell migration and thus angiogenesis is playing a cardioprotective role to

prevent atherosclerosis and myocardial ischemia.

In addition, the AT2R was shown to decrease interleukin 16. Interleukin 16 is a T-

cell specific chemoattractant factor, which plays a major role in trafficking immune cells.

In addition, it has been shown to initiate migration in dendritic cells. 107,10 Thus, the

AT2R may be inhibiting cell migration by decreasing its initiation induced by the

immune system, specifically through the inhibition of interleukin 16 and HMG-CoA.


EC Migration & Angiogenesis AT2R Response

Signal for Angiogenesis
_____ Interleukin 16
i HMG-CoA Reductase
EC Breaks Down ECM
I --IGFBP3
EC Migration and Proliferation
through ECM contacts t Fibrillin 2
I i 4 Decorin
EC Proliferation and Tube Formation PAK-4


New Vessel Formation


Figure 3-8: Graphical representation of the pathway where the AT2R-regulated genes
could exert its actions to inhibit endothelial cell migration and angiogenesis.

Another way the AT2R may inhibit cell migration and angiogenesis is through the

prevention of protease-mediated breakdown of the ECM. One of the first steps in cell

migration and angiogenesis is matrix metalloproteinases (MMP) breakdown the ECM,

allowing room for the endothelial cells to grow and migrate to form new vessels. Insulin

growth-factor binding protein 3 (IGFBP-3) has been shown to be a substrate for some of
109
these MMPs. Our microarray and real-time RT-PCR studies indicate that the AT2R










reduces the expression of IGFBP-3. This would theoretically decrease the amount of

substrate for the MMP to elicit its actions, thus preventing cell migration and

angiogenesis.

In addition to these affect on migration initiation, a number of genes indicate that

the AT2R may be preventing migration through its effects on cell adhesion to the ECM.

Fibrillin-2 is an extracellular matrix glycoprotein that provides an adhesive substrate for

110
migrating cells. In our studies, we see that AT2R expression decreases the expression

of fibrillin 2. Theoretically, this reduction would then reduce cell adhesion and thus

prevent cell migration.

Decorin, which is a proteoglycan associated with tissue development and assembly,

has previously been shown to play a role in angiogenesis. It has been shown that

endothelial cells have increased expression of decorin during angiogenesis.11l2 In

addition, in an in vitro model of angiogenesis, Schonherr et. al. was able to show that

113
adenoviral-mediated overexpression of decorin is sufficient to induce angiogenesis. In

our studies, we see a decrease in expression of decorin with AT2R overexpression,

indicating that the AT2R may be inhibiting cell migration and thus angiogenesis through

the regulation of decorin.

Finally, p21-activated kinase 4 (PAK-4) was shown to decrease with AT2R

expression. PAK-4 is a serine/threonine kinase which plays a role in regulating

cytoskeletal organization through Rac and Cdc-42. This organization promotes the

114
formation of focal complexes needed for migration. In addition, it has been shown that

114
a dominant negative PAK inhibits cell migration. Our results are consistent with this

finding and indicate that AT2R-mediated decreases in PAK-4 may prevent the regulation









of the cytoskeleton needed for the formation of focal adhesions through a Rac and Cdc-

42 pathway.

All of these genes indicate that the AT2R regulates pathways that would be

beneficial to normal CV function, whether it be through the prevention of

cardiomyopathies, or through decreasing AT1R-mediated increases in calcium

mobilization, or through the inhibition of atherosclerosis and myocardial ischemia though

the regulation of migration and angiogenesis. It will be interesting to see how all of these

factors may influence each other to prevent the development of these and other

pathophysiologies.

These studies indicate once again that the AT2R may be acting in a ligand

independent manner. There were a greater number of genes differentially expressed by

overexpressing the AT2R and the AT2R-mediated inhibition of endothelial cell migration

both indicates a constitutive role of this receptor. Future studies will need to address

whether this constitutive activity is due to the overexpression of the receptor or if

endogenous receptors also elicits its effects independent of ligand.

Our migration assay indicated that the AT2R inhibited cell migration independent

of VEGF activation. Previous studies have shown that the AT2R inhibits VEGF-induced

56
cell migration in a different human coronary artery endothelial cell line. In fact, many

cells do not migrate without VEGF induction. We were shown able to show, however,

with our protein array analysis that our HCAEC express endogenous VEGF. This

indicates that the cells did not require additional VEGF to induce migration because of

basal VEGF expression.









Our angiogenesis protein array revealed that the AT2R did not regulate the

activators and inhibitors typically associated with the initiation of angiogenesis. This

indicates that the AT2R is inhibiting this function independent of these cytokines. The

AT2R may have novel mechanism by which it signals to inhibit angiogenesis. Our

microarray data confirms this idea. We described several differentially-expressed genes

which may inhibit migration and angiogenesis by factors not represented on the protein

array. In addition, we performed these experiments on crude protein isolated from the

cells. Since many of the factors represented on the protein array are secreted, it would be

interesting to investigate changes in these factors in the media from these cell.















CHAPTER 4
PREVENTING CARDIAC PATHOPHYSIOLOGIES BY ANGIOTENSIN II TYPE 2
RECEPTOR GENE TRANSFER

Introduction

The conventional concept of cardiac hypertrophy (CH) is that it is an adaptive

response of the heart to sustained increases in BP to preserve cardiac function. In

addition, recent evidence indicates that other non-hemodynamic factors such as the tissue

88,115,116
RAS may also play a role in its development. This sustained and uncontrolled

growth ultimately leads to diminished cardiac performance and cardiac pathophysiologies

such as heart failure.

Numerous studies have shown that the AT1R plays a role in the development of

cardiac hypertrophy. Both ATIR-antisense gene therapy and AT1R antagonists have

resulted in prevention in cardiac hypertrophy and its associated

pathophysiologies.88,115,119 Although the mechanism by which AT1R antagonists

prevent cardiac pathophysiologies is still speculative, it has been suggested that

unopposed stimulation of the AT2R may contribute to their effectiveness. In fact, a study

by Mukawa et al showed that simultaneous administration of an AT2R antagonist with an

47
AT1R antagonist negated the antihypertrophic effects of the AT1R blocker alone. In

addition, studies performed in cultured cardiomyocytes and hypertrophied hearts indicate

a role for the AT2R in the prevention of cardiac pathophysiologies.49,120,121

Despite all of the support for the role of the AT2R in the prevention or regression

of CH, the role of the AT2R in cardiac pathophysiologies remains controversial. Studies









performed in transgenic and knockout animals indicate a different role for the AT2R.

Inagami's group has shown that the absence of the AT2R in knockout mice prevents the

41,42
induction of CH by both AngII-infusion and pressure overload. In contrast to these

studies, other transgenic studies showed no effects of the AT2R on cardiac

45,99,100
hypertrophy.45

These conflicting observations for the role of the AT2R in cardiac

pathophysiologies may be due to inherent problems associated with the experimental

design. Since the AT2R has been found to play a role in embryonic development,

altering the expression levels of the AT2R, as in the development of transgenic and

knockout animals, may result in improper CV development. In addition, AT2R

antagonists are expensive and only short-lived. Therefore, long-term extensive studies

are hard to accomplish. To alleviate from these problems, our laboratory has established

an efficient method of lentiviral vector-mediated gene delivery following the natural

development of the CV system. In the present study, we use this system to determine the

effects of AT2R gene transfer on cardiac pathophysiologies such as CH, HF, and high

BP.

Materials and Methods

Animals and Lentiviral Delivery

All animals used in these studies were purchased from Charles River Laboratories.

Initial studies used to determine the transduction efficiency of 1.5x108 TU and the AngII-

infusion studies used Sprague Dawley (SD) rats, while spontaneously hypertensive rats

(SHR) were used for the other experiments. Offspring from timed-pregnant mothers

were removed at 5-days of age, lightly anesthetized with methoxyflurane (Schering-









Plough), and a bolus of 30-60 [tL of either viral resuspension buffer, 5x109 TU/mL of

Lenti-AT2R-I-Neo or Lenti-PLAP was injected directly into the left ventricular chamber

86 88 122
as previously described.86,88,122 Following this intracardiac delivery, the pups were

lightly coated in peanut oil and returned to their respective mothers until weaning.

All animal procedures were conducted with our Institutional Animal Care and Use

Committee (IACUC) approval and adhered to the guidelines for the care and use of

laboratory animals.

Viral Production and Transgene and AT1R Expression Measurements

Lenti-PLAP or Lenti-AT2R-I-Neo production and titration was performed as

described in Chapter 2.

Histochemical staining was performed on the animals transduced with Lenti-PLAP

86
as previously described. The animals were perfused with cold PBS followed by

perfusion with 4% paraformaldehyde (PFA; Sigma). The tissues were collected and

allowed to post-fix in 4% PFA for an additional 2 hours at 40 C. Following this

incubation, the organs were rinsed in PBS 4 times and heated to 720 C for 3 hours to

inactivate any endogenous alkaline phosphatase activity. Next, the tissues were allowed

to cool to room temperature. Then they were incubated in a pre-incubation BCIP

solution (100 mM Tris, 100 mM sodium chloride, 50 mM magnesium chloride, and

0.5 mM levamisole, pH 9.5) for 1 hour at room temperature. Finally, the tissues were

incubated in BCIP solution containing 100 mM Tris, 100 mM sodium chloride, 50 mM

magnesium chloride, 0.5 mM levamisole, 1 mg/mL nitro blue tetrazolium, and

0.1 mg/mL 5-bromo-4-chloro-3-iodolyl-phosphate, pH 9.5 for 2 hours at room

temperature, followed by an overnight incubation at 40 C. The following day, the tissues









were rinsed in PBS + 50 mM EDTA and whole mount pictures were taken through a

dissecting microscope. Following the pictures, the tissues were cryoprotected in 20%

sucrose overnight at 40 C and frozen in Tissue-Tek freezing medium. Sections at 30

microns were taken and stained for PLAP and/or DAPI.

Total RNA was isolated from the cardiomyocytes of the transduced animals using

Qiagen's RNeasy Fibrous Tissue Mini Kit. Real-time RT-PCR for both the AT1R and

the AT2R was performed as described in Chapter 2. Primers and probe used for the

AT1R were as follows: (forward): 5'-CCATCGTCCACCCAATGAAG-3'; (reverse):

5'-GTGACTTTGGCCACCAGCAT-3'; (probe): 5'-

FAMCTCGCCTTCGCCGCAMGBNFQ-3'.

Physiological Measurements

Indirect blood pressure (BP) was monitored by the tail-cuff method as previously

88
described. Direct BP measurements were monitored using radiotelemetry devices (Data

Sciences, Inc.) according to the manufacturer's protocols. Briefly, the animals were

anesthetized with isoflurane. The abdominal cavity was exposed and the cannula of the

radiotelemetry device was inserted into the abdominal aorta. Once the cannula was

secured into the aorta, the radiotelemetry device was sutured into the abdominal wall and

the wound was closed.

88
Heart weight to body weight ratios were taken as previously described. The

animals were weighed prior to euthanasia; the chest cavity was then opened and the heart

carefully extracted. The whole heart was rinsed in PBS or Kreb's solution, blotted dry

and weighed. Measurements are presented as the heart weight (mg) divided by the body

weight (g).









Echocardiographies were used to monitor wall thickness and ejection fraction of

the hearts. The rats were lightly anesthetized with isoflurane and echocardiographies

were performed using a Hewlett Packard Sonos Model 5500 with a 12-MHz transducer.

Parastemal long and short-axis images and end-diastolic diameter, end-systolic diameter,

ejection fraction and wall thickness were obtained by standard echocardiographic

measurements.

The magnetic resonance imaging (MRI) of the in vivo rat cardiac cycle was

performed at the University of Florida, McKnight Brain Institute's Advanced Magnetic

Resonance Imaging and Spectroscopy Facility. All animals were imaged on a 4.7T

Oxford Magnet using a Bruker Avance console and Para vision software. The rats were

anesthetized by reflexive inhalation of 1.5-2% isoflorane and IL/min oxygen and

monitored using the Small Animal Instrument (SAI) monitoring and gating system for

respiration rate and cardiac triggering. The heart was centered in a custom-built 3.5-5cm

receive-only quadrature saddle surface coil (each element of the coil is a 3.5 x 3.5 cm

rectangle) on a plexiglass cradle and tuned loaded to resonant frequency. The rat and

receive coil were inserted into an 8.8 cm in diameter transmit-only quadrature volume

coil. Following pilot images, dorsal and sagittal images were acquired using a cardiac

gated gradient echo (GEFI_TOMO) sequence with the following parameters: FOV 7.0 x

3.0cm, matrix 256 x 128, TR=12 msec, TE=2.2 msec, 4 AVG, slice thickness 1.5 mm,

and a total of 14 frames. These parameters resulted in a resolution of 273 x 234 microns

in-plane. Based on the sagittal and dorsal views, transverse images were prescribed and

were collected with the GEFI_TOMO sequence with the following parameters: FOV

4.0 x 3.0cm, matrix 256 x 128, TR=12 msec, TE=2.3 msec, 4 AVG, slice thickness









2.0 mm, and a total of 14 frames to capture the entire cardiac cycle. These parameters

resulted in a resolution of 156 x 234 microns.

Pathology of the heart was analyzed by Masson's Trichrome staining through the

UF Molecular Pathology Core Facilities. Portions of the heart were fixed in 4%

paraformaldehyde or PLP solution (2% paraformaldehyde, 75 mM lysine, 37 mM sodium

phosphate, and 10 mM sodium periodate), sectioned and stained.

Statistics

Results are presented as mean +/- SE. All data was analyzed by ANOVA using the

student-Newman-Keuls method for all pairwise multiple comparisons. Values of p<0.05

were considered statistically significant. The number of animals per group is indicated in

the figure legends.

Results

In vivo Gene Transfer

Delivery of 1.5x108 TU of the expression vector, Lenti-PLAP, into the heart of 5-

day old SD animals resulted in efficient transduction of CV-relevant tissues measured at

21 days of age. The lentiviral vector highly transduced the liver, adrenal and heart

(Figure 4-1A, E, I and Figure 4-2B-D) compared to its controls (Figure 4-1B, F, J and

Figure 4-2A), while it only moderately transduced the lungs and the kidneys (Figure 4-

1C,G,K) compared to its controls (Figure 4-1D,H,L). Thin sections of the adrenal shows

that the lentiviral vector can transduce both the medulla and the cortex (Figure 4-1I,J).

Thin sections of the kidney did not reveal any preference for viral transduction (Figure 4-

1K,L), and thin sections of the heart indicated that the lentivirus can efficiently transduce

cardiomyocytes (Figure 4-2D).









The effect of Lenti-AT2R-I-Neo on the expression of both the AT1R and AT2R

was also examined. Real-time RT-PCR was performed on isolated cardiomyocytes from

the hearts of 31 -week-old SHR inj ected into the heart at 5 days of age with either Lenti-


PLAP
~\^^


Cntrl
B ^H


PLAP

I C


Liver


Cntrl


Lung


Adrenal


Kidney


Figure 4-1: In vivo lentiviral transduction efficiency. 1.5x108 lentiviral particles of
Lenti-PLAP (PLAP) or viral resuspension buffer (Cntrl) were injected into the
cavity of the left ventricle at 5-days of age and viral transduction efficiency
was examined by PLAP immunochemistry as described in the materials and
methods at 21 days of age. Examination of the whole tissue revealed
transduction of the A) liver, C) lung, E) adrenal, and G) kidney. Cntrl animals
revealed little to no PLAP staining (B,D,F,H) in the whole tissues. I) Thin
sections of the adrenal showed efficient transduction in the cortex (c) and the
medulla (m) with J) no staining present in either area in the Cntrl animals.
Thin sections of the K) PLAP kidney and the L) Cntrl kidney reveal diverse
staining within the kidney.

AT2R-I-Neo (AT2R) or viral resuspension buffer (Cntrl). Using AT2R-specific

primers and probe, there was negligible expression of the AT2R in the Cntrl animals,

while the AT2R animals had significantly higher AT2R mRNA levels (Figure 4-3A). In







77


contrast to this, the AT1R was abundantly expressed in all samples and its expression

was not altered by AT2R transduction (Figure 4-3B).


Figure 4-2: Lentiviral transduction efficiency in the heart. B-D) Lenti-PLAP (PLAP) or
A) viral resuspension buffer (Cntrl) was delivered and examined as described
in Figure 4-1. Whole hearts stained for PLAP in A) Cntrl and B) Lenti-PLAP.
C) A section of the Lenti-PLAP transduced heart, looking into the ventricles.
D) A thin section of the Lenti-PLAP transduced heart reveals cardiomyocyte
morphology.


AT2R


15

r 10

5


AT2R NRT


AT1R


Cntrl AT2R NRT


Figure 4-3: AT2R and AT1R expression in isolated cardiomyocytes. Viral resuspension
buffer (Cntrl) or Lenti-AT2R-I-Neo (AT2R) was delivered into the heart of
5-day old SHR. At 31 weeks of age, the SHR were sacrificed and
cardiomyocytes were isolated. Total RNA was collected and examined for A)
AT2R and B) AT1R mRNA levels by real-time RT-PCR. No reverse
transcriptase (NRT) was used as a negative control. (n = 4/group; = p<0.05
vs. the other two groups).


A
6

5

4-

3

2

1


Cntrl









Pathophysiology in the SHR

SHR animals were administered with either Lenti-AT2R-I-Neo (AT2R) or viral

resuspension buffer (Cntrl) at 5 days of age. Previous studies in our laboratory indicated

no differences between the HW/BW of the animals injected with a control lentiviral

vector versus viral resuspension buffer (data not shown). At 12, 18, and 22 weeks of age,

echocardiographies (ECHO) were performed to characterize the effects of AT2R

transduction on the heart. At 12 weeks of age, the control ECHO revealed that none of

the groups had begun to develop cardiac hypertrophy as the wall thickness (WT) of both

the Cntrl and AT2R SHR (Cntrl 1.41+/-0.03 mm; AT2R 1.36+/-0.03 mm) animals were

comparable to age-matched normotensive WKY animals (Figure 4-4A; WKY 1.42+/-

0.03 mm). At 18 and 22 weeks, however, we saw a significant increase in WT of the

Cntrl SHR (18 wks 1.78+/-0.03 mm; 22 wks 2.0+/-0.11 mm) animals but not of the

AT2R-treated SHR (18 wks 1.53+/-0.03 mm; 22 wks 1.54+/-0.09 mm) or the

age-matched WKY (Figure 4-4A; 18 wks 1.52+/-0.09 mm; 22 wks 1.52+/-0.09 mm). In

fact the Cntrl-treated SHR had a significantly larger WT compared to both the WKY and

the AT2R-treated SHR (Figure 4-4A). These results were supported by HW/BW which

revealed that the AT2R-treated SHR (3.7+/-0.02 mg/g) had a significantly lower ratio

than the Cntrl-treated SHR animals (Figure 4-4B; 4.0+/-0.1 mg/g). In contrast to these

results, there were no differences observed in the ejection fraction or the BP of the Cntrl

and AT2R-treated SHR (Figure 4-4C,D). Comparison of the BP with age-matched WKY

animals, however, revealed that both the Cntrl and AT2R-treated SHR animals had

elevated BP (Figure 4-4D) at both 18 (Cntrl 184 +/-5 mmHg; AT2R 184+/-6 mmHg) and

22 weeks (Cntrl 187+/-13 mmHg; AT2R 193+/-14 mmHg). These data indicate that







79


AT2R transduction of cardiomyocytes alters cardiac hypertrophy without influencing

high BP.


A B
2.5 4.5

C 280-


S35
7 1



0 2.5
~ cCtrl AT2R

12 weeks 18 weeks 22 weeks

80 D
250

7 -2 0 0
766
150*
S74-
.2 100

70-

68 0


12 weeks 18 weeks 22 weeks 18 weeks 22 weeks


Figure 4-4: Effect of AT2R transduction on CV pathologies in the STR. Five-day-old
SHR animals were injected into the heart with either viral resuspension buffer
(Cntrl) or Lenti-AT2R-J-Neo (AT2R). Age matched WKY animals were used
as a control where possible. At 12, 18, and 22 weeks of age, the animals were
subjected to ECHOs (n = 4 Cntrl and 4 AT2R) to measure both A) wall
thickness and C) ejection fraction (EF). D) At 18 and 22 weeks indirect blood
pressures were measure by the tail cuff method (n = 5 Cntrl and 3 AT2R). B)
Finally, at the end of the study (31 weeks) the animals were sacrificed and
heart weight to body weight ratios were determined (n = 4/group). ( =
p<0.05 compared to 12 weeks. p<0.05 compared to the other groups in
that timepoint).










Pathophysiology in the Angiotensin II-Infusion Model

SD animals were injected at 5 days of age with either viral resuspension buffer

(Cntrl) or Lenti-AT2R-I-Neo (AT2R). BP was monitored both before AngII-infusion

(Pre) or following AngII-infusion either indirectly by the tail-cuff method or directly

through the use of radiotelemetry devices. Following the implantation of osmotic mini-

pumps to continuously deliver either saline or 200 ng/kg/min AngII, the BP was

monitored at 1 and 2 weeks. By 2 weeks, the BP was significantly increased by

-70 mmHg compared to the Pre values in both the Cntrl and the AT2R-treated animals

infused with AngII. In addition, both groups treated with AngII had a significantly

higher BP then those treated with saline (Cntrl Saline 94+/-3 mmHg; Cntrl AngII 172+/-

5 mmHg; AT2R AngII 160+/-8 mmHg; Figure 4-5).

200
0 Cntrl-Saline
E Cntrl-Angll
180 *AT2R-Angll

16G

S 140

a. 120

10a

80

60
Pre 1 week 2 weeks

Figure 4-5: Blood pressure response to AngII infusion. SD animals injected into the
heart at 5-days of age with either viral resuspension buffer (Cntrl) or Lenti-
AT2R-I-Neo (AT2R). BP was taken at 8 weeks of age (Pre) and 1 week and 2
weeks following the start of AngII infusion. (n = 3-4/group; = p<0.05 vs.
Pre; t = p<0.05 vs. Cntrl-Saline).









ECHOs were taken both before (Pre) and 2 weeks after minipump implantation.

After 2 weeks of AngII infusion, it appears that the AT2R-treated animals infused with

AngII have a reduced wall thickness compared to the Cntrl-treated animals infused with

AngII (Cntrl AngII 2.2+/-0.3 mm; AT2R AngII 1.9+/-0.2 mm; Figure 4-6A). This is

better represented when the change in wall thickness from before infusion and 2 weeks

post infusion is presented (Figure 4-6B). This was confirmed by studies using MRI.

These studies showed that AT2R-transduced animals had a reduced wall thickness

following 2 weeks of AngII-infusion (Cntrl AngII 2.1+/-0.06 mm; AT2R AngII 1.8+/-

0.05 mm; Figure 4-7A-B,D). In fact, the WT of the AT2R transduced animals was

similar to that of saline-infused controls (Cntrl Saline 1.7+/-0.04 mm; Figure 4-7B-D).

In addition to these changes in WT, the change in ejection fraction was

significantly improved with the AT2R-treated animals infused with AngII (Cntrl AngII -

28+/-6%; AT2R AngII -3+/-6%; Figure 4-6C). Finally, at the end of the experiments,

HW/BW was determined. Again we see a similar trend with the SHR experiment in that

the AT2R-treated animals exhibited a decrease in HW/BW (Cntrl AngII 10.9+/-1.1 mg/g;

AT2R AngII 9.3+/-0.4 mg/g; Figure 4-6D).

Metabolic parameters, heart rate, and activity were also monitored in these animals.

Two weeks following the implantation of the osmotic mini-pumps infusing either saline

or 200 ng/kg/min AngII, the animals' metabolic parameters were observed. Following 2

weeks of AngII infusion, both the Cntrl and the AT2R-treated animals showed a

significant increase in water intake (Figure 4-8A) and urine output (Figure 4-8B) while

significantly decreasing the body weight (Figure 4-8C) compared to the saline-infused

Cntrl animals. There were, however, no differences in these parameters observed










between the Cntrl and the AT2R-treated animals infused with AngII. In contrast to these

effects, there were no effects of AngII infusion or AT2R-treatment on food intake (Figure

4-8D), fecal output (Figure 4-8E), activity (Figure 4-8F) or heart rate (Figure 4-8G).




A B
0.3 EO Cntrl-Saline

0.25 Cntrl-Angll
SAT2R-Angll 1.3
1.2
0.2

0.15.
0.9
0.1 -0.87
0.7
0.5 0.6
0.05
0.5I
0.4
0Pre 2 weeks Cntrl-Saline Cntrl-All AT2R-AII


Sntrl-Saline Cntrl-All AT2R-AIIl

-51


2 15
S-20 6
u -25 L I
-= 0 .5 5 4
-30 Cntrl-Saline Cntrl-All AT2R All

Figure 4-6: Role of the AT2R in the CV pathologies associated with AngII infusion.
The hearts of SD animals injected with either viral resuspension buffer (Cntrl)
or Lenti-AT2R-I-Neo (AT2R) were monitored by ECHOs both before and 2
weeks following the start of a constant infusion of either Saline or
200 ng/kg/min AngII. A) ECHOs revealed trends towards changes in wall
thickness. B) This is highlighted when the change in wall thickness from Pre
to 2 weeks post infusion is represented. C) In addition, ECHOs reveal that the
AT2R-treated AngII infused animals had a significant prevention of the
observed reduction in ejection fraction of the Cntrl animals infused with
AngII. D) The effects on CH is supported by the observed differences in
HW/BW taken at the end of the experiment after 4 weeks of AngII infusion.
(n = 3-4/group; = p<0.05 vs Cntrl-Angll).










A B











Cntrl-AngII AT2-AngII
C D
0.25

0.2 *

E 0.15
I-
0.1

0.05


Cntrl-Saline Cntrl-Saline Cntrl-All AT2R-AII
Figure 4-7: MRI analysis indicates AT2R transduction prevents cardiac hypertrophy.
SD animals injected with either Lenti-AT2R-I-Neo (AT2R) or viral
resuspension buffer (Cntrl) were subjected to a constant infusion of
200 ng/kg/min of AngII infusion. Following 2 weeks of infusion, MRI
images were taken. A) Cntrl animals infused with AngII elicited an increased
wall thickness compared to both the B) AT2R treated animals infused with
AngII and the C) Cntrl animals infused with saline. D) A graphical
representation for all the animals is also presented (n = 3/group; = p<0.05
vs. Cntrl-AngII).

Finally, at the end of the experiment, the pathology of the heart was examined.

After the animals were sacrificed after 4 weeks of infusion, the hearts were fixed and

stained with Masson's trichrome. Analysis of the sections indicate that the AT2R-treated

animals infused with AngII have decreased myocardial fibrosis (Figure 4-9A) compared

to the Cntrl-treated animals infused with AngII (Figure 4-9B). In contrast, the








84



AT2R-treated animals appeared to have increased perivascular fibrosis (Figure 4-9C)


compared to the Cntrl animals (Figure 4-9D).


B
250

200

150
a
100

*- 50
-


p=0.197










Cntrl-Saline Cntrl-Angll AT2R-Angll


A
350

.300
E250


150 -



0


C
0.6

0.5

0.4 -

0.3

0.2

0.1

0


Cntrl-Saline Cntrl-Angll AT2R-Angll


- 4.5
S4
h 3.5
j 3
2.5

1.5

0.5
0


p=0.637










Cntrl-Saline Cntrl-Angll AT2R-Angll


Cntrl-Saline Cntrl-Angll AT2R-Anglll


Cntrll Saline Cntrll-Anglll AT2R-AII


.400

S300

S200

t100


Pre 2 wks Pre 2 wks Pre 2 wks
Cntrl-Saline Cntrl-Angll AT2R-Angll


Figure 4-8: Effect of the AT2R and AngII on other CV physiologies. SD animals treated
as described in Figure 4-6 were used to monitor A) water intake; B) urine
output; C) body weight; D) food intake; E) fecal output through the use of
metabolic cages and F) activity and G) heart rate through radiotransmittors.


Cntrl-Saline Cntrl-Angll AT2R-Angll









Masson's Trichrome Staining

















AT2R Control

Figure 4-9: Effect of the AT2R on the pathology of the heart following AngII infusion
for 4 weeks. SD animals treated as described in Figure 4-6 were analyzed for
pathologies of the heart at the end of the experiment, following the full 4-
week infusion of AngII. Hearts were isolated from the animals, post-fixed,
and stained with Masson's Trichrome. A,B) Analysis of the tissue indicates
that the AT2R prevents the development of myocardial fibrosis within the
tissue of the heart while C,D) increasing perivascular fibrosis. (Pictures
represent 1 animal from each group. Similar results were seen in the other
animals; n = 3-4/group).

Discussion

These results show that we were able to overcome the inherent problems with

transgenic and knockout animals through the use of lentiviral vector-mediated gene

transfer injected into the ventricular space of 5-day-old animals. Using such techniques

we were able to show: (1) 1.5x108 TU of lentiviral vector was able to transduce CV

relevant tissues. (2) This transduction was able to overexpress the AT2R without

effecting the expression of the AT1R. (3) AT2R overexpression lead to beneficial effects

on cardiac hypertrophy (CH) and ejection fraction (EF). (4) These effects on CH and EF

appear to be regulated by the local/tissue RAS because the AT2R overexpression has no









effect on BP regulation. (5) It appears that only -40% transduction of the

cardiomyocytes is efficient to produce these significant effects.

These studies show a complete inhibition of cardiac hypertrophy despite only

-40% transduction of the heart. This result indicates that the AT2R may be acting in a

autocrine/paracrine manner to propagate its effects. In addition, recent evidence indicates

that new cardiomyocytes can form from stem-like cells, which could play a role in the

123,124
development of CH. Therefore, transduction of a few cells can propagate to elicit

larger effects.

125
Previous studies have shown an increase in AT2R expression in response to CH,

yet the present results do not show an increase in AT2R expression in the control SHR

despite the development of CH. There are several explanations for this observation. In

this study we only examined the AT2R expression levels in isolated cardiomyocytes,

while the previous studies looked at AT2R binding in the whole heart. Therefore, the

observed increases in AT2R expression could be occurring either at a post-transcriptional

level or in cell types other than cardiomyocytes, such as endothelial cells and/or

fibroblasts.

Evidence indicates that all of the components of the RAS exist in the heart. This

tissue RAS appears to regulate normal cardiac functions. ACE inhibitors, AT1R

antagonists and ATIR-antisense have shown a reduction in CV pathologies independent

of changes in arterial pressures.88,126,127 This study indicates that AT2R expression in the

heart is exerting its effects on CH and EF independent of BP. This can be explained

several ways. First, the lentiviral transduction of the vasculature is not high enough to









elicit any effects on BP. Secondly, the AT2R in the heart is exerting its effects through

the tissue RAS rather than the systemic RAS.

Many previous studies using AT2R antagonists and transgenic and knockout

animals showed conflicting data as to the role of the AT2R in cardiac function. We

believe that our gene transfer model has an advantage over these more traditional

methods since the genetic manipulations do not occur until after the cardiac development

has occurred. This raises some questions as to whether or not the AT2R overexpression

prevents the development of CH or simply delays it. In addition, it would be interesting

to determine if AT2R overexpression can reverse the development of CH.

Masson's trichrome staining of the AngII-infused hearts indicates that the AT2R

may be playing opposing roles to prevent myocardial fibrosis while increasing

perivascular fibrosis. The significance of this observation is yet to be determined. It is

possible that the AT2R is playing opposing roles in different cell types. It is also possible

that the prevention in myocardial fibrosis is an AT2R effect while the perivascular

fibrosis is an inflammatory response to lentiviral delivery. Lentiviral controls were not

used in the present studies because previous studies did not show any differences in

HW/BW and BP between lentiviral controls or viral resuspension buffer. These controls

may be needed, however, to address whether the observed effects on perivascular fibrosis

is an effect of the AT2R or lentiviral transduction.

We have shown that the AT2R prevents the development of cardiac pathologies.

We do not know, however, the mechanisms by which the AT2R elicits these effects. Is it

though the prevention of AngII binding to the AT1R? Are the effects we observed

through AT2R overexpression in the heart, or from AT2R overexpression in other tissues