MACROPHAGE-DERIVED SUPEROXIDE DISRUPTS PERVASCULAR MESENTERIC ARTERIAL SYMPATHETIC NERVES IN A RAT MODEL OF DOCA-SALT HYPERTENSION By Loc Vinh Thang A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Pharmacology and Toxicology – Doctor of Philosophy 2013 ! """! ABSTRACT MACROPHAGE-DERIVED SUPEROXIDE DISRUPTS PERVASCULAR MESENTERIC ARTERIAL SYMPATHETIC NERVES IN A RAT MODEL OF DOCA-SALT HYPERTENSION By Loc Vinh Thang Major risks for stroke, heart, and kidney diseases have been linked to hypertension in more than 65 million Americans. Hypertension is a multi-organ disease that involves changes in nervous and immune system function. Sympathetic nerve activity is elevated in some hypertensive humans and in some animal models of hypertension, including the DOCA-salt model. DOCA-salt hypertension in rats is - associated with the impairment of a !2R function, and increased level of O2 and M" number in the MA adventitia. However, the relationships between M" infiltration, O2 - production and !2R impairment are unknown. This dissertation tested the hypothesis that as blood pressure increases in DOCA-salt rats, M" infiltrate into the adventitia of - MA. M" then release O2 that disrupts !2R function, causing an increase in NE release which further increases blood pressure. A time-course study was used to determine the temporal relationship between impaired function of sympathetic nerve terminal !2R and adventitial infiltration of proinflammatory M" in MA from DOCA-salt hypertensive rats. LEC was used to deplete adventitial M". The results of these studies revealed that pro-inflammatory M" - infiltration and increased O2 level in MA of DOCA-salt rats occurred after 10 days of ! "#! initial blood pressure increase, but !2R impairment did not occur until a week after the infiltration of M". Furthermore, LEC prevented the development of the later phases of DOCA-salt hypertension by blocking the infiltration of M" into the MA adventitia, - reducing the O2 level in the MA, and preventing the !2R dysfunction. Focal nerve stimulation and amperometry with microelectrodes to measure NE oxidation currents at the adventitial surface of MA were used to elucidate the mechanism of !2R function impairment. The results suggested that there is no alteration in the amount of NE release from the RRP vesicles, and in the activity of free G#$ +2 function in tonic inhibition of voltage gated Ca channels in DOCA-salt hypertensive MA. The !2R function impairment occurs upstream from the G#$ protein. The balance between Gi/o and Gs is shifted to the Gs, hence the increase in sensitivity of Gs and PKA activity in DOCA-salt hypertensive MA. The novel aspect of this study is that it tested the hypothesis that M"-derived O2 disrupts !2R function, which further contributes to the increase in blood pressure in DOCA-salt rats. Hypertension is a major public health concern. Therefore, clarifying the mechanism that leads to enhanced neurogenic vasoconstriction is important for new discoveries relevant to anti-hypertensive drug development. ! #! - DEDICATIONS To my beloved wife KimHang whom I live, laugh and love with every moment of life together. To my amazing son Isaiah whose smile outshines the sun and whose cheerfulness warms my heart. To my respectful parent, Bay and Tuyet, who gave me the breath of life and taught me to be an indepdent thinker. (%&n b' m( kính m&n c)a tôi, B*y & Tuy&t, ng+,i -ã cho tôi h.i th/ c)a cu0c s'ng và d1y tôi tr/ thành m0t nhà t+ t+/ng -0c l2p.) To my remarkable sister and brother, Loan & Tho, who have challenged me to be a better role model everyday. American Poet Haniel Long once said, “So much of what is best in us is bound up in our love of family, that it remains the measure of our stability because it measures our sense of loyalty.” Love and Gratitude Loc Vinh Thang "#! ACKNOWLEDGEMENTS Whatever I have achieved would not have been possible without the help and support of the kind people around me, to only some of whom it is possible to give particular mention here. Above all, I would like to express my deepest gratitude to my principal supervisor, Dr. James Galligan, who is a thoughtful and caring mentor and scientist. Everything I have done would have not been possible without your help, support and patience. Your advice and unsurpassed knowledge of the sympathetic nervous system was invaluable. I was inspired by the ways you approach science and life in general. You are a great model for all young and aspiring scientists I sincerely appreciate my guidance committee members Drs. Anne Dorrance, Norbert Kaminski, and Donna Wang. Your excellent guidance has made this thesis work possible. It would have not been possible without Dr. Kaminski’s expertise in immunology and Mr. Bob Crawford’s support with the flow cytometry. Likewise, my work with the macrophage-depletion using clodronate would have not been possible without Dr. Anne Dorrance’s expert guidance. Equally important was Dr. Donna Wang who took me into her lab and introduced me to the field of hypertension research. I feel it’s only fitting to quote the great American Journalist, Dan Rather who once said, "the dream begins, most of the time, with a teacher who believes in you, who tugs and pushes, and leads you onto the next plateau, sometimes poking you with a sharp stick called truth.” #! I can’t say enough how lucky I was to be a part of the program project grant. I would like to thank Dr. Fink and his lab, especially Hannah Garver who have always been there to support the in vivo studies. I also would like to thank Dr. Stephanie Watts and her lab for providing space and aid in conducting Western Blot experiments. Last, but by no means least, I would like to thank Dr. Dave Kreulen and his lab especially Amit Shah who helped collect the celiac ganglion. My journey through the MD/PhD program would have not been possible without three important ladies: Dr. Elahe Crockett, Dr. Cindy Arvidson, and Mrs. Margo Smith. I would also like to acknowledge the financial support from MSU CHM and the Graduate School, Spectrum Health MD/PhD Fellowship, PhRMA Paul Calabresi Medical Student Fellowship, and The Gates Millennium Fellowship. One of the best parts of working and studying at MSU was my colleagues in the department and the lab. I would like to thank: Dr. Hui Xu, Dr. Xiaochun Bian, Emmy Sansigri, Matt Fhaner, Eileen Rodriguez, Yogesh Bhattarai, Jasmina Jakupovic, Marion France, Roxanne Fernandes and David Fried for your friendship and support. #"! TABLE OF CONTENTS LIST OF TABLES xi LIST OF FIGURES xii KEY TO ABBREVIATIONS xv CHAPTER 1: GENERAL INTRODUCTION 1 Hypertension: An Epidemiological Perspective Blood Pressure Regulation: The Mosaic Theory of Hypertension Genetics of Hypertension Environmental Factors in the Development of Hypertension Obesity and Hypertension Sodium and Hypertension Salt-Sensitivity Potassium and Hypertension Physical Inactivity and Hypertension Alcohol Consumption and Hypertension Psychosocial Stress and Hypertension Hemodynamics in the Development of Hypertension Anatomical Factors in the Development of Hypertension Coarctation of the Aorta Obstructive Sleep Apnea Syndrome and Hypertension Renal Stenosis and Hypertension Endocrine and Hypertension Mineralocorticoid Aldosterone Effects in Renal Epithelium Aldosterone Effects Outside Renal Epithelium Aldosterone – Heart Aldosterone – Vasculature Aldosterone – M" Glucocorticoid Thyroid Hormone Parathyoid Hormone Humoral Agents in Hypertension Renin Angiotensin Aldosterone System Endothelin Endogenous Ouabain The Nervous System and Hypertension The Properties of Adrenergic Receptors in Cardiovascular System NE Synthesis, Release, Reuptake and Metabolism Adrenergic Receptors !2-adrenergic Receptors #""! 2 4 6 8 8 9 10 12 12 13 13 14 18 18 19 19 20 20 21 24 24 24 26 27 27 28 28 29 33 35 35 40 40 48 48 !2A-adrenergic Receptors and Cardiovascular Functions Oxidative Stress, Inflammation and Hypertension Reactive Oxygen Species Inflammation and Hypertension Lymphocytes and Hypertension M" and Hypertension CD163 and Its Biological Function CD11b and Its Biological Functions Deoxycorticosterone acetate (DOCA)-salt Hypertension G-Protein-Coupled Receptors G Protein RESARCH GOALS & SPECIFIC AIMS Research Goals Overall Hypothesis Specific aim 1 Specific aim 2 Specific aim 3 Bibliography CHAPTER 2: EXPERIMENTAL DESIGN & METHODOLOGIES 48 49 49 52 55 55 56 57 59 60 60 63 65 65 66 66 67 68 86 Animals General Anesthesia Induction and Maintenance DOCA-salt Hypertension Induction Blood Pressure Measurement Immunohistochemistry Dihydroethidium (DHE) Staining Amperometric Measurement of Norepinephrine Liposomal-encapsulated Clodronate (LEC) Depletion of M" Flow-cytometry Drugs Statistics Bibliography 87 87 87 90 91 92 92 96 99 101 101 103 CHAPTER 3: ADVENTITIAL MACROPHAGE INFILTRATION INTO MESENTERIC ARTERIES AND !2 ADRENERGIC AUTORECEPTOR IMPAIRMENT DURING DOCA-SALT HYPERTENSION DEVELOPMENT 105 Abstract Introduction Results Time dependent M! infiltration and O2 production in MA of DOCA-salt rats "2R impaired function begin at day 18 in DOCA-salt hypertensive rats Time course of M! activation in DOCA-salt hypertensive rats #"""! 106 107 109 109 109 110 Phox Activated M! express high levels of TNF-" and p22 subunit of NADPH oxidase The impairment of "2R fuction, and the elevation of BP, M! 110 - number, and O2 level required the synergistic effect of both DOCA and high salt Discussion Bibliography CHAPTER 4: MACROPHAGE DEPLETION REDUCES VASCULAR OXIDATIVE STRESS, RESTORES !2 ADRENERGIC AUTORECEPTOR FUNCTION AND ATTENUATED HYPERTENSION DEVELOPMENT IN DOCA-SALT HYPERTENSION 111 127 130 134 Abstract Introduction Results LEC attenuated the development of later phases of DOCA-salt hypertension LEC depleted activated peritoneal M! LEC depleted M! in the MA adventitia of DOCA-salt rats LEC reduced the levels of O2- in the MA adventitia of DOCA-salt rats LEC prevented the dysfunction of "2R in DOCA-salt rats 135 136 138 Discussion Bibliography 149 152 CHAPTER 5: MECHANISMS OF PRESYNAPTIC NOREPINEPRHINE RELEASE AUTOREGULATION ALTERATION IN SYMPATHETIC PERIVASCULAR NERVES SUPPLYING MESENTERIC ARTERIES FROM DOCA-SALT HYPERTENSIVE RATS Abstract Introduction Results The amount of NE release from the RRP vesicles was the same in sympathetic perivascular nerves from DOCA-salt hypertensive and control MA Inhibition of G#$ equally increased NE release from sympathetic perivascular nerves from DOCA-salt hypertensive and control MA "2R function impairment occurred upstream from the G-protein Low concentration of AlF4- increased NE release from sympathetic perivascular nerves of DOCA-salt hypertensive but not control MA and this effect was not block by PTX "$! 138 138 138 139 139 155 156 158 160 160 160 160 161 High concentration of AlF4- decreased NE release from sympathetic perivascular nerves from DOCA-salt hypertensive much more when compared to control MA and this effect was block by PTX Forskolin increased the release of NE much more in sympathetic perivascular nerves from DOCA-salt hypertensive compared to control MA H-89 was less effective in inhibiting NE release from sympathetic perivascular nerves of DOCA-salt hypertensive compared to control MA Discussion Bibliography CHAPTER 6: GENERAL CONCLUSION AND PERSPECTIVES Summary Novelty and Significance Limitations of Experiments Perspective Future Work Bibliography 161 162 162 169 173 176 177 184 188 190 193 195 $! LIST OF TABLES Table 1 Blood Pressure Classification 3 Table 2 Monogenic Forms of Human Hypertension 7 Table 3 Tissue Distribution and Functions of Adrenergic Receptor Subtypes 47 Table 4 Source of Primary and Secondary Antibodies and the Working Dilutions 91 The Treatment Groups for the M"-depletion Experiment 99 Table 5 $"! LIST OF FIGURES Figure 1 The Mosaic theory proposed by Irvine Page M.D. in 1967 5 Figure 2 Control mechanisms responsible for autoregulation in non-renal tissues during the development of hypertension 17 Hormonal pathways of MR activation in renal cortical collecting duct cells. (For interpretation of the references to color in this and all other figures, the reader is referred to the electronic version of this dissertation) 23 Enzymatic pathways for Ang-II synthesis, metabolism, and functions 32 The circuitry of central nervous system modulation of sympathetic outflow 38 Enzymatic pathways for NE synthesis, release, reuptake, and metabolism 41 Figure 7 Vesicular trafficking and release in presynaptic nerve terminal 45 Figure 8 Enzymatic pathways for ROS synthesis and metabolism 50 Figure 9 Proposed hypotheses describing the role of adaptive immunity in hypertension 54 Figure 10 The Proposed Hypothesis 64 Figure 11 Deoxycorticosterone Acetate-Salt (DOCA)-Salt Model 89 Figure 12 Experimental set up for the focal stimulation and real-time measurement of NE 94 Liposomal-encapsulaed clodronate structure and cellular mechanism of apoptosis 97 Figure 14 Mechanisms and principles of flow-cytometry 100 Figure 15 Identification of M" and sympathetic perivascular nerve in rat MA but not skeletal muscle arteries 112 Figure 3 Figure 4 Figure 5 Figure 6 Figure 13 $""! Figure 16 Detection of superoxide anions in rat MA adventitia 114 Figure 17 !2R is impaired during DOCA-salt day 18-21 115 Figure 18 Time-course of peritoneal M" activation in DOCA-salt hypertensive rats 117 Figure 19 Co-localization of M" and TNF-! in rat MA adventitia at day 28 119 Figure 20 Co-localization of M" and p22Phox subunit of NADPH Oxidase in rat MA adventitia at day 28 121 Figure 21 - The elevation of blood pressure, M" number, and O2 level required the synergistic effect of both DOCA and high salt 123 The impairment of !2R required the synergistic effect of both DOCA and high salt 125 M" depletion lower blood pressure during the late phase of DOCA-salt hypertension 141 Figure 24 Flow-cytometry dot-plots of peritoneal M" 142 Figure 25 Identification of M" and apoptotic M" cell ghosts 144 Figure 26 Detection of O2 in MA adventitia of DOCA-salt rats 146 Figure 27 Analysis of !2R function in DOCA-salt rats 147 Figure 28 Frequency-response-curves for NE release from sympathetic perivascular nerves of DOCA-salt hypertensive and control MA in the presence of PTX, idazoxan, and cocaine 164 The effects of M119, a G#$ inhibitor, on NE release from sympathetic perivascular nerves of DOCA-salt hypertensive and control MA 165 Figure 22 Figure 23 Figure 29 Figure 30 Figure 31 - - The effects of AlF4 on NE release from sympathetic perivascular nerves of DOCA-salt hypertensive and control MA 166 The effects of forskolin and H-89 on NE release from sympathetic perivascular nerves of DOCA-salt hypertensive and control MA 168 $"""! Figure 32 Figure 33 Proposed mechanisms of how M"-derived superoxide anions disrupt !2R function in presynaptic sympathetic perivascular nerve termials from DOCA-salt hypertensive MA 182 The level of sCD163 in human subjects with hypertesion and normotension 191 $"#! KEY TO ABBREVIATIONS 11#-HSD: 11#-hydroxysteroid dehydrogenase 20-HETE: 20-hydroxyeicosatetraenoic acid 3’UTR: 3’ untranslated region 5-HT1A: 5-hydroxytryptamine receptor 1A ACTH: Adrenocorticotropic hormone AME: Syndrome of apparent mineralocorticoid excess Ang-II: Angiotensin II APs: Aminopeptidases AR: Adrenergic receptor ASIC: Acid sensing ion channel AT1: Ang-II receptor type 1 AT2: Ang-II receptor type 2 BMI: Body mass index BP: Blood Pressure CaMK II: Ca +2 calmodulin-dependent protein kinase II CAPP: Ceramide-activated protein phosphatase CHIF: Channel inducing factor COX-2: Cyclooxgenase-2 CSP: Cysteine string protein DAG: Diacylglycerol DBP: Diastolic blood presure DHE: Dihydroethidium DM: Diabetes mellitus DOPA: L-3,4-dihydroxyphenylalanine $#! D#H: Dopamine #-hydroxylase ECE: Endothelin converting enzyme EETs: Epoxyeicosatrienoic acids EGFR: epidermal growth factor receptor ELISA: Enzyme-linked immuno sorbent assay + ENaC: Amiloride-sensitive epithelial Na channels EO: Endogenous ouabain EO: Endogenous Ouabain EPHESUS: Epleronone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study EPs: Endopeptidases ERK: Extracellular signal-regulated kinase ET-1: Endothelin-1 ETA: Endothelin receptor type A ETB: Endothelin receptor type B FAK: Focal adhesion kinase G6PD: Glucose 6 phosphate dehydrogenase GILZ: Glucocorticoid induced leucine-zipper protein GIRK: G protein-coupled inwardly-rectifying potassium channels GPCR: G-protein coupled receptor GR: Glucocorticoid receptor GRA: Glucocorticoid-remediable aldosteronism GRK2: G-protein receptor kinase 2 INTERSALT: International Study of Salt and Blood Pressure IP3: Inositol triphosphate JG: Juxtaglomerular cells JNK: c-jun N terminal kinase $#"! KRH: Krebs-Ringers-HEPES LEC: Liposomal-encapsulated clodronate LHRH: Luteinizing hormone-releasing hormone Lipo-PBS: Liposomal-encapsulated PBS LPS: Lipoposaccharide LTB4: Leukotriene B4 MA: Mesenteric arteries MAP: Mean arterial pressure MAPK: Mitogen-activated protein kinase MnPO: Median preoptic nucleus MR: Mineralocorticoid receptor MR: Mineralocorticoid receptors M": Macrophages NADPH: Nicotinamide adenosine dinucleotide phosphate NE: Norepinephrine Nedd4-2: Neural precursor cell-expressed, developmentally downregulated gene 4 isoform 2 NEP: Neprilysin NET: NE transporter NF3B: Nuclear factor kappa B nNOS: Neuronal nitric oxide synthase NO: Nitric oxide NOS-1: Nitric oxide synthase 1 NR3C2: Nuclear receptor subfamily 3, group C, member 2 NSF: N-ethylmaleimide sensitive factor NTS: Nucleus tractus solitarius - O2 : radical superoxide anion $#""! OSAS: Obstructive sleep apnea syndrome OVLT: Organum vasculosum of the lamina terminalis PAI-1: Plasminogen activator inhibitor-1 PBS: Phosphate buffer saline PDGF: Platelet-derived growth factor PGE2: Prostaglandin E2 PKC: Protein kinase C PLC: Phospholipase C PTH: Parathyroid hormone PTX: Pertussis toxin RAAS: Renin Angiotensin Aldosterone System RALES: Randomized Aldactone Evaluation Study RAP: Right atrial pressure ROS: Reactive oxygen species RP: Reserved pool RRP: Readily releasable pool RVLM: Rostral ventrolateral medulla SBP: Systolic blood pressure SFO: Subfornical organ SGK1: Serum-and glucocorticoid-regulated kinase 1 SGT: Small glutamine-rich protein SHR: Spontaneously hypertensive rats SNA: Sympathetic nervous system activity SNAP-25: Synaptosomal associated protein of 25 kDa SNAPs: Soluble NSF adaptor proteins SNARE: SNAP receptor SR: Salt resistant $#"""! SS: Salt sensitive TGF-#: Tissue growth factor # TNF-!: Tumor necrosis factor alpha TPR: Total peripheral resistance TRP: Transient receptor potential channel VEGF: Vascular endothelial growth factor VMAT: Vesicular monoamine transporter WNK: With-no-lysine kinase !2AR: !2A-adrenergic receptor !2R: !2-adrenergic receptor $"$! CHAPTER 1 GENERAL INTRODUCTION !" Hypertension: An Epidemiological Perspective Blood pressure is a measurement of the pressure exerted against the arterial walls as the heart pumps blood through the body. Hypertension is the term used to describe high blood pressure. There are 65 millions Americans with hypertension (1). This is a major health concern because hypertension is major risk factor for heart disease and stroke, which are the leading causes of death in the United States. In fact, for every 20 mmHg systolic or 10 mmHg diastolic increase in blood pressure there is a doubling of mortality from both ischemic heart disease and stroke (2). In the world, the prevalence of hypertension in adults is estimated at approximately 1 billion individuals, and approximately 7.1 million deaths per year may be attributable to hypertension (3). The prevalence of hypertension increases with advancing age; recent study showed that the lifetime risk of hypertension is about 90% for men and women who were nonhypertensive at age 65 years and survived to age 85 (4). DBP is a stronger cardiovascular risk factor than systolic blood pressure until age 50 (5); thereafter, SBP is more important. Hypertension is defined as sustained SBP ! 140 mmHg or DBP ! 90 mmHg. Table 1 provides a classification of BP for adults (6). Pre-hypertensive individuals are those who have high risk of developing hypertension and are advised to modify their lifestyle in order to reduce the risk of developing hypertension in the future. The overall goal of hypertension management is to lower BP. Pre-hypertensive individuals should achieve this via lifestyle changes, and for all people with hypertension should be treated with drug therapy in addition to lifestyle modifications. !" Table 1: Blood Pressure Classification Blood Pressure Classification Systole Blood Pressure Diastole Blood Pressure < 120 mmHg and < 80 mmHg Pre-hypertension 120-139 mmHg or 80-89 mmHg Stage 1 hypertension 140-159 mmHg or 90-99 mmHg Stage 2 hypertension ! 160 mmHg or ! 100 mmHg Normal There are two categories of hypertension. Essential hypertension is high BP that has no identifiable causes, with it’s the most common form, affecting about 95% of hypertensive individuals (7). High BP that is caused by another medical condition or medication is called secondary hypertension. Secondary hypertension may be due to chronic kidney disease, coarctation of the aorta, pheochromocytoma, Cushing’s syndrome, hyperparathyroidism or medication such as birth control, diet pills, and some cold and migraine medications (8). A new class of hypertension is resistant hypertension. It’s defined as the persistence of BP above 140/90 mmHg despite the treatment of three or more anti-hypertensive drugs from different classes at full concentration, one of which is a diuretic (9). Considerable success has been achieved in the last five decades. The median SBP for individuals ages 60-74 declined by approximately 16 mmHg between 1960-1991 (10). Better treatment of hypertension has been associated with a significant reduction in the hospital case-fatality rate for heart failure. However, 30% of adults are still unaware of their hypertension and greater 40% of individuals with hypertension are not treated (6). Furthermore, the prevalence of resistant hypertension is increasing, and no new anti!" hypertensive drugs have been introduced in the last two-decades. Although the research in the past decades has significant impact in the treatments of hypertension, more research is still needed. Blood Pressure Regulation: The Mosaic Theory of Hypertension Hypertension is a complex medical condition with multi-factorial contributors. Dr. Irvine Page was one of the first pioneers who recognized and proposed that multiple “forces” interdigitate to cause hypertension. He later called this The Mosaic Theory of hypertension (Fig. 1), which states that the etiology of most cases of human hypertension is multi-factorial including genetics, environment, anatomical, adaptive, neural, endocrine, humoral and hemodynamics (11). In this theory, the crisscross of lines indicates that when a factor changes, some other must change concurrently to maintain the equilibrium and consequently the level of BP. While, the Mosaic Theory of hypertension is basic, it serves as a good framework for future studies to further understand the complex nature of the pathophysiology of hypertension. !" Figure 1: The Mosaic theory proposed by Irvine Page M.D. in 1967 The etiology of most cases of human hypertension is multi-factorial, including genetics, environment, anatomical, adaptive, neural, endocrine, humoral, and hemodynamics. !" Genetics of Hypertension First-degree relatives of hypertensive indivuduals have twice the risk of developing hypertension compared to the general population, and the risk increases to four-fold when two or more family members have hypertension (12). Although BP has significant heritability (30-60%), the genes conferring susceptibility to hypertension remain largely unknown because hypertension is a heterogenous disease with multiple phenotypic and genotypic subtypes (13). The genetic determinants of hypertension are polygenic with each gene having only a small effect on the BP. However, the additive effect of these genes results in hypertension. Despite this, there are few cases of monogenic forms of human hypertension (Table 2). The most common form of monogenic human hypertension is GRA, an autosomal dominant disorder characterized by overproduction of aldosterone. People with GRA have two normal copies of aldosterone synthase gene and have moderate to severe hypertension. Cortisol, amiloride and spironolactone are effective treatments to lower BP in GRA (14). Various abnormalities of hydroxylase enzymes including 11!-hydroxylase and 17"-hydroxyase deficiencies cause hypertension in human. 11!-hydroxylase deficiency causes hypertension and hypokalemia because impaired conversion of 11-deoxycorticosterone to corticosterone results in the accumulation of 11-deoxycorticosterone, a potent mineralocorticoid. Mutation causing 11!-hydroxylase deficiency cluster in exons 6-8 of the CYP11B1 gene (15). Similar to 11!-hydroxylase deficiency, 17"-hydroxyase deficiency is characterized by hypokalemia and hypertension. This disorder causes decreased and increased cortisol and 11-deoxycorticosterone production respectively. A large number of random mutations can cause 17"-hydroxyase deficiency (16). !" Table 2: Monogenic Forms of Human Hypertension Disorder Genes Alteration Glucocorticoid-remediable aldosteronism Aldosterone synthase is under the control of CYP11B1 promoter, so aldosterone is also synthesized in zona fasiculata 11!-Hydroxylase, 17"-hydroxyase deficiencies CYP11B1; CYP17 Hypertension exacerbated in pregnancy Mineralocorticoid receptor Liddle’s syndrome ! or # subunit of ENaC genes Syndrome of apparent mineralocorticoid excess 11!-Hydroxysteroid dehydrogenase gene Pseudohypoaldosteronism type II With-no-lysine kinase (WNK) Pregnancy-related hypertension is a serious health concern to the mother and baby. A S810L mutation in the MR causes early-onset hypertension and markedly exacerbated during pregnancy (17). This mutation results in constitutive MR activity, with progesterone--normally a MR antagonists--becoming a potent agonists. Liddle’s syndrome, an autosomal dominant disorder, is characterized by excess sodium retention, low potassium and plasma-renin activity. The problem in Liddle’s syndrome results from constitutive active ENaC on distal renal tubules. The defects have been localized to gene mutations on chromsome 16 that encode for ! and # subunits of ENaC (18). Cortisol and aldosterone can equally activate MRs in vitro; however, aldosterone is the primary activator of renal MR in vivo. Normally in the kidney, cortisol is metabolized to cortisone by 11!-HSD2, which prevents cortisol from activating MR. In states of 11!HSD2 deficiency like that in AME, cortisol can reach and activate type I renal MR, !" causing sodium retention, suppression of the RAAS and hypertension (19). Lastly, in the familial hypertension of pseudohypoaldosteronism type II, there are mutations in the + genes encoding the WNK family causing overactivity of the thiazide-sensitive Na /Cl - co-transporter in the distal nephron (20). Environmental Factors in the Development of Hypertension Genetic susceptibility accounts for much of the variation in blood pressure within population studies. However, environmental factors determine the variation in mean blood pressure between populations. Some of the most important environmental factors in the development of hypertension at the population level are excess calories intake, high sodium intake, low potassium intake, physical inactivity, heavy alcohol consumption, and psychosocial stress (21). Obesity and Hypertension 2 Obesity is clinically defined as BMI ! 30 kg/m . In prospective studies, obesity has been shown to be an independent predictor of subsequent hypertension and weight reduction was an effective way to lower blood pressure (22). While obesity is strongly linked to hypertension, the causal relationship is not as clear. In fact, BMI alone is less tightly associated with cardiovascular disease risk than visceral fat mass. Using computer tomography, it is shown that visceral fat is much more metabolically active compared to subcutaneous fat. The mechanisms by which obesity and body fat distribution lead to higher BP are not well understood. However, increased fat distribution is usually associated with insulin resistance, which may contribute to the development of !" hypertension (23). Multiple mechanisms have been proposed to explain a possible relationship between insulin resistance and hypertension, including increased sympathetic nervous activity, vascular smooth muscle hypertrophy, alteration of cation transport, and salt sensitivity (24). The relationship between insulin and BP is complex. Short-term insulin infusion raises catecholamines but not BP; and it causes vasodilation in humans and dogs (25). While the effect of chronic hyperinsulinemia is not known, ecological data do not support the relationship between insulin and hypertension. For example, Pima Indians and Mexican Americans have high rates of type 2 DM, hyperinsulinemia, and insulin resistance and yet have a lower prevalence of hypertension (26). Although, insulin resistance and hyperinsulinemia may be an important factor in the link between obesity and hypertension, the precise mechanisms have not been established. Perhaps, it is time to look at adipocytes from a different perspective. Recently, research has shown that adipose tissue functions not as a passive fat storage, but rather as an endrocine organ, producing factors that can affect appetite, energy storage, insulin signaling and sensitivity, inflammation, and vascular function (27). Sodium and Hypertension The relationship between sodium intake and BP has been extensively examined in adults and children by epidemiologic observations and experimental studies. For example, in the INTERSALT study that was conducted with 10,000 participants in 52 population samples and 32 countries demonstrated a highly significant relationship between sodium intake and BP as well as with increase in BP with age (28). Several meta-analyses of interventional studies of sodium intake in humans have demonstrated !" significant but small reduction in BP (29). The magnitude of the BP reduction may be explained by the variation in the study. Early exposure to high sodium may be critical in the initiation of hypertension later in life. In a randomized study, sodium restriction in neonates has been shown to associate with lower blood pressure (30). In many sodiumloading studies in human, a progressive and significant rise in BP was observed when comparing the BP at the end of the study to the initial period of low sodium intake. In these studies, MAP of some individuals was increased by 5 mmHg, whereas others had a 35-mmHg increase (31). These observations suggest a difference in susceptibility to the BP-raising effect of sodium in the population. In fact, individuals with BP increases or decreases significantly in response to a positive or negative salt balance exhibited salt-sensitivity. Salt-Sensitivity The prevalence of salt-sensitivity increases in African American, elderly, obese, diabetes, and chronic renal failure populations. Salt-sensitive individuals have a worse cardiovascular prognosis than SR individuals, regardless of BP level (32). Linkage studies of gene polymorphisms about BP responses to salt support the genetic determination of SS hypertension, including studies about haptoglobin, adducin, !2 and "-adrenergic receptors, " subunit of ENaC, angiotensinogen, and CYP4a11 (33). The best evidence of genetic determinant of SSBP comes from inbreeding rodents for SS including Dahl-SS, Milan-SS, and Sabra-SS rats. Abnormalities in renal regulatory mechanisms of salt and water can cause SS hypertension in animals (34). When the SR animals are given a salt-load, depressor and natriuretic mechanisms are activated !"# while pressor and antinatriuretic ones are simultaneously inhibited resulting in no change in BP. However, SS hypertension may result from abnormal responses to the depressor/natriuretic mechanisms and/or pressor/antinatriuretic mechanisms. Salt loading inhibits and salt deprivation stimulates RAAS, which prevent large changes in BP during salt intake. Keeping RAAS at very low or high level of Ang-II or aldosterone produces SS hypertesion in animals. ET-1 production is increased in the endothelium and the kidney in salt-dependent models of hypertension (35). ET-1 is a potent vasoconstrictor, a stimulator of cell growth and an elicitor of inflammatory responses by increasing oxidative stress in the vascular wall; it induces vascular remodeling and endothelial dysfunction. Antioxidant tempol attenuates the hypertension in the SHR, which supports that there is an imbalance between oxidant and antioxidant in SS hypertension. The production of nNOS-derived NO is increased in high salt condition. NO is a powerful vasodilator and a regulator of renal blood flow and natriuresis. Inhibition of nNOS leads to SS hypertension (36). With these combined factors, O2 - generate during oxidative stress can deplete NO and lead SS hypertension. Salt loading increases the SNA and high SNA can impact renal and systemic hemodynamics, volume homeostasis and BP levels in SS hypertension (37). Vast amount of evidences support arachidonic acid products: cyclooxygenation, epoxygenation, and !hydroxylation as a major role in SS hypertension. Inhibition of cyclooxygenase 1 prevents the production of PGE2, a vasodilatory and natriuretic active compound, in response to salt loading and BP becomes SS. Possible explanation for this is PGE2 plays role in removing ENaC from the cell membrane of renal tubule cells for !!" proteasome recycling. Likewise, cyclooxygenase 1 knockout mouse and SS hypertensive individuals have a common feature of impairment BP reduction during sleep cycle. Salt loading increase the level of EETs, the products of epoxygenation of arachidonic acid, which inhibit distal sodium reabsorption altering the gating properties of ENaC. Reduction of EETs synthesis produces SS hypertension. 20-HETE, the major product of !-hydroxylation of arachidonic acid, can reduce BP. Perhaps, this happens through the blockade of potassium channels, thereby inhibiting the action of + + - + + K /Na /2Cl co-transporter and Na /K ATPase in the kidney. There is strong evidence that diminished level of renal 20-HETE is linked with SS hypertension (38). Like hypertension, SS hypertension is a complex medical condition with multi-factorial contributors. Potassium and Hypertension Many studies have shown that potassium intake is inversely related to systolic and diastolic blood pressure (39). In fact, in many of salt loading studies in humans, it was observbed that the net potassium loss occurred when sodium intake increased. In the same studies, when potassium was replaced, the sodium-induced hypertension was reduced compared to experiments without potassium supplement (31). Some of the proposed mechanisms of how potassium lowering BP are natriuretic effects, RAAS and SNA suppression, arterial vasodilation, baroreceptor function improvement, and effect on eicosanoids levels. Physical Inactivity and Hypertension !"# The British Regional Heart Study of over 7000 middle-aged men found that there was significant inverse relationship between amount of physical activity, BP level and cardiovascular events (40). This association was independent of age, BMI, social class, smoking status, and lipid profile. There are numbers of suggested mechanisms linking physical activity and lower BP, including effects on body weight and insulin sensitivity, SNA and baroreceptor function, and vascular structure. Therefore, physical activity is an effective anti-hypertensive therapy; 150 minutes per week of moderate-intensity aerobic activity is recommended (6). Alcohol Consumption and Hypertension Cross-sectional epidemiologic studies have shown a direct relationship between excess alcohol intake (> 28g of ethanol/day, ~ 2, 12 oz of beer) and hypertension (41). Randomized trials and meta-analysis have shown that reduction in alcohol intake is associated with lowering BP (42). Each reduction by one drink per day reduces SBP and DBP by approximately 1 mmHg (43). Some of the proposed mechanisms of the hypertensive effects of alcohol are increase RAAS activity and SNA, depletion of NO, Ca +2 +2 +2 and Mg , increase acetaldehyde, endothelin, and intracellular Ca in vascular smooth muscle (44). Due to the negative effects of alcohol on BP and other health/psychosocial functions, it is recommended by health advisors to limit the maximum average intake of 2 drinks/day for men and 1 drink/day for women (6). Psychosocial Stress and Hypertension Stress is defined as a situation perceived as an uncontrollable threat to the individual’s well being (45). Acute stress that occurs for example during fear or anxiety can cause !"# rapid, large and transient increase in BP and heart rate. The role of chronic stress in contributing to the development of hypertension is not clear. However there is evidence that stressful situations like a stressful job or living in poverty are associated with hypertension (46). People who work in high demands and low control jobs have elevated BP not only during the time at work, but also while at home during sleep. The mechanisms for how stress increases BP are not clear. Perhaps, stress increases the SNA or altering the levels of corticotropin hormones. Hemodynamics in the Development of Hypertension Contractions of the left ventricle propel blood into the systemic circulation that begins at the aorta and flows to the large arteries, small arteries, arterioles, capillaries, venules, veins and back to the right atrium. The aorta and large arteries have high elastic property, therefore they distend and recoil between each ventricular contractions providing a pulsatile pressure that drives blood further away from the heart. The small arteries and arterioles are known as the resistance vessels because of their thick smooth muscle walls that can contract and relax. They provide most of the total vascular resistance in the body. Capillaries are small and have thin walls, which allow for rapid exchange of nutrients and waste products. For this reason, they are called exchange vessels. Lastly, the venules and veins have larger diameters and thinner walls therefore they can hold a large amount of blood. These vessels are referred to as capacitance vessels. Compliance (C) = !V/(Pinside – Poutside). Because the venous side of the systemic circulation is approximately 20 times more compliant than the arterial side, the drop in volume on the venous side has little effect on its pressure. !"# However, an equivalent increase in volume on the low compliant side, the arterial side, causes a 20-fold larger change in the arterial pressure. The left ventricle contractions create a pressure gradient in systemic circulation, which allows for the flow of blood. Although it does not exactly describe the blood flow through elastic, tapering blood 4 vessels, Poiseuille’s law (Q = !P"r /8#L) can be used to understand blood flow. The flow (Q) is proportional to the pressure gradient (!P) between the two ends of the tube 4 and inversely proportional to the TPR (TPR = 8#L/ "r , r is the radius of the tube, L is its length, # is the viscosity of the fluid, and " is a geometric constant). In the body, changes in the radius of the small arteries and arterioles are responsible for most of the changes in the TPR, and flow (Q) is defined as cardiac output (CO) and !P = MAP – RAP where MAP is normally close to zero; Thus CO = MAP/TPR Early hemodynamic changes in hypertension often include increased CO with normal TPR. However, in 60% of chronic human hypertension, TPR is increased and the elevation of TPR is uniformly distributed throughout the body (47). Although the TPR is increased in most patients with sustained hypertension, CO and blood flow in most tissues remain relatively normal. The local regulatory mechanisms including myogenic constriction, metabolic vasodilation, and endothelial-mediated relaxation may play a role in maintaining normal blood flow during increased BP (Figure 2) (48). Limited observational studies under the conjunctiva and nailfold indicate that the capillaries are narrowed and sparse in distribution. Many of hypertensive patients exhibit increased vascular reactivity to stressful stimuli and decreased responses to vasopressor agents. For example, exercise-induced vasodilation is impaired in essential hypertension (49). !"# There is also evidence that capacitance vessels may be contracted in chronic hypertension, which results in the redistribution of blood from the venous to arterial side and increased MAP (47). Moreover, hypertension is associated with the loss of arterial distensibility, which results in higher pulse pressure that is transmitted farther into the peripheral circulation. !"# Figure 2: Control mechanisms responsible for autoregulation in non-renal tissues during the development of hypertension To maintain constant blood flow, myogenic tone is increase during high BP. Increased blood flow removes the metabolic vasodilators. High BP results in endothelial dysfunction and decrease vasodilation. Chronic hypertension leads to vasoconstriction and rarefaction. !"# Anatomical Factors in the Development of Hypertension Coarctation of the Aorta Aortic coarctation is a congenital condition whereby the aorta narrows to an abnormal width. There are three different types of hypertension associated with aortic coarctation: prerepair hypertension, postrepair paradoxical hypertension and late postrepair hypertension (50). Mechanical, neural and renal artery stenosis are three main theories used to explain prerepair hypertension in aortic coarctation. Mechanical theory proposes that the increased BP proximal to the narrowed segment is because of high impedance to left ventricular emptying. The neural theory proposes that hypertension is the result of readjustment of the baroreceptors in the aortic arch, such that an increased proximal pressure becomes necessary to ensure an adequate blood flow to distal organs. Lastly, the renal artery stenosis theory explains that the narrowed aorta causes low renal perfusion, which stimulates RAAS activity and impairs salt/water homeostasis (51). Postrepair paradoxical hypertension occurs during the first week after surgical repair of the coarctation. It has been postulated that loss of cardiopulmonary baroreflex afferent fibers lead to the activation of SNA and RAAS. Balloon angioplasty and !-blocker pretreatment can prevent postrepair paradoxical hypertension (52). Late postrepair hypertension occurs much later after the surgical repair of the coarctation. It often shows in the form of upper extremity hypertension with treadmill exercise but not with arm exercise. There is an increased vascular reactivity to exogenous NE in the arm, normal vascular reactivity in the legs, and abnormal !"# aortocarotid baroreceptor activity. Different patterns of arterial remodeling, either caused by the narrowed aorta or differential postsurgical vascular remodeling in upper and lower extremity, may be the cause of postrepair hypertension (53). Obstructive Sleep Apnea Syndrome and Hypertension OSAS is defined as ! 15 apneas or hypopnea/hour of sleep plus daytime sleepiness. OSAS is associated with pulmonary and systemic hypertension, myocardial infarction, stroke and metabolic disorders (54). Peripharyngeal fat deposition, enlargement of the soft palate or tongue, craniofacial abnormalities and loss of respiratory muscle tone can cause partial or full collapse of the airway during sleep. OSAS triggers increase in SNA, and augmented SNA can be seen during sleep and during wakefulness when breathing is stable (55). High SNA increases RAAS activity and hypertension in individual with OSAS. Excessive aldosterone release may contribute to drug-resistant hypertension in these individuals (56). Suppression of RAAS activity by increasing salt intake can prevent the rise in BP of OSAS individuals. Chronic intermittent hypoxia in rats and in humans with OSAS can cause oxidative stress and vascular dysfunction specifically endothelium-dependent vasodilation impairment (57). Lastly, vascular remodeling is also documented in people with OSAS such as carotid intima media thickness, and arterial stiffness. The proposed mechanisms for the vascular remodeling include hypoxic stimulation of mitogenic factors such as ET-1, PGF, and VGEF (58). Renal Stenosis and Hypertension Renovascular hypertension is high blood pressure due to the narrowing of renal arteries or renal artery stenosis. Atherosclerotic disease of the renal artery, fibromuscular !"# dysplasia and narrowing at the anastomotic site of a transplanted kidney are some of the most common causes of renal artery stenosis. In animals, the two-kidney/one-clip (Goldblatt), the two-kidney/two-clip, and the one-kidney/one-clip are experimental models for renovascular hypertension. Initially, the narrowing of renal arteries decreases renal perfusion, which increases the activity of the afferent nerves, the baroreceptors, and the chemoreceptors in the kidney; together they lead to the activation of RAAS. Increased level of Ang-II promotes renal vasoconstriction and salt/water retention that result in the expansion of the extracellular and intravascular compartments. Volume expansion increases cardiac pre-load and stroke volume, which restores poststenoic perfusion pressure. Chronically, salt and water balance is restored but systemic BP is higher. Activation of renal chemoreceptors, baroreceptors and afferent nerves increase SNA and BP (59, 60). Endocrine and Hypertension Mineralocorticoid Aldosterone and its precursor 11-deoxycorticosterone are the mineralocorticoid in humans. Aldosterone is mainly synthesized by the zona glomerulosa of the adrenal cortex in response to Ang-II, ACTH, and hyperkalemia. The principal epithelial cells of the renal cortical collecting duct have been considered as the main cellular target of aldosterone. Under physiological conditions, glucocorticoid --cortisol--is approximately 1000 times more abundant than aldosterone and can also bind to MR; thus, the level of intracellular cortisol must be decreased in order for aldosterone to access MR. 11!HSD2 enzymes confer this mechanism because they have high affinity for cortisol and !"# efficiently convert it to the inactive cortisone, which allow aldosterone to bind MR (61). If 11-HSD2 activity is reduced (licorice ingestion or by carbenoxolone administration), cortisol can activate MR and GR; in either case, it has indistinguishable effects on urinary electrolyte fluxes. The ability of GR to mimic the effects of MR suggests that the action of both receptors is mediated by a relatively nondiscriminating hormone response element. In addition to nondiscrimating hormone response element, MR also has specific mineralocorticoid response element. For example, MR activation by aldosterone leads to BP elevation; MR occupancy by corticosterone/cortisol, or by the MR antagonist RU28318, is without agonist effect but blocks the action of aldosterone. The findings that neither agonist nor antagonist effects via MR are mimicked or blocked by GR occupancy is interpreted as evidence for MR action via a specific mineralocorticoid response element. NR3C2, the systematic name of MR gene, encodes for a 107 KDa protein MR that shares a close homology with the GR (62). Aldosterone Effects in Renal Epithelium The putative genomic mechanisms of MR action are shown in Figure 3. Upon activation of MR by aldosterone, the receptor undergoes a conformational change and translocates into the nucleus where it functions as a transcriptional factor. MR activation + + leads to an enhanced activity of ENaC and Na /K ATPase that results in sodium reabsorption and potassium excretion. MR activation increases the expression of SGK1, which results in the phosphorylation of ubiquitin-protein Nedd4-2 and consequently the inactivation of Nedd4-2. Thus, SGK1 activation results in decreased ubiquitin-mediated internalization and degradation of ENaC while an increased in its half-life at the plasma !"# + membrane. Activated MR also up-regulates CHIF that enhances the affinity of Na /K + + + ATPase for Na and K . Aldosterone has also been shown to induce the expression of the GTP dependent signaling molecule K-ras2, which enhances the opening probability of ENaC. The aldosterone-activated MR induced the expression of GILZ, and GILZ enhanced ENaC activity by antagonizing ERK signaling, a potent negative regulator of ENaC. Excess aldosterone induces hypertension due to volume expansion and activation of SNA. The most common cause of excess aldosterone is primary aldosteronism, which is also the most common form of secondary hypertension. However, mild elevations of aldosterone levels have also been shown in essential hypertension in humans (63). !!" Figure 3: Hormonal pathways of MR activation in renal cortical collecting duct cells. (For interpretation of the references to color in this and all other figures, the reader is referred to the electronic version of this dissertation) 11!-HSD2 converts cortisol into cortisone, which allows aldosterone to bind to the MR + in renal cortical collecting cells. Aldosterone-induced Na reabsorption by increases the + + activity Na /K ATPase through the action of CHIF. It decreases ENaC degradation by the action of SgK1 and Nedd4-2, and enhances ENaC activity by stimulating K-ras 2 and GILZ. Overall, aldosterone increases the expression and activity of ENaC in renal cortical collecting cells. !"# Aldosterone Effects Outside Renal Epithelium Besides the epithelial cells of the renal cortical collecting duct, aldosterone also works on non-epithelial tissues, such as the heart, the vessels, adipose tissue, and M!. Aldosterone – Heart The heart tissues produce aldosterone and express MR (64). Atrial MR expression is increased in patients with atrial fibrillation (65). It was suggested that aldosterone causes atrial fibrillation because it induces atrial cardiomyocytes hypertrophy, apoptosis and fibrosis. Spironolactone prevents atrial fibrosis and dilation in an experimental model of atrial fibrillation (66). Aldosterone-induced MR activation changes the +2 intracellular Ca T-type Ca +2 signaling of cardiomyocytes and causes rhythm disorders by altering + channel expression, K and L-type Ca +2 channel, and ryanodine receptor activity (67). In fact, the RALES and EPHESUS showed that 50% of the benefits from blocking MR were related to the reduction of sudden death. Aldosterone – Vasculature In the blood vessels, aldosterone induces stress fiber formation and migration in smooth muscles by activating ERK, MAPK, c-Src, JNK, EGFR, and Rho-kinase (68). Furthermore, aldosterone increases aortic fibronectin expression without altering elastin and collagen density, which causes increased stiffness in large arteries (69). In fact, there is an inverse relationship between plasma aldosterone and large vessel compliance, independent of age and blood pressure, in hypertensive individuals. Interestingly, the effects of aldosterone on the cardiovascular system are dependent on !"# salt. For example, individuals with Gitelman or Bartter syndrome are characterized with high level of aldosterone and renal loss of sodium; they have normal BP and no vascular remodeling (70). Aldosterone also induces oxidative stress and inflammation in the vascular wall (71), endothelial cells, and vascular smooth muscles. In animal models, aldosterone infusion causes increased ICAM-1 (72), MCP-1, and TNF-! and infiltration of M" (73) and lymphocytes in the blood vessels. Aldosterone causes vascular oxidative stress by increasing NADPH-oxidase activity through c-Src activation (74). The increase in oxidative stress secondary to aldosterone infusion results in - vascular hypertrophic remodeling and fibrosis. The generation of O2 by NADPH can reduce NO bioavailability and endothelial-dependent vasorelaxation in large and small arteries. However, aldosterone infusion also decreased vascular expression of G6PD, which may also play a role in endothelial dysfunction (75). Aldosterone can alter endothelium’s electrolyte composition and increase endothelial cells’ volume by affecting amiloride-sensitive sodium-proton exchanger (76). It also increases ET-1 expression in the blood vessels, which can cause inward hypertrophic remodeling of resistance arteries, vascular inflammation, and endothelial dysfunction (77). Recently, it has been shown that aldosterone exerts a synergistic effect with Ang-II and results in vascular smooth muscle proliferation and migration. What is interesting is that the effects of aldosterone and Ang-II exhibit a fast and slow non-genomic and genomic response. The genomic effects of aldosterone are all mediated via the activation of the MR whereas the non-genomic effects may be MR dependent or independent e.g. GPR30. In humans and animals, aldosterone injection is associated with an increased vascular resistance within 10 minutes (78). AT1 and transglutaminase inhibitors prevent !"# this constrictive effect of aldosterone but not MR antagonist, which suggests the role of aldosterone in intracellular transglutaminase activity and AT1 dimer formation independent of MR receptor (79). Similarly, aldosterone induces a rapid and concentration dependent increase in phospho-ERK in endothelium-denuded aortic rings, which was partly attenuated by the MR (eplerenon) and GPR30 (G15) antagonist (80). Lastly, aldosterone (independent of MR) has been shown to activate amiloride+ + sensitive Na /H exchanger in vascular smooth muscles that causes increase intracellular IP3 and Ca +2 levels (81). Aldosterone – MΦ M! expresses both MR and GR but not 11"-HSD2. Given that cortisol circulates at much higher concentrations than aldosterone, under normal circumstances the MR in M! would be occupied by cortisol (82). LPS stimulates the expression of GR but suppresses MR. This supports that GR is anti-inflammatory and MR is proinflammatory. Similarly, low concentration of corticosterone enhances peritoneal M! immune functions, whereas high concentrations are immune suppressive (83). The immuno-stimuatory effects produces by low dose of corticosterone are mediated via MR activity, whereas the immunosuppressive effects of high dose are produced by GR. Aldosterone enhances the expression of inflammatory and oxidative stress markers: p22 phox and PAI-1 in isolated human M! (84). Treating ApoE-deficient mice with aldosterone increases oxidative stress in the M! (85). Blocking MR reduces M! !"# accumulation in a number of disease models, including peritoneal fibrosis, myocardial infarction, and vascular inflammation. Glucocorticoid Cortisol, the glucocorticoid in humans, is mainly synthesized by the zona fasciculata of the adrenal cortex in response to ACTH, and stress. Excess cortisol can increase BP as seen in Cushing’s syndrome. The pathogenesis of glucorticoid-induced hypertension remains undetermined, although the increase in BP is independent of salt intake. Its been suggested that the following factors are involved in the pathogenesis of glucocorticoid-induced hypertension. Cortisol activates the RAAS by increasing circulating angiotensinogen. It also enhances NE and Ang-II pressor responses by altering number of receptors e.g. AT1 receptor. However, it suppresses the depressor response of kallikrein-kinin systems and reduces the production of prostaglandins and NO (86). Thyroid Hormone Triiodothyronine (T3) and thyroxine (T4), the main thyroid hormones, are produced by the thyroid gland, which are primarily responsible for regulation of metabolism. T4 is the major circulating thyroid hormone in the blood, and has a longer half-life than T3. The ratio of T4 to T3 in the blood is roughly 20 to 1. The prevalence of hypertension in thyrotoxicosis is approximately 20%-30%. Systolic hypertension is the predominant finding because of the increase CO and cardiac contractility and decrease TPR. It also !"# been postulated that T4 increases !-adrenergic receptors, which increases tissues e.g. the heart sensitivity to catecholamines. Similar to cortisol, thyroid hormones also activate RAAS by stimulate the synthesis and release of angiotensinogen (87). Unlike, thyrotoxicosis, hypothyroidism is associated with diastolic hypertension because of increased catecholamines and TPR (88). Parathyoid Hormone The chief cells of the parathyroid glands secrete PTH, an 84 amino acids long polypeptide. It acts to increase the concentration of Ca +2 in the blood. Excess PTH is associated with primary hyperparathyroidism (causes by adenoma or hyperplasia of the gland), pseudohypoparathyroidism (causes by resistance to PTH), or secondary hyperparathyroidism (causes by CKD). The prevalence of hypertension in primary hyperparathyroidism is approximately 10%-70% and in pseudohypoparathyroidism is approximately 40%-50%. The mechanisms for PTH-induced hypertension are not clear. +2 Perhaps hypercalcemia increases free intracellular Ca , which increases vascular smooth muscle contractility. However, individuals with pseudohypoparathyroidism have +2 low Ca but high PTH, suggesting that an increase in PTH itself may be responsible for hypertension. Many studies also show that RAAS activity is increased in hyperparathyroidism (89). Humoral Agents in Hypertension Studies of cross-circulation between hypertensive and normotensive animals reveal factors in blood that can increase BP. For example, when injecting plasma from dogs !"# with one-kidney Goldblatt hypertension or from essential or renovascular hypertensive patients into the jugular vein of a rat, the animal will slowly increases its BP (90, 91). Some of these humoral agents are endothelin, vasopressin, EO and members of RAAS. Renin Angiotensin Aldosterone System The renin angiotensin aldosterone system plays an important role in regulating blood volume and systemic vascular resistance, which together influence BP. There are three important components to this system: renin, angiotensin, and aldosterone. A schematic pathway and functions of RAAS is depicted in Figure 4. Renin is the rate-limiting step in the RAAS. It is an aspartyl proteolytic enzyme whose only known substrate is angiotensinogen. The JG cell in the kidneys is the primary location where renin is synthesized, stored and released. Renin is synthesized as a preprorenin (404 amino acids); in the rough endoplasmic reticulum, it is cleaved to prorenin (381 amino acids) which is transported to lysosomal granules and cleaved by cathepsin B to an active enzyme renin (339 amino acids). Prorenin is detected in circulation, and under basal conditions its concentration is 2-10 times greater than renin (92). It is also found in extra-renal tissues e.g. adrenal glands, pituitary, and submandibular glands. As an active area of curiosity, more research is needed to learn more about Prorenin. Sodium restriction and hormones e.g. androgens and T4 have been shown to induce renin gene expression, whereas Ang-II suppressed renin expression (93). Renal intrabaroreceptors, chemoreceptors and renal sympathetic efferent nerves are the classic stimuli of renin release (94). On the one hand, reduction in renal perfusion pressure stimulates the JG cell’s baroreceptors and increases its intracellular cAMP levels, which !"# triggers renin release. On the other hand, reduction in NaCl delivery to the macula densa stimulates the chemoreceptors and increases COX-2 production of PGE2, which acts on the JG cells through PGE-4 receptor to increase cAMP production and consequently renin release (95). In addition to stimulating COX-2, chemoreceptors also stimulate NOS-1 production of NO and eventually cGMP, which acts on phosphodiesterase-3 in the JG cells to prevent the degradation of cAMP and sustains the stimulus for renin release (96). Sympathetic nerves directly innervate JG cells, and stimulation of renal efferent nerves releases NE that can activate !-adrenergic receptors on JG cells to increase cAMP production and renin release. Several central neural reflexes, including the cardiac baroreceptors, the aortocarotid baroreceptors and chemoreceptors, and the vagal afferent nerves can stimulate the renal sympathetic +2 nerves. Unlike cAMP, intracellular Ca in the JG cells causes decrease in renin release. Some humoral factors e.g. Ang-II, ET-1, atrial natriuretic peptide, adenosine, and thromboxane can inhibit renin secretion. Angiotensinogen is the only known protein from which renin cleaves to generate a family of angiotensin: Ang-I, Ang-II, Ang-III, Ang-IV, Ang-(1-7), Ang-(1-5), and Ang-(1-4) (97). Figure 4 illustrates the enzymatic pathways responsible for the production and degradation of active angiotensins. The systemic source of angiotensinogen is from the hepatocytes but other tissues e.g. heart and M" can also synthesize angiotensinogen for local usage (98). The half-life of circulating angiotensinogen is about 16 hours. Several hormones e.g. cortisol, estrogens, T4, insulin and selected cytokines can induce angiotensinogen expression in haptocytes. Renin cleaves circulating !"# angiotensinogen at its N-terminus to release Ang-I decapeptide. ACE converts Ang-I into active Ang-II octapeptide by cleaving the C-terminus dipeptide from Ang-I. However, ACE can also cleaves other peptides e.g. bradykinin (99), LHRH, enkephalins, and substance P. Therefore, ACE does not just generate vasoconstrictive Ang-II but it also removes vasodilator bradykinin. In fact, ACE has higher affinity for bradykinin than Ang-I. Most ACE is found on the plasma membrane of various cell types e.g. epithelial lining of small intestine, the choroid plexus, and the placenta. In the vascular beds, ACE is found in the plasma membrane of endothelial cells lining the lung, the retina, and the brain, but the renal proximal tubular brush border has 5-6 times more ACE per unit of wet weight (100). Chymase, mast cell-derived chymotrypsin-like serine protease, is another enzyme that can convert Ang-I into Ang-II; it provides another mechanism for the generation of Ang-II during chronic inhibition of ACE. Its roles in hypertension and especially in hypertensive patients who are taking ACE inhibitors are not clear; hence further research is needed. In addition to Ang-II, Ang-I can also be converted into Ang-(1-7) heptapeptide by EPs (101). Ang-(1-7) is a competitive inhibitor of Ang-II at AT1 receptor but a substrate for AT2 and mas receptor (102). In the fetus, AT2 is the predominant subtype and plays an important role in renal development. In adult, AT2 is found mostly in the brain, the uterus, adrenal medulla, and the kidneys. The functions of AT2, a Gi-protein coupled receptor, are to antagonize those of AT1, including vasodilation, natriuresis, and apoptosis (prevent growth and fibrosis). Mas is a GPCR whose signal transduction leads to protein phosphorylation and the release of NO and eicosanoids (103). Ang-(1-7) can also be formed when !"# Figure 4: Enzymatic pathways for Ang-II synthesis, metabolism, and functions. Ang-II is formed from the hepatocyte-derived angiotensinogen through the actions of renin and ACE. Ang-II can be metabolized into Ang-III/IV, Ang-(1-7), Ang-(1-5), and Ang-(1-4) through the actions of CHYM, AP, EP/NEP and ACE. + Ang-II has a wide range of functions including increase SNA, Na reabsorption, vasopression secretion, ROS production, + + vasoconstriction, and stimulation of Na /H exchanger. !"# ACE2 converts Ang-II into Ang-(1-7) or NEP converts Ang-I into Ang-(1-7); ACE2 is found in the heart, blood vessels, and the kidneys but NEP is found mostly in epithelial cells e.g. renal proximal tubule brush borders, fibroblasts, and neutrophils.NEP can also inactive Ang-II and Ang-(1-7) by converting them into inactive Ang-(1-4). Ang-(1-7) can also be inactivated by ACE by converting it to Ang-(1-5) (104). Inhibition of ACE has three main preventive effects: 1) the formation of vasoconstrictive Ang-II and 2) the degradation of Ang-(1-7), a vasodilation agent and 3) degradation of bradykinin. Together these two account for the BP-lowering effect of ACE inhibitors. Lastly, Ang-II can be metabolized into inactive Ang-III and Ang-IV by APs. The AT1 is a Gq/11 protein coupled receptor. Upon activation of the receptor, the Gq/11 protein stimulates PLC to produce IP3 and DAG. IP3 binds to IP3 receptors and triggers the release of +2 intracellular Ca +2 storage. AT1 also triggers Ca +2 cell membrane. Together Ca influx by open Ca +2 channels in the and DAG activate PKC, which phosphorylates and activates downstream signaling proteins to cause vascular smooth muscles contraction, + aldosterone synthesis and release, increased SNA, ROS production and Na /H + exchanger activation (105). Endothelin There are three endothelins found in mammals: ET-1, ET-2 and ET-3, and all three are 21-amino acids peptides (106). They are found in many different organs but often associated with vascular endothelial cells. They are important regulators of various organ systems including the cardiovascular, digestive, endocrine, nervous, renal and !!" genitourinary system. ET-1 is synthesized in endothelial cells as preproendothelin (203 amino acids), converted into proendothelin (183 amino acids), and sequentially cleaved by ECE into active ET-1 (39 amino acids) (107). Various factors e.g. Ang-II, vasopressin, catecholamines, TGF-!, and high pressure/low shear stress situation stimulate ET-1 secretion from endothelial cells (108). ET-1 can binds to ETA and ETB receptor, and both are GPCR. Upon activation by ET-1, G-protein stimulates PLC, +2 induces intracellular Ca + mobilization (109), and activates PKC and MAPK, and also + Na /H exchanger. ET-1 acts in a paracrine manner on the ETA and ETB2 receptors on the vascular smooth muscle cell membrane to induce contraction, proliferation, migration, and cell hypertrophy (110). However, ET-1 can also activate endothelial ETC and ETB1 receptors to induce the release of NO and prostacyclin which are vasodilators. ET-1 acts on ETA receptors can also increase NADPH oxidase level and oxidative stress (111). In the heart ET-1 has positive chronotropic and inotropic effects on cardiomyocytes. In addition, ET-1 stimulates ETA receptors on cardiomyocytes and fibrobasts to mediate extensive cardiac fibrosis and microvascular remodeling. In the kidney, endothelin receptors are mainly present in the blood vessels and the mesangial cells; and they stimulate the RAAS by increasing the levels of renin, Ang-II, and aldosterone (112). In the nervous system, through ETB receptors, ET-1 modulates presynaptic neurotransmitters release, which increases central and peripheral SNA (113). The endothelin system is activated in many hypertensive models, including !"# DOCA-salt hypertension (114), Dahl salt-sensitive hypertension, Goldblatt hypertension, and cyclosporine-induced hypertension. Endogenous Ouabain 45% of Caucasians who are diagnosed with essential hypertension have elevated EO in their blood. EO is a mammalian steroid counterpart of the plant glycoside ouabain. It + + specifically and reversibly binds to mammalian Na /K ATPase with high-affinity. Upon + + + binding, EO inhibits the Na /K ATPase causes increase in intracellular Na , lowering + +2 the Na gradient across the cell membrane and reduces the efflux of Ca + Na /Ca +2 +2 exchanger (115). This change in intracellular Ca through concentration may increase the arterial myogenic tone and the neurogenic activity (116). High salt intake raises plasma EO in both humans and rats (117). EO synthesis is mediated by the hormone aldosterone. In fact, approximately 50% of patients with primary hyperaldosteronism have elevated plasma EO. EO secretion is stimulated by Ang-II and during volume depletion. EO is an on going area of research. Rostafuroxin, currently in + + phase III trials for essential hypertension, blocks EO binding to Na /K ATPase and reduces BP in hypertensive animal models. The Nervous System and Hypertension The nervous system has short-term and long-term regulations on BP. In several animal models and in subsets of human essential hypertension, chronic activation of the nervous system appears to contribute to persistent hypertension (118). The sympathetic !"# nervous system and the neurohormonal system (primarily regulated by the hypothalamus) of the nervous system contribute the most to arterial blood pressure control. Figure 5 shows the central modulation of the sympathetic outflow by salt, circulating humoral factors e.g. Ang-II, and the afferent nerves. The brain continuously monitors BP through arterial baroreflex or stretch receptors attached to the vagal and glossopharyngeal axons innervating the carotid sinuses and the aortic arch. The mechanism of arterial baroreflex activation is thought to involve opening ENaC, ASIC, and TRP channels during vascular distention (119). The baroreflex provides second-tosecond negative feedback to prevent too much BP fluctuation. In addition to responding to changes in pressure, it also has a tonic sympathoinhibitory effect during resting condition, because in the baroreceptor denervation condition, there is a profound transient increased sympathetic activity and BP but eventually BP returns back to normal. The mechanism underlying the normalization of BP after denervation is not entirely clear. Perhaps, loss of input from baroreceptor afferents may result in central remodeling of neural pathways. This is supported when destroying the NTS in baroreceptor-denervated animals produce no change in BP but in control animals it causes hypertension. Acute rise in BP initially increases baroreceptor activity, but within a minute the baroreceptor activity declines despite the elevated BP. This acute + adaptation involves the activation of 4-aminopyridine-sensitive K channels (120). In hypertension, the baroreceptors are reset to operate at higher-pressure levels. An acute resetting happens within the first 5-15 minutes after BP increased. This resetting is only partial because the increase in pressure threshold for baroreceptor activation represents a fraction of the total pressure increase. Furthermore, acute resetting doesn’t !"# affect the sensitivity of the baroreceptor (121). However sustained increase in BP will lead to completed resetting in which the pressure threshold increase equals the total pressure increase, and the baroreceptor sensitivity is decrease. Baroreceptor resetting + is caused in part by activation of an electrogenic Na pump, which hyperpolarizes !"# Figure 5: The circuitry of central nervous system modulation of sympathetic outflow RVLM generates tonic sympathetic activity output to different parts of the body via IMT/L regions of the spinal column or the prevertebral ganglions e.g. celiac ganglion. Afferent neuronal inputs from the arterial baro/chemoreceptors arrive to the NTS modulate RVLM activity. Similarly, PVN descending fibers can also modulate RVLM activity. Humoral factors e.g. salt and Ang-II can influence RVLM activity via the AP and circumventricular organ. !"# baroreceptor nerve endings. Humans with essential hypertension exhibit impaired baroreceptor reflex, and the baroreceptor is reset to higher-pressure levels (122). Humoral factors e.g. Ang-II acting at the area of postrema and circumventricular region of the brain can decrease the sensitivity and reset the baroreceptor independently of BP (123). ROS acting at the baroreceptor endings may also contribute to decreased baroreceptor sensitivity. Integration and processing of afferent information from the baroreceptor is accomplished by the NTS. Its projections modify preganglionic sympathetic and parasympathetic activity and modulate the release of vasopression from the hypothalamus. Abnormality in NTS causes acute fulminating hypertension in rats, less severe hypertension in cats and dogs, and increased BP in humans because of higher SNA. The NTS neurons provide inhibitory inputs to the RVLM neurons. Cardiovascular neuronal projections of the NTS onto the RVLM are organized in distinct regions e.g. lumbar RVLM projection and renal RVLM projection. RVLM contains efferent neurons that provide tonic drive to the preganlionic efferent neurons that directly regulate peripheral sympathetic nervous system. Activation of the RVLM increases SNA and thereby vasoconstriction, cardiac contractility, and catecholamine release. Increased RVLM firing rate increases BP. RVLM is the hub for the regulation of SNA. It receives excitatory input from the PVN of hypothalamus. Salt and circulating humoral factors e.g. Ang-II, and aldosterone can stimulate neurons in the forebrain circumventricular organs (SFO and OVLT) and subsequently activate PVN neurons through direct or indirect + projections via the MnPO. Benzamil-sensitive Na channels are thought to be involved in this salt signaling. Like the NTS, PVN projections onto the RVLM are organized in !"# distinct areas (124). There is a hypothesis that in hypertension e.g. Ang-II-salt hypertension, there is an enhanced central drive of PVN onto the RVLM and it exhibits a differential pattern of sympathetic outflow such that splanchnic SNA is increase, renal SNA is decrease and lumbar SNA is not change. Sympathetic nerves originating from the celiac and superior mesenteric ganglia densely innervate the splanchnic vascular bed. Therefore, a high splanchnic SNA causes vasoconstriction in arterioles, and small veins, which contribute to increase TPR, decrease vascular capacitance, and increase BP (125). Furthermore, celiac ganglionectomy has been shown to protect against some form of hypertension (126). In some essential hypertensive patients, there is direct relationship between BP and SNA; the higher the SNA the higher the BP. The Properties of Adrenergic Receptors in Cardiovascular System NE Synthesis, Release, Reuptake, and Metabolism Figure 6 illustrates the enzymatic pathways for NE synthesis, release, reuptake and metabolism. The enzymatic rate-limiting step in NE synthesis is the conversion of tyrosine into DOPA by tyrosine hydroxylase. DOPA is converted into dopamine by dopamine decarboxylase. Dopamine is transported into the vesicle by VMAT, and inside the vesicle D!H catalyzes the conversion of dopamine into NE (127). !"# Figure 6: Enzymatic pathways for NE synthesis, release, reuptake, and metabolism NE is synthesized from tyrosine through a series of enzymes, including TH, DD and +2 D!H. It is transported into the vesicles by VMAT. Upon stimulus of Ca , NE containing vesicles are exocytosis. NE are reuptake mostly by NET and broken down by MAO and COMT. Once NE is released, it binds to "1R on the smooth muscle cells and "2R on the presynaptic terminals causing vasoconstriction and inhibition of further release of NE, respectively. !"# Release of NE from sympathetic nerve endings is by vesicles exocytosis. It involves a series of steps, including docking of synaptic vesicles at the active zone, priming the vesicles ready for release, and fusion of the vesicle and plasma membranes. Figure 7 shows the schematic overview of vesicular trafficking and release. Vesicles in the synaptic terminal are grouped into three pools: the RRP, the recycling pool, and the RP. The RRP are docked to the cell membrane and activated for exocytosis and are the first group of vesicles to be released upon stimulation. The recycling pool is close to the cell membrane; under moderate stimulation, these vesicles are cycling between exocytosis and endocytosis. The RP constitutes the vast majority of vesicles in the nerve terminal where they cluster farther away from the plasma membrane. Synapsins are a family of neuronal phosphoproteins that function to cluster the vesicles into the RP (128). Under dephosphorylated state, synapsins form homodimers and heterodimers to anchor the vesicles into a cluster farther away from the membrane forming a RP. Upon phosphorylation by PKA, MAPK, and CaMK II, phosphorylated synapsins mobilize the vesicles to the RRP (129). Before the vesicles can be exocytosis, they must dock to the active zone on the presynaptic terminal. At the plasma membrane, vesicle docking involves a large tethering complex called the exocyst, which comprises eight proteins: Sec3p, Sec5p, Sec6p, Sec8p, Sec 10p, Sec15p, Exo70p, and Exo84p (130). At the vesicular membrane, CSP!/SGT/Hsc70 molecular chaperone facilitate the interaction of the vesicle with the plasma membrane bound SNAP-25 (131). The interaction between RIM and GTP bound Rab3A may also contribute to the docking reaction. At this stage, Munc18 is associated with the closed conformation of syntaxin-1. GTP hydrolysis !"# causes the dissociation of Rab3A from RIM and the synaptic vesicle. RIM then binds Munc13 and displaces Munc18 from syntaxin-1, which changes syntaxin-1 into the open state. Once the syntaxin-1 is in its open state, vesicular priming begins with syntaxin-1, synaptobrevin, and SNAP-25 assembles into the trans-SNARE complex, which pulls synaptic vesicle and plasma membrane into close contact (132). Finally, complexin binds to the fully assemble SNARE complex stabilizes the primed vesicle. The influx of Ca+2 triggers the binding of synaptotagmin to the SNARE complex and penetration of synaptotagmin into the plasma membrane, leading to membrane fusion. After fusion, the cis-SNARE complex is dissociated by NSF/!-SNAP, and the SNARE proteins are recycled for more exocytosis (133). Protein phosphorylation is an important regulatory mechanism that controls the secretory pathway. Phosphorylation of syntaxin-1 by PKA and PKC and desphosphorylation by CAPP inhibits and stimulates the assembly of SNAP-25 and syntaxin-1 complex, respectively. Even so, PKA was shown to phosphorylate threonine138 of SNAP-25 in adrenal chromaffin cells, which is required for the RRP of vesicles to be in a primed and releasable state. PKA phosphorylation of SNAP-25 activates the refilling of recycling pool vesicles and increases the size of the RRP. Munc18 plays a central role in membrane fusion through its interaction with syntaxin-1. Munc18 binds tightly to syntaxin-1 holding it in a closed formation that prevents assembly into a SNARE complex. PKA and PKC phosphorylate Munc18 and inhibit it from binding to syntaxin-1. Thus, the phosphorylation of Munc18 may release syntaxin-1 from an inhibitory interaction in order to promote fusion and increase exocytosis (134). In addition to phosphorylation, G proteins interaction with vesicle fusion machinery can !"# also regulate secretory pathway. It has been shown that G!" subunits interact with CSP and N-type Ca +2 channels resulted in a tonic G protein inhibition of the channels (135). Similarly, G!" subunits directly bind SNAP-25 and interfere with the binding of SNAP-25 to synaptotagmin and prevent vesicle fusion (136). After vesicular exocytosis, NE is reuptake into the presynaptic terminal using NET, and this is the main mechanism of inactivation of NE released from sympathetic nerves. Cocaine and tricyclic antidepressants e.g. Desipramine block NET. Most of NE taken up into the synaptic terminals is transported back into the vesicles by VMAT, and reserpine inhibits VMAT. A small fraction of the non-reuptake NE spills into the circulation, but the majority are metabolized by MAO and COMT into DHPG and MHPG, respectively (127). !!" Figure 7: Vesicular trafficking and release in presynaptic nerve terminal Vesicles exocytosis involves a series of steps, including docking, priming, and fusion of the vesicles. Vesicles in the synaptic terminal are grouped into 3 pools: the RRP, the recycling pool, and the RP. Synapsins are important proteins that hold the vesicles in the RRP. SNARE proteins e.g. synaptobrevin, synaptotagmin, SNAP-25 and syntaxin-1 and SNARE chaperone proteins e.g. CSP-!, HSC-70 and SGT, and SNARE accessory proteins e.g. MUNC18-1 and MUNC13-1 are some of the major components of the exocytosis machinery. SNAPS and NSF are two important proteins involve in the +2 recycling of the vesicles. G"#-CSP! has a tonic inhibition on the Ca !"# channels. Adrenergic Receptors Adrenergic receptors were originally divided into two different classes: !-adrenergic and "-adrenergic. In 1948, Ahlquist was the first to observe the opposing effects of catecholamine in smooth muscle cells, and he suggested that the excitatory effects were mediated by !-adrenergic receptors, whereas the inhibitory actions were caused by "-adrenergic receptors (137). Now through pharmacological, molecular, and cloning techniques, we know there are three types and each has 3 subtypes: !1-adrenergic receptors (!1A, !1B, !1D), !2-adrenergic receptors (!2A, !2B, !2C), and "-adrenergic receptors ("1, "2, "3) (138). All three !1-adrenergic receptors couple to the Gq pathway, resulting in stimulation of PLC, generation of IP3 and DAG, mobilization of intracellular +2 + + + Ca , activation of PKC, Na /H exchanger and Na /Ca +2 exchanger, and inhibition of + K channel. All three !2-adrenergic receptors are couple to Gi/o, which decreases +2 cAMP production, inhibition of Ca channels and PKA (139). All three "-adrenergic receptors are couple to Gs, which increase cAMP and activate PKA. The functions of adrenergic receptors are listed in Table 3. In addition to type- and subtype-specific signaling, adrenergic receptor signaling pathway is a complex multidimensional “signalome” (140). Adrenergic receptors can form homodimers or heterodimers. They can couple to multiple G-proteins, signaling pathways, and scaffold proteins in a temporally and spatially regulated manner. Together these result in different pharmacological and functionally distinct receptor populations (141). !"# Table 3: Tissue Distribution and Functions of Adrenergic Receptor Subtypes Receptor !1A,B,D Tissue Response Smooth muscle: vascular, Contraction iris, ureter, uterus, sphincters of bladder and GI Smooth muscle of GI Heart Gluconeogenesis, sodium reabsorption Presynaptic sympathetic nerve terminals Inhibition of NE release Platelets Aggregation, granule release Pancreas Inhibition of insulin release Adiocytes Inhibition of lipolysis Vascular smooth muscle Contraction Kidney Inhibition of renin release Heart Positive inotropic and chronotropic, cell growth and hypertrophy Adiocytes Lipolysis Kidney Release rennin Hepatocytes Glycogenolysis, gluconeogenesis Skeletal muscle Glycogenolysis, lactate release Smooth muscle: vascular, bronchi, uterus, GI Relaxation Pancreas Insulin secretion Salivary glands "3 Glycogenolysis Kidney "2 Secretion Adiocytes "1 Positive inotropic, cell growth, hypertrophy Salivary, sweat gland !2A,B,C Relaxation Amylase secretion Adipocytes Lipolysis Skeletal muscle Thermogenesis !"# !2-adrenergic Receptors Stimulation of !2R elicits a wide range of effects including hypotension, bradycardia, analgesia, hypothermia, sedation, hypnosis, and anesthetic-sparing (142). !2B plays an important role in the development of the placenta and the lungs during embryonic development (143). It also regulates the vascular tone. !2C were identified as the major feedback receptors of catecholamine release from chromaffin cells in the adrenal medulla (144). , !2A inhibit insulin release from pancreatic islets, facilitate working memory, sedation, hypnosis, and mediates hypotension, bradycardia, and modulate baroreflex sensitivity (145). The completed knockout of !2R is embryonic lethal (146). !2A-adrenergic Receptors and Cardiovascular Functions Data from genome-wide association study have recently linked a single nucleotide polymorphism within the 3’UTR of the human !2A-adrenergic receptor gene ADRA2A +2 with increase BP (147). !2AR activation inhibits voltage-gated Ca channels, activates GIRK, and MAPK in sympathetic neurons, which results in presynaptic inhibition of neurotransmitter release. !2AR deficient animals were more susceptible to the development of cardiac fibrosis, hypertrophy, and heart failure in chronic cardiac pressure overload condition (148). Furthermore, high level of NE concentrations in the synaptic cleft activates smooth muscle and cardiac myocyte adrenergic receptors, which cause hypertension. Numerous studies have shown that !2R function is impaired !"# in human hypertension and in DOCA-salt hypertension (149, 150). Nevertheless, the exact mechanism that causes the receptor impairment is unknown. Clinically, !2R agonist clonidine is used to lower BP and treat hypertension. Following stimulation, !2AR is desensitized and downregulated. A major mechanism for desensitization is initiated by GRK2 where it binds to activate !2AR and phosphorylates several serine residues within the third intracellular loop (151). PKC-dependent phosphorylation is also implicated in !2AR desensitization (152). Phosphorylated !2AR recruits "-arrestins, which uncouples the receptors from the G-proteins and induces receptor endocytosis. Spinophilin was shown to block GRK2 interaction with !2AR and prevent "-arrestin signaling. !2AR in sympathetic neurons have a high receptor reserve and therefore, changes in the receptor density in sympathetic neurons do not correlate with functional changes (153). Oxidative Stress, Inflammation and Hypertension Reactive Oxygen Species Reactive oxygen species are metabolites of oxygen that possess an unpaired electron - in their outer orbital. These molecules include the radical superoxide (O2 ), hydroxyl radical (HO•), and nitric oxide (NO•), and non-radical hydrogen peroxide (H2O2) and - peroxynitrite (ONOO ). Figure 8 depicts the pathways for ROS synthesis and metabolism. !"# Figure 8: Enzymatic pathways for ROS synthesis and metabolism ROS are metabolites of oxygen that possess an unpaired electron; they include !O2-, - HO!, NO!, H2O2, ONOO and are generated from xanthine oxidase, NADPH oxdiase, and mitochondrial dysfunction. ROS can be removed by catalase or glutathione peroxidase. !"# - Enzymes that generate cellular O2 include NAPDH oxidase, xanthine oxidase, NOS, heme oxygenase, cyclooxygenase, lipoxygenase peroxidases, and mitochondrial oxidases (154). In cardiovascular biology, low ROS levels modulate vascular tone and structure (155). Under physiological conditions, ROS are maintained at a low level by the enzymes superoxide dismutase (SOD), catalase, thioredoxin, and glutathione peroxidase. Perturbation of the balance between ROS production and removal results in oxidative stress. In many pathological conditions there is an increase ROS which upregulates many signaling pathways including those that involve in smooth muscle cells growth, endothelial dysfunction, extracellular matrix deposition, angiogenesis and inflammation (156). Uncontrolled ROS causes cellular damage and eventually apoptosis because ROS can damage proteins, lipids, and DNA (157). Interestingly, there is a large body of literature correlating ROS to hypertension.. The SOD mimetic, tempol, lowers blood pressure and sympathetic nerve activity in DOCA-salt hypertension (158). - A major source of O2 is a multi-subunit enzyme, NADPH oxidase. In phagocytes such phox as M!, NADPH oxidase has intracellular p47 , p67 phox phox , p4O subunit and a cytochrome b558 catalytic core composed of membrane bound subunits Nox2/gp91 phox and p22 phox (159). Although NADPH oxidases were originally found in phagocytic cells, the discovery of gp91 phox homologs indicates that there is an entire family of NADPH oxidases. The family includes Nox1, 2, 3, 4, 5, Duox 1 and 2, which is also found in many tissues and mediates diverse functions. Hypertensive stimuli upphox regulate p22 subunit and enzyme activity. Mice deficient of Nox-1 have blunted !"# DOCA-salt hypertension (160). In the brain, increased NADPHD oxidase activity in the circumventricular organs and the NTS increase sympathetic outflow and inhibits its activity in the same brain area prevent hypertension. Despite the ample of evidence suggesting that ROS contributes to hypertension, there is not a clear understanding of exactly how this happens. Furthermore, treatment with antioxidant e.g. vitamin C and E has been found to be effective in some animal models and small clinical trials but data from large clinical studies have not demonstrated benefits (161). Inflammation and Hypertension Many academic publications indicate that inflammation may be involved in the development of hypertension. Transferring of splenic cells from DOCA-salt hypertensive and renal hypertensive rats into normotensive rats caused hypertension in the recipient rats (162). Animals that have thymectomy also have blunted delayed phase of DOCAsalt hypertension (163). Interestingly, a recent analysis of 6000 people with AIDS showed that they have lower prevalence of hypertension comparing to the general population. In addition, further treatment with retroviral therapy for two years restored the prevalence of hypertension to that of the control population (164). Immunosuppressive therapy e.g. mycophenolate mofetil prevents hypertension in some animal models (165). Inhibition of proinfammatory transcription factor, NF!B, prevents nitric oxide inhibitor-induced hypertension. Etanercept, a TNF-" antagonist, prevents hypertension and vascular dysfunction in Ang-II induced hypertension and fructose-fed hypertension (166). IL-17 has been shown to induce chemokines and adhesion molecules in tissues; mice lacking IL-17 have reduced BP when compared to wild-type mice treated with Ang-II (167). A hypothesis for mechanism underlying inflammation!"# induced hypertension is the formation of a neo-antigen (168). Perhaps, the modest increase in BP during early stage of hypertension causes some cellular damage, neoantigen formation, and inflammatory response that serve as stimuli to recruit immune cells e.g. M! and T-cells. Hypertensive animal models that lack the immune -/- components e.g. RAG mice lacking lymphocytes (169), and Op/Op mice lacking M! only increase BP to about 135 mmHg (170), (modest hypertension) even when treated with maximal stimuli e.g. Ang-II and DOCA-salt. Rats that are immune tolerant to HSP70 developed minimal renal inflammation and were protected from the development of salt-sensitive hypertension (171). This supports the hypothesis which suggests that inflammation plays role in the development of hypertension. Figure 9 illustrates the hypothesis for the underlying role of the immune system in hypertension. !"# Figure 9: Proposed hypothesis describing the role of adaptive immunity in hypertension Humoral factors e.g. salt and Ang-II elevate BP. This increased in BP causes tissue damage and the formation of neo-antigen, which triggers the adaptive immune system activation. Activated M! and T-cells infiltrate into the blood vessels and kidneys cause further damage and severe hypertension. !"# Lymphocytes and Hypertension Although it is known that hypertension involves inflammation, most studies focuse on the contribution of lymphocytes. Mice lacking T and B cells have blunt hypertension and do not develop vascular remodeling during angiotensin II infusion or DOCA-salt treatment; adoptive transfer of T, but not B cells restored these changes (169). Furthermore (WC), T cells express angiotensinogen, angiotensin I-converting enzyme, and renin. AT1 receptors are expressed intracellularly by T-cells and angiotensin II activates T-cells (172). However, it is unclear how angiotensin II activates T cells in DOCA-salt hypertension when DOCA-salt hypertension is characterized by low levels of circulating angiotensin II. Even so, none of the studies involving lymphocytes focuse on how these cells affect the periarterial sympathetic nerve function in hypertension. M! and Hypertension The role of inflammation through recruitment, activation and proliferation of M! in the vascular adventitia has been recognized in hypertension (173). In some models of hypertension M!/monocyte infiltrate into the arterial wall (174). Alterations in the number of circulating monocytes and their activation occur in hypertensive patients and animals (175, 176). It is unclear how M! is activated. Leukocyte adhesion molecules, chemokines, specific growth factors and endothelin-1 and angiotensin II can modulate M! activity (177). An interesting question is whether circulating monocytes can sense increased blood pressure. Adventitial M! in MA is exposed to increased cyclic mechanical strain by high blood pressure. Cyclic strain on in vitro monocytic cells induces transcription of cytokine IL-8, NF-"B-inducible IEX-1, and an apoptosis related !!" PAR-4, in an amplitude-dependent manner (178). This suggests that high blood pressure can induce the expression of cytokines and adhesion molecules in the vascular wall causing M! infiltration. It is logical then to ask what is/are the source(s) of infiltrating M!. A current paradigm states that monocytes circulate freely and patrol blood vessels but differentiate irreversibly into M! or dendritic cells. A recent publication showed that bona fide undifferentiated monocytes reside in the spleen and outnumber their equivalents in the circulation. The reservoir monocytes assemble in clusters in the cords of subcapsular red pulp. In response to ischemic myocardial injury, splenic monocytes increase their motility, exit the spleen and accumulate in injured tissue (179). Whether a similar pathophysiology in which splenic monocytes leave the spleen and infiltrate the adventitia of MA in hypertensive animal is unknown. The focus of the contribution of macrophages to the pathophysiology of salt-sensitive hypertension is a novel aspect of this proposal. CD163 and Its Biological Functions CD163 is a member of a scavenger receptor cysteine-rich super family class B (180). In human, it is mapped to the region p13 on chromosome 12 (181). In rats, CD163 has been identified as ED2 antigen and found on the rat chromsome 6 (182). CD163 gene encodes for 1076 amino acid protein with 1003 amino acids as the extracellular part, 24 amino acids as a single transmembrane segment, and 49 amino acids as cytoplasmic domain (183). In general, CD163 is exclusively found on cells of monocytes M! lineage, but only 5-30% of monocytes experesses the receptor (184). CD163 is expressed at high levels in mature tissue M! e.g. splenic red pulp M!, Kupffer cells, dust cells, lymph node M!, thymic M!, and peritoneal M! (180). CD163 have been !"# shown to be involved in pro- and anti-inflammation. TNF-! Interferon-", LPS, or TGF-# causes decrease in the CD163 expression, whereas glucocorticoids, IL-6, or IL-10 induces the increase of CD163 expression (185-189). Some studies show that soluble CD163 may inhibit human T lymphocytes activation and proliferation in vitro (190). However, there are studies reporting that cross-linking human CD163 either with antibodies or hemoglobin-haptoglobin complexes induces the production of NO, IL-1#, IL-6 and TNF-! (191). The specific function of CD163 on M$ is still unclear. It has also been showed that CD163 is involved in the adhesion of monocytes to endothelial cells (192). The best-known function of CD163 is the clearance of fell free hemoglobin from circulation (193). Oxidative stress or inflammatory stimuli e.g. activation of TLR2 or 5 causes the extracellular domain of this receptor to shed from the cell surface and generate a soluble CD163 or sCD163 (194). sCD163 is a new class of M$ specific biomarkers to the detection of coronary artery disease, transplantation, atherosclerosis, and rheumatoid arthritis (195). It has been documented in inflammatory conditions characterized by monocytic infiltration. CD11b and Its Biological Functions CD11 is an integrin !M subunit that forms the heterodimeric integrin !M#2 molecule. It is also known as Mac-1 or CR3. !M#2 belongs to the #2 subfamily integrins and is expressed on the surface of many leukocytes, including monocytes, M$, granulocytes, and natural killer cells (196). The !M subunit of !M#2 integrin is directly involved in causing the adhesion and spreading of the cells but alone, without the #2 subunit, it !"# cannot mediate cellular migration (197). !M"2 integrins are stored in intracellular granules and are rapidly translocated to the cell membrane during cell activation, significantly increasing their expression on the cell surface. Different agonists are able to induce !M"2 integrin expression, including phorbol esters, bacterial formylated peptides, TNF-!, C5a, and LTB4 (198, 199). In addition, the signal-transduction of Land E-selectin can also induce !M"2 integrin expression by neutrophils (200). The ligands for !M"2 integrin are ICAM-1, fibrinogen and iC3b. !M"2 is involved in the phagocytosis of particles that are coated with iC3b. Under inflammatory condition, the level of ICAM-1 expression on endothelial cells, M#, and lymphocytes are upregulated. Integrins are a very important family of cell adhesion molecules involved in both extracellular matrix/cell and cell/cell interactions. As a result, !M"2 integrin is under strict control with multiple levels of regulation. The first level of regulation is the translocation of !M"2 integrin from the granules to the cell membrane. The second level of regulation is through increase avidity in which the receptors are clustered to a specific cell membrane microdomain and activated to increase its affinity for the ligand (201). Both !M and "2 subunit have intracellular tails that interact with cytoskeleton components and cytoplasmic receptors and allows !M"2 integrin to function as a signaltransduction receptor. Two main pathways are triggered through !M"2 are activation of protein kinases (e.g. Src kinases, FAK, Syk-Zap-70 family) and activation of MAPK (202-204). The binding of !M"2 integrin to its ligand triggers MAPK signaling pathways !"# and results in the activation of different transcriptional factors e.g. AP-1 and NF!B (205). These transcriptional factors can upregulate pro-inflammatory cytokines such as IL-1 and TNF-" (206). "M#2 mediates inflammation through its ability to regulate leukocyte chemotaxis, phagocytosis, cell-mediated cytotoxicity, and activation. Deoxycorticosterone acetate (DOCA)-salt Hypertension The proposed studies will use the DOCA-salt model of hypertension in rats. This model was chosen because the sympathetic nervous system participates in the etiology of - hypertension and there is oxidative stress (elevated O2 ) in the vasculature of DOCAsalt rats, with important consequences for the development of hypertension. Interestingly, studies of DOCA-salt hypertension have resulted in controversies because there are different determinants at different phases of the hypertension (207). In order to resolve some of this controversy, we propose to study different phases of hypertension. In addition, DOCA-salt treatment reliably produces hypertension in rodents, but has a gradual onset, allowing the investigation of “pre-hypertensive” changes in blood pressure control systems. We will capitalize on this latter characteristic extensively in our research. Finally, current research indicates excessive mineralocorticoid action contributes to a larger number of cases of clinical hypertension than generally appreciated. The increase in sympathetic nerve activity in DOCA-salt hypertension suggests that there may be alterations in the local mechanisms that modulate sympathetic neurotransmission (208, 209). There is an impaired function of "2R on sympathetic nerves associated with MA in DOCA-salt hypertension (149, 150). In addition, the function of the NE transporter (NET) from sympathetic nerves associated !"# with MA is impaired in DOCA-salt hypertension resulting in an increase in neurogenic constrictions (210). Lastly, purinergic neurotransmission to MA in DOCA-salt hypertensive rats is also impaired due to decreased ATP bioavailability in periarterial sympathetic nerves (211). Recently, a growing number of studies indicate that vascular inflammation may be involved in both the initiation and development of hypertension (212-214). The Nox-based NADPH oxidases produce reactive oxygen species, - especially O2 , contributing to hypertension (215, 216). In a flow-augmented common carotid artery model, there is an increase in M! infiltration into the sustained high blood flow artery and M! depletion reduced vascular remodeling (217). Mice deficient in M! colony-stimulating factor exhibit reduced vascular inflammation and they are protected against damage caused by DOCA-salt hypertension (170). However, it is unclear whether high blood pressure induces M! infiltration into the adventitia of MA and if - infiltrated M! can release O2 that disrupts sympathetic nerve functions causing further increase in blood pressure. This issue will be addressed in the proposed studies. G-Protein-Coupled Receptors Guanine nucleotide-binding protein (G-protein)-coupled receptors (GPCRs) are the largest and most versatile group of cell surface receptors. GPCRs are a superfamily of integral membrane proteins, and possess seven transmembrane domains, three extracellular loops, and three cytosolic loops. They have periplamisc N-terminal domain, and cytosolic C-terminal domain (218). G Proteins !"# The G proteins are a family of signal-coupling proteins that play key roles in many signal transduction processes in cell. They consist of three polypeptides: a guanyl-nucleotide binding ! chain (39-52 kDa), a " chain (35-36 kDa), and a # chain (8 kDa). G proteins are cycle between an inactive GDP state and an active GTP state. When GDP is bound, ! associates with " and # to form a G!"# complex that is membrane bound. When GTP is bound, the G!-GTP dissociates from the G"#. The conversion from GDP to GTP state is slow in the absence of the activated receptor. The activated receptor stimulates the G protein to speed up the rate of exchange of GTP for bound GDP. The ! chain is also a GTPase that converts bound GTP to GDP to terminate activation. The role of G"# is to bind G!-GDP to the receptor. G proteins are classified based on their function and ! subunits. The Gs proteins couples to a stimulatory !-subunit which activates adenylyl cyclase, leading to an increase in cAMP. Gs are ADP-ribosylated by cholera toxin, and Gs alone suffice to propagate the ligand signal from the receptor to the effector. ADP-ribosylation of Gs by cholera toxin inhibits the hydrolysis of bound GTP to GDP, keeping the Gs in its - activated state. Activation of Gs by GTP analog or AlF4 causes reversible dissociation of the "# subunit from the activated ! subunit. This increase in "# subunit slows down the activation of ! subunit. The Gi and Go proteins (Gi/o) proteins activates inhibitory !subunit that blocks adenylyl cyclase. The inhibitory action of Gi/o is mediated by both ! and "# subunits. Gi/o are abundant G protein in the brain and Go comprises about 1% of !"# the membrane bound protein in the brain. Gi/o are targets of PTX ADP-ribosylated. However, Gi is more rapidly ADP-ribosylated by PTX than Go, and Go hydrolyzes GTP more rapidly than does Gi. Gi/o proteins ADP-ribosylated by PTX are permanently trapped in the GDP inactive state because they are unable to bind to the excited receptor. Gq/11 proteins activate PLC and its downstream effectors. Lastly G12 proteins cause activation of the Rho small G-protein. The ! chains of G proteins are nearly identical, whereas their " chains show some differences. The !" subunits of Gs, Gi, Go, are functionally interchangeable (219). !"# RESEARCH GOALS & SPECIFIC AIMS !"# Figure 10: The Proposed Hypothesis As BP increases in DOCA-salt rats, M! infiltrate into the adventitia of mesenteric - arteries. M! release O2 that disrupts "2R function causing increased NE release and further increases in blood pressure. !"# Research Goals More than 65 million Americans have hypertension, which is a major risk for stroke, heart and kidney disease. Hypertension is a complex medical condition but sympathetic nerve activity is elevated in some hypertensive humans and in some animal models of hypertension including the DOCA-salt model. The DOCA-salt model is a salt dependent but rennin-angiotensin independent model of hypertension. In the DOCA-salt model in rats, the function of the !2R regulating NE release from sympathetic nerves supplying arteries is impaired. The cause of this impairment is unknown but this impairment contributes to the increase in sympathetic activity in this model. Overall hypothesis The proposed studies will test the hypothesis that as BP increases in DOCA-salt rats, - M" infiltrate into the adventitia of mesenteric arteries. M" release O2 that disrupts !2R function causing an increase in NE release and further increaseing the blood pressure. This study is novel because it is the first to examine the relationship between M"- derived O2 and the impairment of the !2R function. The overall hypothesis will be tested in 3 specific aims: !"# Specific aim 1 These studies will test the hypothesis that there is a time dependent infiltration of activated M! into the adventitia of MA of DOCA-salt hypertensive rats. This infiltration - is associated with a progressive increase in O2 and impaired "2R function beginning in phase 2. - Measurements of O2 , M! infiltration and activation and sympathetic neuroeffector transmission will be made in rats in the pre-hypertensive stage: Phase 1, Phase 2 and established hypertension. These studies are important for two reasons. Firstly, they will establish when "2R function is impaired. Does this occur early in the onset of DOCA-salt hypertension and therefore is a contributor to hypertension progression or does it occur later in hypertension and therefore is a consequence of the blood pressure increase? Preliminary data indicate that "2R impairment will be first detected during Phase 2 hypertension. Secondly, these studies will establish a relationship between M! infiltration into the vasculature and the development of hypertension. These studies will determine if the vascular inflammatory response is a cause or a consequence of hypertension. Specific aim 2 These studies will test the hypothesis that blockade of M! infiltration into MA reduces blood pressure in phase 2 and established hypertension and preserves "2R function in established hypertension. !!" The studies in Aim 2 will show that there is a requirement for M! infiltration into the vasculature for development of the late phases of DOCA-salt hypertension and impairment of "2R function. These studies will use liposomal clodronate to deplete M! beginning at the start and throughout the period of development of DOCA-salt hypertension. Radiotelemetry will be used to continuously monitor blood pressure in - controls and DOCA-salt rats. Measurements of O2 , M! infiltration and activation and sympathetic neuroeffector transmission will be made in untreated and liposomal clodronate treated DOCA-salt rats at day 28 (established hypertension). Specific aim 3 - These studies will test the hypothesis that O2 uncouples the receptor from its Gprotein. - The studies in Aim 3 will show that O2 disrupts sympathetic nerve function in DOCA salt rats by uncoupling the receptor from the target G-protein. Idazoxan, cocaine, and M119, G#$ inhibitor will be used to show NE vesicular filling/trafficking and G#$-Ca +2 channel coupling is not affected in DOCA-salt rats at day 28 (established hypertension). !"# BIBLIOGRAPHY !"# BIBLIOGRAPHY 1. Hajjar I, Kotchen TA. Trends in prevalence, awareness, treatment, and control of hypertension in the United States, 1988-2000. JAMA 2003;290:199-206. 2. Vasan RS, Larson MG, Lip EP, et al. Impact of high-normal blood pressure on the risk of cardiovascular disease. N Engl J Med 2001;345:1291-1297. 3. World Heath Report 2002: Reducing risks, promoting health life. Geneva, Switzerland: World Health Organization. 2002. 4. Vasan RS, Beiser A, Seshadri S, et al. Residual life-time risk for developing hypertension in middle-aged women and me: The Framingham Heart Study. JAMA 2002;287:1003-1010. 5. Franklin SS, Larson MG, Khan SA, et al. Does the relation of blood pressure to coronary heart disease risk change with aging? The Framingham Heart Study. Circulation 2001;103:1245-1249. 6. Chobanian AV, Bakris GL, Black HR, et al. Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure. Hypertension 2003;42:1206-1252. 7. Carretero OA, Oparil S. Essential hypertension. Part I: definition and etiology. Circulation 2001;101:329-335. 8. Sukor N. Secondary hypertension: A condition not to be missed. Postgrad Med J. 201;1032:706-713. 9. Hyman DJ, Pavlik VN. Characteristics of patients with uncontrolled hypertension in the United States. N. Engl J Med 2001;345:479-488. 10. Whelton PK, He J, Appel LJ, et al. Primary prevention of hypertension: clinical and public health advisory from The National High Blood Pressure Education Program. JAMA 2002;288:1882-1888. 11. Page IH. The mosaic theory of arterial hypertension-its interpretation. Perspect Biol Med 1967;10:325-333. 12. Thomas J, Semenya K, Neser WB, et al. Parental hypertension as a predictor of hypertension in black physicians: the Meharry Cohort Study. J. Natl Med Assoc 1990;82:409-412. !"# 13. Levy D, DeStefano AL, Larson MG, et al. Evidence for a blood pressure gene on chromosome 17: genome scan results fro longitudinal blood pressure phenotypes in subjects from the Framingham Heart Study. Hypertension 2000;36:477-483. 14. Lifton RP, Dluhy RG, Powers M, et al. A chimeric 11!-hydroxylase/aldosterone synthase gene causes glucocorticoid-remediable aldosteronism and human hypertension. Nature 1992;355:262-265. 15. Curnow KM, Slutsker L, Vitek J, et al. Mutations in the CYP11! 1 gene causing congenital adrenal hyperplasia and hypertension cluster in exons 6, 7, and 8. Proc Natl Acad Sci 1993;90:4552-4556. 16. Yanase T, Simpson ER, Waterman MR. 17"-Hydroxylase/17, 20 lyase deficiency: from clinical investigation to molecular definition. Endocr Rev 1991;12:91-108. 17. Geller DS, Farhi A, Pinkerton N, et al. Activating mineralocorticoid receptor mutation in hypertension exacerbated by pregnancy. Science 2000;289:119-123. 18. Shimkets RA, Warnock DG, Bositis CM, et al. Liddle’s syndrome: heritable human hypertension caused by mutations in the !-subunit of the epithelial sodium channel. Cell 1994;79:407-414. 19. Mune F, Rogerson FM, Nikkila H, et al. Human hypertension is caused by mutations in the kidney isozyme of 11!-hydroxysteroid dehydrogenase. Nat Genet 1995;10:394399. 20. Wilson FH, Disse-Nicodeme S, Choate KA, et al. Human hypertension caused by mutations in WNK kinases. Science 2001;293:1107-1112. 21. Perry IJ, Whincup PH, Shaper AG. Environmental factors in the development of essential hypertension. British Medical Bulletin 1994;50:246-259. 22. Skafors ET, Lithell HO. Selinus I. Risk factors for the development of hypertension: a 10-year longitudinal study in middle-aged men. J Hypertension 1991;9:217-223. 23. Ferrannini E, Natali A, Capaldo B, et al. Insulin resistance, hyperinsulinemia, and blood pressure. Hypertension 1997;30:1144-1149. 24. Reaven GM, Hoffman BB. A role for insulin in the etiology and course of hypertension? Lancet 1987;ii:435-437. 25. Creager MA, Liang CS, Coffman JD. Beta adrenergic-mediated vasodilator response to insulin in the human forearm. J Pharmacol Exp Ther. 1985;235:709-714. 26. McKeigue PM. Coronary heart disease in Indians, Pakistanis, and Bangladeshis: etiology and possibilities for prevention. Br Heart J 1992;67:341-342. !"# 27. Kershaw EE, Flier JS. Adipose tissue as an endocrine organ. J Clin Endocrinol Metab 2004;89:2548-2556. 28. INTERSALT Cooperative Research Group. INTERSALT: an international study of electrolyte excretion and blood pressure. Results for 24 hour urinary sodium and potassium excretion. BMJ 1988;297:319-328. 29. Sacks FM, Svetkey LP, Vollmer WM, et al. The DASH-Sodium Collaborative Research Groups. Effects on blood pressure of reduced dietary sodium and the Dietary Approaches to Stop Hypertension (DASH) diet. N. Engl J Med 2001;344:3-10. 30. Prineas RJ, Gomez-Marin O, Sinaiko AR. Electrolytes and blood pressure levels in childhood hypertension: measurement and change. Clin Exp Hypertens A. 1986;8:583604. 31. Weinberger MH. Salt-sensitivity of blood pressure in humans. Hypertension 1996;27:II481-II490. 32. Weinberger MH, Fineberg NS, Fineberg SE, et al. Salt sensitivity, pulse pressure, and death in normal and hypertensive humans. Hypertension 2001;37:429-432. 33. Nakagawa K, Holla VR, Wei Y, et al. Salt-sensitive hypertension is associated with dysfunctional Cyp4a10 gene and kidney epithelial sodium channel. J Clin Invest 2006;116:1696-1702. 34. Roman RJ, Hoagland KM, Lopez B, et al. Characterization of blood pressure and renal function in chromosome 5 congenic strains of Dahl S rats. Am J Physiol 2006;290:F1463-F1471. 35. Elijovich F, Laffer CL. Participation of renal and circulating endothelin in saltsensitive essential hypertension. J Hum Hypertens 2002;16:459-467. 36. Wilcox CS. Oxidative stress and nitric oxide deficiency in the kidney: a critical link to hypertension? Am J Physiol 2005;289:R913-R935. 37. Campese VM, Romoff MS, Levitan D, et al. Abnormal relationship between sodium intake and sympathetic nervous system activity in salt-sensitive patients with essential hypertension. Kidney Int 1982;21:371-378. 38. Laffer CL, Laniado-Schwartzman M, Wang MH, et al. Differential regulation of natriuresis by 20-hydroxyeicosatetraenoic acid in human salt-sensitive versus saltresistant hypertension. Circulation 2003;107:574-578. 39. Whelton PK, He J, Cutler JA, et al. Effects of oral potassium on blood pressure: meta-analysis of randomized controlled clinical trials. JAMA 1997;277:1624-1632. !"# 40. Shaper AG, Wannamethee G. Physical activity and ischemic heart disease in middle-aged British men. Br Heart J 1991;66:384-394. 41. Beilin LJ, Puddey IB. Alcohol and hypertension: an update. Hypertension 2006;47:1035-1038. 42. Xin X, He J, Frontini MG, et al. Effects of alcohol reduction on blood pressure: metaanalysis of randomized controlled trials. Hypertension 2001;38:1112-1117. 43. Cushman WC, Cutler JA, Hanna E, et al. PATHS Group. The Prevention And Treatment of Hypertension Study (PATHS): effects of an alcohol treatment program of blood pressure. Arch Intern Med 1998;152:1197-1207. 44. MacMahon S. Alcohol consumption and hypertension. Hypertension 1987;9:111121. 45. Yan LL, Liu K, Matthews KA, et al. Psychosocial factors and risk of hypertension: the Coronary Artery Risk Development in Young Adults (CARDIA) study. JAMA 2003;290:2138-2148. 46. Schnall PL, Schwarts JE, Landsbergis PA, et al. A longitudinal study of job strain and ambulatory blood pressure: results from a three-year follow-up. Psychosom Med 1998;60:697-706. 47. Freis ED. Hemodynamics of Hypertension. Physiol Rev 1960;40:27-54. 48. Lombard JH. Special issue on microcirculatory adaptations to hypertension. Microcirculation 2002;9:221-223. 49. Amery A, Julius S, Whitlock LS, et al. Influence of hypertension on the hemodynamic response to exercise. Circulation 1967;36:231-237. 50. Cambell M. Natural history of coarctation of the aorta. Br Heart J 1970;32:63-69. 51. Scott HW, Bahnson HT. Evidence for a renal factor on the hypertension of coarctation of the aorta. Surgery 1951;30:206-217. 52. Gidding SS, Rocchini AP, Beekman RH, et al. Therapeutic effect of propranolol on paradoxical hypertension after repair of coarctation of the aorta. N Engl J Med 1985;312:1224-1228. 53. Clarkson P, Nicholson M, Barratt-Boyes BG, et al. Results after repair of coarctationof the aorta beyond infancy. A 10 to 28 year follow-up with particular reference to late systemic hypertension. Am J Caridol 1983;51:1481-1488. !"# 54. Peppard P, Young T, Palta M, et al. Prospective study of the association between sleep-disordered breathing and hypertension. N Engl J Med 2000;342:1378-1384. 55. Carlson JT, Hedner J, Elam M, et al. Augmented resting sympathetic activity in awake patients with obstructive sleep apnea. Chest 1993;103:1763-1768. 56. Goodfriend TL, Calhoun DA. Resistant hypertension, obesity, sleep apnea, and aldosterone: theory and therapy. Hypertension 2004;43:518-524. 57. Phillips SA, Olson EB, Morgan BJ, et al. Chronic intermittent hypoxia impairs endothelium-dependent dilation in cerebral and skeletal muscle resistance arteries. Am J Physiol Heart Circ Physiol 2004;286:H388-H393. 58. Phillips C, Hedner J, Berend J, et al. Diurnal and obstructive sleep apnea influences on arterial stiffness and central blood pressure in men. Sleep 2005;28:604-609. 59. Ploth DW. Renovascular hypertension. In: Jacobson HR, Striker GE, Klahr S, eds. The principles and practice of nephrology, 2nd ed. St. Louis: Mosby-Year Book, 1995:379-386. 60. Pohl MA. Real artery stenosis, renal vascular hypertension and ischemic nephropathy. In: Schrier RW, ed. Disease of the kidney, 7th ed. Philadelphia: Lippincott Williams & Wilkins, 2001:1399-1457. 61. Funder JW, Pearce PT, Smith R, et al. Mineralocorticoid action: target tissue specificity is enzyme, not receptor, mediated. Science 1988;242:583-585. 62. Robinson-Rechavi M, Escriva Garcia H, Laudet V. The nuclear receptor superfamily. J Cell Sci 2003;116:585-586. 63. Odermatt A, Atanasov A. Mineralocorticoid receptors: Emerging complexity and functional diversity. Steroids 2009;74:163-171. 64. Gomez-Sanchez CE, Warden M, Gomez-Sanchez MT, et al. Diverse immunostaining patterns of mineralocorticoid receptor monoclonal antibodies. Steroids 2011;76:1541-1545. 65. Milliez P, Girerd X, Plouin PF, et al. Evidence for an increased rate of cardiovascular events in patients with primary aldosteronism. J Am Coll Cardiol 2005;45:1243-1248. 66. Zhao J, Li J, Li W, et al. Effects of spironolactone on atrial structural remodeling in a canine model of atrial fibrillation produced by prolonged atrial pacing. Br J Pharmacol 2010;159:1584-1594. !"# 67. Lalevee N, Rebsamen MC, Barrere-Lemaire S, et al. Aldosterone increases T-type calcium channel expression and in vitro beating frequency in neonatal rat cardiomyocytes. Cardiovasc Res 2005;67:216-224. 68. Xiao F, Puddefoot JR, Barker S, et al. Mechanism for aldosterone potentiation of angiotensin II-stimulated rat arterial smooth muscle cell proliferation. Hypertension 2004;44:340-345. 69. Lacolley P, Labat C, Pujo A, et al. Increased carotid wall elastic modulus and fibronectin in aldosterone-salt-treated rats: effects of eplernone. Circulation 2002;106:2848-2853. 70. Calo LA, Puato M, Schiavo S, et al. Absence of vascular remodeling in a high angiotensin-II state (Bartter’s and Gitelman’s syndromes): implications for angiotensin II signaling pathways. Nephrol Dial Transplant 2008;23:2804-2809. 71. Nakano S, Kobayashi N, Yoshida K, et al. Cardioprotective mechanisms of spironolactone associated with the angiotensin-converting enzyme/epidermal growth factor receptor/extracellular signal-regulated kinases, NAD(P)H oxdiase/lectin like oxidized low-density lipoprotein receptor-1, and Rho-kinase pathways in aldosterone/salt-induced hypertensive rats. Hypertension Res 2005;28:925-936. 72. Caprio M, Newfell BG, La Sala A, et al. Functional mineralocorticoid receptors in human vascular endothelial cells regulate intercellular adhesion molecule-1 expression and promote leukocytes adhesion. Circ Res 2008;102:1359-1367. 73. Kasal DA, Barhoumi T, Li MW, et al. T regulatory lymphocytes prevent aldosteroneinduced vascular injury. Hypertension 2012;59:324-330. 74. Iwashima F, Yoshimoto T, Minami I, et al. Aldosterone induces superoxide generation via Rac1 activation in endothelial cells. Endocrinology 2008;149:1009-1014. 75. Leopold JA, Dam A, Maron BA, et al. Aldosterone impairs vascular reactivity by decreasing glucose-6-phosphate dehydrogenase activity. Nat Med 2007;13:187-197. 76. Michea L, Delpiano AM, Hitschfeld C, et al. Eplerone blocks nongenomic effects of + + +2 aldosterone on the Na /H exchanger, intracellular Ca levels, and vasoconstriction in mesenteric resistance vessels. Endocrinology 2005;146:973-980. 77. Lariviere R, Thibault G, Schiffrin EL. Increased endothelin-1 content in blood vessels of deoxycorticosterone acetate-salt hypertension but not in spontaneously hypertensive rats. Hypertension 1993;21:294-300. 78. Wehling M, Spes CH, Win N, et al. Rapid cardiovascular action of aldosterone in man. J Clin Endocrinol Metab 1998;83:3517-3522. !"# 79. Yamada M, Kushibiki M, Osanai T, et al. Vasoconstrictor effect of aldosterone via angiotensin II type 1 (AT1) receptor: possible role of AT1 receptor dimerization. Cardiovasc Res 2008;79:169-178. 80. Gros R, Ding Q, Sklar LA, et al. GPR30 expression is required for the mineralocorticoid receptor-independent rapid vascular effects of aldosterone. Hypertension 2011;57:442-451. 81. Christ M, Douwes K, Eisen C, et al. Rapid effects of aldosterone on sodium transport in vascular smooth muscle cells. Hypertension 1995;25:117-123. 82. Barish GD, Downess M, Alaynick WA, et al. A nuclear receptor atlas: macrophage activation. Molecular Endocrinology 2005;19:2466-2477. 83. Lim HY, Muller N, Herold MJ, et al. Glucocorticoids exert opposing effects on macrophage function dependent on their concentration. Immunology 2007;122:47-53. 84. Calo LA, Zaghetto F, Pagnin E, et al. Effect of aldosterone and glycyrrhetinic acid on the protein expression of PAI-1 and p22(phox) in human mononuclear leukocytes. J of Clin Endo and Metab 2004;89:1973-1976. 85. Keidar S, Kaplan M, Pavlotzky E, et al. Aldosterone administration to mice stimulates macrophage NADPH oxidase and increase atherosclerosis development: a possible role for angiotensin-converting enzyme and the receptors for angiotensin II and aldosterone. Circulation 2004;109:2213-2220. 86. Saruta T. Mechanism of glucocorticoid-induced hypertension. Hypertens Res 1996;19:1-8. 87. Akpunonu BE, Mulrow PJ, Hoffman EA. Secondary hypertension: evaluation and treatment: thyrotoxicosis and hypertension. Dis Mon 1996;42:689-703. 88. Biondi B, Klein I. Hypothyroidism as a risk factor for cardiovascular disease. Endocrine 2004;24:1-13. 89. Gennari C, Nami R, Gonnelli S. Hypertension and primary hyperparathyroidism: the role of adrenergic and renin-angiotensin-aldosterone systems. Miner Electrolyte Metab 1995;21:77-81. 90. Michelakis AM, Mizukoshi H, Huang CH, et al. Further studies on the existence of a sensitizing factor to pressor agents in hypertension. J Clin Endo and Met 1975;41:9096. 91. Mizukoski H, Michelakis AM. Evidence for the existence of a sensitizing factor to pressor agents in the plasma of hypertensive patients. J Clin Endocrin 1972;34:10161024. !"# 92. Hsueh WA, Baxter JD. Human prorenin. Hypertension 1991;17:469-477. 93. Sigmund CD, Gross KW. Structure, expression and regulation of the murine renin genes. Hypertension 1991;18:446-457. 94. Keeton TK, Campbell WB. The pharmacologic alteration of renin release. Pharmacol Rev 1980;32:81-227. 95. Kurtz A, Wagner C. Cellular control of renin secretion. J Exp Biol 1999;202:219-225. 96. Kurtz A, Wagner C. Role of nitric oxide in the control of renin secretion. Am J Physiol Renal Physiol 1998;275:F849-F862. 97. Ramaha A, Celerier J, Patston PA. Characterization of different high molecular weight angiotensinogen forms. Am J Hypertens 2003;16:478-483. 98. Lavoie JL, Liu X, Bianco RA, et al. Evidence supporting a functional role for intracellular renin in the brain. Hypertension 2006;47:461-466. 99. Gafford JT, Skidgel RA, Erdos EG, et al. Human kidney “enkephalinase,” a neutral metalloendopeptidase that cleaves active peptides. Biochemistry 1983;22:3265-3271. 100. Skidgel RA, Erdos EG. Angiotensin converting enzyme (ACE) and neprilysin hydrolyze neuropeptides: a brief history, the beginning and follow-ups to early studies. Peptides 2004;25:512-525. 101. Ferrario CM, Trask AJ, Jessup JA. Advances in biochemical and functional roles of angiotensin-converting enzyme 2 and angiotensin-(1-7) in regulation of cardiovascular function. Am J Physiol Heart Circ Physiol 2005;289:H2281-H2290. 102. Santos RA, Simoes E Silva AC, Maric C, et al. Angiotensin-(1-7) is an endogenous ligand for the G protein-coupled receptor mas. Proc Natl Acad Sci 2003;100:8258-8263. 103. Hunyady L, Catt KJ. Pleiotropic AT1 receptor signaling pathways mediating physiological and pathogenic actions of angiotensin II. Mol Endocrinol 2006;20:953-970. 104. Shaltout HA, Westwood BM, Averill DB, et al. Angiotensin metabolism in renal proximal tubules, urine, and serum of sheep: evidence for ACE2-dependent processing of angiotensin II. Am J Physiol Renal Physiol 2007;292:F82-F91. 105. Johren O, Dendorfer A, Dominiak P. Cardiovascular and renal function of angiotensin II type 2-receptors. Cardiovasc Res 2004;62:460-467. !"# 106. Inoue A, Yanagisawa M, Kimura S, et al. The human endothelin family: three structurally and pharmacologically distinct isopeptides predicted by three separate genes. Proc Natl Acad Sci 1989;86:2863-2867. 107. Keller PM, Lee CP, Fenwick AE, et al. Endothelin converting enzyme: substrate specificity and inhibition by novel analogs of phosphoramidon. Biochem Biophys Res Commun 1996;223:372-378. 108. Griffith TM, Edwards DH, Davis RL, et al. Endothelium-dependent responses in the peripheral microcirculation. In: Vanhoutte PM. Ed. Relaxing and Contracting Factors. Clifton NJ: Human, 1988;389-416. 109. Hirata Y, Yoshimi H, Takata S, et al. Cellular mechanism in cultured rat vascular smooth muscle cells. Biochem Biophys Res Commun 1988;154:868-875. 110. Schiffrin EL. Endothelin: potential role in hypertension and vascular hypertrophy. Hypertension 1995;25:1135-1143. 111. Pu Q, Fritsch Neves M, Virdis A, et al. Endothelin antagonism on aldosteroneinduced oxidative stress and vascular remodeling. Hypertension 2003;42:49-55. 112. Rakugi H, Tabuchi Y, Nakamura M. Endothelin activates the vascular reninangiotensin system in rat mesenteric arteries. Biochem Int 1990;21:867-872. 113. Wong-Dusting HK, La M, Rand MJ, Mechanism of the effects of endothelin on responses to noradrenaline and sympathetic nerve stimulation. Clin Exp Pharmacol Physiol 1990;17:269-273. 114. Lariviere R, Day R, Schiffrin EL. Increased expression of endothelin-1 gene in blood vessels of deoxycorticosterone acetate-salt hypertensive rats. Hypertension 1993;21:916-920. 115. Blaustein MP. The physiological effects of endogenous ouabain: control of cell responsiveness. Am J Optom Physiol Opt 1993;264:C1367-C1387. 116. Huang BS, Leenen FH. Brain renin-angiotensin system and ouabain-induced sympathetic hyperactivity and hypertension in Wistar rats. Hypertension 1999;34:107112. 117. Manunta P, Hamilton BP, Hamlyn JM. Salt intake and depletion increase circulating levels of endogenous ouabain in normal men. Am J Physiol Regul Integr Comp Physiol 2006;290:R553-R559. 118. Oparil S, chen YF, Berecek KH, et al. The role of the central nervous system in hypertension. In: Laragh JH, Brenner BM, eds. Hypertension: pathophysiology, diagnosis and management, 2nd ed. New York: Raven Press, 1995;713-740. !!" 119. Chapleau MW, Lu Y, Abboud FM. Mechanosensitive ion channels in blood pressure-sensing baroreceptor neurons. In: Hamill OP, ed. Current topics in membranes, Vol. 59. Elsevier Science, 2007;541-567. 120. Korner P. Baroreceptor resetting and other determinants of baroreflex properties in hypertension. Clin Exp Pharmacolo Physiol Suppl 1989;15:45-64. 121. Chapleau MW, Li Z, Meyrelles SS, et al. Mechanisms determining sensitivity of baroreceptor afferents in health and disease. In: Chapleau MW, Abboud FM, eds. Neuro-cardiovascular regulation: from molecules to man, Vol. 940. The New York Academy of Sciences. 2001:1-19. 122. Abboud FM. The sympathetic system in hypertension: State of the art review. Hypertension 1982;4:208-225. 123. Brooks VL. Chronic infusion of angiotensin II resets baroreflex control of heart rate by an arterial pressure-independent mechanism. Hypertension 1995;26:420-424. 124. Loewy AD. Anatomy of the autonomic nervous system. In: Loewy AD, Speyer KM, eds. Central regulation of autonomic functions. New York: Oxford University Press, 1990:3-16. 125. Osborn JW, Fink GD, Kuroki MT. Neural Mechanisms of Angiotensin II-Salt hypertension: Implications for therapies targeting neural control of the splanchnic circulation. Curr Hypertens Rep 2011;13:221-228. 126. King AJ, Osborn JW, Fink GD. Splanchnic circulation is a critical neural target in angiotensin II salt hypertension in rats. Hypertension. 2007;50:547-556. 127. Eisenhofer G, Kopin IJ, Goldstein DS. Catecholamine metabolism: a contemporary view with implications for physiology and medicine. Pharmacol Rev 2004;56:331-349. 128. Shupliakov O, Haucke V, Pechstein A. How synapsin I may cluster synaptic vesicles. Seminars in Cells & Developmental Biology 2011;22:393-399. 129. Bykhovskaia M. Synapsin regulation of vesicle organization and functional pools. Seminars in Cells & Developmental Biology 2011;22:387-392. 130. Heider MR, Munson M. Exorcising the Exocyst Complex. Traffic 2012;13:898-907. 131. Li L, Chin LS. The molecular machinery of synaptic vesicle exocytosis. Cell. Mol. Life Sci. 2003;60:942-960. !"# 132. Rizo J, Sudhof TC. The membrane fusion enigma: SNAREs, Sec1/Munc18 proteins, and their accomplices-guilty as charged? Annu. Rev. Cell Dev. Biol. 2012;28:279-308. 133. Fernandez-Chacon R, Sudhof TC. Genetics of synaptic vesicle function: Toward the complete functional anatomy of an organelle. Annu. Rev. Physiol. 1999;61:753-776. 134. Weinberger A, Gerst JE. Regulation of SNARE assembly by protein phosphorylation. In: Keranen S, Jantti J eds. Regulatory Mechanisms of Intracellular Membrane Transport, Vol. 10. Springer Berlin Heidelberg. 2004:#145-170. 135. Magga JM, Jarvis SE, Arnot MI, et al. Cysteine string protein regulates G protein modulatin of N-type calcium channels. Neuron 2000;28:195-204. 136. Gerachshenko T, Blackmer T, Yoon E, et al. G!" acts at the C terminus of SNAP25 to mediate presynaptic inhibition. Nature Neuroscience 2005;8:597-605. 137. Ahlquist RP. A study of the adrenotropic receptors. Am J Physiol 1948;153:586600. 138. Bylund DB, Eikenberg DC. Hieble JP, et al. International Union of Pharmacology nomenclature of adrenoceptors. Pharmacol Rev 1994;46:121-136. 139. Cussac D, Schaak S, Gales C, et al. #2B-Adrenergic receptors activate MAPK and modulate the proliferation of primary cultured proximal tubule cells. Am J Physiol Renal Physiol 2002;282:F943-F952. 140. Smith NJ, Luttrell LM. Signal switching, crosstalk and arrestin scaffolds: novel G protein-coupled receptor signaling in cardiovascular disease. Hypertension 2006;48:173-179. 141. Angers S, Salahpour A, Joly E, et al. Detection of !2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc Natl Acad Sci 2000;97:3684-3689. 142. Sanders RD, Maze M. Alpha 2 adrenergic agonists. Curr Opin Investig Drugs 2007;8:25-33. 143. Haubold M, Gilsbach R, Hein L. Alpha 2B adrenoceptor deficiency leads to postnatal respiratory failure in mice. J Biol Chem 2010;285:34213-34219. 144. Gilsbach R, Hein L. Presynaptic metabotropic receptors for acetylcholine and adrenaline/noradrenaline. In: Sudhof TC, Starke K, eds. Handb Exp Pharmacol, Vol. 184. 2007:261-288. !"# 145. Gilsbach R, Roser C , Beetz N, et al. Genetic dissection of alpha 2 adrenoceptor functions in adrenergic versus nonadrenergic cells. Mol Pharmacol 2009;75:1160-1170. 146. Philipp M, Brede ME, Hadamek K, et al. Placental alpha 2 adrenoceptors control vascular development at the interface between mother and embryo. Nat Genet 2002;31:311-315. 147. Sober S, Org E, Kepp K, et al. Targeting 160 candidate gees for blood pressure regulation with a genome-wide genotyping array. PLoS ONE 2009;4:e6034. 148. Brede M, Nagy G, Philipp M, et al. Differential control of adrenal and sympathetic catecholamine release by !2-adrenoceptor subtypes. Mol Endocrinol 2003;17:16401646. 149. Dechamplain J, bouvier M, Drolet G. Abnormal regulation of sympathoadrenal system in deoxycorticosteron acetate salt hypertensive rats. Can J Physiol Pharmacol 1987;65:1605-1614. 150. Park J, Galligan JJ, Fink GD, Swain GM. Alterations in sympathetic neuroeffector transmission to mesenteric arteries but not veins in DOCA-salt hypertension. Auton Neurosci. 2010;152:11-20. 151. Jewell-Motz EA, Liggett SB. G protein-coupled receptor kinase specificity for phosphorylation and desensitization of alpha 2 adrenergic receptor subtypes. J Biol Chem 1996;271:18082-28087. 152. Liang M, Eason MG, Theiss CT, et al. Phosphorylation of Ser360 in the third intracellular loop of the alpha 2A adrenoceptor during protein kinase C-mediated desensitization. Eur J Pharmacol 2002;437:41-46. 153. Agneter E, Singer EA, Sauermann W, et al. The slope parameter of concentrationresponse curves used as a touchstone for the existence of spare receptors. Naunyn Schmiedebergs Arch Pharmacol 1997;356:283-292. 154. Droge W. Free radicals in the physiological control of cell function. Physiol Rev 2001;82:47-95. 155. Griendling KK, Sorescu D, Lassegue B, Ushio-Fukai M. Modulation of protein kinase activity and gene expression by reactive oxygen species and their role in vascular physiology and pathophysiology. Arteriosclerosis Thrombosis and Vascular Biology 2000;20(10):2175-2183. - 156. Dai XL, Galligan JJ, Watts SW et al. Increased O2 production and upregulation of ETB receptors by sympathetic neurons in DOCA-salt hypertensive rats. Hypertension 2004;43:1048-1054. !"# 157. Hare JM. Nitroso-redox balance in the cardiovascular system. N Engl J of Med 2004;351(20):2112-2114. 158. Xu H, Fink GD, Galligan JJ. Tempol lowers blood pressure and sympathetic nerve activity but not vascular O2 in DOCA-salt rats. Hypertension 2004;43:329-334. 159. Lambeth JD. Nox enzymes and the biology of reactive oxygen. Nature Reviews Immunology 2004;4:181-189. 160. Matsuno K, Yamada H, Iwata K et al. Nox1 is involved in angiotensin II-mediated hypertension - A study in Nox1-deficient mice. Circulation 2005;112:2677-2685. 161. Touyz RM. Reactive Oxygen Species, Vascular Oxidative Stress, and Redox Signaling in Hypertension: What is the Clinical Significance? Hypertension 2004;44:248252. 162. Olsen F. Transfer of arterial hypertension by splenic cells from DOCA-salt hypertensive and renal hypertensive rats to normotensive recipients. Acta Path Microbiol Scand Sect 1980;88:1-5. 163. Svendsen UG: Evidence for an initial, thymus independent and a chronic, thymus dependent phase of DOCA and salt hypertension in mice. Acta Path Microbiol Scand 1976;84:523-528. 164. Seaberg EC, Munoz A, Lu M, et al. Association between highly active antiretroviral therapy and hypertension in a large cohort of men followed from 1984 to 2003. AIDS 2005;19:953-960. 165. Bravo Y, Quiroz Y, Ferrebuz A, et al. Mycophenolate mofetil administration reduces renal inflammation, oxidative stress, and arterial pressure in rats with leadinduced hypertension. Am J Physiol Renal Physiol 2007;293:F616-F623. 166. Tran LT, Macleod KM, McNeill JH. Chronic etanercept treatment prevents the development of hypertension in fructose-fed rats. Mol Cell Biochem 2009;330:219-228. 167. Madhur MS, Lob HE, McCann LA, et al. Interleukin 17 promotes angiotensin IIinduced hypertension and vascular dysfunction. Hypertension 2010;55:500-507. 168. Harrison DG, Vinh A, Lob HE. Role of adaptive immune system in hypertension. Curr Opin in Pharmacol 2010;10:203-207. 169. Guzik TJ, Hoch NE, Brown KA et al. Role of the T cell in the genesis of angiotensin II-induced hypertension and vascular dysfunction. Journal of Experimental Medicine 2007;204:2449-2460. !"# 170. Ko EA, Amiri F, Pandey NR et al. Resistance artery remodeling in deoxycorticosterone acetate-salt hypertension is dependent on vascular inflammation: evidence from m-CSF-deficient mice. Am J of Physiol Heart and Cir Physiol 2007;292:H1789-H1795. 171. Pons H, Ferrebuz A, Quiroz Y, Romero-Vasquez F, et al. Immune reactivity to heat shock protein 70 expressed in the kidney is cause of salt sensitive hypertension. Am J Physiol Renal Physiol 2010; [Epub ahead of print]. 172. Hoch NE, Guzik TJ, Chen W et al. Regulation of T-cell function by endogenously produced angiotensin II. Am J of Physiol Reg Integ and Comp Physiol 2009;296:R208R216. 173. Libby P, Ridker PM, Maseri A. Inflammation and atherosclerosis. Circulation 2002;105:1135-1143. 174. Clozel M, Kuhn H, Hefti F, Baumgartner HR. Endothelial Dysfunction and Subendothelial Monocyte Macrophages in Hypertension - Effect of Angiotensin Converting Enzyme-Inhibition. Hypertension 1991;18:132-141. 175. Schmid-Schonbein GW, Seiffge D, Delano FA et al. Leukocyte Counts and Activation in Spontaneously Hypertensive and Normotensive Rats. Hypertension 1991;17:323-330. 176. Dorffel Y, Franz S, Scholze S et al. Preactivated peripheral monocytes in hypertensive patients. Am J of Hyperten 1999;12:19A. 177. Amiri F, Virdis A, Neves MF et al. Endothelium-restricted over expression of human endothelin-1 causes vascular remodeling and endothelial dysfunction. Circulation 2004;110:2233-2240. 178. Ohki R, Yamamoto K, Mano H et al. Identification of mechanically induced genes in human monocytic cells by DNA microarrays. J of Hyperten 2002;20:685-691. 179. Swirski FK, Nahrendorf M, Etzrodt M et al. Identification of Splenic Reservoir Monocytes and Their Deployment to Inflammatory Sites. Science 2009;325:612-616. 180. Fabriek BO, Dijkstra CD, van den Berg TK. The macrophage scavenger receptor CD163. Immunobiology. 2005;210:153-160. 181. Ritter M, Buechler C, Langmann T, et al. Genomic organization and chromosomal localization of the human CD163 (M130) gene: a member of the scavenger receptor cysteine-rich superfamily. Biochem Biophys Res Commun. 1999;260:466-474. !"# 182. Schaer DJ, Schoedon G, Schaffner A. Assignment of the CD163 antigen (CD163) to mouse chromosome 6 band F2 by radiation hybrid mapping. Cytogenet Genome Res. 2002;98:231B. 183. Schaer DJ. The macrophage hemoglobin scavenger receptor (CD163) as a genetically determined disease modifying pathway in atherosclerosis. Atherosclerosis. 2002;163:199-201. 184. Högger P, Dreier J, Droste A, et al. Identification of the integral membrane protein RM3/1 on human monocytes as a glucocorticoid-inducible member of the scavenger receptor cysteine-rich family (CD163). J Immunol. 1998;161:1883-1890. 185. Bächli EB, Schaer DJ, Walter RB, et al. Functional expression of the CD163 scavenger receptor on acute myeloid leukemia cells of monocytic lineage. J Leukoc Biol. 2006;79:312-318. 186. Hintz KA, Rassias AJ, Wardwell K, et al. Endotoxin induces rapid metalloproteinase-mediated shedding followed by up-regulation of the monocyte hemoglobin scavenger receptor CD163. J Leukoc Biol. 2002;72:711-717. 187. Schaer DJ, Boretti FS, Schoedon G, et al. Induction of the CD163-dependent haemoglobin uptake by macrophages as a novel anti-inflammatory action of glucocorticoids. Br J Haematol. 2002;119:239-243. 188. Sulahian TH, Högger P, Wahner AE, et al. Human monocytes express CD163, which is upregulated by IL-10 and identical to p155. Cytokine. 2000;12:1312-1321. 189. Weaver LK, Pioli PA, Wardwell K, et al. Up-regulation of human monocyte CD163 upon activation of cell-surface Toll-like receptors. J Leukoc Biol. 2007;81:663-671. 190. Högger P, Sorg C. Soluble CD163 inhibits phorbol ester-induced lymphocyte proliferation. Biochem Biophys Res Commun. 2001;288:841-843. 191. Polfliet MM, Fabriek BO, Daniëls WP, et al. The rat macrophage scavenger receptor CD163: expression, regulation and role in inflammatory mediator production. Immunobiology. 2006;211:419-425. 192. Vila JM, Padilla O, Arman M, et al. The scavenger receptor cysteine-rich superfamily (SRCR-SF). Structure and function of a group B members. Immunologia 2000;19:105-121. 193. Kristiansen M, Graversen JH, Jacobsen C, et al. Identification of the haemoglobin scavenger receptor. Nature. 2001;409:198-201. !"# 194. Timmermann M, Högger P. Oxidative stress and 8-iso-prostaglandin F(2alpha) induce ectodomain shedding of CD163 and release of tumor necrosis factor-alpha from human monocytes. Free Radic Biol Med. 2005;39:98-107. 195. Kolackova M, Kudlova M, Kunes P, et al. Early expression of FcgammaRI (CD64) on monocytes of cardiac surgical patients and higher density of monocyte antiinflammatory scavenger CD163 receptor in "on-pump" patients. Mediators Inflamm. 2008;2008:235461. 196. Stewart M, Thiel M, Hong N. Leukocyte integrins. Curr. Opin. Cell Biol. 1996;7:690696. 197. Todd RF, Petty HR. Beta 2 (CD11/CD18) integrins can serve as signaling partners for other leukocyte receptors. J. Lab. Clin. Med. 1997;129:492-498. 198. Jutila MA, Rott L, Berg EL, et al. Function and regulation of the neutrophil MEL-14 antigen in vivo: comparison with LFA-1 and Mac-1. J. Immunol. 1989;143:3318-3324. 199. Carlos TM, Harlan JM. Membrane proteins involved in phagocyte adherence to endothelium. Immunol. Rev. 1990;114:5-30. 200. Kuijpers TW, Hakkert BC, Hoogerwerf M, et al. Role of endothelial leukocyte adhesion molecule-1 and platelet-activating factor in neutrophil adherence to IL-1prestimulated endothelial cells. Endothelial leukocyte adhesion molecule-1-mediated CD18 activation. J. Immunol. 1991;147:1369-1376. 201. Gahmberg CG, Tolvanen M, Kotovuori P. Leukocyte adhesion structure and function of human leukoctye !2-integrins and their cellular ligands. Eur J Biochem. 1997;245:215-232. 202. Clark EA, Brugge JS. Integrins and signal transduction pathways: the road taken. Science. 1995;268:233-238. 203. Dedhar S. Integrins and signal transduction. Curr. Opin. Hematol. 1999;6:37-43. 204. Lowell CA, Berton G. Integrin signal transduction in myeloid leukoctyes. J Leukocyte Biol. 1999;65:313-320. 205. Lin TH, Rosales C, Mondal K, et al. Integrin-mediated tyrosine phosphorylation and cytokine message induction in monocytic cells: A possible signaling role for the Syk tyrosine kinase. J. Biol. Chem. 1995;270:16189-16197. 206. McGilvray ID, Lu Z, Wei AC, et al. MAP-kinase dependent induction of monocytic procoagulant activity by !2-integrins. J. Surg. Res. 1998;188:1267-1275. !"# 207. Yemane H, Busauskas M, Kurris S, Knuepfer M. Neurohumoral mechanisms in deoxycorticonsterone acetate (DOCA)-salt hypertension in rats Exp Physiol 2009;95.1:51-55. 208. Schenk J, Mcneill JH. The Pathogenesis of DOCA Salt Hypertension. J of Pharmacol and Toxicol Methods 1992;27:161-170. 209. Tsuda K, Tsuda S, Nishio I, Masuyama Y. Inhibition of Norepinephrine Release by Presynaptic Alpha-2-Adrenoceptors in Mesenteric Vasculature Preparations from Chronic DOCA-Salt Hypertensive Rats. Japanese Heart Journal 1989;30:231-9. 210. Luo M, Fink GD, Lookingland KJ et al. Impaired function of alpha(2)-adrenergic autoreceptors on sympathetic nerves associated with mesenteric arteries and veins in DOCA-salt hypertension. Am J of Physiol Heart and Cir Physiol 2004;286:H1558H1564. 211. Demel SL, Galligan JJ. Impaired purinergic neurotransmission to mesenteric arteries in deoxycorticosterone acetate-salt hypertensive rats. Hypertension 2008;52:322-329. 212. Grundy SM. Inflammation, hypertension, and the metabolic syndrome. JAMA 2003;290:3000-3002. 213. Schillaci G, Pirro M, Gemelli F et al. Increased C-reactive protein concentrations in never-treated hypertension: the role of systolic and pulse pressures. J of Hyperten 2003;21:1841-1846. 214. Dorffel Y, Latsch C, Stuhlmuller B et al. Preactivated peripheral blood monocytes in patients with essential hypertension. Hypertension 1999;34:113-117. 215. Landmesser U, Cai H, Dikalov S et al. Role of p47(phox) in vascular oxidative stress and hypertension caused by angiotensin II. Hypertension 2002;40:511-515. 216. Landmesser U, Dikalov S, Price SR et al. Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell nitric oxide synthase in hypertension. J of Cli Invest 2003;111:1201-1209. 217. Nuki Y, Matsumoto MM, Tsang E et al. Roles of macrophages in flow-induced outward vascular remodeling. J of Cerebral Blood Flow and Metab 2009;29:495-503. 218. Sanders RD, Brian D, Maze M. G-Protein-Coupled Receptors. Handb Exp Pharmacol. 2008;182:93-117. 219. Stryer L, Bourne HR. G Proteins: A family of signal transducers. Ann. Rev. Cell Biol. 1986;2:391-419. !"# CHAPTER 2 EXPERIMENTAL DESIGN & METHODOLOGIES !"# Animals All animals use were approved by the Institutional Animal Care and Use Committee at Michigan State University. Male Sprague-Dawley rats weighing 200 g (Charles River Laboratories, Portage, MI) were acclimated for 5 days before entry into experimental protocols. General Anesthesia Induction and Maintenance The rat was placed in an anesthesia chamber and allowed to breathe a mixture of 2-4% isoflurane in oxygen (2.0 L/min) until a surgical plane of anesthesia is achieved. The animal was positioned on a heated surgical field in dorsal recumbency with its nose inserted into an anesthesia mask and allowed to breathe a mixture of 0.5-2% isoflurane and oxygen (2.0 L/min). The animal was maintained on this anesthesia protocol for the duration of the surgery and closely monitored for the depth of anesthesia. Deoxycorticosterone Acetate (DOCA)-salt Hypertension Induction The animal’s fur on the left flank and the back of the neck, between shoulder blades, was shaved and the skin was disinfected with chlorhexadine scrub and alcohol. A 1.5 cm vertical incision was made at the left flank, through the skin and underlying muscle just caudal to the rib cage. The left kidney was exteriorized and perirenal fat was separated from renal blood vessels. The renal artery, vein and ureter were ligated with 5-0 silk sutures and the kidney was removed. The muscle and skin layers were closed separately with 5-0 silk sutures. A 0.5-1.0 cm incision was made in the shaved area on the back of the neck, and the DOCA-pellet was implanted subcutaneously. Control animals only had uninephrectomy without DOCA-pellet implantation. The DOCA pellet !"# is made up of silastic and DOCA powder in a ratio of two parts silastic to one part DOCA. The DOCA pellet administered is 150 mg per implant per Kg body weight. Post surgery, animals received enrofloxacin antibiotic (5 mg/Kg, i.m.) and carprofen analgesic (5 mg/Kg, s.c.). Rats were housed under standard conditions for 4 weeks. All rats received standard rat chow (Harlan/Teklad 8640 Rodent Diet); however, DOCA and control rats received (ad libitum) salt water (1% NaCl + 0.2% KCl), and distilled water, respectively. Figure 11 summarizes the DOCA-salt hypertension experimental design. !!" Figure 11: Deoxycorticosterone Acetate-Salt (DOCA)-Salt Model DOCA Pellet Silastic: 2 parts DOCA: 150mg/250g BW Right Kidney Ligated Renal Vein and Artery 2 cm dorsal flank incision High Salt Water 1% NaCl + 0.2% KCl # Deoxycorticosterone Acetate The chemical structure at the bottom right is that of DOCA. Male Spraque-Dawley rats are uninephrectomized and received DOCA pellet 150 mg/implant/250 g body weight. Control animals only had uninephrectomy without DOCA pellet implantation. DOCA and control rats received (ad libitum) salt water (1% NaCl + 0.2% KCl), and distilled water, respectively for 4 weeks. !"# Blood Pressure Measurement Tailed-Cuff Plethysmography: Blood pressure in conscious rats were measured using the CODA rat tail-cuff system (Kent Scientific Corporation, Torrington, CT). The animals were restrained and allowed to sit quietly to pre-warm for 10 minutes. The entire measurement process was automated via a computer program that controlled the system. The tail-cuff was inflated five times to 250 mmHg and slowly deflated over a period of 15 seconds. Blood pressure was obtained during each inflation cycle by a volume recording sensor. The reported blood pressure was the average of the five readings. Radiotelemetry: Rat was anesthetized following the protocol in the General Anesthesia Induction and Maintenance section. The anesthetized rat’s fur over the ventral abdomen and left inner thigh region was shaved and the skin was surgically prepared with 3 alternating rounds of chlorhexadine scrub and alcohol. The surgical site was draped with sterile gauze. The catheter of a radiotelemetry-based pressure transmitter (TA11PA-D70, DSI, St. Paul, MN) was implanted into the femoral artery and the body of the transmitter placed subcutaneously at the inner thigh. Rats were allowed 4 days to recover postoperatively; with free access to food and water, each rat was housed in individual cages on top of a radiotelemetry receiver (RPC-1, DSI) that was connected to a data exchange matrix and computerized data acquisition program (Dataquest ART 3.0, DSI) to monitor arterial pressure remotely. Mean arterial pressure was sampled for 10 seconds every minute for the 24 hours period. !"# Immunohistochemistry Second order MA from DOCA-salt and sham rats were excised and cleaned of perivascular fat. MA (1 cm) were fixed in 4% paraformaldehyde overnight at 4°C, blocked with 0.1% Triton X-100 blocking serum for 1 hour. All incubations were done at room temperature, incubated with appropriate 1°antibodies for 2-hour followed by a 1nd hour incubation with appropriate 2 antibodies (Table 4). MA were washed with 0.01 M PBS (composition mM: NaCl 13, KCl 2.7, Na2HPO4 10, and KH2PO4 2) between each incubation. MA were mounted under a glass coverslip with anti-fade gold solution (Vector Laboratories, Burlingame, CA), and images were acquired using confocal microscope (Leica Microsystems, Buffalo Grove, IL). Table 4: Source of Primary and Secondary Antibodies and the Working Dilutions Primary Antibodies Antigen Target CD163 M! p22Phox NADPH Oxidase TNF-" Inflammatory cytokine NPY Sympathetic nerves Secondary Antibodies Target Species Host Species Mouse Donkey Rabbit Donkey Goat Donkey Source AbD Serotec Santa Cruz Santa Cruz Amersham Biosciences Conjugated to FITC Cy3 Cy3 !"# Host Mouse Species Rabbit Goat Rabbit Dilutio 1:200 n 1:400 1:200 1:200 Dilution 1:50 1:400 1:400 Dihydroethidium (DHE) Staining - When DHE reacts with O2 , ethidium bromide is formed and intercalated into DNA yielding a red fluorescent signal when excited at 488 nm. MA were removed from euthanized rats in chilled KRH solution (composition mM: NaCl 130, KCl 1.3, Ca2Cl2 2.2, MgSO4 1.2, KH2PO4 1.2, HEPES 1.0, glucose 0.09; pH = 7.4). Blood vessels were incubated with 2 µM DHE solution for 1 hour at 37°C. Following DHE incubation, blood vessels were washed with KRH solution and mounted for microscopy. Confocal fluorescence images were obtained with 488 nm excitation wavelength and collected at >560 nm wavelengths. Amperometric Measurement of Norephinephrine Carbon Fiber Microelectrode Preparation Heat-treated (3000°C) pitch-base type carbon fiber (Specialty Materials Incorporation, Lowell, MA), with nominal diameter of 35 µm, were attached to a copper wire (2 inches) by silver epoxy. The carbon fiber-copper wire assembly was then inserted into a polypropylene pipette tip (PF2411; Dot Scientific, Burton, MI) and the tapered end was carefully heated in a micropipette puller. The heat softened the polypropylene which caused it to flow over the carbon fiber surface insulating the electrode. The resulting microelectrode possessed a cylindrical architecture with an exposed length of 700-900 µm. Ionic composition in Krebs solution can deteriorate the surface of carbon fiber microelectrode. The oxidation current is found to decay over time after used for several !"# days. The microelectrode may require reconditioning with purified isopropyl alcohol to improve microelectrode sensitivity. Focal Stimulation and Real-time Measurement of NE from Perivascular Nerves Secondary or tertiary MA were isolated, cleaned of fat and connective tissue; they were placed into a small silicone chamber and perfused with 37°C, oxygenated Krebs’ buffer (composition mM: NaCl, 117; KCl, 4.7; CaCl2, 2.5; MgCl2, 1.2; NaHCO3, 25; NaH2PO4, 1.2; dextrose, 11) at flow rate 4 ml/min. Tissues were allowed to equilibrate for 1 hour before beginning experiments. If the experiments required drug treatment, the drug solution was perfused for 40 minutes prior to the recording of NE oxidation currents. The carbon fiber electrode was fixed to a micromanipulator and positioned parallel to the blood vessel so that it detected the NE flux from nearby release sites on its surface. Ag-AgCl reference electrode (Cypress Systems Inc., Fresno, CA) was positioned in the chamber and oxidation currents were recorded with a BioStat Multi-mode potentiostat (ESA Products, Chelmsford, MA). 600 mV applied potential was used to detect NE, and short train of electrical stimulation (10Hz, 5 s, 80V, Grass Instruments; Quincy, MA) was applied to trigger NE release using a bipolar focal stimulation electrode (2 AgCl-coated Ag wires inserted into a double-barreled capillary glass wit tip diameter ~180 µm) positioned along the surface of the vessel. Figure 12 illustrates the experimental set up for the focal stimulation and real-time measurement of NE. !"# Figure 12: Experimental set up for the focal stimulation and real-time measurement A !"# Figure 12 (cont’d) B V VI VII VIII I: 700-900 µm tip II: carbon fiber III: polypropylene pipet tip IV: silver wire V: fiber carbon electrode tip VI: silver epoxy VII: expoxy glue VIII: un-insulated silver wire I II III IV ° A) MA is superfused with 37 C in a flow chamber. A bipolar stimulating electrode (10Hz, 5S, 80V) is used to evolk NE release from perivascular nerves. A recording electrode (holding at 600 mv) is used to detect the oxidation current of NE. The potentiostat converts the current into digital signal, which is displayed on the computer screen. B) The picture of a representive fiber carbon electrode and its parts. !"# Liposomal-encapsulated Clodronate (LEC) Depletion of M!: Clodronate is a member of the family of bisphosphonates that lacks a nitrogen group at the R2 position, and it is considered the best drug to deplete MΦ from organs and tissues in vivo because of its maximum efficacy and minimal toxicity. Clodronate can formed an AppCCl2p-ATP analog intracellularly, which is cytotoxic to M! in vitro. AppCCl2p-ATP analog inhibits mitochondrial oxygen consumption by a mechanism that involves competitive inhibition of the ADP/ATP translocase, and eventually causes the collapse of the mitochondrial membrane potential. By itself clodronate is not a toxic drug; free clodronate will not easily pass phospholipid bilayers. However, when capsulated in a liposome, MΦ will phagocytose clodronate and it will not escape from phagocytic cells. Once the LEC are inside the MΦ lysosomal phospholipases disrupt the membrane and the clodronate accumulates intracellularly until a threshold concentration is reached where the cell is irreversibly damaged initiating apoptosis. Free clodronate released from dead MΦ has very short half-life (15 minutes) in the circulation and it is quickly removed by the kidney. In short, clodronate can be used to remove MΦ in the liver, spleen, lung, peritoneal cavity, and lymph nodes (1). Systemic injections of LEC in mice deplete 90% of peripheral monocytes and tissue M! within 24 hours, and M! reappearance does not take place until around 1 week later (2). Moreover, LEC appears to have a very selective effect on M! and phagocytic dendritic cells, and not on neutrophils and lymphocytes (3, 4). Figure 13 shows the structure of clodronate, its AppCCl2p-ATP analog, the mechanism of cellular damage, and apoptosis. !"# Figure 13: Liposomal-encapsulaed clodronate structure and cellular mechanism of apoptosis Fig. 13. Clodronate, a member of non-nitrogenated bisphosphonates, is encapsulated inside a double lipid bilayer. Upon macrophage phagocytosis of the LEC, clodronate is release by phospholipases. Inside the cell, it forms an AppCCl2p-ATP analog that disrupts the ADP/ATP translocase and causes apoptosis. !"# LEC Prepration: Clodronate was a gift of Roche Diagnostics GmbH. Clodronate (0.6 M) or PBS are encapsulated in liposomes composed of phosphatidylcholine (100 mg/ml) and cholesterol (0.8 mg/ml)(52). ~1% of the clodronate will be encapsulated in the liposomes. The non-encapsulated clodronate will be removed by centrifugation. After washing with sterilized PBS, the LEC are re-suspended into sterilized PBS at 5 mg clodronate/ml. M! Depletion Treatment Plan: The treatment groups to be used for the M!-depletion experiment are outlined in table 5. To remove peritoneal M!, rats were treated with LEC. Control groups received liposome-encapsulated PBS (lipo-PBS). After the animals have been implanted with a radiotemeter for 1 week, DOCA-salt animals received subcutaneously (250 mg/kg) DOCA pellets + either intravenously LEC or lipo-PBS; and similarly sham animals received sham pellets + either LEC or lipo-PBS. The initial dose was 5 mg clodronate/ml per 100 g body weight. Thereafter 2.5 mg clodronate/ml per 100 g body weight was administered intraperitoneally every 7 days. All rats received standard rat chow; however, DOCA, and sham rats received (ad libitum) salt water (1% NaCl + 0.2% KCl), and distilled water, respectively. !"# Table 5: The Treatment Groups for the M!-depletion Experiment Primary Secondary Treatment Sham Treatment PBS liposomes Sham Clodronate liposomes DOCA-salt PBS liposomes DOCA-salt Clodronate liposomes Flow-cytometry After anesthesia, the rat abdomen was exposed and peritoneal cells were collected with 1x HBSS calcium free (Invitrogen). The cells were spun-down at 300-xg centrifugation in 4°C. Erythrocytes were removed by ACK lysing buffer (Invitrogen). Total number of viable leukocytes was determined using Trypan Blue and Automatic Cell Counter (BioRad). In 1 million cells suspension, the Fc receptors were block with Fc Block (BD Pharmingen), and M! were identified with a CD11b-FITC and CD163-PE antibody (AbD Serotec). Lastly, cells were fixed with Cytofix (BD Biosciences). Data was acquired on an LSRII flow cytometer (BD Biosciences). Figure 14 illustrates the mechanisms and principles of how flow-cytometry identified subtypes of leukocytes. !!" Figure 14: Mechanisms and principles of flow-cytometry Forward Scatter Channel (FSC) describes the light that is scattered in the forward direction, typically up to 20° offset from the laser beam’s axis and its intensity roughly equates to the cell’s size. Whereas, the Side Scatter Channel (SSC) defines the light that is measured approximately at a 90° to the excitation line, and it provides information about the granular content within a cell. Base on these two parameters, we could identify types of leukocytes: granulocytes, M!, or lymphocytes. In addition, using fluorescent-labeled antibodies e.g. CD11b-FITC and CD163-PE, we could identify the specific subpopulation within a type of leukocytes, and the intensity of the fluorescent also measures the levels of that protein. !""# Drugs: Idazoxan, UK 14304, H-89, cocaine, forskolin, PTX, NaF and AlCl3 were obtained from Sigma Chemical (St. Louis, MO). M119 was obtained from the chemical diversity set from the Developmental Therapeutics Program from the NCI/NIH. M119 is referenced as compound NSC 119910 within that series. Idazoxan, H-89, cocaine, M119 and NaF and AlCl3 were diluted in deionized water, while UK-14304 and forskolin were dissolved in DMSO to make a concentrated stock solution. Working solution of UK-14304 contained <0.01% of DMSO. Final solutions were made in Kreb’s buffer at the time of experiment. For the PTX experiments, 60 µl of the stock PTX (dissolved in water) is mixed with 2 ml OPTI-MEM buffer to make a PTX working solution (50 µg/500 µl). The MA with its perivascular fats removed is pinned down and incubated in the PTX working solution at 37°C for 2 hours prior to experimental studies. Statistics Data are presented as mean ± SEM and “n” is the number of animals from which the data were obtained. Data were analyzed with Graphpad Prism using Student’s t test, paired t test, one- or two-way ANOVA with Bonferonni’s post hoc test, and nonparametric data were analyzed with Mann-Whitney or Kruskal-Wallis’ test. For multivariate analysis of flow cytometry data, Flowjo 8.8.6 was used for probability binning comparison. Probability binning comparison is nonparametric test designed specifically for analysis of flow-cytometry, particularly when multiple parameters are !"!# measured simultaneously. In order to carry out probability binning comparison, multivariate data must first be divided into multidimensional bins such that the control sample has the same number of events in each bin. Therefore, when selecting an event at random from the control population, there is an equal probability that it will fall into any given bin. The bins defined by the control population are then applied to a comparison sample. The number of events falling within each bin is determined, and the normalized chi-squared value is calculated (5). For all tests, differences were considered significant when P<0.05. $ $ $ $ $ $ $ $ $ !"#$ $ $ $ $ BIBLIOGRAPHY !"#$ BIBLIOGRAPHY 1. VanRooijen N, Sanders A, vandenBerg TK. Apoptosis of macrophages induced by liposome-mediated intracellular delivery of clodronate and propamidine. J Immunol Methods 1996;193:93-9. 2. Sunderkotter C, Nikolic T, Dillon MJ et al. Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response. Journal of Immunology 2004;172:4410-7. 3. Van Rooijen N, Sanders A. Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications. J Immunol Methods 1994;174:83-93. 4. Alves-Rosa F, Stanganelli C, Cabrera J, et al. Treatment with liposome-encapsulated clodronate as a new strategic approach in the management of immune thrombocytopenic purpura in a mouse model. Blood. 2000;96:2834-2840. 5. Roederer M, Moore W, Treister A, et al. Probability Binning Comparision: a metric for quantitating multivariate distribution difference. Cytometry 2001;45:47-55. !"#$ CHAPTER 3 ADVENTITIAL MACROPHAGE INFILTRATION INTO MESENTERIC ARTERIES AND !2 ADRENERGIC AUTORECEPTOR IMPAIRMENT DURING DOCA-SALT HYPERTENSION DEVELOPMENT !"#$ Abstract DOCA-salt hypertension in rats is associated with the impairment of !2- adrenergic autoreceptor (!2R) function, and increased levels of superoxide (O2 ) and macrophage (M") number in the mesenteric arterial (MA) adventitia. However, the - relationship between M" infiltration, O2 production and !2R impairment are unknown. This study tests the hypothesis that there is a time dependent infiltration of activated M" into the MA adventitia of DOCA-salt hypertensive rats. This infiltration is associated - with an increase in O2 and impaired !2R function in later phases of hypertension development in rats. DOCA-salt hypertension developed biphasically with phase 1 - occurring during days 5-10 and phase 2 after day 10. O2 levels in DOCA-salt MA were twice that in control MA during day 10-28. M" numbers in MA from DOCA-salt rats were 4-5x higher than in controls during the same time period. There was no detection of M" infiltration into skeletal muscle arteries from DOCA-salt rats. Impairment of !2R function did not occur until after day 18. Infiltrated M" were in close apposition with phox perivascular sympathetic nerves, and they expressed TNF-! and the p22 subunit of NADPH oxidase. Finally peritoneal M" were activated beginning at day 10; thus, they could be the source of M" in the MA adventitia. The data show that as DOCA-salt hypertension develops peritoneal M" were recruited to the MA adventitia where they - - release O2 . O2 disrupts the !2R function causing increased NE release further increasing blood pressure. !"#$ Introduction One billion people worldwide have hypertension and 60% of Americans are prehypertensive (1). This is a major health concern because hypertension increases the risk for cardiovascular diseases (2). The causes of hypertension are complex, but sympathetic nerve activity is elevated in many hypertensive humans and some animal models of hypertension including the deoxycorticosterone acetate (DOCA)-salt model (3). Increased sympathetic nerve activity in DOCA-salt hypertension suggests that there may be alterations in the local mechanisms that modulate sympathetic neurotransmission (4, 5). !2- adrenergic autoreceptor (!2R) function is impaired at sympathetic nerve endings of mesenteric arteries (MA) in DOCA-salt hypertension (6, 7). Interestingly, there is some controversy about the mechanisms causing DOCA-salt hypertension as these vary over the time course of hypertension development (8). - Reactive oxygen species (ROS) particularly O2 contribute to hypertension development. This conclusion is supported by studies showing that the superoxide dismutase mimetic, tempol, lowers blood pressure and sympathetic nerve activity in DOCA-salt hypertension (9). A major source of O2- is a multi-subunit enzyme, Noxbased nicotinamide adenosine dinucleotide phosphate (NADPH) oxidases, which contributes to hypertension (10, 11). In phagocytes such as macrophages (M!), phox NADPH oxidase has an intracellular p47 subunit and a cytochrome b558 catalytic core composed of membrane bound subunits, Nox-2/gp91 phox Hypertensive stimuli up-regulate p22 phox and p22 phox (12). subunit expression, and enzyme activity. !"#$ Interestingly, mice deficient in this enzyme have a blunted level of DOCA-salt hypertension (13) and inhibition of this enzyme prevented up-regulation of ICAM-1, and M! infiltration into the aorta of Angiotensin II-induced hypertensive rats (14). Despite - the ample evidence that ROS contributes to hypertension, the source of O2 is not entirely clear. Vascular inflammation may be involved in both the initiation and development of hypertension (15, 16, 17). Although it is known that hypertension involves inflammation, most studies focus on the contribution of lymphocytes (18) and there have been no studies of how these cells might interact with periarterial sympathetic nerves. Thus, there is a need to understand the interaction between inflammatory mechanisms and sympathetic nerve function in controlling blood pressure. Recruitment, activation and proliferation of M! in the vascular adventitia occurs in hypertension and this response may contribute to the vascular consequences of hypertension (19, 20). For example, mice deficient in M! colony-stimulating factor exhibit reduced vascular inflammation and they are protected against damage caused by DOCA-salt hypertension (21). However, it is unclear whether high blood pressure induces M! infiltration into the - adventitia of MA and if infiltrated M! release O2 to disrupt sympathetic neuroeffector transmission causing further increases in blood pressure. This study tests the hypothesis that there is a time dependent infiltration of activated M! into the adventitia of the MA of DOCA-salt hypertensive rats. This infiltration is associated with a - progressive increase in O2 and impaired !2R function, which contributes to increased NE release and further increased blood pressure. !"#$ Results - Time dependent M! infiltration and O2 production in MA of DOCA-salt rats The number of CD163 positive M! in DOCA-salt MA adventitia was higher than in control MA (Fig. 15A-B). M! infiltration began at day 10-13 and remained high to day 28 (Fig. 15C). Some M! (Fig. 15E) were located spatially close to perivascular sympathetic nerve fibers (Fig 15D & F). While we detected sympathetic nerve fibers in arteries that supply abdominal skeletal muscle (Fig. 15G), there were no CD163 positive M! (Fig. 15H-I). - The relative level of O2 in the adventitia of MA from DOCA-salt rats (Fig. 16A) was significantly higher compared to controls (Fig. 16B). Semi-quantification of O2 - - levels using DHE fluorescence intensity, we found that O2 level increased in DOCA-salt rats relative to controls at day 10 through day 21 (Fig. 16C). "2R Impaired Function Begins at Day 18 in DOCA-salt Hypertensive Rats Electrical stimulation and amperometry were used to measure real-time NE release from MA. Idazoxan, an "2R antagonist, caused an increase in NE oxidation current equally in DOCA and control during day 3-5 (Fig. 17A) and 10-13 (Fig. 17B). However, during day 18-21, it was not able to increase the NE oxidation current to the level of the controls (Fig. 17C). UK 14304, an "2R agonist, decreased the normalized NE current equally in DOCA and controls during day 3-5 (Fig. 17D) and 10-13 (Fig. 17E); however, the normalized NE current in DOCA-salt was significantly higher than !"#$ that of controls during day 18-21 (Fig. 174F). This pharmacological data suggest that the impairment of !2R occurred during day 18-21 which was much later than the infiltration of M". Time course of M! activation in DOCA-salt hypertensive rats. Using flow-cytometry we found that there are three peritoneal M" populations: fluorescent intensity those with low (20020,000) (Fig. 18A). Between days 3-5 there were no differences in the percentage of M" with low, intermediate or high levels of CD11b expression (Fig. 18B). However, between days 10-21 control rats had a significantly lower percentage of high CD11b intermediate and a higher percentage of CD11b M", respectively (Fig. 18B). Furthermore, when comparing control rats to DOCA-salt rats, CD11b high M" expressed significantly higher levels of CD163 (Fig. 18C). CD11b and CD163 are cell membrane integrin and hemoglobin-haptoglobin scavenger receptors, respectively. These two markers are elevated in activated M" (22, 23). Activated M! express high levels of TNF-" and p22Phox subunit of NADPH oxidase The number of infiltrating M" in DOCA-salt was significantly higher in the adventitia of DOCA-salt compared to control rats (Fig. 19A & D). Furthermore, M" in DOCA-salt rats expressed higher levels of the pro-inflammatory cytokine, TNF-! (Fig. 19B, E & G). M" (Fig. 20A) found in the adventitia of MA from DOCA-salt rats !!"# expressed higher levels of p22 Phox subunit of NADPH oxidase compared to those in control rats (Fig. 20B-C). - The impairment of !2R function, and the elevation of BP, M" number, and O2 level required the synergistic effect of both DOCA and high salt When DOCA-treated or sham-treated rats were placed on distilled water and high salt water, respectively, they did not have elevated BP compared to DOCA-treated rats placed on high salt (Fig. 21A). In addition to not having high BP, these animals also did not have increased numbers of M! infiltration in their MA adventitia (Fig. 21B). - Similarly, the levels of O2 in the MA adventitia of these animals were also not different than that of control animals. Idazoxan, an "2R antagonist, increased the NE oxidation current equally in shams on high salt (Fig. 22A) or DOCA on distilled water (Fig. 22C) when compared to control animals. UK 14304, an "2R agonist, decreased the NE oxidation current equally in shams on high salt (Fig. 22B) or DOCA on distilled water (Fig. 22D) when compared to control animals. !!!" A CD163 B CD163 60 µm D Mesenteric Artery G 60 µm NPY 30 µm Relative Ratio DOCA:Sham Mean # of M!/0.1 mm2 Figure 15: Identification of M! and sympathetic perivascular nerve in rat MA but not skeletal muscle arteries E C Control 3-5 10-13 18-21 >28 Time (days) CD163 Mesenteric Artery H 30 µm F Mesenteric Artery I !!"# NPY+CD163 30 µm Figure 15 (cont’d) G NPY CD163 H I NPY+CD163 Whole-mount immunohistochemical labeling of CD163 positive M! (arrows) in DOCA Skeletal Muscle Artery !"#$%# Skeletal Muscle Artery 30 µm Skeletal Muscle Artery 30 µm 2 (A) and control MA adventitia (B). Normalized mean number of M! /0.1 mm showing 4-5X more M! in DOCA-salt MA adventitia compared to control starting day 10-28 DOCA-salt hypertension (C). The mean number of M! was calculated 2 from 5 areas of 0.1 mm . Data are mean ± SEM and analyzed by one-way ANOVA and Bonferonni's post hoc test. * P<0.05 vs. Control, # P<0.05 vs. Day 3-5, (n=5). Whole-mount immunohistochemical labeling of perivascular sympathetic nerve (arrows) (D), M! (E) in DOCA-salt MA adventitia. F) Overlay of photomicrographs in D and E show a close spatial relationship between M! and sympathetic nerves. Whole-mount immunohistochemical labeling of perivascular sympathetic nerve (arrows) (G), but no M! (H) in arteries supplying abdominal skeletal muscle arteries a. I) Overlay of photomicrographs G and H. !!"# Figure 16: Detection of superoxide anions in rat MA adventitia Photomicrographs showing fluorescence of dihydroethidium (DHE) (arrows) in DOCA (A) and Sham rat MA adventitia (B). 2 Normalized mean fluorescence intensity of DHE/0.1 mm showing 2X higher in DOCA MA adventitia comparing to control starting day 10-21 DOCA-salt hypertension (C). The mean fluorescence intensity of DHE was calculated from 5 areas of 2 0.1 mm . Data are mean ± SEM and analyzed by one-way ANOVA & Bonferonni's post hoc test. * P<0.05 vs. Control, # P<0.05 vs. Day 3-5, (n=5). !!"# Figure 17: !2R is impaired during DOCA-salt day 18-21 A B C !!"# Figure 17 (cont’d) D E F Idazoxan, an !2R antagonist, increased the normalized NE current equally in DOCA and control during DOCA-salt day 35 (A) and 10-13 (B), however, DOCA normalized NE current was significantly less than that of control during DOCA-salt day 18-21 (C). UK 14304, an !2R agonist, decreased the normalized NE current equally in DOCA and control during DOCA-salt day 3-5 (D) and 10-13 (E), however, DOCA normalized NE current was significantly higher than that of control during DOCA-salt day 18-21 (F). Data are mean ± SEM and analyzed by two-way ANOVA & Bonferonni's post hoc test. * P<0.05 vs. Control, (n=5). !!"# Figure 18: Time-course of peritoneal M! activation in DOCA-salt hypertensive rats A C 5 10 CD11b 104 3 10 102 0 0 102 103 CD163 104 105 !!"# Figure 18 (cont’d) B Dot-plot of Day 28 DOCA-salt hypertensive peritoneal M!, showing three different high populations of M!: CD11b low, intermediate, and high (A). Percentage of CD11b M! was significantly higher in DOCA than control during DOCA-salt day 10-13 and 18intermediate 21 (B). In contrast, percentage of CD11b was significantly lower in DOCA than control during the same time periods (B). There were no significant changes in the low CD11b M! population. Data are mean ± SEM and analyzed Kruskal-Wallis' test. # high P<0.05 vs. Control, (n=5). CD163 Fluorescence intensity histogram of CD11b M! population showed a significantly higher express CD163 in DOCA comparing to control (C). Data are analyzed with Flowjo 8.8.6 by using probability binning comparison !!"# Figure 19: Co-localization of M! and TNF-" in rat MA adventitia at day 28 CD163 10 µm DOCA 40 µm 10 µm TNF-! 10 µm DOCA 10 µm 40 µm 10 µm DOCA 40 µm 10 µm CD163 SHAM 40 µm CD163+TNF-! TNF-! SHAM !!"# 40 µm CD163+TNF-! SHAM 40 µm Figure 19 (cont’d) G Whole-mount immunohistochemical labeling of CD163 positive M! (A), TNF-" (B), and overlay of M! with TNF-" (C) in Day 28 DOCA-salt hypertensive rat. Whole-mount immunohistochemical labeling of CD163 positive M! (D), TNF-" (E), and overlay of M! with TNF-" (F) in normotensive rat. (G) Mean M! TNF-" fluorescence intensity showing 3X higher in DOCA MA adventitial M! comparing to control. Data are mean ± SEM and analyzed by paired t test. * P<0.05 vs. Control, (n=5). !"#$ Figure 20: Co-localization of M! and p22Phox subunit of NADPH Oxidase in rat MA adventitia at day 28 CD163 10 µm 40 µm 10 µm CD163+p22Phox p22Phox 10 µm 40 µm 10 µm 10 µm 40 µm 10 µm p22Phox 40 µm !"!# CD163+p22Phox 40 µm CD163 40 µm Figure 20 (cont’d) G Whole-mount immunohistochemical labeling of CD163 positive M! (A), p22Phox (B), and overlay of M! with p22 Phox (C) Phox in Day 28 DOCA-salt hypertensive rat. Whole-mount immunohistochemical labeling of CD163 positive M! (D), p22 (E), and overlay of M! with p22 Phox Phox (F) in normotensive rat. (G) Mean macrophage p22 fluorescence intensity: showing 3X higher in DOCA MA adventitial M! comparing to control. Data are mean ± SEM and analyzed by paired t test. * P<0.05 vs. Control, (n=5). !""# - Figure 21: The elevation of blood pressure, M! number, and O2 level required the synergistic effect of both DOCA and high salt A $$$$ $ $ $ $ $ $ $ $ $B $ $ $ $ !"#$ Figure 21 (cont’d) C Rats that had DOCA pellet implantation or high salt water alone did not show an increase in BP (A). Similarly, treating - with DOCA or high salt alone did not show an increase in the number of M! (B) or O2 level (C) in MA adventitia. * P<0.05 vs. Sham, (n=5). !"#$ Figure 22: The impairment of !2R required the synergistic effect of both DOCA and high salt ! A B !"#$ Figure 22 (cont’d) ! C D Idazoxan, an !2R antagonist, increased the NE oxidation current equally in shams on high salt (A) or DOCA on distilled water (C) when comparing to shams. UK 14304, an !2R agonist, decreased the NE oxidation current equally in shams on high salt (B) or DOCA on distilled water (D) when comparing to shams. Data are mean ± SEM and analyzed by two-way ANOVA & Bonferonni’s post hoc test, n = 4. !"#$ Discussion Our data show that: 1) pro-inflammatory M! infiltrate in the adventitia of MA after - the initial BP increase, 2) high-levels of M! derived O2 disrupts !2R function leading to further increases in BP, 3) peritoneal M! are activated and may be the source of MA adventitial M! 4) lastly, the impairment of !2R function, the elevated BP, the number of - M!, and O2 level required the synergistic effects of DOCA and high salt. Previous studies showed that there is an increased sympathetic nerve activity in DOCA-salt hypertension. In addition to increased sympathetic nerve activity, there is impaired function of !2R on sympathetic nerve terminals, which provides feedback inhibition of NE release (7, 24). !2R impairment may be due to the effects of vascular - oxidative stress, particularly O2 (25). Many human and animal studies show an increase in ROS production during hypertension (9, 10, 11, 12, 13). ROS causes lipid peroxidation, receptor uncoupling and cellular damage. Vascular ROS are primarily - derived from NADPH oxidase, an enzyme catalyzing O2 production (26). M! are a major source of NADPH oxidase derived O2- (10, 11). This study established the - association between M!-derived O2 and !2R function on perivascular sympathetic nerve terminals. Our time-course study shows that there are two phases to DOCA-salt hypertension development. Perhaps, the initiation of hypertension is triggered by sodium retention, which then activates the sympathetic nervous system (27). This modest increase in BP could lead to cellular damage, neo-antigen formation, and !"#$ inflammatory response that further increase BP. This model is supported by studies done in rats that were immuno-tolerant to HSP70 and that developed minimal renal inflammation and were protected from the development of salt-sensitive hypertension (28). Possibly the formation of neo-antigen e.g. damaged-HSP70 is the signal that recruits M! into the MA adventitia of DOCA-salt rats. These M! are pro-inflammatory - and capable of releasing O2 because they express a high level of NADPH oxidase. - Although, our time-course study suggests O2 is derived from M!, there is evidence - suggesting that NADPH oxidase-derived O2 is a stimulus that recruits M! into the aorta of Ang II-induced hypertensive rats (14). However, DOCA-salt model is a renninangiotensin independent model of hypertension. Perhaps, ET-1 is the humoral factor that enhances vascular NADPH oxidase activity and causes a low-level oxidative stress. This low-level of oxidative stress triggers activated M! infiltration and further releases of - O2 that contribute to cellular damage in MA of DOCA-salt rats. - O2 is a reactive and short-lived molecule. However, we show that some infiltrated M! come in close contact with the perivascular sympathetic nerves, which - could allow the O2 to disrupt !2R function. Damage to G-protein receptor signaling by oxidative stress has been demonstrated in renal proximal tubules in primary culture - (29). Thus, a possible mechanism of O2 disruption of !2R function is through uncoupling the G-protein from its receptor. It appears that the infiltration of M! is location specific because we only found M! in the MA and not in skeletal muscle arteries. Perhaps, the reason for this is because the MA bed is located in the peritoneal !"#$ cavity. We also found that the peritoneal M! expresses high level of the adhesion molecule, CD11b. This transmembrane receptor may mediate the attachment of M! to the MA adventitia. Previously, we showed that rats treated with apocynin had reduced vascular oxidative stress and improved !2R function (25). Hence, the later phases of DOCA-salt hypertension may be due to oxidative damage of !2R on the perivascular sympathetic nerves. The data support the hypothesis that M! NADPH oxidase is the - source for vascular O2 . !"#$ BIBLIOGRAPHY !"#$ BIBLIOGRAPHY 1. Chobanian AV, Bakris GL, Black HR et al. Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure. Hypertension 2003;42:1206-1252. 2. Lewington S, Clarke R, Qizilbash N et al. Age-specific relevance of usual blood pressure to vascular mortality: a meta-analysis of individual data for one million adults in 61 prospective studies. Lancet 2002;360:1903-1913. 3. Grassi G. Assessment of Sympathetic Cardiovascular Drive in Human Hypertension Achievements and Perspectives. Hypertension 2009;54:690-697. 4. Schenk J, Mcneill JH. The Pathogenesis of Doca Salt Hypertension. Journal of Pharmacological and Toxicological Methods 1992;27:161-170. 5. Tsuda K, Tsuda S, Nishio I, Masuyama Y. Inhibition of Norepinephrine Release by Presynaptic Alpha-2-Adrenoceptors in Mesenteric Vasculature Preparations from Chronic Doca-Salt Hypertensive Rats. Japanese Heart Journal 1989;30:231-239. 6. Dechamplain J, Bouvier M, Drolet G. Abnormal Regulation of the Sympathoadrenal System in Deoxycorticosterone Acetate Salt Hypertensive Rats. Canadian Journal of Physiology and Pharmacology 1987;65:1605-1614. 7. Luo M, Fink GD, Lookingland KJ, Morris JA, Galligan JJ. Imparied function of !2adrenergic autoreceptors on sympathetic nerves associated with mesenteric arteries and veins in DOCA-salt hypertension. Am J Physiol Heart Circ Physiol 2004;286:H1558-H1564. 8. Yemane H, Busauskas M, Kurris S, Knuepfer M. Neurohumoral mechanisms in deoxycorticonsterone acetate (DOCA)-salt hypertension in rats. Exp Physiol 2010;95:51-55. 9. Xu H, Fink GD, Galligan JJ. Tempol lowers blood pressure and sympathetic nerve activity but not vascular O2- in DOCA-salt rats. Hypertension 2004;43:329-334. 10. Landmesser U, Cai H, Dikalov S et al. Role of p47(phox) in vascular oxidative stress and hypertension caused by angiotensin II. Hypertension 2002;40:511-515. 11. Landmesser U, Dikalov S, Price SR et al. Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell nitric oxide synthase in hypertension. Journal of Clinical Investigation 2003;111:1201-1209. !"!# 12. Lambeth JD. Nox enzymes and the biology of reactive oxygen. Nature Reviews Immunology 2004;4:181-189. 13. Matsuno K, Yamada H, Iwata K et al. Nox1 is involved in angiotensin II-mediated hypertension - A study in Nox1-deficient mice. Circulation 2005;112:2677-2685. 14. Liu J, Yang F, Yang X, Jankowski M, Pagano P. NAD(P)H Oxidase Mediates Angiotensin II-induced Vascular Macrophage Infiltration and Medial Hypertrophy. 2003;23:776-782. 15. Grundy SM. Inflammation, hypertension, and the metabolic syndrome. JamaJournal of the American Medical Association 2003;290:3000-3002. 16. Schillaci G, Pirro M, Gemelli F et al. Increased C-reactive protein concentrations in never-treated hypertension: the role of systolic and pulse pressures. Journal of Hypertension 2003;21:1841-1846. 17. Dorffel Y, Latsch C, Stuhlmuller B et al. Preactivated peripheral blood monocytes in patients with essential hypertension. Hypertension 1999;34:113-117. 18. Harrison DG, Gongora MC. Oxidative Stress and Hypertension. Medical Clinics of North America 2009;93:621-635. 19. Libby P, Ridker PM, Maseri A. Inflammation and atherosclerosis. Circulation 2002;105:1135-1143. 20. Clozel M, Kuhn H, Hefti F, Baumgartner HR. Endothelial Dysfunction and Subendothelial Monocyte Macrophages in Hypertension - Effect of Angiotensin Converting Enzyme-Inhibition. Hypertension 1991;18:132-141. 21. Ko EA, Amiri F, Pandey NR et al. Resistance artery remodeling in deoxycorticosterone acetate-salt hypertension is dependent on vascular inflammation: evidence from m-CSF-deficient mice. American Journal of Physiology-Heart and Circulatory Physiology 2007;292:H1789-H1795. 22. Polfliet MM, Fabriek BO, Daniëls WP, Dijkstra CD, van den Berg TK. The rat macrophage scavenger receptor CD163: expression, regulation and role in inflammatory mediator production. Immunobiology 2006;211:419-425. 23. Kataru RP, Jung K, Jang C, Yang H, Schwendener RA, Baik JE, Han SH, Alitalo K, Koh GY. Critical role of CD11b+ macrophages and VEGF in inflammatory lymphangiogenesis, antigen clearance, and inflammation resolution. Blood 2009;113:5650-5659. 24. Park J, Galligan JJ, Fink GD, Swain GM. Alteration in sympathetic neuroeffector transmission to mesenteric arteries but not veins in DOCA-salt hypertension. Autonomic Neuroscience 2010;152:11-20. !"#$ 25. Demel SL, Dong H, Swain GM, Wang X, Kreulen DL, Galligan JJ. Antioxidant treatment restores prejunctional regulation of purinergic transmission in mesenteric arteries of deoxycorticosterone acetate-salt hypertension rats. Neuroscience 2010;168:335-345. 26.Touyz RM. Reactive oxygen species, vascular oxidative stress, and redox signaling in hypertension what is the clinical significance? Hypertension 2004; 44:248-252. 27. Osborn JW, Fink GD, Kuroki MT. Neural Mechanisms of Angiotensin II-Salt Hypertension: Implications for Therapies Targeting Neural Control of the Splanchnic Circulation. 2011;13:221-228. 28. Pons H, Ferrebuz A, Quiroz Y, Romero-Vasquez F, Parra G, Johnson RJ, Rodriguez-Iturbe B. Immune reactivity to heat shock protein 70 expressed in the kidney is cause of salt sensitive hypertension. Am J Physiol Renal Physiol 2012 Oct 24. [Epub ahead of print]. 29. Asghar M, Banday AA, Fardoun RZ, Lokhandwala MF. Hydrogen peroxide causes uncoupling of dopamine D1-like receptors from G proteins via a mechanism involving protein kinase C and G-protein-coupled receptor kinase 2. Free Radic Biol Med. 2006;40:13-20. !""# CHAPTER 4 MACROPHAGE DEPLETION REDUCES VASCULAR OXIDATIVE STRESS, RESTORES !2 ADRENERGIC AUTORECEPTOR FUNCTION AND ATTENUATED HYPERTENSION DEVELOPMENT IN DOCA-SALT HYPERTENSION !"#$ Abstract There is temporal relationship between impaired function of sympathetic nerve terminal !2-adrenergic autoreceptor (!2R) and adventitial infiltration of pro-inflammatory macrophages (M") in mesenteric arteries (MA) from DOCA-salt hypertensive rats. We - tested the hypothesis that M" release O2 , which disrupts !2R function causing increased norepinephrine (NE) release and further increase of blood pressure in DOCAsalt rats. Liposome-encapsulated clodronate (LEC) was used to deplete adventitial M" 2 in rat MA, (average # M"/0.1 mm , DOCA PBS: DOCA LEC, 39.96: 4.16, p<0.05). M" - depletion reduced vascular O2 (measured using dihydroethidium) (#$ DOCA PBSDOCA LEC: 23.85, p<0.05). !2R function was also restored in M" depleted animals. To establish this we used focal nerve stimulation and amperometry with microelectrodes to measure NE oxidation currents at the adventitial surface of MA in the presence !2R agonist, UK 14304, and antagonist, idazoxan. (Normalized 1%M UK 14304 and idazoxan NE current fold changes respectively, DOCA LEC: DOCA PBS, 0.326: 0.611, & 2.74: 2.18, P<0.05). Lastly, M" depletion attenuated DOCA-salt blood pressure development. (MAP on day 25-28,DOCA LEC: DOCA PBS, 154.8 mmHg: 197.4 mmHg, p<0.05). These data support the hypothesis that M" increased blood pressure in - DOCA-salt rats by releasing O2 , which disrupted !2R function and enhanced sympathetic nerve activity. !"#$ Introduction Blood pressure (BP) regulation is complex but the nervous system plays an essential role, and the sympathetic nerves innervating the splanchnic circulation are particularly important (1). In the periphery, norepinephrine (NE) is released from postganglionic sympathetic nerves and binds to postjunctional !1-adrenergic receptor causing vasoconstriction. It also binds to !2-adrenergic autoreceptor (!2R), a Gi/oprotein coupled receptor, on the prejunctional sympathetic nerve terminal inhibiting further release of NE (2). In the DOCA-salt model in rats, the function of !2R regulating NE release from sympathetic nerves supplying arteries is impaired (3). The cause of this impairment is unknown, but this impairment contributes to the increase in sympathetic activity in this model. Reactive oxygen species (ROS) have been shown to impair G-protein coupled receptor function by uncoupling the receptor from its G-protein (4). Perhaps, vascular - ROS e.g. O2 impairs the function of !2R by a similar mechanism. One of the major - sources of O2 is macrophage (M"). The recruitment, activation and proliferation of M" in the vascular adventitia have been recognized in hypertension (5). In some models of hypertension M" infiltrate into the arterial wall (6). Alterations in the number of circulating monocytes and their activation occur in hypertensive patients and animals (7, 8). Mice deficient in M" colony-stimulating factor exhibit reduced vascular inflammation and they are protected against damage caused by DOCA-salt hypertension (9). Furthermore, INCB3344, an antagonist of CCR2, prevented the infiltration of vascular !"#$ M! and reduced blood pressure in DOCA-salt mice (10). However, it is unclear whether - M!-derived O2 can disrupt !2R and further increase BP in DOCA-salt model. This study tests the hypothesis that M! depletion blocks the infiltration of M! into - mesenteric arteries (MA) adventitia, reduces vascular O2 , preserves the !2R function and prevents the development of BP in the later phases of DOCA-salt hypertension. !"#$ Results LEC attenuated the development of later phases of DOCA-salt hypertension. Using radiotelemetry we examined the effect of LEC on the development of DOCA-salt hypertension. Development of DOCA-salt hypertension was biphasic, phase 1: day 5-18 and phase 2: day >18 (Fig. 23A). Mean arterial pressure (MAP) of DOCAsalt rats treated with LEC was significantly lower during days 23-28 compared to DOCAsalt rats treated with lipo-PBS (Fig. 23A). LEC treatment did not affect the MAP of sham rats (Fig. 23A). Furthermore, there was no difference in heart rate between the two treatment groups of DOCA and of Sham groups (Fig. 23B). These in vivo data show that depletion of perivascular M! prevented the development of the late phase of DOCA-salt hypertension. LEC depleted activated peritoneal M!. M! can phagocytose cellular debris and pathogens. We capitalized on this characteristic and injected LEC into our DOCA-salt rats to deplete the M!. Upon encountering LEC, M! engulfed the molecules, and the cells were killed by clodronate via apoptosis. Using flow-cytometry we found that the percentage of peritoneal M! in DOCA-salt rats treated with LEC was significantly lower than in DOCA-salt rats treated Lipo-PBS (Fig. 24A & B). Further gating on the peritoneal M! population, we found that + + LEC depleted the activated, CD11b /CD163 , peritoneal M! from DOCA-salt rats treated with LEC (Fig. 24C & D). LEC depleted M! in the MA adventitia of DOCA-salt rats. !"#$ Using whole-mount immunohistochemistry, we found that the number of CD163 positive M! in the MA adventitia of DOCA-salt rats treated with LEC was significantly lower than DOCA-salt rats treated with lipo-PBS (Fig. 25A, B & D). This validated that depletion of peritoneal M! would reduce perivascular M! in DOCA-salt rats. Most interestingly, in the MA adventitia of DOCA-salt rats treated with LEC, we detected CD163 positive apoptotic M! cell ghosts (Fig. 25C), and we did not find any M! cell ghosts in DOCA-salt rats treated with lipo-PBS (Fig. 25A). - LEC reduced the levels of O2 in the MA adventitia of DOCA-salt rats. - Using DHE-fluorescence we found that relative levels of O2 in MA of DOCA-salt rats treated with LEC was much lower than in DOCA-salt rats treated with lipo-PBS (Fig. - - 26A & B). Semi-quantification of O2 levels revealed that the levels of O2 in MA of DOCA-salt rats treated with LEC was significantly lower than in DOCA-salt rats treated - with lipo-PBS. Furthermore, LEC also reduced the baseline levels of O2 in control rats - (Fig. 26C). Thus, the depletion of perivascular M! reduced the levels of O2 in DOCAsalt and sham rats. LEC prevented the dysfunction of !2R in DOCA-salt rats. Previously, we have shown that !2R on sympathetic nerve terminals regulating feedback inhibition of NE release in MA is impaired. Using electrical stimulation and amperometry, we measured real-time NE release from MA. Idazoxan, an !2R !"#$ antagonist, failed to increase the NE oxidation current in DOCA-salt rats treated with lipo-PBS (Fig. 27B & C). However, in the DOCA-salt rats that were treated with LEC, idazoxan increased the NE oxidation current (Fig. 27A & C). UK 14304, an !2R agonist, decreased the NE oxidation current in DOCA-salt rats treated with LEC (Fig. 27D & F) but failed to reduce the current in DOCA-salt rats treated with lipo-PBS (Fig. 27E & C). These pharmacological data confirmed that the depletion of perivascular M! prevented !2R dysfunction in DOCA-salt rats. !"#$ Figure 23: M! depletion lower blood pressure during the late phase of DOCA-salt hypertension A B A) MAP of DOCA-salt rats treated with LEC was significantly lower during day 23-28 comparing to DOCA-salt rats treated with lipo-PBS. LEC treatment did not affect the blood pressure in Sham groups. B) There was no significant change in heart rate between two treatment groups of DOCA, and similarly in Shams groups. Data are mean ± SEM and analyzed by two-way ANOVA & Bonferonni’s post hoc test. * P<0.05 vs. LEC, (n=6-7). !"!# Figure 24: Flow-cytometry dot-plots of peritoneal M! !" 5 10 )-%" ./0!12345" #" 9:;<1=#6" ./0!12345" 9>0" )104'" 6601!" SSC-A $ *+&,()-&," 63.5 10 +" )-3 6601!" SSC-A )-4'" 10 5 10 )-%" !"# 10 )-3+" $%&'()'&'" 27.8 )-$" 102 !"# )-$" 102 0 -" 0 -" =?@:5