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Mechanisms of vascular dysfunction in diabetes and hypertension Nagareddy, Prabhakara Reddy 2009

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MECHANISMS OF VASCULAR DYSFUNCTION IN DIABETES AND HYPERTENSION by PRABHAKARA REDDY NAGAREDDY B.Pharm., Bangalore University, 1998 M.Pharm., Rajiv Gandhi University of Health Sciences, 2001 M.Sc., The University of British Columbia, 2005  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in The Faculty of Graduate Studies (Pharmaceutical Sciences)   THE UNIVERSITY OF BRITISH COLUMBIA (VANCOUVER) June 2009  !Prabhakara Reddy Nagareddy, 2009 ! ""! ABSTRACT Cardiovascular complications of diabetes are related in part to abnormal vascular function, a manifestation of the many changes induced in the arterial wall by the metabolic abnormalities accompanying diabetes and insulin resistance. We investigated the biochemical and functional consequences of diabetes and metabolic abnormalities, particularly insulin resistance, on vascular function, using two different animal models, the streptozotocin (STZ)-diabetic rat model of type 1 diabetes and the high fructose diet-fed rat model of insulin resistance and hypertension (FHR).  In STZ diabetic rats, we found that complex biochemical interactions led to changes in hemodynamic variables. Specifically, we found that increased activation of PKC!2 leads to induction of inducible nitric oxide synthase (iNOS), which results in increased production of both nitric oxide and peroxynitrite, causing pressor hypo-responsiveness, depressed cardiac function, mean arterial blood pressure and heart rate and impaired endothelial function in STZ- diabetic rats. Further, we found that hyperglycemia-induced activation of PKC!2 is antecedent to increases in oxidative stress, activation of ERK1/2, NF-"B, and iNOS expression, and the protective effects of PKC! or iNOS inhibition in STZ diabetic rats are associated with inhibition of iNOS-mediated peroxynitrite formation. In contrast to STZ diabetic rats, endothelial dysfunction in FHR is associated with hypertension. In FHR, we investigated the role of matrix metalloproteinases (MMP) and epidermal growth factor receptor (EGFR) transactivation, a novel pathway that has been proposed to link all the pathological features of hypertension including endothelial dysfunction, enhanced vascular tone and hypertrophic growth of cardiovascular tissue. We found that in normal arteries, MMP- EGFR pathway modulates vascular tone, at least in part, via activation of PI3-kinase and mitochondrial ATP synthesis. In FHR arteries, inhibition of MMPs by doxycycline improved ! """! endothelial function while EGFR inhibition by AG1478 promoted vasorelaxation. Further, in insulin resistant vascular smooth muscle cells and arteries from FHR, pharmacological or siRNA inhibition of MMP-EGFR signaling normalized the increased expression and activity of contractile proteins (MLCK, MLC II) and their transcriptional activators (P90RSK and SRF) in addition to the prevention of hypertension in FHR. Our data suggests that the MMP-EGFR pathway could be a potential target in the treatment of hypertension.                   ! "#! TABLE OF CONTENTS Abstract ....................................................................................................................................... ii Table of Contents....................................................................................................................... iv List of Tables .............................................................................................................................. xi List of Figures ........................................................................................................................... xii List of Abbreviations ............................................................................................................... xvi Acknowledgements .................................................................................................................. xxi Co-authorship Statement ....................................................................................................... xxii  1 Introduction ............................................................................................................................. 1 1.1 Diabetes mellitus .................................................................................................................. 1 1.2 Diabetic complications: Acute and chronic ......................................................................... 1 1.2.1 Cardiovascular complications of diabetes .................................................................... 2 1.2.2 Potential mechanisms underlying cardiovascular complications in diabetes............... 3 1.3 Cardiovascular dysfunction in type 1 diabetes..................................................................... 3 1.3.1 Cardiac dysfunction...................................................................................................... 4 1.3.2 Vascular dysfunction .................................................................................................... 5 1.3.3 Potential mechanisms of cardiovascular dysfunction in type 1 diabetes...................... 6 1.3.3.1 Nitric oxide, iNOS and nitrosative stress ................................................................ 6 1.3.3.2 iNOS- a missing link between diabetes and cardiovascular dysfunction?.............. 6 1.3.4 Animal models to study cardiovascular dysfunction in type 1 diabetes:  the streptozotocin (STZ) diabetic model..................................................................... 8 1.4 Vascular dysfunction in insulin resistance (pre-diabetes).................................................... 9 1.4.1 The metabolic syndrome .............................................................................................. 9 1.4.2 Insulin resistance ........................................................................................................ 10 1.4.3 Insulin resistance and hypertension............................................................................ 10 1.4.4 Mechanisms of blood pressure regulation in insulin resistance ................................. 11 1.4.4.1 Nitric oxide and the endothelium.......................................................................... 11 1.4.4.2 Sympathetic nervous system ................................................................................. 12 1.4.4.3 Kidney ................................................................................................................... 12 1.4.4.4 Vascular smooth muscle cell growth promoting pathways................................... 12 1.4.5 Vascular smooth muscle and growth factor receptors................................................ 13 ! "! 1.4.6 Transactivation of the EGFR by GPCR agonists ....................................................... 15 1.4.7 The matrix-metalloproteinases (MMPs)..................................................................... 15 1.4.8 MMP transactivation of the EGFR –missing link in hypertension?........................... 16 1.4.9 Animal models to study vascular dysfunction in insulin resistance: the fructose hypertensive rat ...................................................................................... 17 1.5 Overall rationale, hypothesis and research objectives ....................................................... 19 1.5.1 Overall rationale ......................................................................................................... 19 1.5.2 Study 1: Investigation of the mechanisms and consequences of increased expression of iNOS in STZ-diabetic rats ..................................................................................... 20 1.5.2.1 Hypothesis............................................................................................................. 20 1.5.2.2 Rationale ............................................................................................................... 20 1.5.2.3 Specific research objectives .................................................................................. 21 1.5.3 Study 2: Role of MMPs and the EGFR transactivation pathway in the etiology of hypertension in insulin resistance............................................................ 23 1.5.3.1 Hypothesis............................................................................................................. 23 1.5.3.2 Rationale ............................................................................................................... 23 1.5.3.3 Specific research objectives .................................................................................. 25 1.6 Bibliography....................................................................................................................... 26  2 Selective Inhibition of Protein Kinase C !2 Attenuates Inducible Nitric Oxide    Synthase Mediated Cardiovascular Abnormalities in Streptozotocin -Diabetic Rats .... 40 2.1 Introduction ........................................................................................................................ 41 2.2 Methods.............................................................................................................................. 42 2.2.1 Study design and induction of diabetes ..................................................................... 42 2.2.2 Surgical procedures and hemodynamic measurements .............................................. 43 2.2.3 Collection of tissue samples ....................................................................................... 43 2.2.4 Cell culture studies ..................................................................................................... 44 2.2.4.1 Preparation of isolated rat ventricular cardiomyocytes......................................... 44 2.2.4.2 Preparation and culture of rat aortic vascular smooth muscle (VSMC) cells ....... 44 2.2.5 Preparation of VSMC membrane and cytosol fractions............................................. 45 2.2.6 PKC!2 siRNA studies in rat aortic VSM cells ........................................................... 45 2.2.7 Immunoprecipitation and Western blot analysis ........................................................ 46 ! "#! 2.2.8 Immunohistochemistry and quantification of iNOS and NT immunostain in the heart and SMA sections ................................................................................... 46 2.2.9 Measurement of oxidative stress (superoxide anion formation) in VSM cells .......... 47 2.2.10 Statistical analysis .................................................................................................... 47 2.3 Results ................................................................................................................................ 48 2.3.1 Effect of LY333531 on PKC!2 and iNOS expression in cardiomyocytes and diabetic hearts...................................................................................................... 48 2.3.2 Effect of LY333531 on PKC!2 and iNOS expression in VSM cells and SMAs…….49 2.3.3 Induction of iNOS is PKC!2 dependent in VSM cells............................................... 49 2.3.4 Induction of iNOS by PKC!2 involves ERK1/2 and NF-"B activation in VSMC .... 49 2.3.5 LY333531 inhibition of PKC! reduces oxidative stress in VSM cells...................... 51 2.3.6 In vivo effects of LY333531 on PKC!2, iNOS and nitrotyrosine expression in the heart and mesenteric arteries............................................................................ 51 2.3.7 Effect of PKC! and iNOS inhibition on cardiovascular function in diabetic rats ..... 52 2.4 Discussion .......................................................................................................................... 53 2.5 Tables and figures .............................................................................................................. 57 2.6 Bibliography....................................................................................................................... 74  3 Chronic Inhibition of Inducible Nitric Oxide Synthase Ameliorates Cardiovascular Abnormalities in Streptozotocin Diabetic Rats ................................................................... 79 3.1 Introduction ........................................................................................................................ 80 3.2 Methods.............................................................................................................................. 81 3.2.1Study design and induction of diabetes ....................................................................... 81 3.2.2 Surgical procedures .................................................................................................... 82 3.2.3 Isolated working heart procedure ............................................................................... 83 3.2.4 Western blot studies ................................................................................................... 83 3.2.5 Immunohistochemistry of iNOS and NT in the heart and SMA sections .................. 84 3.2.6 iNOS activity assay in the mesenteric arterial bed ..................................................... 85 3.2.7 Statistical analysis ...................................................................................................... 85 3.3 Results ................................................................................................................................ 85 3.3.1 General characteristics................................................................................................ 85 3.3.2 Effect of L-NIL on mean arterial blood pressure and heart rate ................................ 86 ! "##! 3.3.3 Effect of L-NIL treatment on pressor responses to methoxamine.............................. 86 3.3.4 Effect of L-NIL treatment on cardiac performance.................................................... 86 3.3.5 Effect of L-NIL treatment on eNOS, iNOS and NT expression  in the heart and SMA ................................................................................................ 87 3.4 Discussion .......................................................................................................................... 88 3.5 Tables and figures .............................................................................................................. 93 3.6 Bibliography..................................................................................................................... 103  4 Maintenance of Adrenergic Vascular Tone by Matrix Metalloproteinase Transactivation of the Epidermal Growth Factor Receptor Requires Phosphoinositide-3-Kinase and Mitochondrial ATP Synthesis ....................................... 107 4.1 Introduction ...................................................................................................................... 108 4.2 Methods............................................................................................................................ 109 4.2.1 Microperfusion experiments to study changes in vascular tone............................... 109 4.2.2 Cell culture studies ................................................................................................... 110 4.2.2.1 Preparation of rat aortic vascular smooth muscle cells ....................................... 110 4.2.2.2 siRNA suppression of EGFR, MMP-2 and MMP-7 in VSM cells ..................... 110 4.2.3 Measurement of MMP activity by substrate zymography........................................ 111 4.2.4 Western blotting ....................................................................................................... 111 4.2.5 ATP measurements................................................................................................... 112 4.2.6 Confocal immunofluorescence imaging of phospho-Akt (Ser 473)......................... 112 4.2.7 Preparation of detergent resistant plasma membrane fractions for detection of GLUT4 in VSM cells ........................................................................... 112 4.2.8 Statistical analysis .................................................................................................... 113 4.3 Results .............................................................................................................................. 114 4.3.1 Agonist (PE) induced activation of PI3-kinase is required for maintenance of adrenergic vascular tone in rat small mesenteric arteries .................................... 114 4.3.2 Inhibition of the MMP-EGFR pathway suppress PI3-kinase activation of Akt in VSM cells............................................................................................................. 114 4.3.3 Maintenance of adrenergic vascular tone requires mitochondrial ATP synthesis in VSM cells .................................................................................... 115  ! "###! 4.3.4 Phenylephrine-induced GLUT4 translocation is reduced by the inhibition of PI3-kinase and the MMP-EGFR pathway in VSM cells ..................................... 116 4.3.5 Phenylephrine-induced mitochondrial ATP synthesis activates MMP-7 in vascular tissue ...................................................................................................... 116 4.3.6 Activation of MMP-7 by ATP involves purinergic P2X receptors and calcium...... 117 4.4 Discussion ........................................................................................................................ 118 4.5 Tables and figures ............................................................................................................ 123 4.6 Bibliography..................................................................................................................... 137  5 Inhibition of Matrix Metalloproteinases and the Epidermal Growth Factor Receptor Transactivation Prevents the Development of Hypertension in Insulin Resistant  Rats ....................................................................................................................................... 141 5.1 Introduction ...................................................................................................................... 142 5.2 Methods............................................................................................................................ 144 5.2.1 Experimental design ................................................................................................. 144 5.2.1.1 Animal studies .................................................................................................. 144 5.2.1.2 Ex vivo vascular reactivity studies ................................................................... 145 5.2.1.3 Cell culture studies............................................................................................ 146 5.2.2 Biochemical measurements ...................................................................................... 147 5.2.3 Determination of MMP activity by zymography ..................................................... 147 5.2.4 Western blotting ....................................................................................................... 147 5.2.5 Chemicals and reagents ............................................................................................ 148 5.2.6 Statistical analysis .................................................................................................... 148 5.3 Results .............................................................................................................................. 149 5.3.1 General physical and biochemical characteristics .................................................... 149 5.3.2 Doxycycline prevents PE-induced MMP activation in the cardiovascular tissues of control and FHR .................................................................................................. 149 5.3.3 Doxycycline and AG1478 inhibit the activity but not the expression of MMPs in the arteries ............................................................................................................ 150 5.3.4 Doxycycline and AG1478 prevents the development of hypertension in FHR ....... 151 5.3.5 Doxycycline and AG1478 do not improve insulin sensitivity in FHR .................... 151 5.3.6 Doxycycline but not AG1478 prevents endothelial dysfunction.............................. 152 ! "#! 5.3.7 MMP-2 decreases agonist-induced NO release from endothelial cells.................... 153 5.3.8 Inhibition of the EGFR causes vasorelaxation in PE constricted arteries ................ 154 5.4 Discussion ........................................................................................................................ 154 5.5 Tables and figures ............................................................................................................ 161 5.6 Bibliography..................................................................................................................... 178  6 Agonist Induced Activation of Matrix Metalloproteinases (MMP) Promotes Increased Expression of Contractile Proteins in Insulin Resistant Vascular Smooth Muscle Cells through the Epidermal Growth Factor Receptor (EGFR) Pathway .......... 185 6.1 Introduction ...................................................................................................................... 186 6.2 Methods............................................................................................................................ 188 6.2.1 Cell culture ............................................................................................................... 188 6.2.1.1 SiRNA experiments in VSM cells .................................................................... 189 6.2.1.2 Measurement of MMP activity by substrate zymography................................ 189 6.2.1.3 Western blotting................................................................................................ 190 6.2.2 Animal studies .......................................................................................................... 190 6.2.3 Statistical analysis .................................................................................................... 191 6.3 Results .............................................................................................................................. 191 6.3.1 Modeling insulin resistance in VSM cells................................................................ 191 6.3.2 Effect of PE-stimulation on MMP-EGFR signaling in insulin resistant VSM cells................................................................................................................. 192 6.3.3 Inhibition of MMPs reduces EGFR phosphorylation in insulin resistant VSM cells................................................................................................................. 193 6.3.4 Effect of PE- stimulation on MAP-kinases in insulin resistant VSM cells .............. 193 6.3.5 Inhibition of MMP-EGFR pathway reduces PE-induced ERK activation in insulin resistant VSM cells .................................................................................. 194 6.3.6 Effect of PE-stimulation on the expression of contractile proteins in insulin resistant VSM cells .................................................................................................. 194 6.3.7 Inhibition of MMP-EGFR pathway reduces SRF activation induced by insulin resistance and PE-stimulation in VSM cells ........................................... 195 6.3.8 Treatment of FHR with AG1478 decreases the activation of the EGFR, ERK, SRF and MLC II and prevents the development of hypertension............................ 196 ! "! 6.4 Discussion ........................................................................................................................ 197 6.5 Tables and figures ............................................................................................................ 204 6.6 Bibliography..................................................................................................................... 217  7 Summary and conclusions .................................................................................................. 222 7.1 Summary and conclusions................................................................................................ 223 7.2 Important findings ............................................................................................................ 232 7.3 Future directions............................................................................................................... 234 7.4 Bibliography..................................................................................................................... 238  Appendices .............................................................................................................................. 242 List of publications ................................................................................................................... 243 Animal care certificate.............................................................................................................. 245                ! "#! LIST OF TABLES Table 2.1 General characteristics following 3 weeks of LY333531 and L-NIL treatment in control and diabetic rats ................................................................................................57 Table 3.1 General characteristics of rats following 8 weeks of L-NIL treatment .........................93 Table 5.1 Effect of doxycycline and AG1478 on general physical and plasma biochemical markers in control and high fructose diet fed rats.......................................................161                  ! "##! LIST OF FIGURES Figure 1.1 Schematic representations of two major branches of insulin signal transduction pathway ............................................................................................................... 14 Figure 1.2 Model showing the proposed mechanisms of iNOS induction and its  consequences in STZ diabetic rats ...................................................................... 22 Figure 1.3 Schematic representation of agonist induced MMP-dependent transactivation pathway in vascular smooth muscle cell............................................................. 24 Figure  2.1 LY333531 treatment decreases PKC!2 phosphorylation and inducible nitric oxide synthase expression  in cardiomyocytes ................................................... 58 Figure 2.2 Effect of LY333531 on PKC!2 phosphorylation and iNOS expression in control and diabetic heart tissues ........................................................................ 59 Figure 2.3 Effect of LY333531 on iNOS expression in isolated SMA and VSM cells ......  60 Figure 2.4       Effect of LY333531 on PKC! translocation cultured rat aortic VSM cells ...... 61 Figure 2.5       Effect of siRNA suppression of PKC!2 on iNOS expression in VSM cells....... 62 Figure 2.6       Effect of LY333531 on ERK1/2 and NF-"B activation in VSM cells ............... 63 Figure 2.7 In vivo effects of LY333531 on ERK1/2 phosphorylation in SMAs ................. 64 Figure 2.8       Effect of PKC!2 silencing on ERK1/2 and NF-"B activation in VSM cells ...... 65 Figure 2.9       Effect of mannitol on the expression of iNOS and PKC! .................................. 66 Figure 2.10 Effect of LY333531 on superoxide formation in VSM cells…………………...67 Figure 2.11 Effect of in vivo LY333531 administration on iNOS expression in the heart and SMAs of diabetic and control rats....................................................... 68 Figure 2.12 Effect of in vivo LY333531 administration on nitrotyrosine (NT) levels in the heart and SMAs of diabetic and control rats…………………………………….70 Figure 2.13 Effect of 3 weeks of LY333531 (1 mg/kg/day) or L-NIL (3 mg/kg/day) treatment on hemodynamic parameters in freely moving conscious rats ........... 72  ! "###! Figure 3.1 Effect of 8 weeks of L-NIL treatment on mean arterial blood pressure, MABP and heart rate in freely moving conscious rats.................................................... 94 Figure 3.2 Effect of 8 weeks of L-NIL treatment on pressor responses to bolus doses of methoxamine in freely moving conscious rats.................................................... 95 Figure 3.3 Effect of 8 weeks of L-NIL treatment on cardiac function of isolated working hearts from control and diabetic rats................................................................... 96 Figure 3.4 Effect of L-NIL treatments on eNOS expression in the heart and SMA ............ 97 Figure 3.5 Effect of L-NIL treatments on iNOS expression in the heart and SMA............. 98 Figure 3.6 Photomicrographs illustrating the immunohistochemical localization of iNOS in sections of SMAs of control and diabetic rats.................................. 99 Figure 3.7       Effect of L-NIL treatment on iNOS activity in the mesenteric arterial bed...... 100 Figure 3.8 Effect of L-NIL treatment on NT levels in the heart and SMA........................ 101 Figure 3.9 Photomicrographs illustrating the immunohistochemical localization of NT in sections of heart from control and diabetic rats ................................. 102 Figure 4.1       Maintenance of PE-induced adrenergic vascular tone requires PI3-kinase ...... 123 Figure 4.2 Inhibition of the MMP-EGFR pathway suppresses PE-induced activation of PI3K in VSM cells........................................................................................ 125 Figure 4.3        Effect of EGFR suppression on PE-induced activation of Akt in VSM cells.. 126 Figure 4.4       Effect of MMP-2 suppression on PE-induced activation of Akt in VSM cells 128 Figure 4.5       Effect of MMP-7 suppression on PE-induced activation of Akt in VSM cells 129 Figure 4.6 Stimulation of !1-adrenoceptors increases mitochondrial ATP synthesis in time and concentration dependent manner in VSM cells ............................. 130 Figure 4.7 Effect of the inhibitors of MMP, the EGFR and PI3-kinase on PE- stimulated mitochondrial ATP synthesis in VSM cells...................................................... 131 Figure 4.8 Maintenance of adrenergic vascular tone requires mitochondrial ATP synthesis ................................................................................................... 132 ! "#$! Figure 4.9 Phenylephrine-stimulated GLUT4 translocation is reduced by the inhibitors of MMPs, the EGFR and PI3-kinase in VSM cells .......................................... 133 Figure 4.10 Phenylephrine-stimulated activation of MMP-7 requires mitochondrial ATP synthesis and release ......................................................................................... 134 Figure 4.11 Activation of MMP-7 by ATP involves purinergic P2X receptors and calcium ....................................................................................................... 135 Figure 4.12 Proposed mechanism for agonist-induced signaling of vascular tone, hypertrophic growth and remodeling................................................................ 136 Figure 5.1       MMP-7 activities in control and fructose rats................................................... 162 Figure 5.2 Effect of doxycycline and AG1478 on the expression of MMPs in the arteries of control and FHR............................................................................... 164 Figure 5.3 Effect of doxycycline and AG1478 on the expression of TIMPs in the SMAs of control and FHR ................................................................................ 166 Figure 5.4       Effect of doxycycline and AG1478 on the activation of the EGFR in SMA.... 167 Figure 5.5       Effect of doxycycline and AG1478 on systolic blood pressure in FHR ........... 168 Figure 5.6 Effect of doxycycline and AG1478 treatment on plasma glucose and insulin profiles following an OGTT in control and FHR ............................................. 169 Figure 5.7 Effect of doxycycline and AG1478 treatment on vascular responses to PE and endothelial function in SMAs isolated from control and FHR............. 171 Figure 5.8 Effect of doxycycline and AG1478 on the expression of eNOS in the arteries of control and FHR............................................................................... 173 Figure 5.9 Effect of MMP-2 on A-23187 stimulated NO release in BCAE cells .............. 174 Figure 5.10 Effect of MMP and the EGFR inhibition on the maintenance of adrenergic vascular tone in SMA isolated from control and FHR...................................... 176 Figure 6.1       Insulin resistance induced by high glucose in rat aortic VSM cells.................. 204  ! "#! Figure 6.2 Effect of GM6001 on PE-induced activation of MMP-2 and the EGFR in insulin resistant VSM cells ............................................................................... 205 Figure 6.3 Effect of MMP suppression on PE-induced EGFR transactivation in  insulin resistant VSM cells ............................................................................... 206 Figure 6.4 Effect of EGFR suppression on PE-induced EGFR transactivation in  insulin resistant VSM cells ............................................................................... 207 Figure 6.5 Effect of MMP and EGFR inhibition on PE-induced ERK activation in insulin resistant VSM cells ............................................................................... 208 Figure 6.6 Effect of PE-stimulation on p38MAPK and JNK1 in insulin resistant VSM cells.......................................................................................................... 209 Figure 6.7 Effect of MMP suppression on PE-induced activation of ERK in insulin resistant VSM cells ........................................................................................... 210 Figure 6.8 Effect of MMP and EGFR inhibition on phenylephrine-induced expression  of contractile proteins in insulin resistant VSM cells ....................................... 211 Figure 6.9 Effect of MMP and EGFR inhibition on phenylephrine-induced activation  of transcriptional activators in insulin resistant VSM cells .............................. 213 Figure 6.10 In vivo effects of AG1478 on blood pressure and the expression of  contractile proteins in SMA of FHR................................................................. 215 Figure 7.1 Schematic summary of the MMP-EGFR signaling in VSM cells investigated  in the present thesis ........................................................................................... 231     ! "#$! LIST OF ABBREVIATIONS -dP/dT   Rate of relaxation +dP/dT  Rate of contraction Ach   Acetylcholine ADA   American diabetes association ADAM  A disintegrin and metalloproteinase ADP   Adenosine diphosphate AG1478  4-(3-chloroanillino)-6,7-dimethoxyquinazoline AGE   Advanced glycosylation end product AHA   American heart association Akt   Protein kinase B AMP   Adenosine monophosphate Ang II   Angiotensin-2 ANOVA  Analysis of variance ANS   Autonomic nervous system APMA   Aminophenyl mercuric acetate AR   Adrenergic receptor ATP   Adenosine triphosphate BAPTA 1,2-bis-(o-Aminophenoxy)-ethane-tetra acetic acid BB rat   Biobreeding rat BCAEC  Bovine coronary artery endothelial cells BH4   Tetrahydrobiopterin BP   Blood pressure BSA   Bovine serum albumin cAMP   Cyclic adenosine monophosphate CDA   Canadian diabetes association cGMP   Cyclic guanosine monophosphate CHG   Chronic hyperglycemia CK buffer  Chenoweth-Keolle buffer CMT   Carboxy-methyl transferrin CP1-17  Protein-kinase C-potentiated myosin phosphatase inhibitor CRC   Concentration response curve ! "#$$! DAG   Diacylglycerol DHE   Dihydroethidium DM   Diabetes mellitus DMEM  Dulbecco's modified eagle medium DMSO   Dimethyl sulfoxide DRC   Dose response curve ECM   Extracellular matrix ED 70   Concentration producing 70% for the maximal effect EDHF   Endothelium dependent hyperpolarization factor EDRF   Endothelium derived relaxation factor EDTA   Ethylenediamine tetra acetic acid EGF   Epidermal growth factor EGFR   Epidermal growth factor receptor EGM   Endothelial growth medium EIA   Enzyme immunoassay EIT   S-ethylisothiourea eNOS   Endothelial nitric oxide synthase ERK   Extracellular signal regulated kinase ET-1   Endothelin-1 FAD   Flavin adenine dinucleotide FBS   Fetal bovine serum FHR   Fructose hypertensive rat FMN   Flavin adenine mononucleotide GAPDH  Glyceraldehyde 3-phosphate dehydrogenase GLUT4  Glucose transporter 4 GMP   Guanosine monophosphate GPCR   G-protein coupled receptor GTP   Guanosine triphosphate HB-EGF  Heparin binding- epidermal growth factor HBSS   Hanks balanced salt solution HEPES  4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HG   High glucose ! "#$$$! HR   Heart rate HRP   Horseradish peroxidase IC50   Half maximal inhibitory concentration IF-!   Interferon- ! IGF-1   Insulin like growth factor IgG   Immunoglobulin G IL-1"   Interleukin-1 " IL-6   Interleukin-6 iNOS   Inducible nitric oxide synthase IP3    Inositol triphosphate IR   Insulin resistance IRS-1   Insulin receptor substrate-1 i!B-" Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha i!!B   Inhibitor of nuclear factor kappa B kinase JNK   c-Jun-N-terminal kinase L-NAME  N # -nitro-L-arginine methyl ester hydrochloride L-NIL   L-N6-(1-Iminoethyl)-lysine L-NMMA  N G -monomethyl-L-arginine monoacetate LG   Low glucose LPS   Lipopolysaccharide LVP   left ventricular developed pressure LY333531  (S)-13-[(Dimethylamino) methyl]-tetrahydro-dimetheno, dibenzo pyrrolo- oxadiazacyclohexadecene-1, 3-dione. MABP   Mean arterial blood pressure MALDI-TOF  Matrix-assisted laser desorption/ionization-time of flight MAPK   Mitogen activated protein kinase MEK   Extracellular signal-regulated kinase kinase MLC   Myosin light chain MLCK   Myosin light chain kinase MMP   Matrix metalloproteinase MT-MMP  Membrane type-Matrix metalloproteinase ! "#"! MYPT   Myosin light chain phosphatase NA/ NE  Noradrenaline or norepinephrine NAC   N-acetyl-l-cysteine NAD   Nicotinamide adenine dinucleotide NADH   Nicotinamide dinucleotide reduced NADPH  Nicotinamide adenine dinucleotide phosphate reduced NBF   Neutral buffered formalin NF-!B   Nuclear factor kappa beta NG   Normal glucose NGS   Normal goal serum nNOS   Neuronal nitric oxide synthase NO   Nitric oxide NOD   Non-diabetic mouse NOS   Nitric oxide synthase NOx   Nitrite and nitrate level NT   Nitrotyrosine O2 -    Superoxide anion OGTT   Oral Glucose tolerance test OH -    Hydroxyl ion ONOO-  Peroxynitrite P2X   Purinergic receptor X P2Y   Purinergic receptor Y P90RSK  P90 ribosomal S6 kinase PAGE    Polyacrylamide gel electrophoresis PBS   Phosphate buffered saline PDGF   Plate derived growth factor PDK   Phosphoinositide-dependent kinase PE   Phenylephrine PE-50   Polyethylene-50 PI3K   Phosphatidylinositol -3-kinase PIP2    Phosphatidylinositol biphosphate PKC   Protein Kinase C ! ""! PKG   Protein kinase G PLA2    Phospholipase A2 PLC   Phospholipase C PMSF   Phenylmethylsulphonyl fluoride p.o   Per oral PPADS  Pyridoxal-phosphate-6-azophenyl-2', 4’-disulfonate PVDF   Polyvinylidene difluoride RAS   Renin aldosterone angiotensin system RIA   Radioimmunoassay assay RLC   Regulatory light chain RNS   Reactive nitrogen species ROCK   Rho kinase ROS   Reactive oxygen species SDS   Sodium dodecyl sulfate SEM   Standard error of the mean sGC   Soluble guanylate cyclase SHR   Spontaneously hypertensive rat siRNA   Small interfering RNA SMA   Superior mesenteric artery SNS   Sympathetic nervous system SOD   Superoxide dismutase SRE   Serum response element SRF   Serum response factor STZ   Streptozotocin TBS-T   Tris buffered saline containing 0.1% Tween 20 TCF   Ternary complex factors TGF-!   Transforming growth factor-! TIMP   Tissue inhibitor of matrix metalloproteinase TNF-"   Tumor necrosis factor TXA2    Thromboxane A2 VSM   Vascular smooth muscle WKY   Wistar Kyoto rat ! ""#! ACKNOWLEDGEMENTS It is with great pleasure, deep satisfaction and gratitude that I express my sincere thanks to my research supervisors Drs. John H McNeill and Kathleen M MacLeod for all their guidance and encouragement throughout the course of my graduate studies. I am especially indebted to Dr. McNeill for his enormous support, encouragement, patience and guidance throughout my studies in UBC. I place on record my sincere thanks to the chair Dr. Kishore Wasan and members of my supervisory committee, Dr. Brian Rodrigues, Dr. Michael Allard and Dr. Ismail Laher for their valuable time, suggestions, constructive criticisms and guidance throughout my Ph.D program. Special thanks are owed to Dr. Carlos Fernandez-Patron, our collaborator at The Department of Biochemistry, Faculty of Medicine, University of Alberta, Edmonton, for providing me an opportunity to work in his lab. I also thank his lab members, Fung, Hao Li and Dong for their help and support. My big thanks to Violet Yuen for her tremendous help, technical assistance and advices. I thank all the McNeill lab members, Mary Battell, Moira, Harish, Vijay, Linda, Brian McClure, Sally, Dr. Linfu Yao and Dr. Zhengyuan Xia. I also thank Drs. MacLeod and Rodrigues lab members, Girish, Ashraf, Fang, Minsuk, Prashanth, Fang, Hesham and Dr. Gurong Lin for their assistance and support. My special thanks to Dr. Ujendra Kumar and his lab members particularly, Padmesh. I sincerely acknowledge the support of the faculty members, the technical and administrative staff of the Faculty of Pharmaceutical Sciences. I am grateful to the Health Research Foundation of CIHR/Rx&D, the Heart and Stroke Foundation, Michael Smith Foundation for Health Research and all other people and agencies who provided me with generous financial support. Last but not the least, no words of appreciation for the love and support of Beatriz, Shobha, Harish, Ramu, Thomas, Jayakumar and my other friends and relatives.    ! ""##! CO-AUTHORSHIP STATEMENT Mr. Hesham Soliman, fellow graduate student in the Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences contributed to the chapter-2. His contributions include design of experiments, performing research and data analysis related to experiments involving isolated cardiomyocytes. He has also revised the manuscript that has been submitted to a journal for publication. Mrs. Hao Li and Fung Lan Chow, members of the Dr Fernandez-Patron’s lab at the University of Alberta, Edmonton contributed to zymography, confocal microscopy and perfusion studies described in the chapter 4 of the thesis. Mr. Harish Vasudevan, fellow graduate student at the Division of Pharmacology, Faculty of Pharmaceutical Sciences contributed to the chapters 5 and 6. His contributions include performing ex vivo vascular reactivity studies and systolic blood pressure measurements by tail cuff method.  !   1 1. Introduction 1.1 Diabetes mellitus Diabetes mellitus (DM) is a chronic disorder affecting carbohydrate, fat and protein metabolism. A characteristic and prominent feature of DM is hyperglycemia, a reflection of deranged carbohydrate (glucose) utilization resulting from defect in insulin action or deficient insulin secretory response 1 . Accordingly DM is classified into 2 major groups, type 1 diabetes and type 2 diabetes. Type 1 DM is caused by pancreatic ! cell destruction leading to absolute insulin deficiency (8-10% of all cases). Type 2 DM results from insulin resistance (IR) with relative insulin deficiency or an insulin secretory defect (~ 90% of all cases). The incidence and prevalence of DM is increasing rapidly. In fact it has increased five fold from 1959 to 1993 2 . It is projected that over 300 million people or 5.4% of the world population will be suffering from DM by 2025 3 . Canada, though a developed country, is no exception to the incidence of DM. Approximately 3.5% of all Canadians or 0.9 million people aged 12 or older were diagnosed as having DM by the year 1999 4 . Given the fact that there are large numbers of undiagnosed cases of DM 5 , it is believed that up to 2.6 million Canadians may presently have DM and this number is projected to reach 3 million by 2010 6 . DM is thus a serious health problem throughout the world. 1.2 Diabetic complications: Acute and chronic Acute complications of DM include polyuria, polyphagia, polydipsia, weakness, weight loss, dry skin and less often ketoacidosis 7 . Prior to the discovery of insulin by Banting and Best in the early 1900s, the outlook for this disease was very grim, as patients would succumb to fatal consequences due to severe acute complications such as ketoacidosis. However, with the introduction of insulin therapy, mortality due to acute diabetic complications decreased dramatically but the prolongation of the survival still carried a high price in terms of morbidity.   2 Two decades after insulin had become available, the clinical impact of complications arising from chronic diabetes became much more evident when a growing number of reports documented the occurrence of chronic complications. These include macrovascular diseases (peripheral, cerebral, cardiovascular), microvascular diseases (retinopathy and nephropathy), neuropathy (peripheral and autonomic), foot problems and a host of other complications. It is of interest to note that all the above complications were observed despite efforts to attain tight metabolic control with insulin therapy 8 . The present understanding is that, regardless of the etiology, long term diabetes often leads to a cluster of abnormalities resulting in damage, dysfunction and organ failure including heart, kidneys, nerves and eyes. Clinically these consequences are manifested in the form of end stage renal failure, neuropathy, occular disorders and cardiac diseases such as angina, atherosclerosis, cardiomyopathy, stroke, myocardial infarction, heart failure, hypertension etc. 1.2.1 Cardiovascular complications of diabetes Cardiovascular disease is the leading cause of mortality among patients with DM 8, 9 . The incidence of cardiovascular disease is three to four fold higher in the presence of diabetes than in patients without diabetes. It is reported that up to 80% of deaths in diabetic patients are due to cardiovascular events. In fact the American Heart Association (AHA) considers diabetes to be a cardiovascular disease 10 . For the sake of simplicity, cardiovascular complications in DM can be categorized into three major groups as follows, • Diseases of large vessels (macroangiopathy) may lead to the development of coronary artery disease, atherosclerosis, peripheral vascular disease, stroke, hypertension and myocardial infarction. • Diseases of microvasculature (microangiopathy), characterized by structural and functional alterations in the vessels such as basement membrane thickening, increased vascular   3 permeability and microaneurysms, may contribute towards the development of diabetic retinopathy, neuropathy, nephropathy, cardiomyopathy and gangrene. • Cardiac muscle disease, also known as cardiomyopathy occurs independently of any major vascular disease in diabetes 11 . 1.2.2 Potential mechanisms underlying cardiovascular complications in diabetes Although chronic hyperglycemia may be the most important cause of cardiovascular disease in DM, a number of equally tenable mechanisms such as lipid abnormalities and inflammation have been proposed to initiate or mediate the cardiovascular complications of DM. While the molecular mechanisms leading to chronic complications have not been conclusively delineated, several abnormal pathways have been suggested. These include increased advanced glycosylation end product (AGE) formation, increased polyol pathway activity, activation of protein kinase C (PKC) isoforms and increased hexosamine pathway activity. The possibility of a common factor linking all these pathways, which was initially a matter of debate has been resolved by a recent finding by Nishikawa et al 12  who suggested that mitochondrial production of superoxide anion is the basis of all cardiovascular complications in diabetes. 1.3 Cardiovascular dysfunction in type 1 diabetes Clinical studies indicate that cardiovascular dysfunction is a major cause of death in patients with Type 1 diabetes. Whether these impairments are a consequence of direct cardiovascular deficits (such as abnormalities of the heart and vasculature apart from neural defects) and /or indirect autonomic neuropathy is still unclear. However, epidemiological studies suggest that cardiac dysfunction, either alone or in combination with vascular injury can significantly increase morbidity and mortality in diabetic patients 13, 14 .      4 1.3.1 Cardiac dysfunction A number of in vitro and in vivo studies have shown depressed cardiac contractility in various models of Type 1 DM. Following injection of streptozotocin (STZ), isolated cardiac myocytes have been shown to exhibit impaired contractility both in short and long term diabetes 15, 16 . Studies using isolated working hearts have demonstrated impaired left ventricular pressure development (LVP), rate of contraction (+dP/dT) and rate of relaxation (-dP/dT) in diabetic rats 17, 18 . In vivo studies from our lab and elsewhere have reported a lower resting heart rate (HR), mean arterial blood pressure (MABP), attenuated pressor responses and impaired cardiac performance in STZ diabetic rats 19-23 . Further, results of insulin treatment of STZ diabetic rats suggest that changes in heart function are due to insulin deficiency or  hyperglycemia 24, 25 . Diabetes associated changes in cardiac function in STZ diabetic rats are not entirely consistent with alterations observed in diabetic patients. The differences observed between the clinical and experimental scenario may be due to the variation in species and method of induction of diabetes. Further, treatment in patients is usually initiated almost immediately following diagnosis of diabetes, which is not the case with diabetic animals. However, some of the cardiac abnormalities in animal models are mirrored in human diabetes. For example, similar to depressed MABP and heart rate in STZ diabetic rats, clinical studies indicate that diabetes is associated with normal or low blood pressure. Contrary to the popular belief that all diabetic patients are hypertensive, hypertension is seen only in type 1 diabetic patients who also have a major renal disease 26 . Other abnormalities that are observed in humans include ventricular dysfunction with diastolic abnormalities 27 , impaired vagal and sympathetic control of heart, blunted tachycardiac responses to exercise 28 , to drug-induced reductions in arterial pressure 29  and to postural changes 30  and a decrease in circadian variation in HR. All the above factors contribute significantly to higher mortality rate in diabetic patients.   5 1.3.2 Vascular dysfunction A large number of studies have looked at the effect of diabetes on vascular morphology and function using a wide variety of animal and human vascular beds 31 . There are considerable discrepancies among the results reported so far. Most of the studies have demonstrated impaired, contractile and relaxant effects to vasoactive agents in diabetic animals. Among these, altered responses to noradrenaline (NA), methoxamine and calcium have been reported in isolated diabetic arteries 32, 33 . In vitro studies utilizing large conductance vessels such as aorta and smaller conductance vessels such as mesenteric arteries have shown either a reduced or enhanced contractility to vasoconstrictor agents 34, 35 . The reasons for these inconsistencies are not apparent, but the variability is generally attributed to the differences in the type of diabetogens used, gender differences, the techniques used to measure contractility and, most important of all, the progression, duration and severity of the disease. In contrast, most of the in vivo studies with STZ diabetic rats have demonstrated attenuated pressor responses to vasoconstrictor agents. Diminished pressor responses to NA, angiotensin-2 (Ang II), phenylephrine (PE) and methoxamine have been reported in conscious STZ diabetic rats 20, 21, 23, 36 . In addition, pressor responses to acetylcholine were also depressed in STZ diabetic rats 21, 37 . These results suggest an impairment of endothelial function in diabetic arteries. Studies assessing endothelial function in humans with DM have also reported impaired vasodilatation to infusions of muscarinic agonists such as acetylcholine and carbachol 38, 39 . These results suggest that vascular relaxation and contraction are both impaired in human and animal models of type 1 diabetes. The impairment of cardiac and vascular function in DM may contribute to a state of cardiovascular depression characterized by impaired cardiac performance, depressed resting heart rate and blood pressure, attenuated pressor responses to vasoactive agents and inadequate organ perfusion. Various mechanisms have been proposed to explain the pathogenesis of   6 cardiovascular dysfunction in DM. Among these inducible nitric oxide synthase (iNOS) mediated formation of NO and associated oxidative stress are being explored as potential mechanisms. 1.3.3 Potential mechanisms of cardiovascular dysfunction in type 1 diabetes 1.3.3.1 Nitric oxide, iNOS and nitrosative stress It is very well known that NO has physiologically beneficial effects on the cardiovascular system including the regulation of vascular tone, cardiac contractility, cell growth, vascular remodeling and baroreflex functions 40 . Most of the evidence for NO deficiency in diabetes has been derived from studies on the endothelium where NO synthesis is regulated by eNOS, which may respond differently to chronic hyperglycemia than does NO produced from other sources. NO in macrophages, monocytes, epithelial cells, VSM cells, hepatocytes and many other tissues of the body is synthesized by iNOS 41 . The gene expression of iNOS is modulated by NF-!B, which in turn can be activated by hyperglycemia and oxidative stress. Overexpression of iNOS results in an increased generation of NO 42 . Enhanced NO production in concert with overproduction of superoxides results in formation of peroxynitrite 43 . Peroxynitrite formation, in turn, leads to increased levels of nitrosative stress and may cause cardiovascular abnormalities such as depressed blood pressure and heart rate and attenuated pressor responses to various vasoactive agents in diabetes 44, 45 . 1.3.3.2 iNOS- a missing link between diabetes and cardiovascular dysfunction? In hyperglycemic conditions, there is an overproduction of both superoxide and NO favoring the production of the toxic reaction product, peroxynitrite anion. Peroxynitrite in turn can oxidize BH4, a iNOS cofactor, to dihydrobiopterin 46 . Under conditions of BH4 deficiency, NOS is in an uncoupled state, which means that electrons flowing from the NOS reductase domain are diverted to molecular oxygen rather than to L-arginine, resulting in the production of superoxide instead of NO. Exposure to peroxynitrite during hyperglycemia also produces an uncoupling   7 state of eNOS, presumably via a zinc depletion of the enzyme, favoring superoxide over production 47 . The cytotoxic actions of peroxynitrite are due to its strong ability to oxidize sulfhydryl groups in proteins and to cause lipid peroxidation and nitration of amino acids such as tyrosine that, in turn, can affect many signal transduction pathways. Nitrotyrosine, an indirect marker of peroxynitrite formation, is associated with increased apoptosis in cardiac myocytes, endothelial cells and fibroblasts in heart biopsies from diabetic patients 48 , in hearts from STZ diabetic rats 49 , and in isolated working hearts exposed to high glucose concentrations 50 . Increased formation of peroxynitrite may cause cardiovascular depression and vascular hyporeactivity in diabetes 51 . The mechanisms by which peroxynitrite causes cardiac depression are not clear but it is suggested that peroxynitrite depresses cardiac contractility by decreasing the Ca 2+ sensitivity of contractile elements 52 . Studies have demonstrated that infusion of peroxynitrite into working hearts impairs cardiac contractile function by decreasing cardiac efficiency 53 . Other studies have reported that peroxynitrite can cause direct oxidation of catecholamines 54  and reduces binding capacity of endogenous agonists to !-adrenergic receptors (!-1A and !-1D) 55  thereby decreasing the vascular reactivity to vasoactive agents. Overall, the results of these studies strongly indicate that, under conditions of increased oxidative stress, iNOS play a crucial role in abetting the formation of peroxynitrite, which in turn causes cardiovascular depression by contributing greatly to nitrosative stress. Interventions with strong antioxidants or inhibitors of iNOS should therefore reduce nitrosative stress and improve cardiovascular function in diabetes 56 .      8 1.3.4 Animal models to study cardiovascular dysfunction in type 1 diabetes: the streptozotocin (STZ) diabetic model Animal models of diabetes are enormously useful in understanding the etiologies of diabetic complications, both acute and chronic. In addition to having a shorter life span, these animals also demonstrate many physiological and pathophysiological characteristics similar to those seen in human diabetes. Various animal models of both genetic and chemically induced diabetes have been developed. Genetic models of Type 1 diabetes include the spontaneously diabetic biobreeding (BB) Wistar rat and the non-obese diabetic (NOD) mouse. Chemically-induced models such as the streptozotocin (STZ) and alloxan models of diabetes allow for better control of the duration and severity of the diabetic state. These chemicals specifically cause !-cell necrosis leading to the development of hyperglycemia and a hypoinsulinemic state and mimic human type 1 diabetes. Streptozotocin is preferred over alloxan as a diabetogenic agent due to its greater selectivity for pancreatic !-cells and lower mortality 57 . Over a dose range of 45-100 mg/kg, STZ (either intravenously or intraperitoneally) produces the characteristic symptoms of Type 1 diabetes such as polydipsia, polyphagia, decreased body weight gain, hyperglycemia, hypoinsulinemia and elevated levels of plasma lipids 17, 58 . In addition to the dose, the severity of diabetes also depends on the strain, gender and age of the rat 59 . In our laboratory, induction of diabetes in male Wistar rats is achieved by a single tail vein injection of 60 mg/kg of STZ 60 . Chronic STZ-diabetic rats exhibit many cardiovascular abnormalities similar to those seen in human diabetic patients 35 . Studies with isolated working heart have demonstrated impaired ventricular performance 24, 61 . In vivo studies employing rodent echocardiography have confirmed many of the cardiac functional aberrations associated with chronic STZ-induced diabetes in rats 62 . Furthermore numerous in vivo and in vitro studies have reported the occurrence of functional abnormalities in vascular tissues of STZ diabetic rats 32, 63-65 . Thus, the   9 STZ-diabetic model offers many advantages and allows for the evaluation of pharmacological interventions to gain an insight into the disease processes and therapeutic modalities that could prevent or reverse the abnormalities present in humans. 1.4 Vascular dysfunction in insulin resistance (pre-diabetes) 1.4.1 The metabolic syndrome The metabolic syndrome is a constellation of abnormalities including obesity, glucose intolerance, insulin resistance, dyslipidemia and hypertension and is a major risk factor in the development of stroke, chronic kidney disease and type 2 diabetes 66, 67 . Although not all humans with metabolic syndrome develop type 2 diabetes, it still is an effective predictor of type 2 DM and cardiovascular disease. A 4-factor model linking high blood pressure, obesity, insulin resistance, and dyslipidemia has been proposed, which relates all the components of the metabolic syndrome 68 . The interrelationships among the different components of the syndrome and their associated disturbances make it difficult to understand the underlying causes. Nevertheless the consequence of metabolic syndrome is very clear; a higher incidence and prevalence of cardiovascular disease. The presence of metabolic abnormalities commonly results in the impairment of endothelial function, which in turn contributes to other cardiovascular diseases such as hypertension and atherosclerosis 69 . Several components of the metabolic syndrome, such as insulin resistance/hyperinsulinemia, are associated with left ventricular hypertrophy and diastolic dysfunction in non-diabetic hypertensive patients 70 . Metabolic syndrome is also associated with many hypertension-related target organ damage, both in diabetic and non-diabetic patients 71 . Among all the components of the metabolic syndrome, the presence of insulin resistance is the most accepted unifying hypothesis to describe the pathophysiology of the metabolic syndrome. The molecular mechanisms linking insulin resistance and the other metabolic risk factors are not fully understood but appear to be very complex.   10 1.4.2 Insulin resistance Insulin-mediated glucose disposal varies widely in humans, and depending on the ability of the tissues to dispose of glucose, a subject is termed either insulin sensitive or resistant. Once present, insulin resistance results in impaired insulin action in insulin-sensitive tissues such as muscle, fat, and liver. Insulin resistance results in abnormal glucose metabolism, with reduced peripheral disposal of glucose in muscle and increased hepatic glucose output in the fasting state. It also impairs disposal of plasma triglycerides and free fatty acids 72 . It is not clear what causes or initiates insulin resistance but combinations of genetic, dietary and environmental factors have been implicated in the development of insulin resistance 73 . Similarly, it is unclear if the compensatory hyperinsulinemia follows or precedes insulin resistance. However, what is clear is that over a period of time, when the pancreas fails to keep up with the increasing demand of insulin production and secretion 74 , a fully blown diabetes characterized by hyperglycemia occurs. 1.4.3 Insulin resistance and hypertension Hyperinsulinemia and insulin resistance are often found to be associated with high blood pressure in both humans and several animal models 75 . Based on this observation, the “insulin hypothesis” was developed which proposes that the underlying metabolic impairment is responsible for the development of hypertension. Although several studies reported a positive correlation between high blood pressure and insulin resistance, yet others have found a weak or no correlation 76, 77 . Despite the conflicting reports, it appears that nearly 50% of hypertensive patients are hyperinsulinemic and insulin resistant 78 . However, the mechanisms responsible for hypertension in insulin resistance are still not clear. Individuals with insulin resistance often manifest changes in the behavior of large blood vessels, with characteristic abnormalities in vascular reactivity, changes in several key regulators of thrombolysis, and an increased risk for   11 vascular inflammation all contributing to vascular dysfunction and the development of hypertension 72, 79 . 1.4.4 Mechanisms of blood pressure regulation in insulin resistance Regulation of blood pressure is accomplished by several different mechanisms including baroreflexes mediated via the sympathetic nervous system (SNS), humoral regulation of vascular tone and renal mechanisms that control intravascular fluid volume. In hypertension, one or more of the normal homeostatic mechanisms that regulate blood pressure may be defective. In conditions of insulin resistance, it is important to note that insulin, in addition to its metabolic effects, also has a significant influence on the cardiovascular and renal systems. Various possible mechanisms by which insulin influences blood pressure regulation have been proposed. These include stimulation of the vascular endothelial nitric oxide system, the SNS, an increase in renal sodium retention and stimulation of vascular smooth muscle cell growth. 1.4.4.1 Nitric oxide and the endothelium Insulin has complex actions (vasodilator and vasoconstrictor) on the circulatory system, including its well-known vasodilatory effect on blood vessels supplying skeletal muscle tissue 80, 81 . It is well established that insulin-mediated vasodilatation is dependent on a functional endothelium and involves the nitric oxide pathway. Inhibition of nitric oxide synthase or removal of the endothelium converts insulin-induced vasodilatation to vasoconstriction 82 . Further, insulin is known to stimulate the synthesis, secretion and gene expression of ET-1 in endothelial and vascular smooth muscle cells 83, 84 . The effect of insulin to activate both NO and ET-1 pathways appear to occur at the same time and in the same vascular tissue 85, 86 . Therefore any defect in endothelial function or NO system may transform vasodilatory properties of insulin into vasoconstrictory actions and thus may contribute to the development of high blood pressure associated with hyperinsulinemia and insulin resistance.    12 1.4.4.2 Sympathetic nervous system A significant increase in the plasma NA levels, sympathetic nerve activity and a consequent increase in the peripheral vascular tone and blood pressure has been observed in chronic hyperinsulinemic conditions as well as in acute insulin infusion studies 87-89 . On the other hand, elevated SNS activity may also contribute to insulin resistance via vasoconstriction, thereby reducing blood flow and glucose disposal 90 . As a result hyperinsulinemia may occur to compensate for insulin resistance, creating a vicious cycle that reinforces the insulin-resistant state and elevated blood pressure. 1.4.4.3 Kidney Insulin has a direct effect on kidney and is known to increase sodium reabsorption, acting on various regions on the nephron 91 . Insulin-resistant humans and animals are characterized by impaired sodium homeostasis 92 . Insulin infusion also has been demonstrated to increase blood pressure in rats and in humans. However it is unclear if it is insulin resistance or increased insulin signaling in the kidney that is important in determining the rise in blood pressure. Some studies suggest that unlike other tissues, the kidney does not develop insulin resistance and that higher levels of circulating insulin may cause inappropriate sodium retention and produce an increase in BP 93 . On the contrary, other studies suggest that defective insulin receptor signaling may be more important in determining the net rise in blood pressure in insulin resistant rats 94 . 1.4.4.4 Vascular smooth muscle cell growth promoting pathways The physiologic actions of insulin in the vasculature serve to couple regulation of metabolic and hemodynamic homeostasis (Fig.1.1). However it should be noted that insulin-signaling pathways in vascular endothelium involved in the activation of eNOS and NO production such as PI3K are completely distinct and independent from the Ras/MAP-kinase (mitogen activated protein kinase) branch that is involved in the growth promoting effects of insulin 95 . Ras/MAP kinase pathways do not contribute significantly to insulin mediated glucose uptake. However,   13 insulin activation of the MAPK pathway leads to vasoconstriction and pathologic vascular cellular growth. In conditions of insulin resistance, insulin activation of PI3K is selectively impaired, whereas the MAPK pathway is unaffected 96 . In endothelium, impaired PI3K activity and increased MAPK signaling in response to insulin or other hormones may lead to decreased production of NO and increased secretion of ET-1 characteristic of endothelial dysfunction. Further, compensatory hyperinsulinemia may also activate the MAPK pathway directly or indirectly by increasing SNS activity, thereby increasing the plasma levels of various catecholamines, Ang II and ET-1. All the above factors are known to initiate vascular remodeling and contribute significantly to the development of hypertension in insulin resistant conditions 95 . 1.4.5 Vascular smooth muscle and growth factor receptors A growing body of evidence suggests that the presence of metabolic and hemodynamic abnormalities activates growth-promoting pathways and contributes to hypertension both in insulin resistant and non-insulin resistant states. Activation of MAPK pathways not only promotes growth but also causes vasoconstriction and involves mechanisms such as oxidative stress 97 . Excess activation of growth promoting pathways by insulin or insulin like growth factors (IGF) results in abnormal cardiovascular remodeling such as LV hypertrophy and arterial remodeling 98 . It is not clear if cardiovascular remodeling is an adaptive response to hypertension or the cause of increased arterial blood pressure. However, increasing evidence suggests that growth-promoting pathways are activated by vasoactive peptides such as Ang II, ET-1 and hormones (which are all elevated in hypertension) acting on their respective G-protein couple receptors (GPCR). Studies have demonstrated that these vasoactive peptides stimulate intracellular signaling pathways of the growth factor receptors similar to those activated by their endogenous ligands such as epidermal growth factor (EGF), IGF, platelet derived growth factor (PDGF) and insulin 99 . This process, which is termed “transactivation”, was first observed by   14 Daub et al 100  who demonstrated phosphorylation of the EGFR by ET-1, thrombin and lysophosphatadic acid. Since then, this phenomenon has been observed in many cell types including cardiomyocytes 101  and vascular smooth muscle cells 102 .              Figure 1.1 Schematic representations of two major branches of insulin signal transduction pathways. PI3-kinase branch regulates GLUT4 translocation and glucose uptake in skeletal muscle and NO production and vasodilatation in vascular endothelium. MAP-kinase branch of insulin signaling generally regulates growth and mitogenesis and controls secretion of ET-1 in vascular endothelium [Adapted from the review by Kim et al 95 ].     15 1.4.6 Transactivation of the EGFR by GPCR agonists Increasing evidence suggests that the EGFR functions as a convergent point for both mitogenic and non-mitogenic signals arising from different stimuli such as GPCR activation by vasoactive peptides and catecholamines in vascular smooth muscle cells 103 . Agonists of vasoactive GPCRs are potent stimuli for activation of the EGFR. Several ligand-dependent and independent pathways have been proposed to explain the transactivation of the EGFR in VSM cells 97 . Supporters of the intracellular or ligand-independent mechanisms propose that EGFR transactivation is exclusively mediated through intracellular signals and implicate various intracellular kinases and second messengers such as Src, PKC, ROS and Ca 2+  in the activation of the EGFR 100, 104, 105 . On the contrary the extracellular or ligand-dependent pathway is a relatively new concept and the supporters of this pathway propose that transactivation of the EGFR may involve extracellular factors such as matrix metalloproteinases (MMPs) 106 . The current understanding is that, agonist stimulation of GPCRs somehow activates MMPs, which in turn cleave a membrane-bound ligand such as pro-heparin bound EGF (pro-HB-EGF) to release a soluble mature factor (e.g. HB-EGF). Similar to EGF, HB-EGF binds to the EGFR and causes phosphorylation of the tyrosine residues to activate the downstream mitogenic signals 106 . 1.4.7 The matrix-metalloproteinases (MMPs) Matrix metalloproteinases constitute a multi-gene family of zinc and calcium dependent endopeptidases secreted by connective tissue cells and inflammatory phagocytes 107 . They are synthesized as inactive proenzymes with a signal sequence and propeptide segment that is cleaved during activation. Breakage of the cysteinyl sulfhydryl bond between a cysteine residue of the propeptide and the zinc catalytic center is necessary for the activation of MMPs from their zymogen forms 108, 109 . This can be achieved by proteolytic cleavage of the propeptide by serine proteinases, active MMP-2 and membrane type (MT)-MMPs or by conformational changes induced by denaturants and oxidants 110 . Cellular control of these enzymes occurs at various   16 levels including synthesis, secretion and activation/inhibition. Under normal conditions their proteolytic activities are precisely controlled by their endogenous protein inhibitors known as tissue specific inhibitors of MMPs (TIMPs) 111 . The main functions of MMPs are tissue remodeling in wound healing, embryonic development, nerve growth, angiogenesis and secretion of defense proteins and growth factors 107 . In addition to their historical role of extracellular matrix (ECM) turnover, shedding of transmembrane proteins to soluble forms is another function of MMPs that was discovered recently 104, 105, 112-114 . Among the various MMPs, MMP- 2, 7 and 9 are expressed in the arteries of diabetic 115, 116  and hypertensive patients 117, 118 . The gelatinases, MMP-2 and MMP-9 are expressed in arteries and have the capacity to shed membrane-anchored molecules 119-121 . The other major MMP involved in shedding of HB-EGF in arteries is MMP-7. Similar to MMP-2, it is also highly expressed in the arteries of spontaneously hypertensive rats (SHR). Further, stimulation of arteries by GPCR agonists such as phenylephrine (PE) and Ang II activates MMP-7 122, 123 . 1.4.8 MMP transactivation of the EGFR –missing link in hypertension? Despite major research efforts, it remains uncertain what causes hypertension particularly in conditions of insulin resistance. Regardless of the cause, the most commonly observed and agreed pathological hallmarks of hypertension, both in humans and animal models of metabolic syndrome are increased vasoconstriction or decreased vasodilatation, endothelial dysfunction associated with oxidative stress and hypertrophy associated with abnormal cardiovascular remodeling. Interestingly, the states of elevated blood pressure, oxidative stress and cardiovascular hypertrophy observed in hypertension can all be explained by the actions of elevated levels of vasoconstrictive GPCR agonists such as NE, Ang II, ET-1 103, 112, 122, 124-127 . Increasing evidence suggests that the GPCR agonist-stimulated, MMP-dependent transactivation of the EGFR might be a unifying mechanism in the etiology of hypertension. It   17 has been proposed that elevated levels of ROS, increased vasoconstriction and hypertrophic growth of cardiac and vascular tissues are all the consequences of excessive and abnormal stimulation of their corresponding GPCRs 97 . Several studies have demonstrated that blockade of MMPs or generation of HB-EGF or abrogation of EGFR tyrosine kinase activity by selective pharmacological inhibition blunts vasoconstriction both in isolated arterial rings in vitro and in various animal models of hypertension in vivo 122, 128, 129 . Additionally, MMP inhibitors prevent cardiac hypertrophy induced by Ang II and PE 112, 130  and are beneficial in the treatment of heart failure 131 . Blockade of MMP-dependent EGFR transactivation inhibits ROS formation, spares NO being scavenged by ROS and hence helps prevent the impairment of endothelial function 123 . Taken together, these results indicate that blockade of MMP-dependent transactivation of the EGFR may provide a therapeutic strategy for hypertension. 1.4.9 Animal models to study vascular dysfunction in insulin resistance: the fructose hypertensive rat Various animal models have been used to study the interaction between insulin resistance and hypertension. Among them, the spontaneously hypertensive rat (SHR) and the high fructose-diet fed hypertensive rat (FHR) are two of the most widely used animal models 132 . The SHR model is a genetic model, which is used not only to study the pharmacological interventions of drugs on blood pressure but also to study the association between insulin resistance, impaired glucose metabolism and hypertension 133 . The SHR model has demonstrated some specific genetic factors affecting traits of the metabolic syndrome as well as a possible genetic relationship between the syndrome and diabetes mellitus 134 . The issue of choosing between predominantly environmental and predominantly genetic models to study hypertension in insulin resistance is difficult because of the complex nature of the disease and the relevance of the model to the clinical setting 135 . Although many experts stress   18 the importance of genetic predisposition as the underlying factor in the development of metabolic syndrome, others argue that dietary factors play a prominent role in its development. They support their arguments by pointing to the evidence linking increased fructose consumption to a parallel elevation in the incidence and prevalence of metabolic syndrome. It is true that due to changes in the dietary habits of the general population, an increased consumption of sucrose and fructose, owing to increased ingestion of corn syrup and sweeteners, has been observed 136 . In fact, glucose, fructose and sucrose are the most consumed sugars in the modern diet 136, 137 . Various animal studies have reported that feeding rats with a high carbohydrate diet (fructose, glucose or sucrose) results in a host of metabolic abnormalities similar to that observed in metabolic syndrome in humans 138-140 . These include hypertension, hyperinsulinemia, hypertriglyceridemia and insulin resistance 75 . Among the various sugars, fructose-feeding has the potential to induce the characteristic symptoms of the metabolic syndrome 141 . This is because intestinal delivery of fructose is two times faster than that of glucose and unlike glucose 142 , which is absorbed by an active energy dependent process, absorption of fructose takes place by facilitated diffusion 143 . Many of the mechanisms linking insulin resistance to hypertension are apparent in rats fed a high fructose diet. These include hyperinsulinemia 144 , hypertriglyceridemia 138 , endothelial dysfunction 145, 146 , increased levels of oxidative stress 147 , defects in insulin signaling 148  and increased SNS activity 149  with elevated levels of various vasoactive agents such as NA, ET-1 144 , Ang II 150  and thromboxane A2 151 . It should be noted that increased levels of vasoactive peptides act as potent stimuli to induce transactivation the EGFR via the activation of MMPs.      19 1.5 Overall rationale, hypothesis and research objectives 1.5.1 Overall rationale Cardiovascular complications are recognized to be the major cause of morbidity and mortality associated with diabetes mellitus 8, 152 , although the underlying mechanisms are still unclear. However, the hemodynamic changes that occur in diabetic patients have been proposed to be related at least in part to abnormalities in vascular reactivity 153 , a possibility that has been extensively investigated in animal models. We have investigated the biochemical and functional consequences of insulin resistance and diabetes on vascular function, using two different models: the FHR rat model of acquired systolic hypertension, and the hypoinsulinemic STZ- diabetic rat model. For the sake of simplicity we have divided our research work into two major studies. In the first study, we investigated the mechanisms involved in cardiovascular dysfunction in type 1 diabetes using the STZ diabetic rat model. In the second study, we investigated the mechanisms of altered vascular reactivity and the etiology of hypertension in insulin resistant conditions using the high fructose diet fed hypertensive rats. In the STZ diabetic rat model, we have investigated the effect of diabetes on vascular function and the ensuing hemodynamic changes. The results of our previous studies demonstrate that complex biochemical interactions leading to changes in endothelial function and pressor responsiveness occur in these rats 23, 154 . Specifically, we have found depressed MABP and HR, attenuated pressor responses to vasoactive agents such as methoxamine and Ang II, and endothelial dysfunction in STZ diabetic rats. We have also found that this was associated with an increased expression of iNOS and nitrotyrosine, and decreased expression of eNOS in the heart and arteries 23 . In our first study, we investigated the mechanisms involved in the “causes and consequences” of increased expression of iNOS in STZ-diabetic rats. In contrast to the STZ-diabetic rat, FHR animals develop systolic hypertension with abnormalities in vascular and endothelial function. Previous studies from our lab and elsewhere   20 have clearly demonstrated impairment of endothelial function and altered vascular responses to insulin in this model. FHR also exhibit various pathological features such as increased SNS activity and elevated levels of various vasoactive peptides, which have the potential to induce transactivation of the EGFR via the MMPs. In our second study, we investigated the role of MMPs and the EGFR transactivation pathway in the etiology of hypertension in fructose hypertensive rats. 1.5.2 Study 1: Investigation of the mechanisms and consequences of increased expression of iNOS in STZ-diabetic rats 1.5.2.1 Hypothesis We hypothesized that “increased activation of PKC! leads to the induction of iNOS, which results in increased production of both nitric oxide and peroxynitrite, leading to impaired pressor responsiveness, endothelial function and cardiovascular function in STZ-diabetic animals” 1.5.2.2 Rationale Three isoforms of nitric oxide synthase (NOS) generate NO from oxygen and L-arginine in the presence of tetrahydrobiopterin (BH4). Neuronal NOS (nNOS) and endothelial NOS (eNOS) are constitutively expressed, Ca 2+ -regulated isoforms, which produce low levels of NO.  iNOS is normally not expressed in vascular tissue, but its expression can be increased in pathological conditions and by inflammatory factors and cytokines 155, 156 , and once induced, iNOS produces prolonged high levels of NO 157 . We were the first lab to demonstrate increased expression of iNOS in the arteries of STZ diabetic rats 158 , although the mechanisms involved in its induction are still unclear. The first objective of this study was to investigate the mechanisms underlying the induction of iNOS in cardiovascular tissues of STZ-diabetic rats. It likely occurs as a result of activation of the redox sensitive transcription factor, NF-"B, which appears to play a critical role in iNOS induction in vascular smooth muscle cells 159-161 . There is considerable evidence that   21 hyperglycemia results in the generation of superoxide anions which in turn activate NF-!B 162 . An important role for PKC in increasing SO production in diabetic arteries has also been shown 163 . Incubation of cultured mesangial cells in elevated glucose has been reported to result in increased iNOS expression, that was blocked by inhibition of PKC 164 . The increased activation of NF-!B produced by exposure of bovine aortic endothelial cells to hyperglycemia was also prevented by PKC inhibition 165 . The isoform of PKC most commonly reported to be activated in cardiovascular tissues is PKC"2, and treatment of STZ-diabetic rats with the PKC"- selective inhibitor ruboxistaurin (LY333531) was shown to reduce vascular dysfunction in these animals 166 . Taken together, these data suggest that PKC" plays a major role in the induction of iNOS. We proposed that increased activation of PKC" results in activation of NF-!B, leading to induction of iNOS in STZ-diabetic rat arteries (Fig.1.2). Further, in STZ-diabetic rats, we investigated the effect of long-term inhibition of iNOS and PKC" on iNOS mediated cardiovascular abnormalities such endothelial dysfunction, depression of cardiac function, mean arterial blood pressure and heart rate and attenuated pressor responses to vasoactive agents. 1.5.2.3 Specific research objectives 1. To determine if PKC" is involved in the induction of iNOS in cardiomyocytes and vascular smooth muscle cells and the mechanisms involved therein. 2. To determine whether treatment of STZ- diabetic rats with a selective PKC" inhibitor improves cardiovascular function by preventing the activation of NF-!B and induction of iNOS in STZ-diabetic rats. 3. To determine whether a short or long-term treatment of diabetic rats with a selective iNOS inhibitor improves cardiovascular function in STZ-diabetic rats.     22                     Figure 1.2 Model showing the proposed mechanisms of iNOS induction and its consequences in STZ diabetic rats.     23 1.5.3 Study 2: Role of MMPs and the EGFR transactivation pathway in the etiology of hypertension in insulin resistance 1.5.3.1 Hypothesis We hypothesized that “increased vascular MMP activity and stimulation of growth promoting pathways via the EGFR impairs endothelial function and contributes to the development of hypertension in insulin resistant conditions” 1.5.3.2 Rationale Apart from the historical role of MMPs in extracellular matrix turnover, increasing evidence now suggests that many MMPs are involved in the regulation of cardiovascular function through transactivation of the EGFR 106 . Many ligands including the HB-EGF are shed from cell surfaces by MMP activation in response to specific signals, leading to phosphorylation of the EGFR and initiation of downstream MAPK activation. As mentioned before, various stimuli including activation of GPCRs by PE, Ang II, ET-1 are known to transactivate EGFR by an MMP- dependent mechanism 101, 130  Intracellular signaling events such as generation of ROS and phosphorylation of Src cause activation of MMPs such as MMP-2 and MMP-7 that leads to the enzymatic conversion of pro HB-EGF to HB-EGF. The latter binds to and activates EGFR leading to the phosphorylation of PI3K, Akt and MAP kinases including ERK1/2, thereby promoting cell growth, proliferation and vasoconstriction (Fig. 1.3). Although there is a wealth of information concerning the role of MMPs and growth promoting effects of EGFR transactivation, very little is known about the role of either MMPs or the MMP-dependent EGFR transactivation pathway in the etiology of endothelial dysfunction or hypertension in insulin-resistant conditions.      24                Figure 1.3 Schematic representation of agonist induced MMP-dependent transactivation pathway in vascular smooth muscle cell. Agonist (catecholamines, Ang II, ET-1) induced activation of the corresponding GPCR leads to the activation of phospholipase C (PLC) and subsequent production of several intermediary signaling molecules such as Ca 2+ , PKC, ROS and tyrosine kinases (e.g. Src). Activation of the MMP by these signaling molecules leads to shedding of mature HB–EGF, which later binds to and phosphorylates the EGF receptor. Activation of EGF receptors initiates a MAPK cascade resulting in activation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2). EGF receptor stimulation also leads to the activation of PI3K and protein kinase B (Akt) pathway, culminating in cell growth and hypertrophy.   25 1.5.3.3 Specific research objectives 1. To determine if vascular MMPs (MMP-2, MMP-9 and MMP-7) are upregulated and if MMP-dependent EGFR transactivation pathway in activated in fructose hypertensive rats. 2.  To study the effect of MMP and EGFR inhibition on insulin resistance, endothelial function and altered vascular responses and elevated blood pressure in high fructose diet fed rats. 3. To examine the role of the MMP-EGFR pathway on the expression of vascular contractile proteins and their contribution to the development of hypertension in conditions of insulin resistance and elevated levels of the GPCR agonists.                  26 1.6 Bibliography 1. Volk BW, Arquilla ER. 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Mechanisms underlying endothelial dysfunction in diabetes mellitus. Circ Res. 2001;88(2):E14-22. 164. Noh H, Ha H, Yu MR, Kang SW, Choi KH, Han DS, Lee HY. High glucose increases inducible NO production in cultured rat mesangial cells. Possible role in fibronectin production. Nephron. 2002;90(1):78-85. 165. Pieper GM, Riaz ul H. Activation of nuclear factor-kappaB in cultured endothelial cells by increased glucose concentration: prevention by calphostin C. J Cardiovasc Pharmacol. 1997;30(4):528-532. 166. Ishii H, Jirousek MR, Koya D, Takagi C, Xia P, Clermont A, Bursell SE, Kern TS, Ballas LM, Heath WF, Stramm LE, Feener EP, King GL. Amelioration of vascular dysfunctions in diabetic rats by an oral PKC beta inhibitor. Science. 1996;272(5262):728-731.     40 2. Selective inhibition of protein kinase C !2 attenuates inducible nitric oxide synthase mediated cardiovascular abnormalities in streptozotocin-diabetic rats 1                    1 A version of this chapter has been submitted for publication. Nagareddy PR, Soliman H, Lin G, McNeill JH and MacLeod KM. Selective inhibition of protein kinase C !2 attenuates inducible nitric oxide synthase mediated cardiovascular abnormalities in streptozotocin - diabetic rats (2009).    41 2.1 Introduction Cardiovascular complications are recognized to be the major cause of morbidity and mortality associated with diabetes mellitus 1 . Activation of protein kinase C (PKC) isoforms, increased glucose flux through the polyol pathway, formation of advanced glycation end products (AGE) and increased levels of oxidative and nitrosative stress are some of the mechanisms believed to be involved in the etiology of these complications 2-4 . Increasing evidence now implicates the abnormal activation of PKC!2, secondary to increased formation of diacylglycerol (DAG) by hyperglycemia, in a number of cardiovascular diabetic complications 3, 5 . Studies from our lab 6, 7  and elsewhere 5  have found preferential increases in expression and/or activation of the PKC!2 isoform in cardiac and vascular tissues of diabetic animals. Further, inhibition of the activity of PKC! has been shown to result in amelioration of diabetic nephropathy and retinopathy in human patients 8, 9  and  was recently reported to improve cardiac function in streptozotocin (STZ)-diabetic rats 10 . However, the mechanism(s) by which activation of this PKC isoform exerts adverse effects in cardiovascular tissues remain unclear. A seminal study suggested that normalizing mitochondrial oxidative stress could prevent hyperglycemia-induced activation of PKC, increased flux through the polyol pathway and formation of AGE 2 , underscoring the importance of oxidative stress in the etiology of diabetic complications. Previous studies from our lab have demonstrated significant improvements in cardiovascular function of STZ diabetic rats treated with the antioxidant, N-acetylcysteine in parallel with inhibition of PKC!2 activation 6  and reduction in iNOS-mediated nitrosative stress 11 . Specifically, we found that improvements in cardiac performance, mean arterial blood pressure (MABP), heart rate (HR), pressor responses to vasoactive agents and endothelial function were associated with improvements in oxidative and nitrosative stress in the heart and arteries of STZ diabetic rats 11-13 . However, it remains unclear whether the increase in iNOS-   42 mediated nitrosative stress is an independent manifestation of hyperglycemic injury or is linked to the activation of PKC!2. In the present study, we tested the hypothesis that diabetes-induced activation of PKC!2 causes cardiovascular abnormalities via induction of iNOS. First, we investigated whether PKC!2 induces iNOS expression in cardiac and vascular tissues and the mechanisms involved therein, using the selective PKC! inhibitor LY333531 or PKC!2 siRNA. Secondly, we investigated the functional significance of this in vivo, by determining the effects of treatment of STZ diabetic rats with LY333531 on iNOS expression, nitrotyrosine formation and hemodynamic abnormalities. Our results strongly suggest that induction of iNOS, and consequently increased nitrosative stress, is one of the mechanisms by which PKC!2 leads to cardiovascular complications in diabetes. 2.2 Methods 2.2.1 Study design and induction of diabetes This study conforms with the Canadian Council on Animal Care Guidelines on the Care and Use of Experimental Animals and was approved by the University of British Columbia Animal Care committee. Forty-eight male Wistar rats weighing between 280 to 300 g were obtained from Charles River Laboratories Inc., Quebec and allowed to acclimatize to the local vivarium. They were randomly divided into 6 equal groups: control (C), control treated with LY333531 (C-LY) or L-NIL (C-LNIL), diabetic (D) and diabetic treated with LY333531 (D-LY) or L-NIL (D-LNIL). Diabetes was induced by a single tail vein injection of 60 mg/kg streptozotocin (STZ) while control animals received equal volume of citrate buffer. The presence of diabetes was confirmed by hyperglycemia (>20 mmol/L) 72 hours after STZ administration. Plasma glucose was measured using an enzymatic colorimetric assay kit (Roche Diagnostics) and a Beckman Glucose Analyzer. One week after the induction of diabetes, animals received vehicle   43 or the selective PKC! inhibitor, LY333531 (1 mg/kg/day)14 or the selective iNOS inhibitor, L- NIL (3 mg/kg/day) by oral gavage. LY333531 is a potent and specific inhibitor of PKC with an IC50 of approximately 5 nM for the ! isozymes, which is 100-fold lower than for the ", # or $ isoforms of PKC 15 . Similarly, L-NIL is a potent and relatively a selective iNOS inhibitor with an IC50 of 5.9 µM for iNOS compared to an IC 50 of 138 µM for eNOS and 35µM for nNOS 16 . A dose of 3 mg/kg/day was selected based on our previous study in which we were able to inhibit iNOS-mediated NO production in heart tissues from STZ diabetic rats 17 . After 3 weeks of treatment each animal was surgically prepared for measurement of MABP, HR and pressor responses to methoxamine. 2.2.2 Surgical procedures and hemodynamic measurements Animals were surgically prepared as described previously 13 . Five hours after surgery, basal MABP and HR were measured in freely moving conscious rats. Subsequently, dose response curves (DRC) to iso-volumic bolus doses of methoxamine (100-300 nmol/kg) were constructed in control and diabetic rats. DRC were constructed by measuring MABP in response to each bolus dose of methoxamine, allowing sufficient time (or 10 min) for MABP to return to normal between each dose. Finally, to determine endothelial function, a single bolus dose of N-nitro-L- arginine methyl ester (L-NAME, 10 mg/kg iv), a nonselective NOS inhibitor, was administered, and %MABP was recorded over a period of 15–30 min. 2.2.3 Collection of tissue samples Following hemodynamic measurements, animals were terminated with an overdose of pentobarbital (100 mg/kg). The heart and superior mesenteric arteries (SMA) were immediately removed and placed in ice-cold Krebs solution (120 mM NaCl, 5.9 mM KCl, 25 mM NaHCO3, 11.5 mM glucose, 1.2 mM NaH2PO4, 1.2 mM MgCl2, 2.5 mM CaCl2) containing 0.1µM water- soluble dexamethasone to prevent induction of iNOS in vitro. The tissues were cleaned of all   44 adherent tissue, snap frozen in liquid nitrogen and stored at -70 °C for Western blot measurements. Some tissue sections were preserved in neutral buffered formalin for immunohistochemistry experiments. 2.2.4 Cell culture studies 2.2.4.1 Preparation of isolated rat ventricular cardiomyocytes Ca 2+  tolerant cardiomyocytes were isolated as described previously 18 . Cardiomyocytes were either snap frozen in liquid nitrogen or were maintained in primary culture in order to assess the effects of drug treatment. For the latter, cardiomyocytes were resuspended in medium 199 supplemented with 1% BSA, 100 units/ml penicillin, 100 µg/ml streptomycin, 1.2 mM L- carnitine and 25 mM HEPES (pH 7.4). Cells prepared from a single rat heart were plated on laminin-coated culture dishes and allowed to recover for 3 hours. Cultured cells were then incubated for 18 hours either in 5.5 mM glucose, 25 mM glucose or 5.5 mM glucose plus 19.5 mM mannitol at 37°C in supplemented medium 199 containing various treatments, and then snap frozen in liquid nitrogen. 2.2.4.2 Preparation and culture of rat aortic vascular smooth muscle cells (VSMC) Rat aortic vascular smooth muscle cells (VSMCs) were grown in complete Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen Life Technologies, Carlsbad, CA) with 10% fetal bovine serum (FBS) and 100-units/mL penicillin-streptomycin at 37ºC and 95% O2/5% CO2. Cells from passages 2-6 were used in all experiments. When cells were 80-90% confluent they were starved overnight using DMEM media without growth factors. On the day of experiments cells were either incubated in low glucose (5.5 mM), high glucose (25 mM) or mannitol (5.5 mM glucose + 19.5 mM mannitol) in serum-free medium for 36 hours in the presence of PKC! inhibitor (LY333531, 20 nM). At the end of the experiment, cells were washed with ice-cold phosphate-buffered saline (PBS), lysed and the cell lysates subjected to fractionation or collected and stored (at -80 o C) for western blotting.   45 2.2.5 Preparation of VSMC membrane and cytosol fractions Following treatment, VSMCs were scraped and lysed by sonication in a buffer composed of the following (mM): Tris HCl 25, NaCl 150, !-mercaptoethanol 50, EGTA 2, Halt protease and phosphatase inhibitor cocktails (Thermo Scientific, IL USA). The lysates were centrifuged at 10,000xg for 10 minutes at 4ºC and the supernatant was fractionated by ultracentrifugation at 100,000xg for 60 minutes at 4ºC. The supernatant was collected as the cytosolic fraction and the residue was washed 3 times with ice-cold saline, followed by resuspension in a buffer composed of (mM where appropriate): Tris HCl 20, !-mercaptoethanol 50, EGTA 2, EDTA 5, Halt phosphatase and protease inhibitor cocktails, Triton-X 100 (0.1% v/v), sodium deoxycholate (0.5% w/v) and SDS (0.1% w/v). This fraction was collected as the membrane fraction. 2.2.6 PKC!2 siRNA studies in rat aortic VSMC Small interference RNA (siRNA) specific to rat PKC!2 was used to knock down the expression of PKC!2 in rat aortic VSMC. The PKC!2 siRNA, which was custom made by Santa Cruz Biotech Inc (CA, USA), consisted of a pool of 3 target-specific 19-25 nt siRNA duplexes. The transfection reagent, medium and scrambled siRNA were also purchased from Santa Cruz Biotech Inc. For optimal siRNA transfection, the manufacturer's protocol was followed. Briefly, VSMC were seeded in a 6 well plate and cultured in 2 ml antibiotic-free Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) until the cells were 60-80% confluent (~36 hours). On the day of transfection, cells were washed with transfection medium and incubated with 1 ml of transfection reagent containing 60 pmols of either MOCK (scrambled) or rat specific PKC!2 siRNA oligonucleotides for 16 hours. The medium was then supplemented with 1 ml of fresh DMEM (containing 2x FBS and antibiotics) for another 24 hours. At this point, the cells were washed with warm PBS and treated with either low or high   46 glucose containing DMEM for another 48 hours in the presence or absence of various drugs as mentioned in the results section. 2.2.7 Immunoprecipitation and Western blot analysis Frozen ventricles, arteries and isolated cardiomyocytes and VSMC cells were homogenized or sonicated in modified RIPA buffer containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM PMSF, 1 mM EDTA, 5 !g/ml aprotinin, 5 !g/ml leupeptin, 1% Triton x-100, 1% sodium deoxycholate and 0.1% SDS at 4°C. The homogenate was centrifuged at 10000"g at 4°C for 15 min and the protein content of the supernatants was determined by the Bradford protein assay. Some samples (hearts and SMAs) underwent immunoprecipitation where 100-200 µg of tissue homogenate protein was diluted to 1 µg/µl and subjected to immunoprecipitation with 1µl anti- iNOS antibody. For western blotting, equal amounts of protein (10-30µg) from each sample were separated by 8-10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked for 1 hour in a solution of 5% skim milk (in TBS containing 0.1% tween 20) and then incubated overnight at 4C with primary antibodies against iNOS (1:1000, BD Transduction Labs), phospho and total PKC!2, ERK1/2 and NF-kB (p65) antibodies (1:1000, Cell Signaling). Membranes were washed then incubated with their corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (1:8,000) for 1 hour. After washing, the membranes were exposed to enhanced chemiluminescence reagents (Amersham Inc, Québec, Canada) and developed on photographic film. The intensity of the bands was determined by using image J software from NIH. 2.2.8 Immunohistochemistry and quantification of iNOS and NT immunostain in the heart and SMA sections The heart and SMA tissues were fixed in 10% NBF overnight and transferred to 70% ethanol. This was followed by paraffin processing through increasing grades of ethanol, xylene and paraplast (Fischer Scientific, ON). Paraffin embedded tissue blocks were sectioned at 3 µm and   47 mounted on slides. The sections were deparaffinized, rehydrated, washed with PBS and blocked with 5% normal goat serum (NGS) in PBS for 60 minutes. The slides were subsequently incubated with primary rabbit polyclonal anti-iNOS (1:100, Abcam) or mouse monoclonal anti- NT (1:200, Cayman Chemicals) antibodies in PBS containing 1% NGS overnight at 4 o C in a humidity chamber. After washing off the primary antibody with PBS, the sections were incubated with biotinylated goat anti-rabbit or anti-mouse secondary antibodies (1 drop in 10 mL PBS, Vectastain ABC kit, Vector Laboratories) for 1 hour followed by avidin-biotinylated HRP complex and color development was performed using 3,3-diaminobenzidine. Some sections incubated without primary antibodies served as negative controls. Using a high power microscope and digital imaging system all images were observed individually and photographed (20X and 40X). Immunostain intensity from each photograph in terms of percent area stained by iNOS or NT antibodies was calculated using image J software. 2.2.9 Measurement of oxidative stress (superoxide anion formation) in VSM cells VSMC seeded in 6-well plates were cultured until they achieved 80-90% confluence after which they were serum starved (overnight) and incubated in low or high glucose for additional 36 hours in the presence or absence of LY333531 (20 nM). On the day of the experiment, VSMC were washed with PBS and treated with 10 !M dihydroethidium (DHE) and incubated at 37°C. Fluorescence intensity was measured using a Biotek Luminescence Spectrometer at excitation/emission wavelengths of 530/620 nm. The intensity of DHE fluorescence in cells was measured every 5 min for the first 15 min, followed by 15-minute readings for another 2 hours. 2.2.10 Statistical analysis All values are expressed as mean ± SEM. “n” denotes the sample size in each group. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by the Newman-Keuls test for multiple comparisons. GraphPad Prism (GraphPad Software, CA)   48 software program was used for statistical analysis. For all results the level of significance was set at P<0.05. 2.3 Results 2.3.1 Effect of LY333531 on PKC!2 and iNOS expression in cardiomyocytes and diabetic hearts We previously have shown increased expression of iNOS in hearts 13  and cardiomyocytes 17  isolated from diabetic rats. In the present study we examined whether incubation of normal cardiomyocytes in high glucose increases iNOS expression, and if so, whether this could be prevented by treatment with the PKC! inhibitor, LY333531. Treatment of cardiomyocytes with high glucose (25 mM) for 18 hours had no effect on total levels of PKC!2 but significantly increased levels of threonine 641-phosphorylated PKC!2 (Fig. 2.1). Phosphorylation of threonine 641 is crucial for the appropriate subcellular localization and catalytic function of PKC!2 and is often used as an index of PKC!2 activation 19 . On the other hand, exposure of cardiomyocytes to mannitol (19.5 mM + 5.5 mM glucose), in order to determine any effects due to hyperosmolarity, did not alter levels of either threonine 641- phosphorylated or total PKC!2 compared to cells treated with low glucose (data not shown). Treatment of cardiomyocytes with LY333531 prevented the high glucose-induced increase in the phosphorylation of PKC!2 without affecting the expression of total PKC!2 (Fig. 2.1). Further, high glucose also increased the expression of iNOS in isolated cardiomyocytes, and this was also prevented by pretreatment with LY333531 (Fig. 2.1). Similarly, there was a significant increase in the expression of iNOS and in levels of threonine 641 phosphorylated PKC!2 without any change in total PKC!2 in hearts from untreated diabetic rats (Fig. 2.2). Treatment of diabetic rats with LY333531 significantly reduced levels of phosphorylated PKC!2 as well as iNOS expression without affecting these parameters in age-matched control rat hearts (Fig. 2.2).   49 2.3.2 Effect of LY333531 on PKC!2 and iNOS expression in VSMC and superior mesenteric arteries We next determined whether iNOS expression was induced in rat aortic VSMC cultured in high glucose as well as in superior mesenteric arteries from diabetic rats. Similar to diabetic rat hearts, there was a significant increase in the expression of iNOS in mesenteric arteries from untreated diabetic rats, that was reduced by treatment with LY333531 (Fig. 2.3 A). Further, incubation of VSMC in high glucose (Fig. 2.3 B) but not mannitol (Fig. 2.9) for 36 hours also significantly increased iNOS expression. Activation of PKC!2 is accompanied by its translocation to the membrane 19 , and levels of PKC!2 in the particulate (membrane) fraction were significantly elevated in high glucose conditions (Fig. 2.4). Treatment of VSMC with LY333531 not only normalized particulate levels of PKC!2, consistent with inhibition of its activation, but also prevented the increase in iNOS expression (Fig. 2.4). 2.3.3 Induction of iNOS is PKC!2 dependent in VSM cells Our experiments so far indicated that exposure of VSMC and cardiomyocytes to high glucose leads to an increase in iNOS expression that is prevented by the non-selective PKC! inhibitor, LY333531. To determine whether PKC!2 is specifically required for induction of iNOS, we suppressed its expression in VSMC using rat specific PKC!2 siRNA and again exposed the cells to low and high glucose conditions. Although, we were not able to suppress the expression of PKC!2 completely, a reduction in PKC!2 protein expression of approximately 40% (Fig. 2.5) prevented the increase in expression of iNOS in VSMC exposed to high glucose while having no effect on iNOS expression in VSMC incubated in low glucose. 2.3.4 Induction of iNOS by PKC!2 involves ERK1/2 and NF-"B activation in VSMC Since ERK1/2 has been reported to mediate increased expression of iNOS in vascular smooth muscle via the NF-"B pathway 20, 21 , we next investigated whether ERK was activated in cells   50 exposed to high glucose. Incubation of VSMC in high glucose (Fig. 2.6) but not mannitol (Fig. 2.9 C) increased ERK activity as measured by increased phosphorylation of both ERK-1 and ERK-2 at Thr 202 and Tyr 204, respectively. Further, treatment of VSMC exposed to high glucose with LY333531 significantly prevented ERK activation (Fig. 2.6). Similarly, in superior mesenteric arteries from untreated diabetic rats, we found a significant increase in ERK activity, which was prevented by treatment with LY333531 (Fig. 2.7). The signaling events that lead to NF-!B activation involve phosphorylation, ubiquitination, and proteolytic degradation of I!B-! from its inactive trimeric complex of I!B/p65/p50 in the cytosol. This is followed by translocation of the active dimer (p65/p50) to the nucleus, where it binds to specific regions within the promoter to initiate iNOS message transcription 22 . To assess NF-!B activation, we measured the phosphorylation of Ser 536 on the p65 subunit of NF-!B. Exposure of VSMC to high glucose but not mannitol increased the phosphorylation of the NF- "B p65 subunit and this was inhibited by pretreatment with LY333531 (Fig. 2.6). The expression of total NF-"B p65 subunit was similar in all groups except the mannitol group where it was increased. However, this increase was not reduced by treatment with LY333531, indicating that the hyper-osmotic signal leading to increased synthesis of NF-"B P65 protein is not PKC#2 dependent (Fig. 2.9 D). To confirm the specific involvement of the PKC#2 isoform in the activation of ERK1/2 and NF- "B, we incubated PKC#2 siRNA-transfected VSMC in high glucose. VSMC in which PKC#2 was suppressed failed to show activation of ERK 1/2 and NF-"B on exposure to high glucose levels (Fig. 2.8). These data clearly suggest that PKC#2 is the predominant isoform involved in the activation of NF-"B by ERK1/2 in VSMC exposed to high glucose.     51 2.3.5 LY333531 inhibition of PKC!  reduces oxidative stress in VSMC VSMC exposed to high glucose produced a significant increase in the generation of superoxide anions free radicals as measured by DHE fluorescence. Treatment of VSMC with LY333531 not only inhibited the high glucose-induced increase in oxidative stress, but also reduced superoxide anion production in low glucose, suggesting an inherent antioxidant effect of the inhibitor (Fig. 2.10). 2.3.6 In vivo effects of LY333531 on PKC!2, iNOS and nitrotyrosine expression in the heart and mesenteric arteries Rats were made diabetic with STZ and were treated with LY333531 (1 mg/kg/day) or L-NIL (3 mg/kg/day) by oral gavage for 3 weeks. Previous studies have shown the effectiveness of these doses of the inhibitors in STZ diabetic rats 17, 23 . At the end of the treatment period, untreated diabetic rats had significantly elevated blood glucose levels and reduced body weights compared to control rats, which were not altered by LY333531 or L-NIL treatment (Table 2.1). As explained in previous sections, heart and mesenteric arteries from untreated diabetic rats showed increased phosphorylation of PKC!2 and iNOS expression, which were significantly inhibited by treatment with LY333531 (Fig. 2.2 and Fig. 2.3). Further, immunohistochemical analysis revealed increased expression of iNOS in the cardiomyocytes of heart sections and the medial and advential layers of superior mesenteric artery sections (Fig. 2.11) from untreated diabetic rats. In superior mesenteric arteries, as opposed to the dense immunostaining in the medial and advential layers, very little staining was observed in the tunica intima or endothelium, suggesting that the major source of iNOS is the media and adventia. Semi- quantitative analysis of iNOS immunostain in the photomicrographs suggests a reduction of iNOS expression by ~ 50% in both the heart and superior mesenteric arteries of diabetic rats treated with LY333531. Similarly, immunohistochemical analysis of nitrotyrosine (NT), an   52 indirect marker of peroxynitrite formation, indicated an increase in the nitration of proteins in the hearts and superior mesenteric arteries (Fig. 2.12) of untreated diabetic rats. As shown, there is a substantial increase in the levels of NT in the diabetic heart (>60%) and arteries (>40%) compared to control rats. Treatment with LY333531 significantly decreased the formation of NT in diabetic rats compared to untreated diabetic rats both in superior mesenteric arteries and in ventricular muscle. 2.3.7 Effect of PKC!  and iNOS inhibition on cardiovascular function in diabetic rats We previously have shown that direct inhibition of iNOS by L-NIL in vivo significantly improved cardiac performance, blood pressure, heart rate, pressor responsiveness and endothelial function in STZ-diabetic rats 13, 17 . In the present study we examined the in vivo effects of PKC! inhibition on iNOS mediated cardiovascular abnormalities in these animals. Four weeks after the induction of diabetes, untreated diabetic rats showed significantly lower MABP (Fig. 2.13 A) and HR (Fig. 2.13 B) compared to age matched control rats. Treatment with LY333531 or L-NIL significantly prevented the depression in both MABP and HR in diabetic rats without affecting these parameters in control animals. Further, administration of bolus doses of methoxamine (100-300 nmoles/kg) increased MABP in both control and diabetic rats in a dose-dependent manner. Compared to the corresponding age matched control rats, responses to methoxamine were significantly attenuated in untreated diabetic rats (Fig. 2.13 D). However, treatment of diabetic rats with LY333531 or L-NIL significantly augmented pressor responses to methoxamine, while having no effect in control rats. Endothelial function was tested using a single bolus dose of L-NAME. As shown (Fig. 2.13 C), untreated diabetic rats exhibited attenuated pressure response ("MABP) to L-NAME suggesting an impairment of endothelial function. Treatment with L-NIL but not LY333531 significantly improved endothelial function in diabetic rats.   53 2.4 Discussion Numerous studies have led to the identification of multiple hyperglycemia-induced alterations in metabolism and signaling that have been linked to activation of PKC and an eventual increase in oxidative /nitrosative stress in diabetes 2-4, 24-26 . It remains unclear whether the increase in nitrosative stress, which is implicated in the etiology of diabetic secondary complications 4 , is an independent manifestation of hyperglycemic injury or is linked to the activation of PKC. In the present study, we tested the hypothesis that high glucose induced-activation of PKC!2 increases iNOS mediated nitrosative stress leading to cardiovascular abnormalities. The results of our investigation provide clear evidence supporting this hypothesis, since treatment with LY333531 or PKC!2 siRNA inhibited the increased expression of iNOS in cardiovascular tissues, and LY333531 administration in vivo protected diabetic animals from the deleterious cardiovascular effects of hyperglycemia. Furthermore, our results demonstrate that PKC!2 is essential for high glucose-induced activation of ERK1/2 and NF-!B and subsequent induction of iNOS, and that the protective effects of PKC!2 inhibition in diabetes are associated with inhibition of iNOS- mediated peroxynitrite formation. High glucose is known to increase de novo synthesis of diacylglycerol (DAG), which is a potent activator of PKC in many cellular types 27, 28 . Elevated DAG levels have been reported in various tissues including the heart, retina, and kidney of diabetic rats 3, 29 . Furthermore, it is now established that increased levels and activity of specific isoforms of PKC occurs in the cardiovascular system in diabetes, and evidence suggests abnormal activation of the PKC system contributes to the development of diabetic cardiovascular complications 3, 5, 30, 31 . However, understanding the PKC signaling system is complicated by the presence of a large number of isoforms each with varying cellular distribution and sometimes opposing functions 32 . The isoform that has been most frequently implicated in diabetic cardiovascular complications 6,   54 30, 31  is PKC!2 5 . Studies from our lab and elsewhere have reported increased membrane levels of PKC!2 in the heart 6  and superior mesenteric arteries 33  from diabetic rats. Consistent with this, in the present study we found evidence for increased activation of PKC!2 both in the heart and superior mesenteric arteries from diabetic rats as well as in cardiomyocytes and VSMC exposed to high glucose. Activation of PKC by pharmacological activators such as phorbol esters or PKC overexpression has been reported to up-regulate cytokine-induced increases in iNOS and NO production in various cell types 34-36 . These studies, most of which were conducted in non-cardiovascular cell types, suggested the involvement of a variety of PKC isoforms in the induction of iNOS in response to proinflammatory cytokines 34-36 . On the other hand, high glucose-mediated increases in PKC have been variously reported to both inhibit 37  and to potentiate 38  cytokine-induced increases in iNOS expression in VSMC. Although the reason for the discrepancy between previous studies is not clear, our data show that inhibition of PKC! attenuates iNOS expression both in isolated cardiomyocytes and VSMC exposed to high glucose in culture, and in the heart and arteries of diabetic rats. This was confirmed using both pharmacological inhibition and silencing of PKC!2 in VSMC. To our knowledge, this study is the first to demonstrate the specific involvement of PKC!2 in the induction of iNOS in cardiovascular tissues, and most importantly its significance in the development of diabetic cardiovascular complications. The mechanism by which PKC!2 induces the expression of iNOS in cardiovascular tissues likely involves ERK1/2 and NF-"B, since the suppressive effect of LY333531 on iNOS expression was accompanied by inhibition of ERK and NF-"B activation. Previous studies have shown that inhibition of ERK activation, either by selective chemical inhibitors or by antisense oligodeoxynucleotides, attenuates NF-"B activation and iNOS expression in VSMC stimulated with cytokines 20, 21, 39 . Several cell types are known to increase ERK and NF-"B activity when   55 exposed to high glucose, and persistently higher levels of NF-!B have been reported in target organs such as the retina, the heart, and the kidney of diabetic rats 40, 41 . In agreement with these observations, we also found increased ERK activity in arteries of STZ diabetic rats. Taken together, these data support the view that high glucose-induced activation of PKC "2 increases iNOS expression by ERK activation of NF-!B 20, 21, 40 . Although it is unclear how PKC"2 activates the ERK - NF-!B pathway, the mechanism may involve the formation of reactive oxygen species (ROS) secondary to PKC activation 2, 42, 43 . In the present study, exposure of VSMC to high glucose produced a significant increase in ROS that was prevented by treatment with LY333531. The evidence that LY333531 inhibits ERK and NF- !B at the same time that it decreases ROS production suggests the possibility that ROS are central to activation of this pathway. Indeed ROS are known to directly activate NF- !B, a pleiotropic oxidant-sensitive transcriptional factor, as well as ERK 44, 45 . Studies from our lab and elsewhere have demonstrated increased levels of oxidative and nitrosative stress in STZ-diabetic rats and treatment with either an antioxidant or iNOS inhibitor has reduced nitrotyrosine levels, an indirect marker of peroxynitrite formation 11, 13 . Peroxynitrite has been shown to cause severe hypotension, profound vasodilatation, cardiac depression and multiple organ failure in various models of septic shock 46, 47 . Increasing evidence suggests that many of the cardiovascular abnormalities in diabetic rats can be prevented by inhibiting nitrosative stress caused by peroxynitrite 48, 49 . For instance, previous studies from our lab 6, 11  showed that inhibition of peroxynitrite using antioxidants such as N-acetylcysteine improves cardiac performance, MABP and heart rate in streptozotocin diabetic rats 6, 11 . In the present study inhibition of nitrosative stress by both LY333531 and L-NIL was associated with improvements in MABP and HR, and pressor responses to vasoactive agents in STZ-diabetic rats.   56 Increasing evidence now suggest that iNOS and nitrosative stress are critical determinants in the development of diabetic complications in humans 4, 50, 51 . Increased generation of NO occurs in patients with type 1 diabetes, and is associated with enhanced peroxynitrite production and lipid peroxidation 52 . Furthermore, a correlation between increased plasma NOx levels and endothelial dysfunction, lower blood pressure and sympathetic nerve dysfunction in type 1 diabetes has also been found 52, 53 . In conclusion, the results of the present study demonstrate that PKC!2 is an obligatory mediator of nitrosative stress and that LY333531 significantly reduced the formation of iNOS and improved cardiovascular abnormalities in STZ-diabetic rats. Moreover, hyperglycemia-induced activation of PKC!2 is antecedent to increases in superoxide, ERK1/2, NF-"B, and iNOS expression in cardiovascular tissues, while inhibition of this pathway suppresses key signaling events that lead to increased nitrosative stress. Collectively, our data suggest that inhibition of PKC!2 may be a useful approach  for correcting abnormal hemodynamics in diabetes by preventing  iNOS-mediated nitrosative stress.        57 2.5 Tables and figures Table 2.1 General characteristics following 3 weeks of LY333531 and L-NIL treatment in control and diabetic rats.    Parameters   C   D   C-LY   C-LNIL  D-LY   D-LNIL Body weight (g)  Before Treatment  After Treatment    278 ± 4.2  423 ± 5.3    253 ± 5.0  360 ± 8.2*   282 ± 6.2  425 ± 6.2   292  ± 6.3  428 ± 11.9   253 ± 4.2  372 ± 9.5*   270 ± 6.0*  372 ±13.2*  Blood Glucose (mM)   8.3 ± 0.23   31.1 ± 0.8*   8.5 ± 0.22  7.7 ± 0.1  31.8 ± 0.75*  27.5 ± 0.8*  All data expressed are mean ± SEM. Data were analyzed using One-Way ANOVA with Newman–Keuls post hoc test. * different from C, C-LY and C-LNIL groups     58           Figure 2.1 LY333531 treatment decreases PKC!2 phosphorylation and inducible nitric oxide synthase expression (iNOS) in cardiomyocytes. A) (Top) Representative Western blot showing phospho-PKC!2 (Thr 641) in comparison with total PKC!2 in cardiomyocytes exposed to low (5.5 mM; LG) or high glucose (25 mM; HG) for 18 hours in the absence or presence of LY3335351 (20 nM; LG+LY and HG+LY). (Bottom) The densitometric values of phospho-PKC!2 were normalized to corresponding total PKC!2 densitometric values and the relative band intensities are expressed as mean ± SEM (n=4 independent experiments). *P<0.05 compared with LG, LG-LY and HG-LY groups. B) Representative Western blot showing iNOS expression, with GAPDH as a loading control, in cardiomyocytes exposed to low (5.5 mM) or high glucose (25 mM) for 18 hours in the presence or absence of LY3335351 (20 nM). iNOS densitometric values were normalized to their corresponding GAPDH densitometric values and expressed as relative band intensities. All data are expressed as mean ± SEM (n=3 to 5 independent experiments). *P<0.05 compared with LG, LG-LY and HG-LY groups.   59            Figure 2.2 Effect of LY333531 on PKC!2 phosphorylation and iNOS expression in control and diabetic heart tissues. A) Representative Western blot showing phospho-PKC!2 (Thr 641) in comparison with total PKC!2 expression levels in ventricular tissues of control (C), diabetic (D), control LY333531- treated (C-LY) and diabetic LY333531- treated (D-LY) rats. The densitometric values of phospho-PKC!2 were normalized to corresponding total PKC!2 expression levels and the relative band intensities are expressed as mean ± SEM (n=5). *P<0.05 compared with C, C-LY and D-LY groups. B) Representative Western blot showing iNOS expression in the ventricular tissues of C, D, C-LY and D-LY groups. iNOS protein was immunoprecipitated from heart ventricular tissue lysates using mouse monoclonal anti-iNOS antibody. Equal amounts of immunoprecipitated complex were loaded on to the gels and were subjected to SDS-PAGE. Densitometric data are expressed as percentage of control. All values are expressed as mean ± SEM (n=4). *P<0.05 compared with C, C-LY and D-LY groups.   60            Figure 2.3 Effect of LY333531 on iNOS expression in isolated superior mesenteric arteries and cultured rat aortic VSMC. A) Representative Western blot showing iNOS expression in superior mesenteric arteries of C, D, C-LY and D-LY groups. iNOS was immunoprecipitated from superior mesenteric artery tissue lysates using mouse monoclonal anti-iNOS antibody. Densitometric data are expressed as percentage of control. All values are expressed as mean ± SEM (n=4). *P<0.05 compared with C, C-LY and D-LY groups. B) Representative Western blot showing iNOS expression, with GAPDH shown as a loading control, in aortic VSMC exposed to low (5.5 mM) and high glucose (25 mM) for 36 hours in the presence or absence of LY3335351 (20 nM). iNOS densitometric values were normalized to their corresponding GAPDH densitometric values and expressed as relative band intensities. All data are expressed as mean ± SEM (n=4 independent experiments). *P<0.05 compared with LG, LG-LY and HG-LY groups.   61              Figure 2.4 Effect of LY333531 on PKC!  translocation cultured rat aortic VSMC. Representative Western blot (top panel) showing total PKC!2 expression in the cytosolic and membrane fractions along with membrane (pan-cadherin) and cytosol (GAPDH) loading controls in VSMC exposed to low (5.5 mM) or high glucose (25 mM) for 36 hours in the presence or absence of LY333531 (20 nM). Densitometric data (bottom panel) for PKC!2 expression in the membrane fractions. The data represented are the mean ± SEM from 3 independent experiments. *P<0.05 compared with all other groups.       62            Figure 2.5 Effect of siRNA suppression of PKC!2 on iNOS expression in VSMC. Cells were transfected with either MOCK or PKC!2 specific siRNA and exposed to low or high glucose for 36 hours. A) Representative Western blot showing total PKC!2 expression, with GAPDH as a loading control in VSMC treated with MOCK or PKC!2 siRNA. The densitometric values of PKC!2 were normalized to corresponding total GAPDH densitometric values and the relative band intensities are expressed as mean ± SEM (n=4). Open bars represent LG and dark bars represent HG treated VSMC. *P<0.05 compared with MOCK siRNA treated VSMC exposed to LG or HG conditions. B) Representative Western blot showing iNOS expression, with GAPDH as a loading control in VSMC treated with MOCK or PKC!2 siRNA. The densitometric values of iNOS were normalized to corresponding total GAPDH densitometric values and the relative band intensities are expressed as mean ± SEM (n=4). Open bars represent LG and dark bars represent HG treated VSMC. * different from all other groups (P<0.05). # different from MOCK siRNA treated VSMC exposed to LG or HG conditions.   63           Figure 2.6 Effect of LY333531 on ERK1/2 and NF-!B activation in VSMC. A) Representative Western blot showing phospho-ERK-1 (Thr 202) and phospho-ERK-2 (Tyr 204) in comparison with total ERK1/2 and with GAPDH as a loading control in VSMC exposed to low (5.5 mM) or high glucose (25 mM) for 36 hours in the presence or absence of LY3335351 (20 nM). The densitometric values of phospho ERK-1 were normalized to corresponding total ERK-1 densitometric values and the relative band intensities are expressed as mean ± SEM (n= 4 independent experiments). *P<0.05 compared with LG, LG-LY and HG- LY groups. B) Representative Western blot showing phospho-NF-!B P65 subunit (Ser 536) in comparison with total NF-!B P65 and with GAPDH as a loading control in VSMC exposed to low (5.5 mM) and high glucose (25 mM) for 36 hours in the presence or absence of LY333531 (20 nM). The densitometric values of phospho-NF-!B were normalized to corresponding total phospho-NF-!B P65 densitometric values and the relative band intensities are expressed as mean ± SEM (n= 4 independent experiments). *P<0.05 compared with LG, LG-LY and HG-LY groups.   64            Figure 2.7 In vivo effects of LY333531 on ERK1/2 phosphorylation in the SMA. Representative western blot showing phospho-ERK-1 (Thr 202) and phospho-ERK-2 (Tyr 204) in comparison with total ERK1/2 in SMA of control (C), diabetic (D), Control LY333531- treated (C-LY) and diabetic LY333531-treated (D-LY) rats. The densitometric values of phospho-ERK-1 were normalized to corresponding total ERK-1 densitometric values and the relative band intensities are expressed as mean ± SEM (n= 4). Data are represented as mean ± SEM (n=4). *different from all other groups; # different from D group (P<0.05).      65            Figure 2.8 Effect of PKC!2 silencing on ERK1/2 and NF-"B activation in VSMC. A) Representative Western blot showing phospho-ERK-1 (Thr 202) and phospho-ERK-2 (Tyr 204) in comparison with total ERK1/2 in VSMC treated with MOCK or PKC!2 siRNA. The densitometric values of phospho-ERK-1 were normalized to corresponding total ERK-1 densitometric values and the relative band intensities are expressed as mean ± SEM (n= 4 independent experiments). Open bars represent LG and dark bars represent HG treated VSMC. *different from all other groups; #different from MOCK siRNA treated VSMC exposed to HG (P<0.05. B) Representative western blot showing phospho-NF-"B P65 subunit (Ser 536) in comparison with total NF-"B P65 in VSMC treated with MOCK or PKC!2 siRNA. The densitometric values of phospho-NF-"B P65 were normalized to corresponding total NF-"B P65 densitometric values and the relative band intensities are expressed as mean ± SEM (n= 4 independent experiments). Open bars represent LG and dark bars represent HG treated VSMC. *different from all other groups.   66                Figure 2.9 Effect of mannitol on the expression of iNOS and PKC! . VSMC were exposed to low glucose (5.5 mM) or high mannitol (5.5 mM glucose plus 19.5 mM mannitol) in the presence or absence of LY333531 (20 nM) for 36 hours. Representative Western blots showing the expression of A) phospho-PKC!2 and total PKC!2, B) iNOS, C) phospho-ERK1/2 and total ERK1/2, D) phospho-NF-"B P65 and total NF-"B P65 and E) GAPDH as loading control.     67                Figure 2.10 Effect of LY333531 on superoxide formation in VSMC incubated in low or high glucose. Formation of superoxide anions as determined by DHE fluorescence in VSMC exposed to low (5.5 mM) and high glucose (25 mM) in the presence or absence of LY333531 (20 nM) for 36 hours. Data are represented as percent increase in DHE fluorescence with respect to the control group (low glucose). All data are represented as mean ± SEM (n=6). *different from all other groups: #different from LG and HG groups (P<0.05).   68 Figure 2.11 Effects of in vivo LY333531 administration on iNOS expression in the heart and superior mesenteric arteries (SMA) of diabetic and control rats. Photomicrographs (X20) illustrating the immunohistochemical localization of iNOS in sections of the heart (top panel, A) and SMAs (bottom panel, B) of control (C), diabetic (D), Control LY333531-treated (C-LY) and diabetic LY333531-treated (D-LY) rats. NC represents a negative control (where the section was incubated with NGS instead of the iNOS antibody) showing no staining. Moreover, careful observation of the diabetic heart sections under higher magnification (D1, X40) clearly suggests that iNOS is predominantly localized in cardiomyocytes that are histologically identified by their elongated brick-like appearance, relatively large size and striations. A1) Semi-quantitative analysis of iNOS immunostain from heart (ventricular) section photomicrographs. iNOS Immunostain was quantified using Image J analysis software. As shown, there is a significant increase in the expression of iNOS in the untreated diabetic heart sections compared to the control group. LY333531 treatment has reduced the expression of iNOS by approximately 50%. Data are represented as mean ± SEM (n=4). *different from all other groups; #different from D group (P<0.05). B) The bottom panel represents the expression of iNOS in SMAs. As shown there is a significant increase in the expression of iNOS particularly in the medial and adventitial layers of the arteries from untreated diabetic rats. As opposed to the dense immunostaining in these layers (see picture D2, X40), very little staining can be observed in the tunica intima or endothelium, suggesting that the major source of iNOS is the media and adventitia. B1) Semi-quantitative analysis of iNOS immunostain in SMA section photomicrographs. As shown, there is a significant increase in the total expression of iNOS in the untreated diabetic SMA sections compared to the control group. LY333531 treatment reduced the expression of iNOS by ~ 50%. Data are represented as mean ± SEM (n=4). *different from all other groups; #different from D group (P<0.05).    69                  70 Figure 2.12 Effects of in vivo LY333531 administration on nitrotyrosine (NT) levels in the heart and SMA of diabetic and control rats. Photomicrographs (X20) illustrating the formation of NT in sections of the heart (top panel, A) and SMAs (bottom panel, B) of control (C), diabetic (D), Control treated (C-LY) and diabetic treated (D-LY) rats. NC represents a negative control (where the section was incubated with NGS instead of the NT antibody) confirming the absence of non-specific binding of anti-NT antibody. A1) Semi-quantitative analysis of NT immunostain from heart (ventricular) section photomicrographs. NT Immunostain was quantified using Image J analysis software. As shown, there is a significant increase (~ 60%) in the formation of NT in the untreated diabetic heart sections compared to the control group. LY333531 treatment reduced the formation of NT by ~ 60%. Data are represented as mean ± SEM (n=4). *different from all other groups; #different from D group (P<0.05). B) The bottom panel represents the expression of NT in the SMAs. As shown there is a significant increase in the formation of NT in all the layers of the diabetic arteries including endothelium. B1) Semi-quantitative analysis of NT immunostain in SMA section photomicrographs. As shown, there is a significant increase (~ 40%) in the formation of NT in the untreated diabetic SMA sections compared to the control group. LY333531 treatment reduced the formation of NT by ~ 70%. Data are represented as mean ± SEM (n=4). *different from all other groups; #different from D group (P<0.05).         71                 72 Figure 2.13 Effect of 3 weeks of LY333531 or L-NIL treatment on hemodynamic parameters in freely moving conscious rats. Data represent means ± SEM (n=8). A) Effect of LY333531 or L-NIL on MABP. *different from all other groups; #different from D group (P<0.05). B) Effect of LY333531 or L-NIL on HR. *different from all other groups; #different from D group (P<0.05). C) Effect of LY333531 or L-NIL on endothelial function as measured by the change in blood pressure (! MABP) in response to a single i.v bolus dose of L-NAME (10mg/kg) in control and diabetic rats. *different from their respective control groups (P<0.05), #different from D and D-LY groups (P<0.05). D) Effect of LY333531 or L-NIL on pressor responses to bolus doses of methoxamine (100-300 nmoles/kg). *different from all other groups (P<0.05); # different from D group (P<0.05).                 73                  74 2.6    Bibliography 1. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. The Diabetes Control and Complications Trial Research Group. 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The extended protein kinase C superfamily. Biochem J. 1998;332 ( Pt 2):281-292.   77 33. Mueed I, Zhang L, Macleod KM. Role of the PKC/CPI-17 pathway in enhanced contractile responses of mesenteric arteries from diabetic rats to alpha-adrenoceptor stimulation. Br J Pharmacol. 2005. 34. Chen CC, Wang JK, Chen WC, Lin SB. Protein kinase C eta mediates lipopolysaccharide-induced nitric-oxide synthase expression in primary astrocytes. J Biol Chem. 1998;273(31):19424-19430. 35. Chen CC, Wang JK, Lin SB. Antisense oligonucleotides targeting protein kinase C- alpha, -beta I, or -delta but not -eta inhibit lipopolysaccharide-induced nitric oxide synthase expression in RAW 264.7 macrophages: involvement of a nuclear factor kappa B-dependent mechanism. J Immunol. 1998;161(11):6206-6214. 36. Hortelano S, Genaro AM, Bosca L. Phorbol esters induce nitric oxide synthase and increase arginine influx in cultured peritoneal macrophages. FEBS Lett. 1993;320(2):135-139. 37. Nishio E, Watanabe Y. Glucose-induced down-regulation of NO production and inducible NOS expression in cultured rat aortic vascular smooth muscle cells: role of protein kinase C. Biochemical and biophysical research communications. 1996;229(3):857-863. 38. Pacheco ME, Beltran A, Redondo J, Manso AM, Alonso MJ, Salaices M. High glucose enhances inducible nitric oxide synthase expression. Role of protein kinase C-betaII. Eur J Pharmacol. 2006;538(1-3):115-123. 39. Lafuente N, Matesanz N, Azcutia V, Romacho T, Nevado J, Rodriguez-Manas L, Moncada S, Peiro C, Sanchez-Ferrer CF. The deleterious effect of high concentrations of D-glucose requires pro-inflammatory preconditioning. J Hypertens. 2008;26(3):478-485. 40. Ramana KV, Friedrich B, Srivastava S, Bhatnagar A, Srivastava SK. Activation of nuclear factor-kappaB by hyperglycemia in vascular smooth muscle cells is regulated by aldose reductase. Diabetes. 2004;53(11):2910-2920. 41. Yerneni KK, Bai W, Khan BV, Medford RM, Natarajan R. Hyperglycemia-induced activation of nuclear transcription factor kappaB in vascular smooth muscle cells. Diabetes. 1999;48(4):855-864. 42. Konishi H, Tanaka M, Takemura Y, Matsuzaki H, Ono Y, Kikkawa U, Nishizuka Y. Activation of protein kinase C by tyrosine phosphorylation in response to H2O2. Proc Natl Acad Sci U S A. 1997;94(21):11233-11237.   78 43. Kitada M, Koya D, Sugimoto T, Isono M, Araki S, Kashiwagi A, Haneda M. Translocation of glomerular p47phox and p67phox by protein kinase C-beta activation is required for oxidative stress in diabetic nephropathy. Diabetes. 2003;52(10):2603-2614. 44. Daou GB, Srivastava AK. Reactive oxygen species mediate Endothelin-1-induced activation of ERK1/2, PKB, and Pyk2 signaling, as well as protein synthesis, in vascular smooth muscle cells. Free Radic Biol Med. 2004;37(2):208-215. 45. Gloire G, Legrand-Poels S, Piette J. NF-kappaB activation by reactive oxygen species: fifteen years later. Biochem Pharmacol. 2006;72(11):1493-1505. 46. Ferdinandy P, Danial H, Ambrus I, Rothery RA, Schulz R. Peroxynitrite is a major contributor to cytokine-induced myocardial contractile failure. Circ Res. 2000;87(3):241-247. 47. Parrillo JE. Pathogenetic mechanisms of septic shock. N Engl J Med. 1993;328(20):1471-1477. 48. Cuzzocrea S, Riley DP, Caputi AP, Salvemini D. Antioxidant therapy: a new pharmacological approach in shock, inflammation, and ischemia/reperfusion injury. Pharmacol Rev. 2001;53(1):135-159. 49. Nassar T, Kadery B, Lotan C, Da'as N, Kleinman Y, Haj-Yehia A. Effects of the superoxide dismutase-mimetic compound tempol on endothelial dysfunction in streptozotocin-induced diabetic rats. Eur J Pharmacol. 2002;436(1-2):111-118. 50. Ceriello A, Mercuri F, Quagliaro L, Assaloni R, Motz E, Tonutti L, Taboga C. Detection of nitrotyrosine in the diabetic plasma: evidence of oxidative stress. Diabetologia. 2001;44(7):834-838. 51. Mabley JG, Soriano FG. Role of nitrosative stress and poly(ADP-ribose) polymerase activation in diabetic vascular dysfunction. Curr Vasc Pharmacol. 2005;3(3):247-252. 52. Hoeldtke RD. Nitrosative stress in early Type 1 diabetes. David H. P. Streeten Memorial Lecture. Clin Auton Res. 2003;13(6):406-421. 53. Ceriello A, Piconi L, Esposito K, Giugliano D. Telmisartan shows an equivalent effect of vitamin C in further improving endothelial dysfunction after glycemia normalization in type 1 diabetes. Diabetes Care. 2007;30(7):1694-1698.    79 3. Chronic inhibition of inducible nitric oxide synthase ameliorates cardiovascular abnormalities in streptozotocin diabetic rats 1 .                     1 A version of this chapter has been accepted for publication. Nagareddy PR, McNeill JH and MacLeod KM. Chronic inhibition of inducible nitric oxide synthase ameliorates cardiovascular abnormalities in streptozotocin diabetic rats (2009). European Journal of Pharmacology; 611(1-3): 53-9.  80 3.1 Introduction Cardiovascular abnormalities, manifested by depressed mean arterial blood pressure, heart rate, cardiac output, endothelial dysfunction and attenuated pressor responses to vasoactive agents are the key pathophysiological events associated with hyperglycemia, particularly in animal models of type 1 diabetes 1, 2 . Although the exact mechanisms by which chronic hyperglycemia contributes to these abnormalities are currently unknown, a number of mechanisms have been proposed including activation of protein kinase C, increased activity of the polyol pathway, formation of non-enzymatic advanced glycosylation end products, oxidative stress and/or possibly by induction of nitric oxide synthase 3, 4 . Studies from our lab and elsewhere have demonstrated an increased expression of iNOS in cardiac, vascular and renal tissues of streptozotocin diabetic rats 5-8 . Physiological concentrations of nitric oxide (NO) maintain the vasculature in a state of active vasodilatation and regulate regional blood flow to tissues such as heart and kidney in response to local environmental changes 9, 10 . NO in large quantities however, is toxic to the cardiovascular system, particularly when associated with increased levels of reactive oxygen species (ROS) such as superoxide anions 11, 12 . Both NO and superoxide anions are highly reactive and can rapidly form peroxynitrite [ONOO - ], a cytotoxic compound 13, 14 . In fact, peroxynitrite is a favored product under conditions of hyperglycemia where cellular production of both NO and reactive oxygen species are increased 11, 12 . Further, peroxynitrite has been shown to cause severe hypotension, profound vasodilatation, cardiac depression and multiple organ failure in various models of septic shock 14, 15 . Previous studies from our lab demonstrated that acute inhibition of iNOS improves vascular reactivity but not depressed mean arterial blood pressure and heart rate in streptozotocin diabetic rats 8 . In the present study we hypothesized that long-term inhibition of iNOS is necessary to prevent endothelial dysfunction and depression of  81 cardiac function, mean arterial blood pressure, heart rate and pressor responses to vasoactive agents. 3.2 Methods 3.2.1 Study design and induction of diabetes This study conforms with the Canadian Council on Animal Care Guidelines on the Care and Use of Experimental Animals. Sixty-four male Wistar rats weighing between 280 to 300 g were obtained from Charles River Laboratories Inc., Quebec and allowed to acclimatize to the local vivarium. They were randomly divided into two study groups each consisting of four equal groups: Control (C), Control treated (CT), Diabetic (D) and Diabetic treated (DT). Diabetes was induced by a single tail vein injection of 60 mg/kg streptozotocin. The presence of diabetes was confirmed by hyperglycemia (>20 mmol/L) 72 h after streptozotocin administration. Plasma glucose was measured by an enzymatic colorimetric assay kit (Roche Diagnostics, Laval, Quebec) using a Beckman Glucose Analyzer. One week after the induction of diabetes, CT and DT groups received the selective iNOS inhibitor, N6-(1-Iminoethyl)-L-lysine dihydrochloride (L-NIL) at a dose of 3 mg/kg/day by oral gavage. L-NIL is a potent and relatively selective iNOS inhibitor with an IC50 of 5.9 µM for iNOS compared to an IC50 of 138 µM for eNOS and 35 µM for nNOS 16 . A dose of 3 mg/kg/day was selected based on our previous study in which we were able to inhibit iNOS derived NO production in heart tissues from streptozotocin diabetic rats 17 . After 8 weeks of treatment each animal in the first study group was surgically prepared for measurement of mean arterial blood pressure and heart rate. In the second study group in each rat, cardiac performance was evaluated using isolated working heart technique.    82 3.2.2 Surgical procedures Rats were anesthetized with halothane and fluid-filled (heparinised saline, 20 U/ml) catheters were placed in the left carotid artery (PE 50) and jugular vein (PE 10) for measurements of blood pressure and for drug administration respectively. All the catheters were exteriorized at the nape of the neck, passed through a harness and tether and connected to swivels (Instec Lab Inc., PA) mounted above the cage for free movement of the animal. The arterial catheter was connected to a disposable pressure transducer (Viggo-Spectramed, CA) mounted on the cage exterior at the level of the rat. Mean arterial blood pressure and heart rate were simultaneously recorded on a Gould TA 2000 Thermal Array Recorder (Gould Instrument System Inc., OH) and a computer, using custom-made data acquisition software. In order to achieve normalization of cardiac baroreflexes, the animals were allowed to recover from anesthesia and surgery for at least 4 hours before recording mean arterial blood pressure and heart rate 18 . Four hours after surgery, basal mean arterial blood pressure and heart rate were measured in all rats. Subsequently, dose response curves (dose response curve) to isovolumic bolus doses of methoxamine  (100-300 nmol/kg) were constructed in control and diabetic rats. Dose response curves were constructed by measuring mean arterial blood pressure in response to each bolus dose of methoxamine, allowing sufficient time (or 10 min) for mean arterial blood pressure to return to normal between each dose. At termination, the heart, and superior mesenteric artery or the whole mesenteric arterial bed were immediately removed and placed in ice-cold Krebs solution (120 mM NaCl, 5.9 mM KCl, 25 mM NaHCO3, 11.5 mM glucose, 1.2 mM NaH2PO4, 1.2 mM MgCl2, 2.5 mM CaCl2) containing 0.1 µM water-soluble dexamethasone to prevent induction of iNOS in vitro. The tissues were cleaned of all adherent tissue, snap frozen in liquid nitrogen and stored at -70°C for Western blot and iNOS activity (in superior mesenteric artery) measurements.   83 3.2.3 Isolated working heart procedure In the second study, following anesthesia, the chest cavity was opened and the heart removed, placed in ice cold buffer and perfused in the isolated working heart apparatus, using warm oxygenated (95% O2) Chenoweth-Keolle (CK) buffer containing (in mM) NaCl, 120; KCl, 5.6; CaCl2, 2.18; MgCl2, 2.1; NaHCO3, 19.2; and glucose, 10.  Perfusion with CK buffer was initiated in a retrograde manner through the aorta. Left ventricular developed pressure (LVP) was measured by means of a Statham P23 AA transducer (Gould Statham Instruments Inc.), which was inserted through the apex of heart into the left ventricle via a 20-gauge needle. Cardiac work was initiated by switching the perfusion system from the retrograde mode to the working heart mode 19 . The perfusion buffer was pumped into left atrium and through to the left ventricle before being ejected out of the aorta. The aortic outflow was subjected to an after-load of a 75-mm column of H2O. Left ventricular pressure and the first derivative of left ventricular pressure (dP/dt) were recorded on a Grass model 79D polygraph. The heart rate was maintained at 300 beats per minute using a stimulator (Grass model SD 90 stimulator) by placing a stainless steel electrode on the left atrium at twice the threshold voltage with a square wave pulse of 5-ms duration. Against a constant afterload (a 19 cm column of water in a polyethylene tube PE 160), the heart was subjected to the changes in preload (left atrial filling pressures) from 3 to 11 mm Hg by varying the peristaltic pump speed. Left ventricular performance was assessed in terms of the rate of contraction (+ dP/dt), rate of relaxation  (- dP/dt) and left ventricular developed pressure (LVP). Data were collected and analyzed using a microcomputer with a custom made software program, as described previously 20 . 3.2.4 Western blot studies Protein expression and NT levels in superior mesenteric arteries and hearts were determined by western blot analysis using specific antibodies directed against eNOS, iNOS and NT. Briefly, aliquots of the pulverized superior mesenteric artery from each rat were homogenized in  84 modified RIPA buffer containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM PMSF, 1 mM EDTA, 5 !g/ml aprotinin, 5 !g/ml leupeptin, 1% Triton x-100, 1% sodium deoxycholate and 0.1% SDS at 4 °C, using a homogenizer. The homogenate was centrifuged at 10000"g at 4 °C for 15 min. Total protein content of the supernatant was determined using the Bio-Rad protein assay, which is based on Bradford method 21 . Aliquots of protein were separated by SDS-7.5% (for eNOS and iNOS) or SDS-10% (for NT) polyacrylamide gel electrophoresis. Immunoblotting was performed either with a mouse monoclonal anti-iNOS (1:200, BD Transduction Labs), anti-NT (1:500, Cayman Chemical Company) or rabbit polyclonal anti- eNOS antibody (Upstate Biotechnology). HRP conjugated goat anti-rabbit and anti-mouse secondary antibodies were obtained from Santa Cruz Biotechnology. The immune complexes were detected using enhanced chemiluminescence  dye (ECL, Amersham Pharmacia Biotech, Piscataway, NJ).  The intensity of the bands was determined using image J software from NIH. 3.2.5 Immunohistochemistry of iNOS and NT in the heart and SMA sections The heart and superior mesenteric artery tissues were fixed in 10% NBF overnight and transferred to 70% ethanol. This was followed by paraffin processing through increasing grades of ethanol, xylene and paraplast (Fischer Scientific, ON). Paraffin embedded tissue blocks were sectioned at 3 µm and mounted on slides. The sections were deparaffinized, rehydrated, washed with PBS and blocked with 5% normal goat serum (NGS) in PBS for 60 minutes. The slides were subsequently incubated with primary rabbit polyclonal anti-iNOS (1:100, Abcam) or mouse monoclonal anti- NT (1:200, Cayman Chemicals) antibodies in PBS containing 1% NGS overnight at 4 o C in a humidity chamber. After washing off the primary antibody with PBS, the sections were incubated with biotinylated goat anti-rabbit or anti-mouse secondary antibody (1 drop in 10 mL PBS, Vectastain ABC kit, Vector Laboratories) for 1 hour followed by avidin- biotinylated HRP complex and color development performed using 3,3-diaminobenzidine. Some sections incubated without primary antibodies served as negative controls. Using a high power  85 microscope and digital imaging system all images were observed individually and photographed (20X or 60X). In each section, 4-5 random fields were selected and staining intensity analyzed using a scale ranging from 1 to 5. 3.2.6 iNOS activity assay in the mesenteric arterial bed iNOS activity in the mesenteric arterial bed was measured by monitoring the biochemical conversion of [ 14 C]L-arginine to [ 14 C]L-citrulline using a commercially available NOS assay kit (Cayman Chemical, Ann Arbor, MI). Briefly, mesenteric arterial bed homogenates in buffer were centrifuged, and supernatant (80 µg protein) was added to a calcium free reaction mixture composed of buffer, 10 mM NADPH, and 10 µl of [ 14 C] L-arginine (50 µCi/µl). The reaction was maintained for 60 min at room temperature and stopped using stop buffer. The remaining [ 14 C] L-arginine was removed from the solution using an equilibrated resin. Finally, the radioactivity was measured using a liquid scintillation counter. 3.2.7 Statistical analysis All values are expressed as mean ± S.E.M. “n” denotes the sample size in each group. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by the Newman-Keuls test for multiple comparisons. GraphPad Prism (GraphPad Software, CA) software program was used for statistical analysis. For all results the level of significance was set at P<0.05. 3.3 Results 3.3.1 General characteristics The general characteristics of rats following 8 weeks of L-NIL treatment are shown in table 3.1. All streptozotocin-injected rats were hyperglycemic (>20 mM) compared to their age matched controls. In addition, body weights of both untreated and treated diabetic rats were significantly  86 lower than their age matched controls. Treatment of diabetic rats with L-NIL did not change either the body weight or blood glucose in any of the groups. 3.3.2 Effect of L-NIL on mean arterial blood pressure and heart rate  Untreated diabetic rats showed significantly lower mean arterial blood pressure and heart rate than matched control rats (Fig. 3.1). Treatment with L-NIL however, improved the depression in both mean arterial blood pressure and heart rate in diabetic rats without affecting these parameters in control animals. 3.3.3 Effect of L-NIL treatment on pressor responses to methoxamine  Administration of bolus doses of methoxamine increased mean arterial blood pressure in both control and diabetic rats in a dose-dependent manner. Compared to the corresponding age matched control rats, the responses to methoxamine were significantly attenuated in untreated diabetic rats. However, treatment of diabetic rats with L-NIL significantly augmented pressor responses without affecting the pressor responses in control rats (Fig. 3.2). 3.3.4 Effect of L-NIL treatment on cardiac performance Cardiac performance was assessed by measuring left ventricular responses to varying left atrial filling pressures in terms of left ventricular pressure (LVP), the rate of contraction, +dP/dT and the rate of relaxation, -dP/dT. As shown in the Fig. 3.3, compared with controls, hearts from  vehicle-treated diabetic rats showed a significant reduction  in LVP, +dP/dT, and –dP/dT at above 5 mmHg, which  is characteristic of the left ventricular dysfunction of diabetic  cardiomyopathy. Administration of L-NIL to diabetic rats prevented  the deterioration of cardiac contractile function and significantly  improved LVP, +dP/dT, and –dP/dT compared with the diabetic  untreated rats. L-NIL did not significantly alter the contractile  function of hearts from control rats except for a small decrease  in LVDP at the highest filling pressure (10 mmHg).    87 3.3.5 Effect of L-NIL treatment on eNOS, iNOS and NT expression in the heart and SMA Decreased eNOS expression was observed both in the heart and superior mesenteric arteries of untreated diabetic rats compared to C and CT rats (Fig. 3.4). L-NIL treatment of diabetic rats improved the expression of eNOS to that in the control rats. On the contrary, untreated diabetic rats exhibited increased expression of iNOS both in the heart and superior mesenteric arteries as determined by immunoblotting (Fig. 3.5). Further, immunohistochemical analysis revealed increased localization of iNOS in the medial and advential layers of superior mesenteric arteries (Fig. 3.6) from untreated diabetic rats which is consistent with our previous findings 5 . As opposed to the dense immunostaining in the medial and advential layers, very little staining can be observed in the tunica intima or endothelium, suggesting that the major source of iNOS is media and adventia. As expected treatment with L-NIL did not change the expression of iNOS (Fig. 3.5) but its activity was significantly reduced in superior mesenteric artery from diabetic rats (Fig. 3.7). Similarly, Western blot analysis of NT, an indirect marker of peroxynitrite formation, indicated an increase in the nitration of proteins in the hearts and superior mesenteric arteries of untreated diabetic rats (Fig.3.8). Among various nitrated proteins, an unidentified protein with a molecular weight of 80 kDa showed increased nitration both in the hearts and superior mesenteric arteries. Further, the detection of elevated levels of NT in whole hearts (Fig. 3.9) suggests that cardiomyocytes rather than inflammatory or endothelial cells are the major sites of iNOS expression and probably the source for increased peroxynitrite formation in the diabetic heart. As shown, there is a substantial increase in the levels of NT in the diabetic heart (Fig. 3.9; panel B) and pictures of higher magnification (x100) clearly demonstrate that the immunostaining is predominantly located in cardiomyocytes which are histologically characterized by their elongated brick-like appearance, relatively large size and striations (Fig. 3.9; panel E). Treatment with L-NIL significantly decreased the formation of NT in the DT group compared to untreated diabetic rat not only in SMA but also heart (Fig.3.9).  88 3.4 Discussion The major findings of the present study are that chronic inhibition of iNOS attenuated the depression of mean arterial blood pressure and heart rate and improved cardiac performance in streptozotocin-diabetic rats. This was associated with normalization of the expression of vascular eNOS, reduced formation of NT and improved pressor responsiveness to !1-AR stimulation. These data, together with the results of our previous studies, highlight an important role for iNOS in mediating cardiovascular depression in the streptozotocin-diabetic rat. Administration of streptozotocin to rats results in the classical symptoms of diabetes such as hyperglycemia, decreased body weight gain, polyuria, and increased food and fluid intake (data not shown). In addition, diabetic rats also demonstrate lower mean arterial blood pressure, heart rate and attenuated pressor responses compared to their age matched control animals. The mechanisms underlying the depressed mean arterial blood pressure and bradycardia in streptozotocin diabetic rats are not well understood. However, previous studies from our lab have shown that although the expression of eNOS is reduced and that of nNOS is unchanged, the expression and activity of iNOS in heart, aorta and superior mesenteric artery from streptozotocin diabetic rats are increased 5, 8 . Since NO plays an important role in cardiovascular homeostasis, we hypothesized that its over-production in diabetes contributes to the depressed cardiac function, mean arterial blood pressure and heart rate. However, we have previously shown that acute inhibition of iNOS, while restoring pressor responses to vasoactive agents, had no effect on the depressed mean arterial blood pressure and heart rate in streptozotocin diabetic rats 8 . Therefore, in the present study we investigated the possibility that chronic inhibition of iNOS would prevent the depression of mean arterial blood pressure, heart rate and cardiac function in streptozotocin diabetic rats.   89 If iNOS is responsible for the depressed mean arterial blood pressure and heart rate observed in streptozotocin diabetic rats, then why did acute inhibition of iNOS not improve resting mean arterial blood pressure and heart rate? Although NO from iNOS does not seem to directly depress mean arterial blood pressure and heart rate, changes secondary to increased expression of iNOS and over-production of NO in cardiovascular tissue may contribute to depressed mean arterial blood pressure and heart rate in these rats. These include increased formation of peroxynitrite in cardiovascular tissue. Increased formation of reactive oxygen species, which occurs in hyperglycemic conditions, can scavenge NO resulting in the formation of peroxynitrite. We previously have found altered levels of plasma and tissue markers of oxidative stress including decreased total antioxidant and superoxide dismutase and increased free 15-F2t- Isoprostane levels in streptozotocin diabetic rats 22 . Our present data demonstrating increased levels of NT (a marker of peroxynitrite formation) both in the heart and superior mesenteric artery suggests increased levels of nitrosative stress in diabetic rats. Further, an improvement in blood pressure and heart rate by L-NIL was associated not only with normalization of iNOS expression and activity, but with attenuation of NT levels in the heart and superior mesenteric artery of diabetic rats. These data suggest that the depressed cardiac function, mean arterial blood pressure and heart rate in untreated diabetic rats may be due to the actions of peroxynitrite on cardiovascular tissues. In addition to reduced peroxynitrite formation, normalization of eNOS expression or iNOS activity in the heart by L-NIL treatment may explain in part, the improvement in bradycardia in DT rats. Studies have demonstrated that knockout of eNOS results in decreased basal heart rate, leading to the suggestion of a positive chronotropic effect of eNOS 23 . Further, low and physiological concentrations of NO have been shown to increase heart rate in vitro by activating cAMP-dependent protein kinase. On the other hand, high concentrations of NO can also reduce heart rate by activating cGMP-dependent protein kinase 24 . Our recent studies demonstrated that  90 increased iNOS expression is associated with elevated NOx levels in the hearts of untreated diabetic rats 17 . This is consistent with the possibility that high concentrations of locally produced NO in the heart can cause bradycardia. Treatment of diabetic rats with L-NIL significantly improved cardiac function in streptozotocin diabetic rats possibly by protecting the heart tissue from the harmful effects of peroxynitrite. Previous studies from our lab have showed that inhibition of peroxynitrite using antioxidants such as n-acetylcysteine also improves cardiac performance in streptozotocin diabetic rats 22 . Although we believe that improvement in cardiac function is due to the direct effects of L-NIL on peroxynitrite formation, it is possible that  other mechanisms such as  inhibition of RhoA might have improved contractile function  in the diabetic rat hearts. We have reported previously that the contractile dysfunction seen in hearts of diabetic rats was accompanied by elevation of iNOS and RhoA activity and that inhibition of either iNOS or RhoA normalized the cardiac contractile function. This implies that iNOS induction, by positively regulating RhoA expression, may contribute to the increased RhoA activity leading to an increase in the RhoA/ROCK pathway activity and contractile dysfunction 17 . Taken together, these data suggests that iNOS mediated cardiovascular abnormalities may involve multiple mechanisms and that inhibition of its activity may be a potential strategy in the treatment of cardiac dysfunction in diabetes. Increased formation of peroxynitrite may reduce the bioavailability of functional NO from eNOS and cause endothelial dysfunction 25 . Previous studies from our lab have reported an improvement in endothelial function when iNOS was inhibited acutely 8 . The data from the present study demonstrate decreased expression of eNOS in the superior mesenteric arteries of untreated diabetic rats that was also improved by pretreatment with L-NIL. Although we do not have any direct evidence to show that peroxynitrite per se is involved in the decreased expression of eNOS, it is possible that L-NIL treatment, by inhibiting the formation of NO,  91 prevented the pro-oxidant actions of peroxynitrite on the eNOS protein. A recent study reported a positive correlation between decreased eNOS expression and endothelial dysfunction with that of an imbalance in [NO]/[ONOO - ] concentrations 26 . In human umbilical vein endothelial (HUVEC) cells, using real time measurements of NO and ONOO - , the authors reported that a shift in the [NO/ ONOO-] towards a high production of ONOO -  causes decreased eNOS protein expression. Further, peroxynitrite can oxidize the zinc thiolate center in eNOS, a modification that results in reduced NO bioactivity and enhanced endothelial O 2-  production. This modification can result in the generation of more peroxynitrite and eNOS uncoupling. Another mechanism of eNOS uncoupling by peroxynitrite is by direct oxidation of tetrahydrobiopterin (BH4), an essential and critical cofactor for NO synthesis 27 . Thus peroxynitrite formation represents an important complement in the mechanisms of endothelial dysfunction. Treatment of diabetic rats with L-NIL also improved pressor responses to methoxamine. These data, in addition to that of our previous study 8  suggest that NO from iNOS contributes significantly to the vascular hyporeactivity in diabetic rats. Although we believe that improvements in pressor responses to methoxamine are due to the direct effects of L-NIL on iNOS, it is possible that L-NIL also protects the vascular tissues from the harmful effects of peroxynitrite by preventing its formation. In the vasculature, it has been reported that peroxynitrite causes direct oxidation of catecholamines 28  and reduces the binding capacity of !- adrenergic receptors, thereby decreasing the vascular reactivity to vasoactive agents 29 . It is therefore possible that L-NIL by inhibiting iNOS and formation of peroxynitrite preserves the pressor responses in diabetic rats. Increasing evidence suggests that many of the cardiovascular abnormalities in diabetic rats can be prevented by inhibiting the formation of peroxynitrite 30, 31 . For instance, previous studies from our lab showed that inhibition of peroxynitrite using antioxidants such as n-acetylcysteine and ascorbic acid improves cardiac performance, mean arterial blood pressure and heart rate in  92 streptozotocin diabetic rats 7, 22 . At the same time, the inability of L-NIL treatment to completely restore the depressed mean arterial blood pressure and heart rate in streptozotocin diabetic rats suggests the possibility of other mechanisms in the etiology of these abnormalities. Recent studies from our lab suggest the involvement of PKC!, since treatment of streptozotocin diabetic rats with a selective inhibitor of PKC! for 3 weeks also improved mean arterial blood pressure, heart rate and pressor responses (see chapter 2). Recent clinical studies have shown that increased generation of NO occurs in patients with type 1 diabetes, and is associated with enhanced peroxynitrite production and lipid peroxidation 32 . Furthermore, a correlation between increased plasma NOx levels and endothelial dysfunction, lower blood pressure and sympathetic nerve dysfunction in type 1 diabetes has also been found 32, 33 . Although a large body of evidence suggests the involvement of peroxynitrite/ nitrotyrosine in the etiology of hypotension, many reports have also linked nitrotyrosine with hypertension in both humans and animal models particularly in type 2 diabetes 34, 35 . This is most likely due to decreased bioavailability of physiological NO because of its quenching by increased levels of reactive oxygen species leading to attenuation of endothelium-dependent vasorelaxation. Further, post-translational modifications of proteins such as nitration by peroxynitrite are also known to be associated with the hypertension 36 . However, in conditions of abnormal expression and activation of iNOS, increased formation of NO/peroxynitrite produces the features of hypotension. In summary, the results of the present study suggest that induction of iNOS contributes significantly to the depressed cardiac function, mean arterial blood pressure, heart rate, endothelial dysfunction and pressor responses to vasoactive agents in type 1 diabetes. Thus therapeutic strategies aimed at inhibiting iNOS or its downstream product, peroxynitrite may be a rational approach to prevent some of the cardiovascular abnormalities that occur in diabetes.  93    3.5 Tables and figures    Table 3.1 General characteristics of rats following 8 weeks of L-NIL treatment.        Data represent means ± S.E.M  a  significantly different from C & CT,  b  significantly different from C & CT (P < 0.05).   Parameters (n=16)  C   D   CT   DT   Body weight (g)       Before Treatment       After Treatment   365 ± 21.4 580 ± 14.9    338 ± 16.1 384 ± 10.8 a    357 ± 20.2 571 ± 9.6   335 ± 14.6 408 ± 9.3 b  Blood Glucose (mM)   7.88 ± 0.13   25.45 ± 1.2 a    7.4 ± 0.13   26.28 ± 0.64 b    94                  Figure 3.1 Effect of 8 weeks of L-NIL treatment on mean arterial blood pressure (top panel) and heart rate (bottom panel) in freely moving conscious rats. Data represent means ± S.E.M. Data were analyzed using One-Way ANOVA with Newman–Keuls post hoc test. * different from C, CT & DT groups; # different from C, D & CT groups (P<0.05). n=16.   95             Figure 3.2 Effect of 8 weeks of L-NIL treatment on pressor responses to bolus doses of methoxamine in freely moving conscious rats. Data represent means ± S.E.M. ! mean arterial blood pressure represents change in blood pressure (compared to basal blood pressure) in response to every bolus dose of methoxamine (100-300 nmol). Data were analyzed using One-Way ANOVA with Newman–Keuls post hoc test. * different from C & CT groups; # different from D group (P<0.05). ! "#!             Figure 3.3 Effect of 8 weeks of L-NIL treatment on cardiac function of isolated working hearts from control and diabetic rats 17 . Increases in left ventricular developed pressure (LVP, left panel), +dP/dt (middle panel), and –dP/dt (right panel) in response to increases in left atria filling pressure (LAFP). Black squares represent untreated control hearts; open squares, untreated diabetic hearts; open triangles, LNIL-treated control hearts; black triangles, L-NIL-treated diabetic hearts (n = 6–7 in each group). Data are expressed as mean ± SEM. * P < 0.05 compared with all other groups.  97                Figure 3.4 Effect of L-NIL treatment on eNOS expression in the heart and SMA. Western blot analysis showing expression of eNOS and the corresponding GAPDH protein in the heart (top panel) and superior mesenteric artery (bottom panel). Densitometric analysis of eNOS expression (corrected by normalizing to the corresponding GAPDH expression) in the heart and superior mesenteric artery (n=8). Data were analyzed using One-Way ANOVA with Newman– Keuls post hoc test. * different from C, CT & DT groups; # different from D group (P<0.05).  98                Figure 3.5 Effect of L-NIL treatment on iNOS expression in the heart and SMA. Western blot analysis showing expression of iNOS and the corresponding GAPDH protein in the heart and superior mesenteric artery. Densitometric analysis of iNOS expression (corrected by normalizing to the corresponding GAPDH expression) in the heart (top panel) and superior mesenteric artery (bottom panel) (n=8). Data were analyzed using One-Way ANOVA with Newman–Keuls post hoc test. * different from C & CT groups (P<0.05). ! ""!   Figure 3.6 Photomicrographs (X20) illustrating the immunohistochemical localization of iNOS in sections of superior mesenteric artery of control (A), diabetic (B), control treated (C) and diabetic treated (D) rats. As shown, there is a significant increase in the expression of iNOS in the medial and advential layers of the untreated diabetic superior mesenteric artery. E represents a negative control (where the section was incubated with NGS instead of the iNOS antibody) showing no staining, indicating that the secondary antibody at the concentration used is devoid of any non-specific binding.     100                 Figure 3.7 Effect of L-NIL treatment on iNOS activity in the mesenteric arterial bed. Data were analyzed using One-Way ANOVA with Newman–Keuls post hoc test. *different from C, CT & DT groups, #different from D group (P<0.05). n=8    101                 Figure 3.8 Effect of L-NIL treatment on NT levels in the heart and SMA.  Western blot analysis showing NT and the corresponding GAPDH protein in the heart and superior mesenteric artery. Densitometric analysis of NT levels (corrected by normalizing to the corresponding GAPDH expression) in the heart and superior mesenteric artery (n=8). Data were analyzed using One-Way ANOVA with Newman–Keuls post hoc test.  *different from C, CT & DT groups, # different from D group (P<0.05). ! "#$!     Figure 3.9 Photomicrographs (X20) illustrating the immunohistochemical localization of NT in sections of heart from control (A), diabetic (B), control treated (C) and diabetic treated (D) rats.  As shown, there is a substantial increase in nitrotyrosine immunostain in the diabetic heart section (B) as well as the L-NIL treated diabetic section (D) compared to the control (A) and L-NIL treated control (C). E represents a photomicrograph of higher magnification (X40) clearly demonstrating NT immunostain predominantly in cardiomyocytes.     103 3.6.    Bibliography 1. 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Long-term N-acetylcysteine and L- arginine administration reduces endothelial activation and systolic blood pressure in hypertensive patients with type 2 diabetes. Diabetes Care. 2008;31(5):940-944.  106 35. Frustaci A, Kajstura J, Chimenti C, Jakoniuk I, Leri A, Maseri A, Nadal-Ginard B, Anversa P. Myocardial cell death in human diabetes. Circ Res. 2000;87(12):1123-1132. 36. Tyther R, Ahmeda A, Johns E, Sheehan D. Proteomic identification of tyrosine nitration targets in kidney of spontaneously hypertensive rats. Proteomics. 2007;7(24):4555-4564.    107 4. Maintenance of adrenergic vascular tone by matrix metalloproteinase transactivation of the epidermal growth factor receptor requires phosphatidylinositol-3-kinase and mitochondrial ATP synthesis 1 .                   1 A version of this chapter will be submitted for publication. Nagareddy PR, Chow F, Hao L, Wang X, Nishimura T, MacLeod KM, McNeill JH and Fernandez-Patron C. Maintenance of adrenergic vascular tone by matrix metalloproteinase transactivation of the epidermal growth factor receptor requires phosphatidylinositol-3-kinase and mitochondrial ATP synthesis (2009).   108 4.1 Introduction Hypertension is a cardiovascular disease characterized by sustained high blood pressure with a complex and multifactorial etiology 1 . Factors initiating this condition in the general population remain unknown despite major research efforts. Whatever the initial cause, the common hallmarks of hypertension both in humans and in animal models are enhanced agonist- stimulated vasoconstriction, decreased vasodilatation, oxidative stress associated endothelial dysfunction and hypertrophic growth of cardiac and vascular tissues 2-5 . Although it is unclear how and why such apparently distinct pathological processes concur in hypertension, increasing evidence now suggests that elevated levels of vasoactive GPCR agonists such as catecholamines, endothelin-1 and angiotensin II may explain, at least in part, the development and progression of many hypertensive disorders 6-13 . We have proposed that increased vascular tone, oxidative stress and hypertrophic growth are interrelated processes largely signaled through a common pathway 11, 14, 15 . An important step in this pathway is transactivation of growth factor receptors, such as the EGFR, by MMPs 16 . MMPs act by shedding mature growth factors such as HB-EGF from transmembrane precursors, which then bind to the EGFR and phosphorylate tyrosine kinase. Activation of the EGFR leads to downstream activation of mitogen activated protein kinases (MAPK) such as p38 (stress activated), JNK (NH2-terminal c-Jun kinase), extracellular signal–regulated kinase (ERK) 1 and Erk2 and protein kinase B/Akt 13 . Once activated, these kinases are translocated to the nucleus where they bind to and phosphorylate nuclear transcription factors, stimulating gene transcription, protein synthesis and cell growth 17-20 . Among these kinases, p38 MAPK and PI3-kinase are known to participate in the signaling events leading to vasoconstriction subsequent to the activation of the EGFR by MMPs. This is because inhibitors of p38 MAPK and PI3-kinase but not ERK1/2, produced vasorelaxation in   109 PE-constricted arteries 11 . Of specific interest is the PI3-kinase pathway because PI3K has been implicated not only in the modulation of contraction 21, 22  but also metabolism and growth 23 . Further, a recent study from our lab suggested that following EGFR transactivation, the ensuing signaling events led to significant increase in the generation of reactive oxygen species, most probably as a result of increased ATP synthesis in mitochondria 15 . These studies support the view that under sustained GPCR stimulation, growth promoting and metabolic pathways culminate to increase oxidative stress, hypertrophic growth and vascular tone contributing to abnormal hemodynamic outcomes such as hypertension. We therefore hypothesized that PI3- kinase, by virtue of its actions on metabolic and growth-promoting pathways, modulates vascular tone by regulating mitochondrial ATP synthesis. These actions in turn are regulated by MMP transactivation of the EGFR secondary to the activation of GPCR by agonists such as PE. 4.2 Methods 4.2.1 Microperfusion experiments to study changes in vascular tone Animal protocols were conducted in accordance with institutional guidelines issued by the Canada Council on Animal Care. Small (OD: ~500 µm, ID: 250-300 µm) mesenteric arteries from male Sprague-Dawley rats were used in studies of vascular tone. The arteries were dissected and mounted on a microperfusion arteriograph (Danish MyoTechnology, Aarhus, Denmark). This perfusion system facilitated the study of vascular reactivity to luminal infusions of drugs as well as to drugs added to the arteriograph bath (adventitia side). The arteries were perfused at constant temperature (37°C) and flow rate (2 µL/min) with standard HEPES-PSS, pH 7.4 supplemented with glucose (5.5 mM). In experiments involving the luminal administration of drugs, small volumes (5 µL) of specified drugs were injected into the perfusion line towards the artery. Changes in arterial outer diameter in response to drugs were monitored using a video camera and processed using VediView software (Danish   110 MyoTechnology). In this system, maximal changes in arterial diameter coincide with the infused drug being in the lumen of the artery. As the drug bolus leaves the artery, arterial diameter returns to its pre-infusion magnitude. The injection of drugs in the line towards the artery, without introducing flow rate change-related artifacts, was facilitated by an HPLC injection valve (Rheodyne Model 9725I, Mandel Scientific Co., ON, Canada). We injected drugs into the lumen to test their effects on the tone of phenylephrine-constricted arteries. 4.2.2 Cell culture studies 4.2.2.1 Preparation of rat aortic vascular smooth muscle cells Rat aortic vascular smooth muscle cells (VSMCs) were grown in complete Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen Life Technologies, Carlsbad, CA) with 10% fetal bovine serum (FBS) and 100-units/mL penicillin-streptomycin at 37 o C and 95% O2/5% CO2. Cells from passages 2-6 were used in all the experiments. When cells were 80-90% confluent they were starved overnight using DMEM media without growth factors. On the day of experiments VSMC cells were washed with PBS (pH 7.4) and replaced with fresh media devoid of growth factors and antibiotics. In experiments involving MMP and EGFR inhibitors, drugs were added to the media 1 hour prior to the stimulation with GPCR agonists. The medium was collected and stored at -80 o C until MMP activities were determined by zymography. At the end of the experiment, cells were washed with ice-cold phosphate-buffered saline (PBS), lysed and the cell lysates collected and stored (at -80 o C) for Western blotting. 4.2.2.2 EGFR, MMP-2 and MMP-7 suppression in VSM cells by siRNA oligonucleotides Small interference RNAs (siRNA) specific to rat EGFR, MMP-2 and MMP-7 were purchased from Santa Cruz Biotech. For optimal siRNA transfection efficiency, the manufacturer’s protocol was followed. Briefly, VSM cells were seeded in a 6 well plate and cultured in 2 ml antibiotic-free normal growth medium supplemented with 10% FBS until the cells were 60-80% confluent (~36 hours). On the day of transfection, cells were washed with transfection medium   111 (sc-36868) and incubated with 1 ml of transfection reagent (sc-29528) containing 60 pmols of either MOCK (scrambled, sc-sc37007) or rat-specific EGFR, MMP-2 or MMP-7 siRNA oligonucleotides for 12 hours. After 12 h, the medium was supplemented with another 1 ml of fresh DMEM (containing 2X FBS and antibiotics) for another 24 hours. At this point, the cells were washed with warm PBS and incubated with normal DMEM (containing normal growth factors and antibiotics) for an additional 48 hours. On the day of the experiment, medium was collected for zymography experiments and the serum-starved cells were stimulated with PE for 30 minutes. At the end of the experiments, cells were lysed and the lysates collected and stored (at -80 o C) for western blotting. 4.2.3 Measurement of MMP activity by substrate zymography MMP-2 and MMP-7 activity in tissue and cell culture releasates was measured using gelatin (for MMP-2) or carboxymethyl-transferrin (CMT) substrate (for MMP-7) zymography assays. For measurement of MMP activity in arteries, pieces of carefully dissected and equilibrated arteries of equal length were incubated (20-60 min) in the presence of various inhibitors (see results section) and stimulated with PE (10 µM) for times ranging from 20-60 minutes. Arterial releasates were subjected to SDS PAGE using CMT (2.5 mg/mL) or 2% gelatin in gels. Following electrophoresis, the gels were washed with Triton X-100 (2.5% for 3x20 min) and incubated overnight at 37°C in zymogram development buffer containing 50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 5 mM CaCl2, 0.02% Brij-35Tris. The gels were stained with coomassie blue and MMPs were detected as transparent bands against the background of coomassie blue-stained undigested CM transferrin. The ratio of pro-MMP to active-MMP was used to represent MMP activity. 4.2.4 Western blotting VSM cells were homogenized either using a sonicator in modified RIPA buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM PMSF, 1 mM EDTA, 5 !g/ml aprotinin, 5 !g/ml   112 leupeptin, 1% Triton x-100, 1% Sodium deoxycholate and 0.1% SDS at 4 °C. The homogenate was centrifuged at 10000!g at 4 °C for 15 min and the protein content of the supernatants was determined by the Bradford protein assay. Equal amounts of protein (15-30µg) from each sample were separated by 8% or 10% SDS PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked for 1 hour in a solution of 5% skim milk (in TBS-T) and then incubated overnight at 4°C with corresponding primary antibodies. Membranes were washed and incubated with their corresponding HRP- conjugated secondary antibodies for 1 hour followed by treatment with chemiluminescence reagents (Amersham Inc, Québec, Canada). Immunoblots were exposed and developed on photographic film. Densitometric analysis was performed using image J software from NIH. 4.2.5 ATP measurements ATP measurements were made using a commercially available luciferin-luciferase based assay kit (Sigma). Rat VSMCs were grown in 96-well luminescence compatible plates. Overnight serum starved VSMCs were washed with PBS (pH 7.4) and incubated with vehicle (HEPES buffer) or inhibitors prepared in HEPES buffer (see results section for times and concentrations) followed by stimulation with PE or Ang II.  Media was collected for measurement of ATP in the releasate, followed by lysis of cells using somatic cell lysis reagent (Sigma). After appropriate sample dilution, both released ATP and intracellular ATP (from cell lysate) were measured using a luminometer (Biotek Synergy™ HT Multi-Detection Microplate Reader, BioTek Instruments Inc, USA). 4.2.6 Confocal Immunofluorescence Imaging of Phospho-Akt (Ser 473) For immunofluorescence imaging of phospho Akt (Ser 473), serial cryostat sections (8µ) of rat mesenteric arteries (following stimulation with PE) were embedded in O.C.T compound (Tissue-Tec, Sakura Finetek USA, Inc., Torrance, CA) and placed on glass slides. The sections were fixed, blocked with normal goat serum (10%) and subsequently incubated with anti   113 phospho-Akt antibody (Cell Signaling). After primary antibody labeling, slides were washed twice with PBS, and the anti-rabbit Alexa Fluor 488 (Invitrogen) secondary antibody (1:500 in 1x PBS + 1% NGS) was added for 1 hour at room temperature. Excess secondary antibody was washed off again with PBS. Confocal imaging was performed with a Zeiss Axiovert 100M coupled with a Zeiss LSM510 laser scanning system (Germany) at the Microscopy Service Facility, Department of Cell Biology, University of Alberta. 4.2.7 Preparation of detergent resistant plasma membrane fractions for detection of GLUT4 in VSM cells For preparation of cell membranes, VSMCs were grown in 75mm 2  culture flasks. At the end of experiments the cells were washed with ice cold PBS and scraped in the presence of chilled TES buffer containing 10mM Tris (pH 7.4), 1mM EDTA, 0.25 M sucrose, 0.1 mM PMSF and protease inhibitor cock tail (sigma). Cell were homogenized on ice by sonication and centrifuged at 16000g for 20 min. The pellet was resuspended in 200 µl of TES buffer and dispersed again by sonication. Cell membranes were  separated from whole lysates using two- layer (0.25/1.2 mol/L, 400µl each)  sucrose-gradient centrifugation (158000g for 20 minutes at 4°C). The interface band containing detergent resistant membranes was harvested into a number of fractions, mixed with SDS buffer and subjected to SDS-PAGE electrophoresis for western blot detection of GLUT4. 4.2.8 Statistical analysis All values are expressed as mean ± SEM. “n” denotes the sample size in each group. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by the Newman-Keuls test for multiple comparisons. GraphPad Prism (GraphPad Software, CA) software program were used for statistical analysis. For all results the level of significance was set at P<0.05.   114 4.3 Results 4.3.1 Agonist (PE) induced activation of PI3-kinase is required for maintenance of adrenergic vascular tone in rat small mesenteric arteries To determine whether GPCR agonists such as PE activate PI3-kinase, we isolated resistance- size small mesenteric arteries and stimulated them with PE (10µM) for 30 minutes. As shown in Fig.4.1A, PE produced a significant increase in the activation of PI3-kinase as determined by the phosphorylation of Akt at ser 473, a PI3-kinase substrate. We next studied the effect of PI3- kinase inhibitors such as LY294002 and wortmannin on PE mediated adrenergic vascular tone. Resistance size small mesenteric arteries were isolated from rat mesenteric arterial bed and mounted in a microperfusion arteriograph, a system where addition of PE (1-10 µM) to the bath (adventitia side) produces long-lasting increases in vascular tone. In such PE-constricted arteries, PI3-kinase inhibitors were slowly infused at a rate of 2 µL/min. As shown, both wortmannin (Fig.4.1B) and LY294002 (Fig.4.1C) produced a concentration-dependent vasodilatation. These observations, which are consistent with our previous work on the vasodilatatory actions of MMP and EGFR inhibitors, suggest that maintenance of adrenergic vascular tone depends on PI3-kinase activity. 4.3.2 Inhibition of the MMP-EGFR pathway suppress PI3-kinase activation of Akt in VSM cells To investigate whether the maintenance of adrenergic vascular tone by PI3-kinase involved MMP transactivation of the EGFR, we stimulated VSM cells with PE (10 µM) in the presence or absence of GM6001 (25 µM) and AG1478 (10 µM) for 30 minutes. As observed in intact arteries, stimulation of !1-adreneroceptors significantly increased the phosphorylation of Akt, and this was blunted in the presence of MMP and EGFR inhibitors (Fig.4.2). Further in VSM cells, suppressing the expression (~40%) of the EGFR by siRNA not only reduced its activation   115 but also significantly prevented the increase in phosphorylation of Akt by PE (Fig.4.3). Likewise suppression of MMP-2 expression by ~60% (Fig.4.4) or MMP-7 expression by ~50% (Fig.4.5) by their corresponding siRNAs prevented PE-induced activation of Akt. 4.3.3 Maintenance of adrenergic vascular tone requires mitochondrial ATP synthesis in VSM cells We next examined the potential mechanisms involved in the maintenance of adrenergic vascular tone downstream of PI3-kinase. We specifically wanted to determine whether stimulation of !1- adrenoceptor signaling triggers mitochondrial ATP synthesis in VSM cells and if inhibition of PI3-kinase and/or the MMP-EGFR pathway can modulate this response. Stimulation of !1- adrenoceptors with PE (10 !M) increased intracellular ATP levels in a concentration and time- dependent manner peaking at 10 min (Fig.4.6A). To confirm the generality of this finding, we compared PE (0.1-10 !M) effects to those of another GPCR agonist (Ang II) on ATP synthesis. Similar to PE, Ang II (0.1–10 mM) increased ATP synthesis in a time and concentration dependent manner (data not shown). Oligomycin (1 !M), a mitochondrial ATP synthase inhibitor, not only inhibited PE and Ang II induced increases in ATP synthesis, but also decreased basal ATP synthesis in resting cells (Fig.4.6B). These findings suggest that mitochondria are the major source of ATP in VSM cells both in normal resting and agonist- stimulated working conditions. Interestingly, the PE induced initial surge in ATP synthesis (until 10 min) was not reduced by inhibitors of PI3-kinase (LY294002, 10!M), the MMPs (GM6001, 25!M) or the EGFR (AG1478, 10!M). However, coinciding with the maximum relaxation of arteries observed in our previous studies with these inhibitors, ATP levels declined more rapidly in the presence of PI3-kinase and MMP-EGFR inhibitors compared to ATP levels in PE-stimulated cells in the absence of these inhibitors (Fig.4.7). Furthermore, inhibition of either PI3-kinase (Fig.4.1) or MMPs or the EGFR 11  or mitochondrial ATP synthesis (Fig.4.8) produced vasodilatation all peaking at ~10 minutes in PE-constricted arteries suggesting that   116 maintenance of adrenergic vascular tone requires ATP synthesis by mechanisms involving activation of MMPs, the EGFR and PI3-kinase. 4.3.4 Phenylephrine-induced GLUT4 translocation is reduced by the inhibition of PI3- kinase and the MMP-EGFR pathway in VSM cells Having determined that agonist induced increase in vascular tone is maintained by MMP transactivation of EGFR via the activation of PI3-kinase and mitochondrial ATP synthesis, we reasoned that a constant and an unlimited supply of fuel substrate may be necessary for ATP synthesis. Because glucose is the predominant source of energy during vascular contraction 24, 25  and GLUT4 is the major glucose transporter 26 , we examined if PE increased GLUT4 translocation to the plasma membrane and the effects of GM6001, AG1478 and LY294002 on its activation. Stimulation of VSM cells with PE significantly increased the translocation of GLUT4 to plasma membrane as determined by the increased expression of GLUT in detergent insoluble membrane fractions. However, the presence of MMP or EGFR or PI3-kinase inhibitors prevented this response as observed by decreased expression of GLUT4 in the membrane compared to cytosol fraction (Fig.4.9). 4.3.5 Phenylephrine-induced mitochondrial ATP synthesis activates MMP-7 in vascular tissue Previously, we have reported that GPCR stimulation by agonists such as PE and Ang II increases MMP-7 activity in arteries. Since these agonists also triggered ATP synthesis in vascular smooth muscle cells, we next examined whether ATP synthesis in response to PE has any influence on further activation of MMP-7 in vascular tissue. When either mitochondrial ATP synthesis was inhibited (by oligomycin, 1 µM) or when extracellularly released ATP was scavenged using apyrase (5 U/mL), the ability of PE to activate MMP-7 was significantly reduced in arteries (Fig.4.10A) suggesting a novel pathway of MMP activation in vascular tissues. Mimicking the effects of PE, direct application of ATP (1 µM) exogenously to arteries   117 promoted a rapid release and activation of vascular MMP-7 that was statistically significant after 40 min of stimulation with ATP (Fig.4.10B). Because ATP can be rapidly degraded to ADP, AMP and other metabolites in the extracellular space by ectoenzymes, we next wanted to clarify whether the observed effect on MMP-7 activation was due to ATP or to its metabolites. The direct application of ADP (0.01 to 100 µM) failed to activate MMP-7 in vascular tissues (data not shown). 4.3.6 Activation of MMP-7 by ATP involves purinergic P2X receptors and calcium ATP activates a family of ionotropic receptors (P2X) as well as metabotropic receptors (P2Y), collectively called purinergic receptors. P2Y receptors couple to intracellular second messenger systems through heterotrimeric G-proteins 27  while the P2X receptors are ligand (ATP) gated ion channel receptors 28 . To clarify the involvement of purinergic receptor signaling in ATP -induced activation of MMP-7 we first tested the effect of ATP in the presence of suramin (100µM), a non-selective blocker of P2X and P2Y receptors and then in the presence of pyridoxal- phosphate-6-azophenyl-2', 4’-disulfonate (PPADS, 100µM), a selective P2Y blocker. ATP mediated MMP-7 activation was abolished when vascular tissues were incubated with suramin but not with PPADS suggesting a possible role for P2X receptors (Fig.4.11A). ATP binding to cell surface P2X receptors produces a rapid influx of divalent cations such as Ca 2+ . Since calcium is an important cofactor for a number of secretory events, we next studied the effect of Ca 2+  inhibition in ATP induced MMP-7 activation. In separate experiments, under conditions of either a Ca 2+  free HEPES buffer (containing EDTA, 50µM + EGTA, 50µM) or in the presence of BAPTA-AM (100µM), an intracellular Ca 2+ chelating agent, ATP did not activate MMP-7 (Fig.4.11B). These observations suggest a novel mechanism by which ATP activates MMP-7 in vascular tissues involving Ca 2+ and purinergic P2X receptors.    118 4.4 Discussion Hypertension is a disease characterized by enhanced agonist-induced vasoconstriction, reduced vasodilatation and increased smooth muscle cell growth. We hypothesized that the MMP-EGFR pathway modulates vascular tone, at least in part, via the activation of PI3-kinase and modulation of mitochondrial ATP synthesis. In the present study we investigated the mechanisms by which MMP-transactivation of the EGFR modulates adrenergic vascular tone in VSM cells. The key findings in the present study are, 1) Phenylephrine-induced stimulation of !1-adrenergic receptors triggers mitochondrial ATP synthesis and as well activates PI3-kinase via the MMP-EGFR pathway in VSM cells. 2) Maintenance of adrenergic vascular tone requires PI3-kinase activation and mitochondrial ATP synthesis. 3) Phenylephrine-induced ATP synthesis promotes further activation of MMPs such as MMP-7. These new findings are consistent with and expand our recent research on the role of MMP-EGFR pathway on adrenergic signaling in vascular smooth muscle 11, 15 . Various mechanisms have been proposed to explain the missing link between the transactivation of the EGFR by MMPs and the intracellular factors that regulate contractile function in the vascular smooth muscle cell. In our previous investigation, we demonstrated the involvement of mitochondrial respiratory chain complexes I and III and proposed a possible role for mitochondrial ATP synthesis in the modulation of adrenergic vascular tone by the MMP-EGFR pathway 11, 15 . In the present study, we examined whether agonist induced mitochondrial ATP synthesis is required for the maintenance of adrenergic vascular tone and if this is modulated by PI3-kinase. We found that the stimulation of vascular GPCRs by PE or Ang II significantly increased intracellular ATP levels in a concentration and time dependent manner. The initial phase of ATP synthesis, characterized by a rapid and profound increase (< 10 min) in ATP   119 levels was probably to meet the immediate energy requirements of actin-myosin filaments during contraction and later phase may help to sustain the contraction initiated by the agonist. Our results showing increased ATP synthesis peaking at 10 minutes following stimulation of !1- adrenoceptors are consistent with our previous observations of increased mitochondrial membrane potential, mitochondrial ROS production, phosphorylation of the EGFR and vasoconstriction all of which also peaked at 10 min 15 . Because oligomycin, a mitochondrial ATP synthase inhibitor, completely abolished both the agonist-induced as well as basal ATP synthesis, the source of ATP could be attributed to mitochondria. Interestingly, MMP-EGFR and PI3K inhibitors did not inhibit the initial surge in mitochondrial ATP synthesis triggered by PE. Further in our previous studies, inhibition of EGFR did not prevent the phosphorylation of myosin light chain (MLC) in response to adrenergic stimulation 11 . Taken together these data suggest that the MMP-EGFR pathway influences neither vascular contraction nor ATP synthesis triggered by vasoconstrictors. However, subsequent to initial increase in ATP synthesis, PI3- kinase and MMP-EGFR inhibitors caused a rapid decline in the intracellular ATP levels. These data suggests that MMP-EGFR-PI3K pathway influences vascular tone as well as ATP synthesis possibly by way of providing substrates for sustained ATP synthesis. Further, it is likely that the MMPs are activated secondary to the initial contraction of vascular smooth muscle cells or mitochondrial ATP synthesis by mechanisms involving intracellular calcium, generation of ROS, phosphorylation of Src and ATP production 14, 29-32 . Activation of P13-kinase is one of the downstream events associated with phosphorylation of the EGFR in VSM cells that potentially could modulate mitochondrial ATP synthesis via Akt- GLUT4 signaling. PI3-kinase is a lipid kinase that converts phosphatidylinositol 4, 5- diphosphate to a more potent second messenger, phosphatidylinositol 3, 4, 5-triphosphate. This compound is essential for the translocation of Akt to the plasma membrane where it is phosphorylated and activated by phosphoinositide-dependent kinases (PDK). Akt is known to   120 regulate energy metabolism by multiple mechanisms, including increased expression and translocation of glucose transporters such as GLUT4 33-35 . The phosphorylation of Akt increases the expression and translocation of GLUT4 to the plasma membrane, which in turn facilitates the uptake of glucose, a major substrate in vascular metabolism. Indeed, our data demonstrate that adrenergic stimulation in isolated rat arteries and VSM cells causes the activation of PI3- kinase downstream of the EGFR, as detected by the increased levels of phospho-Akt. The blockade of MMPs, the EGFR or PI3-kinase using both pharmacological inhibitors and siRNA blunted the phosphorylation of Akt (at Ser 473 ) as well as the synthesis of ATP downstream of adrenergic receptors. Our data are consistent with previous studies that have reported lower ATP levels in Akt- deficient cells 36 . Further, we have also found that inhibition of PI3-kinase or ATP synthesis dose-dependently inhibited adrenergic vascular tone in rat small mesenteric arteries. These findings can be explained by a mechanism (Fig.4.12) whereby, downstream of the adrenoceptors, MMP transactivation of the EGFR results in PI3-kinase phosphorylation of Akt, recruitment and translocation of GLUT4 to the plasma membrane, and increased glucose uptake to provide a continuous source of substrates for ATP synthesis and to maintain vascular tone. In support of our interpretation, previous studies using GLUT4 inhibitors have demonstrated decreased glucose uptake and attenuated vascular responses to adrenergic stimulation in vascular tissues 25, 37 . The detailed study of glucose transport is, however, beyond the scope of the present investigation. Mitochondrial ATP synthesis in response to agonist stimulation was also found to activate MMPs such as MMP-7 although the mechanisms remain unclear. The observation that apyrase, an ATP scavenger, inhibits activation of MMP-7 suggests that activation of MMP-7 requires ATP to be released to the extracellular milieu. In spite of very high concentrations in the cytosol (~3-10 mM), ATP and other nucleotides cannot penetrate cell membranes due to their negative charge. However, due to its high concentration gradient (10 6 fold across the membrane) ATP   121 can activate certain nucleotide transporters and channels to release ATP 38-40 . Further, the released ATP has a very short life and is rapidly degraded to ADP, AMP and adenosine by ectonucleotidases. Our data suggest that it was ATP and not the degradation products that activated MMP-7 because direct stimulation of arteries with ADP did not activate MMP-7. We next studied the potential mechanisms by which ATP activates MMP-7. Because ATP activates both ionotropic P2X and metabotropic P2Y receptors 41 , we studied at the role of these receptors in MMP-7 activation. P2X-purinoceptors are found on VSM cells where they mediate vasoconstriction resulting from ATP released as a co-transmitter with norepinephrine from sympathetic nerves. P2Y-purinoceptors, however are located on the vascular endothelium and mediate vasorelaxation to locally produced ATP 42 . Although, our data suggest that MMP-7 activation is mediated by P2X receptors and involves calcium, it is possible that inhibition of MMP-7 activity may be due to the non-specific effects of the calcium antagonists used. Since MMPs require metal ions such as calcium and zinc for their catalytic activity, the calcium chelating agents used in the present study might have inhibited MMP activity 43 . Further studies are required to elucidate the mechanisms of MMP-7 activation by ATP. Regardless of the mechanism involved, activation of MMP-7 by ATP released as a result of agonist stimulation of vascular GPCRs is of prime importance because it initiates a feed forward cycle involving GPCR-MMP-EGFR-ATP and MMP to sustain vascular tone 11, 15  as well as growth and remodeling 44 . In addition, extracellular ATP is mitogenic and stimulates several pathways either directly or synergistically with other polypeptide growth factors such as platelet derived growth factor (PDGF), insulin like growth factor-1 (IGF-1), EGF and insulin resulting in VSM proliferation and hypertrophy 44 . In conclusion, our data suggest that agonist-induced stimulation of GPCRs such as adrenergic receptors causes vasoconstriction possibly as a result of the initial surge in mitochondrial ATP synthesis. This is followed by the activation of PI3-kinase via the mechanisms involving MMPs   122 and subsequent transactivation of the EGFR. Activation of PI3-kinase results in the phosphorylation of Akt and recruitment of glucose transporters such as GLUT4, which, in turn, provides a continuous supply of substrates for mitochondrial ATP synthesis. The provision of substrates such as glucose might help to sustain the vascular tone initiated by vasoactive agonists for longer periods of time. Additionally, the ATP synthesized and released into the extracellular milieu activates MMPs such as MMP-7 to maintain the feed forward cycle of sustained ATP synthesis and hence vascular tone. In hypertension, the characteristically elevated levels of GPCR agonists such as NE, Ang II, and ET-1 may result in exaggerated transactivation of the vascular MMP-EGFR pathway culminating in pathological features such as enhanced vascular tone and hypertrophic growth. Selective inhibition of the vascular MMP-EGFR transactivation pathway could have therapeutic potential for decreasing agonist-induced activation of PI3-kinase and for combating increased vasoconstriction and hypertrophy in hypertensive disorders.             123 4.5 Tables and figures Figure 4.1 Maintenance of PE-induced adrenergic vascular tone requires PI3K activation. A) Confocal immunoflorescence microscopy of small mesenteric arteries showing Akt phosphorylation (Ser 473 ) after exposure to PE (10 µM) for 30 minutes. B) Rat small mesenteric arteries were mounted on the microperfusion system. PE (10 µM) was added to the bath (advential side) to cause vasoconstriction followed by luminal injection of bolus doses of either wortmannin or LY294002. Representative trace (top panel) and quantitative analysis (bottom panel) of vasorelaxation of PE-constricted arteries in response to the luminal injections of bolus doses of wortmannin (50-500 pM). All data are expressed as mean ± SEM (n= 3-4 independent experiments). C) Representative trace (top panel) and quantitative analysis (bottom panel) of vasorelaxation of phenylephrine-constricted arteries in response to the luminal injections of bolus doses of LY294002 (50-500 pM). All data are expressed as mean ± SEM (n= 3-4 independent experiments).             124                    125                 Figure 4.2 Inhibition of the MMP-EGFR pathway suppresses PE-induced activation of PI3K in VSM cells. Representative Western blot showing the expression of phospho-Akt (Ser473) in VSMC stimulated with PE (10 µM) for 30 minutes in the presence of MMP (GM6001, 25 µM) and EGFR (AG1478, 10 µM) inhibitors. The densitometric values of phospho-Akt (Ser473) were normalized to corresponding total Akt densitometric values and the relative band intensities are expressed as mean ± SEM (n= 4 independent experiments). GAPDH served as a loading control. *P < 0.05 compared with all other groups, # different from PE group.   126 Figure 4.3 Effect of the EGFR suppression on PE-induced phosphorylation of Akt in VSM cells. Rat aortic VSM cells were transfected with either MOCK or rat specific EGFR siRNA and stimulated with PE (10µM) for 30 minutes. A) Representative Western blot showing total EGFR expression, with GAPDH as a loading control in VSMCs treated with MOCK or EGFR siRNA. The densitometric values of EGFR were normalized to corresponding total GAPDH densitometric values and the relative band intensities are expressed as mean ± SEM (n=4). * different from MOCK siRNA treated groups (P < 0.05). B) Representative Western blot showing phospho-EGFR expression, with GAPDH as a loading control in VSMCs treated with MOCK or EGFR siRNA and stimulated with PE (10 µmol/L) for 30 minutes. The densitometric values of phospho-EGFR (Tyr1177) were normalized to corresponding total GAPDH densitometric values and the relative band intensities are expressed as mean ± SEM (n=4). * different from all other groups (P < 0.05), # different from MOCK siRNA and PE treated cells (P < 0.05). C) Representative Western blot showing phospho-Akt (Ser473) levels in VSMCs treated with MOCK or EGFR siRNA and stimulated with PE (10 µM) for 30 minutes. The densitometric values of phospho-Akt (Ser473) were normalized to corresponding total Akt densitometric values and the relative band intensities are expressed as mean ± SEM (n= 4-6 independent experiments). * different from all other groups (P<0.05), # different from MOCK siRNA and PE treated group (P<0.05).        127                   128            Figure 4.4 Effect of MMP-2 suppression on PE-induced phosphorylation of Akt in VSM cells. In all experiments, cells were transfected with either MOCK or rat MMP-2 specific siRNA and stimulated with PE (10µM) for 30 minutes. A) Representative Western blot showing total MMP-2 expression, with GAPDH as a loading control in VSMCs treated with MOCK or MMP- 2 siRNA. The densitometric values of MMP-2 were normalized to corresponding GAPDH densitometric values and the relative band intensities are expressed as mean ± SEM (n=4). * different from MOCK siRNA treated groups (P < 0.05). B) Representative Western blot showing phospho-Akt (Ser473) expression in VSMCs treated with MOCK or MMP-2 siRNA and stimulated with PE (10 µM) for 30 minutes. The densitometric values of phospho-Akt (Ser473) were normalized to corresponding total Akt densitometric values and the relative band intensities are expressed as mean ± SEM (n= 4-6 independent experiments). * different from all other groups (P<0.05), # different from MOCK siRNA PE-treated group (P<0.05).   129            Figure 4.5 Effect of MMP-7 suppression on PE-induced phosphorylation of Akt in VSM cells. Vascular smooth muscle cells were transfected with either MOCK or MMP-7 specific siRNA and stimulated with PE (10µM) for 30 minutes. A) Representative Western blot showing total MMP-7 expression, with GAPDH as a loading control in VSMCs treated with MOCK or MMP- 7 siRNA. The densitometric values of MMP-7 were normalized to corresponding GAPDH densitometric values and the relative band intensities are expressed as mean ± SEM (n=4). * different from MOCK siRNA treated groups (P < 0.05). B) Representative Western blot showing phospho-Akt (Ser473) expression in VSMCs treated with MOCK or MMP-7 siRNA and stimulated with PE (10 µM) for 30 minutes. The densitometric values of phospho-Akt (Ser473) were normalized to corresponding total Akt densitometric values and the relative band intensities are expressed as mean ± SEM (n= 4-6 independent experiments). * different from all other groups (P<0.05), # different from MOCK siRNA PE-treated group (P<0.05).    130            Figure 4.6 Stimulation of !1-adrenoceptors increases mitochondrial ATP synthesis in time and dose dependent manner in VSM cells. Serum starved rat aortic VSMCs grown in 96-well bioluminescent compatible plates were stimulated with PE (0.1 to 10 µM) for 60 minutes in the presence or absence of 1 µM oligomycin. Next, the cells were lysed using a somatic cell lysis reagent and ATP was measured in the lysates using luminometric ATP detection assay. A) Time course of ATP generation following stimulation of !1-adrenoceptors with PE (10 µM). All data are expressed as mean ± SEM (n= 4-6 independent experiments). *different from the control curve at time points, 2, 10 and 20 min (P<0.05). B) Effect of PE (0.1 – 10 µM) on ATP synthesis in the presence of mitochondrial ATP synthase inhibitor, oligomycin (1µM). All data are expressed as mean ± SEM (n= 4-6 independent experiments), * different from control group (P<0.05), #different from all other groups (P<0.05).    131              Figure 4.7 Effect of the inhibitors of MMP, the EGFR and PI3-kinase on PE-stimulated mitochondrial ATP synthesis in VSM cells. Serum starved rat aortic VSMCs were grown in 96-well bioluminescent compatible plates and treated with inhibitors of MMP (GM6001, 25 µM), the EGFR (AG1478, 10µM) and PI3K (LY294002, 10µM) for 20 minutes followed by stimulation with PE (10 µM) for an additional 60 minutes. Next, the cells were lysed using a somatic cell lysis reagent and ATP was measured in the lysates using a luminometric ATP detection assay. All data are expressed as mean ± SEM (n= 4-6 independent experiments), * different from control group at 10 min (P<0.05), # different from PE stimulated group at 20 min (P<0.05).      132                 Figure 4.8 Maintenance of adrenergic vascular tone requires mitochondrial ATP synthesis. Rat small mesenteric arteries were mounted on the microperfusion system. PE (10 µM) was added to the bath (advential side) to cause vasoconstriction followed by luminal injection of bolus doses of oligomycin (50-500 pM). Representative trace (top panel) and quantitative analysis (bottom panel) of vasorelaxation of PE-constricted arteries in response to the luminal injections of bolus doses of oligomycin (50-500 pM). All data are expressed as mean ± SEM (n= 4 independent experiments), * different from PE group in the absence of oligomycin (P<0.05).    133             Figure 4.9 Phenylephrine-stimulated GLUT4 translocation is reduced by the inhibitors of MMPs, the EGFR and PI3-kinase in VSM cells. Serum starved VSM cells grown in 75mm 2  culture flasks were incubated with inhibitors of MMPs (GM6001, 25µM), the EGFR (Ag1478, 10µM) or the PI3-kinase (LY294002, 10µM) for 20 min followed by stimulation with PE (10 µM) for additional 40 min. Cell membranes were separated from whole cell lysates using two- layer sucrose-gradient centrifugation. GLUT4 in the membrane and cytosol fractions were detected by Western blotting with specific antibodies. Representative Western blot showing GLUT4 expression in the membrane and cytosol fractions and pan cadherin as a marker of membrane fractions. The extent of GLUT4 translocation was measured by determining the ratio of GLUT4 expression in the membrane and cytosol fractions. All data are expressed as mean ± SEM (n=4). * different from all other groups (P<0.05), # different from PE group (P<0.05).   134           Figure 4.10 Phenylephrine-stimulated activation of MMP-7 requires mitochondrial ATP synthesis and release. A) Rat superior mesenteric arteries (SMA) were incubated in the presence of oligomycin (1µM) or apyrase (5U/ml) for 20 minutes followed by stimulation with PE (10 µM) for another 40 minutes. MMP-7 activity in the releasates from PE-incubated arteries was assessed by CM- transferrin zymography and quantitated as the ratio of active MMP-7 to pro-MMP-7. All data are expressed as mean ± SEM (n= 4-6 independent experiments). * different from all other groups (P<0.05), # different from PE stimulated VSM cells in the absence of inhibitors (P<0.05). B) Rat arteries (SMA and aorta) were incubated in the presence of ATP (1 µM) for 40 minutes followed by the measurement of MMP-7 activity in the releasates using CM-transferrin zymography. Quantitative analysis of MMP-7 activity as measured by the ratio of active MMP- 7 to pro-MMP-7. All data are expressed as mean ± SEM (n= 4-6 independent experiments).  * different from control groups (P<0.05).   135          Figure 4.11 Activation of MMP-7 by ATP involves purinergic P2X receptors and calcium. Rat arteries (pieces of equal length, were incubated with either non-selective purinergic receptor blocker (suramin) or selective P2Y blocker (PPADS) or calcium chelating agents [cell permeable and intracellular calcium chelating agent, BAPTA-AM, or extracellular calcium chelating agents such as EDTA + EGTA] for 20 minutes and stimulated with ATP (1 µM) for 40 minutes. MMP-7 activity in the releasates was assessed by CM-transferrin zymography. A) MMP-7 activity from the releasates (aorta) in the presence of suramin (20 µM) or PPADS (100 µM). Quantitative analysis of MMP-7 activity as measured by the ratio of active MMP-7 to pro- MMP-7. All data expressed as mean ± SEM and representative of 4-6 independent experiments. * different from control and ATP + suramin groups (P<0.05), # different from ATP and ATP+PPADS group (P<0.05). B) MMP-7 activity from the releasates (aorta) in the presence of EGTA (50 µM) + EDTA (50 µM) or BAPTA-AM (100 µM). Quantitative analysis of MMP-7 activity as measured by the ratio of active MMP-7 to pro-MMP-7. All data expressed as mean ± SEM and representative of 4-6 independent experiments. * different from all other groups (P<0.05), # different from the ATP group (P<0.05).   136              Figure 4.12 Proposed mechanism for agonist-induced signaling of vascular tone, hypertrophic growth and remodeling. Activation of vascular Gq-protein coupled receptors (such as !1-adrenergic receptors or angiotensin receptors) by cognate agonists transactivates MMPs (such as MMP-7) and, thereby, growth factor receptors (such as EGFR). These transactivation events then trigger ATP synthesis in mitochondria thereby regulating vascular tone, growth and remodeling processes.   137 4.6   Bibliography 1. Kakar P, Lip GY. Towards understanding the aetiology and pathophysiology of human hypertension: where are we now? J Hum Hypertens. 2006;20(11):833-836. 2. Cai H, Harrison DG. 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Gen Pharmacol. 1998;31(1):1-8.     ! "#"! 5. Inhibition of matrix metalloproteinases and the epidermal growth factor receptor transactivation prevents the development of hypertension in insulin resistant rats 1 .                1 A version of this chapter will be submitted for publication. Nagareddy PR, Vasudevan H, McClure B, MacLeod KM and McNeill JH. Inhibition of matrix metalloproteinases (MMP) and the epidermal growth factor receptor (EGFR) transactivation prevents the development of hypertension in insulin resistant rats (2009). ! "#$! 5.1 Introduction Hyperinsulinemia and insulin resistance are often found to be associated with high blood pressure in both humans and several animal models 1 . Although several studies have reported a positive correlation between high blood pressure and insulin resistance, others have found a weak or no correlation 2,3 . Despite the conflicting reports, it appears that nearly 50% of hypertensive patients are hyperinsulinemic and insulin resistant 4 . Individuals with insulin resistance often manifest changes in the behavior of blood vessels, with characteristic abnormalities in vascular reactivity, changes in several key regulators in endothelium, and an increased risk for vascular inflammation all of which could contribute to vascular dysfunction and the development of hypertension 5, 6 . Despite major research efforts, it remains uncertain what causes hypertension in insulin resistant conditions. Regardless of the cause, the most commonly observed pathological hallmarks of hypertension, both in humans and animal models are increased vasoconstriction or decreased vasodilatation 7 , endothelial dysfunction associated with oxidative stress 8  and hypertrophy associated with abnormal cardiovascular remodeling 9 . Interestingly, the state of elevated blood pressure and all the above hallmarks of hypertension are found be associated with elevated levels of vasoconstrictory G-protein coupled receptor (GPCR) agonists such as norepinephrine (NE), angiotensin II (Ang II) and endothelin I (ET-1) 10-16 . It has been proposed that elevated levels of reactive oxygen species (ROS), increased vasoconstriction and hypertrophic growth of cardiac and vascular tissues are all the consequences of excess and abnormal stimulation of their corresponding GPCRs 17 . Increasing evidence suggests that matrix metalloproteinase (MMP)-dependent transactivation of the epidermal growth factor receptor (EGFR) by GPCR agonists might be a unifying mechanism in the etiology of hypertension. Several studies have demonstrated that blockade of MMPs or ! "#$! abrogation of EGFR tyrosine kinase activity by selective pharmacological inhibition blunts vasoconstriction both in isolated arterial rings in vitro and in various animal models of hypertension in vivo 15,18,19 . Blockade of MMP-dependent EGFR transactivation inhibits ROS formation, spares nitric oxide being scavenged by ROS and hence helps prevent the impairment of endothelial function 20 . Additionally, MMP inhibitors such as doxycycline have been shown to improve insulin resistance, oxidative stress, endothelial function, blood pressure in spontaneously hypertensive rats (SHR) 21 . Further inhibition of MMPs inhibited proteolytic processing of membrane-bound inactive tumor necrosis factor ! (TNF!) into an active form, a factor that is closely associated with insulin resistance 22 . Taken together, these results suggest that inhibition of MMPs or blockade of MMP-dependent transactivation of the EGFR may provide a potential therapeutic strategy for hypertension. We previously have shown that feeding rats with a high fructose diet produces some of the characteristic symptoms of metabolic syndrome such as insulin resistance, hypertriglyceridemia, hyperinsulinemia and hypertension 23-25 . Although various mechanisms have been proposed to explain the development of hypertension in insulin resistance, it is not clear if MMPs or the MMP-dependent transactivation of the EGFR pathway has a role in the etiology of diet-induced insulin resistant hypertension. In the present study, using the fructose-hypertensive rat model, we investigated the role of MMPs and the EGFR in the etiology of hypertension and explored the cellular and molecular mechanisms involved therein. Further, using drug interventions, we sought to determine the long-term effects of MMP and EGFR inhibition on insulin resistance, endothelial function and blood pressure.    ! "##! 5.2 Methods 5.2.1 Experimental Design 5.2.1.1 Animal Studies. This study conforms with the Canadian Council on Animal Care Guidelines on the Care and Use of Experimental Animals and was approved by the University of British Columbia Animal Care committee. Forty-eight male Wistar rats weighing between 280 to 300 g were obtained from Charles River Laboratories Inc., Quebec and allowed to acclimatize to the local vivarium. They were randomly divided into 6 equal groups: control (C), control treated with doxycycline, a broad spectrum MMP inhibitor (CD) or AG1478, the EGFR tyrosine kinase inhibitor (CA), fructose-fed (F), fructose-fed and treated with doxycycline (FD) or AG1478 (FA). Animals were allowed ad libitum access to food and water. Rats in control groups received normal rat chow while the rats in fructose groups were given a diet containing high fructose (60%) for 10 weeks to render them insulin resistant and hypertensive. Following 6 weeks of fructose feeding, the rats in CD and FD groups were administered doxycycline at a dose of 20 mg/kg, daily 15, 26  and rats in CA and FA groups were given AG1478 (5mg/kg, daily) 27  by oral gavage for 4 weeks until termination. AG1478 is a synthetic tyrphostin compound, a potent and selective EGFR tyrosine kinase inhibitor (IC50=3 nmol/L) 28  and it acts by competing for the substrate-binding site to affect tyrosine kinase activity 29 . Control rats received equal volumes of vehicle. Basal blood pressure (at week 6) and the final blood pressure (at week 10) were measured in conscious rats using the indirect tail cuff method as described previously 23 . At the end of the treatment period an oral glucose tolerance test (OGTT) was performed. At termination, blood was collected from the rats following a 5-h fast for measuring glucose, insulin, and cholesterol. Animals were terminated over a period of 6 days by an i.p administration of pentobarbital (65 mg/kg, bw) and the superior mesenteric artery (SMA) was isolated, removed, and cleaned of excess adipose and connective tissues. Some of ! "#$! the SMA tissues were used immediately for vascular reactivity studies and for determination of MMP activity using zymography, after which they were snap frozen in liquid nitrogen and stored at  - 70 0 C for Western blot studies. Oral glucose tolerance test (OGTT) and insulin sensitivity index (ISI) At the end of treatment an OGTT was performed by administration of glucose at a dose of 1g/kg to overnight fasted rats 30 . Blood samples from tail vein were taken prior to, and 10, 20, 30 and 60 minutes after administration of the glucose load. Plasma was separated by centrifugation and stored at -70 o C until assayed for glucose and insulin. Insulin sensitivity following the OGTT was estimated using the formula of Matsuda and DeFronzo 31 , where ISI = 100/square root [(mean plasma glucose ! mean plasma insulin) ! (fasting plasma glucose ! fasting plasma insulin)]. 5.2.1.2 Ex vivo vascular reactivity studies. In vivo effects of MMP and EGFR inhibition on vascular reactivity and endothelial function Tissue rings each of length 3–4 mm with intact endothelium were dissected from the SMA and appended onto glass hooks, which were then mounted in a 20-ml isolated tissue bath containing carboxygenated (95% O2-5% CO2) Krebs-Ringer buffer at 37°C as described previously (8, 38). Tissues were primed twice with 40 mM KCl followed by assessment of endothelial integrity using acetylcholine (ACh). Later, the tissues were assessed for changes in contraction to phenylephrine (PE) (10 –9  to 10 –4  M), after which they were preconstricted with the 70% of the effective dose (ED70) of PE, and relaxation  responses to increasing concentrations of Ach (10 –9  to 10 –4  M) were obtained. Alterations in responses to  ACh were compared between control and fructose-fed animals treated with or without the MMP and EGFR inhibitors. At the end of experiment, the tissues were blotted onto paper towels and weighed. Responses to PE are reported as mg/sq. mm in the SMA and as a percentage of maximum KCl contraction. ! "#$! Responses to Ach are reported as percent relaxation in tissue precontracted by a ED70 dose of PE. In vitro effects of MMP and EGFR inhibition on vascular reactivity To study the in vitro effects of MMP and EGFR inhibition on vascular contractility, a separate study was conducted. Twelve male Wistar rats were randomly and equally divided into 2 groups and fed with either a normal rat chow or high fructose diet for 10 weeks. At the end of 10 weeks, rats were killed under pentobarbital anesthesia and their SMAs were collected. Similar to the previous experimental protocol, tissues of equal length were mounted in a tissue bath and subjected to dose response curves to increasing concentrations of PE (see the previous section). Following precontraction of the tissues with an ED70 dose of PE, relaxation responses to graded doses of GM6001 (an MMP inhibitor, 10 -7  to 10 -4 M) and AG1478 (10 -7  to 10 -5 M) were obtained. 5.2.1.3 Cell culture studies. Bovine coronary arterial endothelial cells (BCAE, clonetics) were cultured in endothelial growth medium (EGM) supplemented with EGM-MV Bullet Kit (Lonza) at 37 o C in a 5% CO2 humidified incubator. Passages 4-6 were used for all the experiments. BCAE cells were plated in 6-well plates and grown until they reach ~ 70% confluence. A day before the experiment, the cells were placed in growth factor-free media. On the day of the experiment, the cells were washed with Hanks balanced salt solution (HBSS) and preincubated with or without 1mM N G -nitro-L-arginine methyl ester (L-NAME) and 1 to 8 pmoles of human recombinant MMP-2 (activated with p-aminophenyl mercuric acetate, 1 mM APMA) or its vehicle for 2 hours. The medium was removed and the cells in the presence of L-arginine (25µM) were incubated with or without L-NAME and calcium ionophore, A-23187 (5µM) for 30 minutes at 37 0 C.  ! "#$! Measurement of nitric oxide levels NO released into the culture media was evaluated by measuring the total nitrite and nitrate content using a commercially available fluorimetric kit (Cayman Chemical Company, MI) 5.2.2 Biochemical measurements. Plasma glucose was measured using an enzymatic colorimetric assay kit (Roche Diagnostics) and a Beckman Glucose Analyzer. Plasma insulin levels were determined using a double antibody RIA using a kit from Linco Research Diagnostics, St. Charles, MO. Plasma cholesterol and triglycerides were measured using enzymatic colorimetric assay kits from Wako Diagnostics, VA. 5.2.3 Determination of MMP activity by zymography. MMP activities were determined by substrate zymography techniques, using gelatin as a substrate for the gelatinases MMP-2 and MMP-9 and carboxymethylated transferrin (CMT) as substrate for MMP-7. Briefly, several pieces of carefully dissected and equilibrated arteries of equal length were incubated in the presence of various inhibitors (see results section) for 30 min and stimulated with either PE (10 µM) for 30 min. Arterial releasates were subjected to SDS PAGE using CMT (2.5 mg/mL) or 2% gelatin in gels. Following electrophoresis, the gels were washed with Triton X-100 (2.5% for 3x20 min) and incubated at 37°C overnight in zymogram development buffer containing 50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 5 mM CaCl2, 0.02% Brij-35Tris. The gels were stained with coomassie blue and MMPs were detected as transparent bands against the background of coomassie blue-stained undigested gelatin or CMT. The ratio of active-MMP to pro-MMP was used to represent MMP activity. 5.2.4 Western blotting. Tissues were homogenized using a homogenizer in modified RIPA buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM PMSF, 1 mM EDTA, 5 !g/ml aprotinin, 5 !g/ml leupeptin, 1% Triton x-100, 1% sodium deoxycholate, 0.1% SDS at 4 °C. The homogenate was centrifuged at 10000"g at 4 °C for 15 min and the protein content of ! "#$! the supernatants was determined by the Bradford protein assay. Equal amounts of protein (15- 30µg) from each sample were separated by 8% or 10% SDS PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked for 1 hour in a solution of 5% skim milk (in TBS-T) and then incubated overnight at 4°C with corresponding primary antibodies. Membranes were washed, incubated with their corresponding HRP- conjugated secondary antibodies for 1 hour followed by treatment with chemiluminescence reagents (Amersham Inc, Québec, Canada). Immunoblots were exposed and developed on photographic film. Densitometric analysis was performed using “Image J” software from the NIH. 5.2.5 Chemicals and reagents. All chemicals unless otherwise mentioned were of reagent grade and purchased from Sigma (St. Louis, MO). MMP-2, MMP-7 and MMP-9 antibodies were purchased from Millipore, MMP-2 proenzyme (Human Recombinant) from Alexis Biochemicals, AG1478 from LC laboratories, EGFR and eNOS antibodies from Cell Signaling, TIMP antibodies from Calbiochem. PE, ACh, and L-NAME  were dissolved in Krebs-Ringer, while GM6001 and AG1478 were dissolved in DMSO. For oral treatment, AG1478 was made in a suspension with 0.5% sodium carboxymethyl cellulose while doxycycline was dissolved in water. 5.2.6 Statistical analysis. All values are expressed as mean ± SEM. “n” denotes the sample size in each group. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by the Newman-Keuls test for multiple comparisons. GraphPad Prism (GraphPad Software, CA) software program was used for statistical analysis. For all results the level of significance was set at P<0.05.   ! "#$! 5.3 Results 5.3.1 General physical and biochemical characteristics The effect of 4 weeks of doxycycline and AG1478 treatment on general physical and biochemical parameters in control and high fructose diet fed rats are summarized in Table 5.1. Fructose feeding did not affect the body weights of either control or fructose-fed rats. Similarly, fructose feeding for 10 weeks did not affect plasma glucose. However, fasted plasma insulin, triglyceride and cholesterol levels were significantly higher in fructose-fed rats compared to control rats. It is noteworthy that elevated plasma insulin in the presence of normal glucose levels suggests impaired insulin sensitivity. Treatment with doxycycline or AG1478 did not affect any of these parameters in either control or fructose-fed rats. 5.3.2 Doxycycline prevents PE-induced MMP activation in the cardiovascular tissues of control and FHR To determine the efficacy and suitability of doxycycline as a MMP inhibitor activity, control and fructose-fed rats were treated with doxycycline (20 mg/kg, p.o) or its vehicle one hour before injection of PE (3mg/kg i.p). Thirty-minutes following administration of PE, rats were killed under pentobarbital anesthesia and their tissues collected. Pieces of heart, aorta and SMA tissues were collected, weighed and incubated in Krebs ringer buffer for 30 minutes. Gelatinolytic and MMP-7 activity from the tissue releasates was measured by gelatin and CMT substrate zymography respectively. Stimulation of !1 adrenergic receptors by PE increased both gelatinolytic (data not shown) and MMP-7 activity in the heart, aorta and SMA tissues of both control and fructose-fed rats (Fig.5.1A). However, fructose-fed rats demonstrated a significantly higher MMP activity in all tissues as determined by the ratio of active to pro-MMPs. Pretreatment with doxycycline significantly prevented PE induced MMP activation both in control and fructose-fed rats. These ! "#$! data suggests that doxycycline is not only an effective inhibitor of MMPs but also validates the dose of doxycycline (20 mg/kg, p.o) selected for our chronic studies. 5.3.3 Doxycycline and AG1478 inhibit the activity but not the expression of MMPs in the arteries At termination, the arteries (aorta and SMAs) from control and fructose-fed rats were carefully dissected out, weighed and incubated in Krebs ringer buffer to determine MMP activity. The remaining tissues were snap frozen and processed for Western blotting to determine the expression of MMPs and the EGFR. Fructose feeding significantly increased MMP-7 activity as determined by substrate zymography of tissue releasates both in SMA (Fig.5.1B) and aorta (Fig.5.1C) compared to control rats. Similarly, MMP-2 and MMP-9 activity were also increased in the arteries of fructose-fed rats (data not shown). However treatment of fructose-fed rats with doxycycline significantly inhibited MMP activity in both arteries. Surprisingly, in SMA from fructose-fed rats, AG1478 also inhibited MMP-7 activity. SMA from fructose-fed rats showed a significant increase in the expression of MMP-2, MMP-9 and MMP-7 (Fig.5.2). Treatment with either doxycycline or AG1478 did not change the expression of MMPs in arteries from either control or fructose-fed rats. However doxycycline treatment significantly upregulated the expression of endogenous MMP inhibitors, the TIMPs particularly in fructose-fed rats (Fig.5.3). Further, the ratio of TIMP-1/MMP-9 and TIMP- 2/MMP-2 was also increased in FD rats (Fig.5.3) suggesting that doxycycline inhibits MMP activity by increasing TIMPs levels 26 . Similarly, SMA from fructose-fed rats exhibited increased activation of the EGFR as determined by increased phosphorylation of tyr1177 on the EGFR (Fig.5.4). Treatment of fructose-fed rats with either doxycycline or AG1478 reduced the levels of pEGFR without affecting total EGFR expression. These data suggest that fructose ! "#"! feeding increases MMP-dependent transactivation of the EGFR in SMA and that both doxycycline and AG1478 attenuated the increased activation of the EGFR. 5.3.4 Doxycycline and AG1478 prevent the development of hypertension in FHR The basal blood pressure at week 6, before the treatment of rats with doxycycline and AG1478 was not significantly different between control and fructose rats. However, feeding rats with fructose for a further 4 weeks resulted in a significant increase in their systolic blood pressures (Fig.5.5). Treatment of fructose rats with either doxycycline or AG1478 prevented the increase in systolic blood pressure without affecting blood pressure in control rats. 5.3.5 Doxycycline and AG1478 do not improve insulin sensitivity in FHR It has been shown previously both in our laboratory and elsewhere that several drug interventions, that improve insulin sensitivity, including metformin, vanadium compounds and thiazolidinediones, can ameliorate the hypertension in this animal model 32-35 .  We therefore wanted to determine if the improvement in hypertension in fructose-fed rats treated with doxycycline and AG1478 are due to changes in insulin sensitivity. The effect of MMP and EGFR inhibition on insulin resistance was determined by an OGTT. Changes in glucose and insulin levels subsequent to glucose challenge are summarized in Fig.5.6. Fructose feeding resulted in insulin resistance as demonstrated by increased insulin secretion to glucose challenge compared to control rats (Fig.5.6B). Further, analysis of ISI values calculated from the OGTT revealed a significant attenuation in insulin sensitivity in fructose-fed rats (Fig.5.6C). Treatment with either doxycycline or AG1478 did not improve insulin sensitivity in fructose-fed rats. These data suggest that inhibition of MMPs or the EGFR tyrosine kinase activity does not change insulin sensitivity, and that improvement of hypertension with these drug interventions in fructose-fed rats is independent of insulin resistance.  ! "#$! 5.3.6 Doxycycline but not AG1478 prevents endothelial dysfunction One of the pathological hallmarks of hypertension in insulin resistance is increased vasoconstrictor responses to contracting factors 25  or decreased vasorelaxation to vasodilators 36 . Further, previous studies from our lab and elsewhere have demonstrated impairment in endothelial function in fructose-fed rats, while reduction in blood pressure is associated with improved endothelial function 36,37 . We therefore wanted to investigate the effects of MMP and EGFR inhibition on vascular function in isolated mesenteric arteries with an intact endothelium. Subsequent to the testing of functional integrity of endothelium, cumulative concentration responses curves to PE and acetylcholine were constructed in the mesenteric arteries from control and fructose-fed rats. The vascular responses to PE and acetylcholine are summarized in Fig.5.7. As previously reported, fructose feeding did not change the responses to PE in these arteries 24 . Further, as expected treatment of fructose-fed rats with doxycycline or AG1478 did not affect the PE responses (Fig.5.7A). Similar to the results from our previous studies 24,36 , fructose-fed rats exhibited impaired endothelial function as demonstrated by attenuated vasorelaxant responses to acetylcholine (Fig.5.7B). However, doxycycline but not AG1478 prevented the impairment of endothelial function as demonstrated by improved vascular responses to acetylcholine in FD rats compared to untreated fructose-fed rats (Fig.5.7B). To investigate whether doxycycline improved endothelial function by preserving endothelial nitric oxide synthase (eNOS) activity, we determined the expression of phospho and total eNOS expression in the SMA. Fructose feeding significantly decreased the phosphorylation of eNOS without affecting total eNOS expression in SMA (Fig.5.8). However, treatment of fructose-fed rats with doxycycline preserved the phosphorylation status of eNOS as determined by the increased ratio of phospho eNOS to total eNOS. These data suggest that MMPs might be involved in the impairment of endothelial function in fructose-fed rats. ! "#$! 5.3.7 MMP-2 decreases agonist-induced NO release from endothelial cells It has long been known that endothelium dependent vasorelaxation depends on a functional NO system and defects in vasodilatory mechanisms in fructose-fed rats could be due to decreased NO release or function 24,36 . To further explore the cellular mechanisms underlying the MMP- driven endothelial dysfunction, the interaction between MMP-2 and the eNOS-NO system was examined in vitro using BCAE cells. Among the various MMP subtypes, MMP-2, which is highly expressed in VSM cells and endothelium 38,39 , also possesses proteolytic activity. It has been shown to cleave various vascular peptides including endothelium-derived big endothelin 40  and calcitonin gene-related peptide 41 . In the present experiment we investigated whether MMP- 2 impairs the capacity of eNOS to produce NO. The effect of MMP-2 on agonist-stimulated NO release in BCAE cells is summarized in Fig.5.9. Stimulation of BCAE cells with calcium ionophore A-23187, which dissociates eNOS from caveolin and activates eNOS 42 , resulted in greater than 2 fold increase in NO levels compared to control cells. To determine whether the source of NO production was eNOS, the effect of A- 23187 to increase NO production was determined in the presence of eNOS inhibitor, L-NAME. Treatment with L-NAME alone did not change NO levels compared to control cells (Fig.5.9A). However, pretreatment with L-NAME significantly reduced A-23187-stimulated NO release suggesting that NO release was due to activation of eNOS. We next determined the effect of MMP-2 on eNOS derived NO production in response to A-23187. As shown in Fig 5.9B, active MMP-2 (1 to 8 pM, treated with APMA) significantly decreased NO release in a concentration dependent manner in response to agonist stimulation. On the other hand, inactive MMP-2 (untreated with APMA) fail to inhibit A-23187-stimulated NO release suggesting the possibility that MMP-2 interferes with eNOS activity either by inactivation or proteolytic degradation of functional eNOS or its cofactors 40, 43 . ! "#$! 5.3.8 Inhibition of the EGFR causes vasorelaxation in PE constricted arteries Doxycycline prevented the development of hypertension in fructose-fed rats possibly by preserving the functional integrity of vascular endothelium. On the other hand the mechanism by which AG1478 prevented the development of hypertension remains unclear and may involve inhibition of EGFR transactivation induced by vasoconstrictory factors. To understand the mechanisms by which AG1478 improves blood pressure, we conducted a separate study using control and fructose-fed rats. In these rats, SMA were isolated, mounted in a tissue bath and a cumulative concentration response curve for PE was constructed (10 -9  to 10 -4  M). The direct vasorelaxant effects of GM6001 (10 -7  to 10 -4  M) and AG1478 (10 -7  to 10 -5  M) were tested on PE constricted arteries using a concentration equivalent of ED70 of PE. As expected the contractile responses to PE were similar in the arteries of both control and fructose-fed rats. GM6001 and its vehicle DMSO produced a concentration dependent vasorelaxation and the responses were similar in the arteries of control and fructose-fed rats, a response that may be due to solvent effect (Fig.5.10A). Similar to GM6001, AG1478 also produced a vasodilatation in both control and fructose-fed rats. However, AG1478 produced a significantly higher vasorelaxation in the arteries of fructose-fed rats (Fig.5.10C) compared to vehicle (Fig.5.10B). These data suggest that EGFR activation is essential to maintain the adrenergic vascular tone initiated by PE and that, fructose arteries depends more on the EGFR activation to maintain vascular tone similar to control rats. 5.4. Discussion Several mechanisms have been proposed to explain the pathogenesis of hypertension associated with hyperinsulinemia and insulin resistance 44 . We hypothesized that in insulin resistance, the characteristically elevated levels of GPCR agonists such as catecholamines and other vasoactive peptides overstimulate MMP-dependent transactivation of the EGFR resulting in the ! "##! pathological features of hypertension such as enhanced vascular tone, decreased vasodilatation or endothelial dysfunction. The important findings in the present study are 1) hypertension in insulin resistance was associated with increased expression and activity of vascular MMPs and the EGFR, 2) Inhibition of MMP activity by doxycycline prevented the development of hypertension and impairment of endothelial function without improvement in insulin resistance and 3) blockade of the EGFR by AG1478 prevented the development of hypertension without an improvement in endothelial function or insulin resistance. In the present study, using the fructose hypertensive rat (FHR) model, we studied the effect of inhibition of MMPs and the EGFR tyrosine kinase activity on vascular reactivity, endothelial function, insulin sensitivity and development of hypertension. We chose the FHR model because it represents a host of metabolic abnormalities similar to that observed in the metabolic syndrome in humans 45,46 . Further, many of the mechanisms linking insulin resistance to hypertension are apparent in rats fed a high fructose diet including hyperinsulinemia 25 , endothelial dysfunction 36,37 , increased levels of oxidative stress 47 , and increased sympathetic nervous system (SNS) activity 48  with elevated levels of catecholamines and other vasoactive peptides. Further, we previously have found that chronic fructose feeding is also associated with increased levels of plasma NE, Ang II 49,50 , thromboxane A2 (TXA2) 51 , and ET-1 25  which are all known to transactivate the EGFR via MMPs. Feeding rats a high fructose diet not only produced the classical symptoms of metabolic syndrome such as insulin resistance, hyperinsulinemia and hypertension 23-25  but also resulted in increased expression and activity of MMP-2, MMP-9, MMP-7 and the EGFR in arteries. Further, in mesenteric arteries, the increase in MMP-2 and MMP-9 expression paralleled a decreased expression of their endogenous inhibitors TIMP-2 and TIMP-1 respectively. It should be noted that a relative paucity of the corresponding TIMPs in relation to their MMPs favors ! "#$! increased proteolytic activity 52 . Our findings are in agreement with previous studies that also have reported an increased expression of MMP- 2, 7 and 9 in the arteries of diabetic 53,54  and hypertensive patients 55, 56 . The mechanisms underlying excessive activation of vascular MMPs in insulin resistance could be multifactorial but may involve stimulation by GPCR agonists such as NE 57 , Ang II 49,50 , ET- 1 25 and TXA2 51 , all of which are elevated in fructose-fed rats. Consistent with this idea, we found that stimulating !1 adrenergic receptors (AR) with PE increased MMP activity in the heart and arteries and this was inhibited by pretreatment with doxycycline. The observation that MMP activity was increased in fructose rats even in the absence of external stimuli suggest that insulin resistance is associated with increased MMP activity. Interestingly, stimulation of !1-ARs in fructose-fed rats produced an even greater activation of MMPs suggesting the possibility that the presence of insulin resistance exaggerates the proteolytic activity of MMPs. Further supporting our results, in vitro studies have shown that stimulation of GPCRs with agonists such as PE and Ang II enhances MMP-2 activity in insulin resistant VSM cells (see next chapter). Treatment of fructose-fed rats with doxycycline decreased MMP activity, a property that could be attributed to doxycycline binding to the zinc/calcium at their catalytic sites, blocking the active site or inducing conformational changes that render the proenzyme susceptible to fragmentation during activation 58,59 . Alternatively, doxycycline could inhibit MMP activity by upregulating the levels of the TIMPs that can bind and inactivate MMPs 60  a mechanism that is supported by the findings of our present study, where doxycycline increased the levels of both TIMP-1 and TIMP-2 as well as ratio of TIMP-1/MMP-9 and TIMP-2/MMP-2. It is not clear if increased MMP activity contributes to the development of insulin resistance or vice versa. A recent study reported that elevated MMP activity could lead to the development of insulin resistance by proteolytic cleavage of the insulin receptor-binding domain in SHR 21 . ! "#$! Further, it was also demonstrated that doxycycline inhibition of MMP-9 normalized endothelial function, reduced oxidative stress and elevated blood pressure with improvements in blood glucose and glycohemoglobin levels in SHRs 21 . In contrast, we did not observe any improvement in insulin sensitivity with doxycycline treatment. This could be due to the differences in dosage of doxycycline used, the subtype of MMP enzyme involved or, most importantly, the animal model used. We used the FHR model, a diet-induced model of acquired systolic hypertension while in the other study the SHR model, a genetic model of essential hypertension was used. The inherent differences between these two models, particularly in the etiology of insulin resistance, could explain the differential effects of MMP inhibition on insulin resistance 61 . Another mechanism that could be relevant to this discrepancy is related to the inhibition of MMP-dependent transactivation of the EGFR, as observed in the present study. It should be noted that the insulin receptor and the EGFR have some common and shared downstream signaling pathways in the vascular endothelium including the phosphatidylinositol 3-kinase (PI3K) and the mitogen activated protein kinase (MAPK) pathways. In the presence of metabolic insulin resistance, many insulin target tissues including vascular cells display a significant defect of PI3K signaling but retain normal sensitivity to insulin via the MAPK signaling pathway 62,63 . Further, it has also been shown that disruption of MMPs, HB-EGF, or the extracellular part of the EGFR will disrupt insulin-mediated EGFR phosphorylation 64 . These data suggests the possibility that in the present study the beneficial effects of MMP-9 inhibition on improving insulin sensitivity via the prevention of insulin receptor degradation 21  are outweighed by inhibitory effects on insulin signaling via blockade of the EGFR. The inhibitory effect of doxycycline on insulin signaling could be related to the differences in the concentration of doxycycline required to inhibit MMP-9 activity and that required to block MMP-2-dependent transactivation of the EGFR. For example, MMP-9 activity in human aortic VSM cells was ! "#$! inhibited using a low concentration of doxycycline but MMP-2 inhibition required a much higher concentration 60 . The concentration of drug required for MMP inhibition has varied widely, with the IC50 ranging from 5 to 500 µM depending on specificity and the type of MMP involved 65 . Regardless of the changes in insulin resistance, treatment of fructose-fed rats with doxycycline significantly improved endothelial function in both models. It has been long known that improving endothelial function improves insulin resistance and vice versa 44 . This has led to the expectation that insulin-resistant states would be associated with reduced eNOS expression, and that improving insulin sensitivity promotes NO-mediated vasodilatation by activating eNOS. In line with this hypothesis a recent study demonstrated that introducing the eNOS gene in fructose-fed rats reduces hypertension and improves insulin resistance by correcting defects in PI3K and Akt pathways 66 . However, in the present study doxycycline treatment of FHRs prevented endothelial dysfunction but surprisingly, did not improve insulin resistance. As discussed previously this could be due to the blockade of MMP- dependent transactivation of the EGFR and inhibition of the downstream PI3K/Akt pathway by doxycycline. Further, in insulin resistant VSM cells we found that inhibition of MMPs or the EGFR significantly attenuates agonist-induced activation of the EGFR and the downstream P13K-Akt pathway (next chapter). Doxycycline also did not improve any of the metabolic parameters suggesting that it’s beneficial effects on endothelial function are confined to protecting the eNOS-NO system possibly from the deleterious effects of MMPs. We found that improvement in endothelial function in doxycycline treated rats was associated with an upregulation of active eNOS in the arteries. Reduced eNOS activation in insulin resistant rats could be due to direct interaction of MMPs with eNOS resulting in its inactivation or degradation by proteolytic cleavage 40,41  in the endothelium and may include mechanisms such as oxidative stress 20,21,67 . Alternatively, increased proteolytic activity could lead to the ! "#$! destruction of elastic fibers or degradation of matrix resulting in the loss of eNOS activity 68, 69 . Further studies in BCAE cells revealed that MMP-2 decreased NO production possibly by interfering with eNOS activity and/ or increasing oxidative stress 20 . To corroborate this, we recently found that pharmacological inhibition of MMPs (by GM6001) or the EGFR (by AG1478) reduced the PE-induced increase in oxidative stress in insulin resistant VSM cells (see next chapter). The prevention of hypertension in doxycycline treated fructose-fed rats may be due to an overall improvement in endothelial function, rather than endothelium independent mechanisms as suggested by other studies 70 . Although MMP inhibition like in many other studies 15,40,41,71,72  produced a concentration dependent vasodilatation in PE constricted arteries, the responses observed in our study were not due to MMP inhibition but due to solvent effect. Nevertheless, our data suggests that improvement in blood pressure in fructose-fed rats is not related to direct vasodilatation by MMP inhibition. However, unlike the effect of MMP inhibition on blood pressure and endothelial function, inhibition of the EGFR prevented the development of hypertension without any improvement in endothelial function or insulin resistance. Because the EGFR shares many of the downstream signaling pathways with the insulin receptor, particularly the PI3K-Akt pathway, we did not anticipate improvement in either insulin resistance or endothelial function in AG1478-treated rats. A recent study however, reported an improvement in endothelial function in diabetic rats treated with AG1478 although the mechanism remains unclear 73 . Despite no significant improvement in endothelial function, AG1478 inhibition of the EGFR prevented hypertension, possibly by producing direct vasodilatation, a property that has been observed previously 15 . The mechanism by which AG1478 produces vasodilatation is not clear but may involve inhibition of the p38 MAPK and the PI3-kinase downstream of the EGFR 15  and may include mechanism such as prevention of Ca 2+  entry 74 . ! "#$! Taken as a whole, the results of our experiments suggest that vascular MMPs either acting directly or in concert with the EGFR contribute to the development of hypertension in insulin resistance conditions. Specifically, doxycycline seems to exert its beneficial effects by protecting the vascular endothelium and AG1478 by promoting vasodilatation. Further studies are required to elucidate the mechanisms by which MMPs impair NO production in endothelial cells and doxycycline normalizes eNOS activity. Perspectives A growing body of evidence suggests that the EGFR could function as a convergent point for both mitogenic and non-mitogenic signals arising from various stimuli in vascular smooth muscle cells 14 . Abnormal stimulation of the EGFR such as in conditions of insulin resistance and hypertension could lead to increased activation of MAP kinases that promote cell growth, proliferation and vasoconstriction 17 . Several studies have demonstrated that inhibition of the activities of MMP or the EGFR promotes vasodilatation 15,17,40 , reduces oxidative stress 20,21 , improves insulin resistance 21,22 , and reduces elevated blood pressure 15,21  and cardiovascular hypertrophy 75-77 . Unpublished data from our lab have revealed an important role for the MMP- EGFR pathway in the enhanced synthesis and activation of contractile proteins in insulin resistant VSM cells (see next chapter). Taken together, these findings indicate potential therapeutic strategies to control cardiovascular function and to treat cardiovascular diseases such as hypertension and heart failure. The extent to which inhibition of this pathway is clinically applicable to the prevention and treatment of hypertension in human patients remains to be determined. ! "#"! 5.5 Tables and Figures Table 5.1 Effect of doxycycline and AG1478 on general physical and plasma biochemical markers in control and high fructose diet - fed rats.             All values are expressed as mean ± SEM. * different from all control groups (P<0.05) Characteristics C F CD FD CA FA Body Weight (Gms) Basal Final  284 ± 4.4 489 ± 2.3  290 ± 6.5 490 ± 11.3  287 ± 7.6 503 ± 27.1  300 ± 5.9 528 ± 16.2  286 ± 7.1 500 ± 16.2  301 ± 7.2 507 ± 17.9 Glucose (mM) 8.9 ± 0.4 10 ± 0.4 9.5 ± 0.2 9.3 ± 0.5 9.6 ± 0.3 9.3 ± 0.7 Insulin (ng/ml) 0.73 ± 0.07 1.22 ± 0.12* 0.94 ± 0.2 1.3 ± 0.06 0.75 ± 0.11 1.34 ± 0.12  Cholesterol (mg/dl)  21.5 ± 3.8  43.6 ± 5.3*  17.1 ± 2.6  38.8 ± 8.5  23.9 ± 4.2  38.5 ± 4.1  Triglycerides (mg/dl)  47.6 ± 3.2  79.5 ± 8.9  38.2 ± 4.1  72.4 ± 7.1  40.4 ± 4.32  66.7 ± 12.9 ! "#$! Figure 5.1 MMP-7 activities in control and fructose-fed rats.  A) Effect of doxycycline on phenylephrine (PE) - induced MMP-7 activity in the heart and arteries. Representative zymograms showing pro- and active MMP-7 in the tissues of control and fructose-fed rats. For experiments in panel A, control and fructose-fed rats were treated with doxycycline (20 mg/kg, p.o) or its vehicle and 1 hour later PE was administered (3mg/kg i.p). Thirty-minutes following PE injection, rats were euthanized and pieces of heart, aorta and SMA tissues were collected, weighed and incubated in Krebs ringer buffer for 30 minutes. MMP-7 activity in the releasates from PE-incubated tissues was assessed by CM-transferrin zymography. B) Representative zymograms and quantitative analysis of MMP-7 activity as measured by the ratio of active MMP-7 to pro-MMP-7 in the B) aorta C) and SMA. For experiments in panel B, arteries (aorta and SMA) of equal length from control and fructose-fed rats (treated with doxycycline or AG1478 for 4 weeks) were incubated in Krebs ringer buffer and the basal MMP-7 activity in the releasate was determined by CMT zymography. All data are expressed as mean ± SEM (n=4). *different from all other groups (P<0.05), # different from F group (P<0.05). Open bars represent control groups and solid bars represent fructose-fed rats.        ! "#$!  ! "#$! Figure 5.2 Effect of doxycycline and AG1478 on the expression of MMPs in the arteries of control and fructose-fed rats. Representative Western blot showing A) MMP-7, B) MMP-2 and C) MMP-9 expression in the SMAs, with GAPDH as a loading control. The densitometric values of active MMPs were normalized to their corresponding pro-MMP densitometric values and the relative band intensities are expressed as mean ± SEM (n=8). * different from C, CD and CA groups (P < 0.05). Open bars represent control groups and solid bars represent fructose-fed rats. For all experiments involving doxycycline and AG1478 treatment, rats were treated with these inhibitors for 4 weeks, from week 6 through week10. At termination, their SMAs were collected for Western blot experiments.            ! "#$!                  ! "##!  Figure 5.3 Effect of doxycycline and AG1478 on the expression of tissue inhibitors of matrix metalloproteinases (TIMPs) in the SMA of control and fructose-fed rats. A) Representative Western blot showing the expression of TIMP-2 in relation to expression of active MMP-2. The densitometric values of TIMP-2 were normalized to their corresponding MMP-2 densitometric values and the relative band intensities are expressed as mean ± SEM (n=8). * different from all groups (P < 0.05). Open bars represent control groups and solid bars represent fructose-fed rats. B) Representative Western blot showing the expression of TIMP-1 in relation to expression of active MMP-9. The densitometric values of TIMP-1 were normalized to their corresponding MMP-9 densitometric values and the relative band intensities are expressed as mean ± SEM (n=8). * different from all groups (P < 0.05). Open bars represent control groups and solid bars represent fructose-fed rats  ! "#$!  Figure 5.4 Effect of doxycycline and AG1478 on the activation of the EGFR in SMA. Representative Western blot showing phospho-EGFR expression, with GAPDH as a loading control. The densitometric values of phospho-EGFR were normalized to their corresponding total EGFR densitometric values and the relative band intensities are expressed as mean ± SEM (n=8). * different from all groups (P < 0.05), # different from the F group (P < 0.05). Open bars represent control groups and solid bars represent fructose-fed rats.   ! "#$!                 Figure 5.5 Effect of doxycycline and AG1478 on systolic blood pressure in fructose-fed rats. Control and fructose-fed rats were treated orally with doxycycline (20 mg/kg/day) or AG1478 (5 mg/kg/day) for 4 weeks starting at week 6 (basal, open bars) until week 10 (final, solid bars). All values are expressed as mean ± SEM (n=8). * different from all groups (P < 0.05).    ! "#$! Figure 5.6 Effect of doxycycline and AG1478 treatment on plasma glucose and insulin profiles following an OGTT in control and fructose-fed rats. A) Plasma glucose (mM) and B) plasma insulin (ng/ml) before, and 10, 20, 30 and 60 min after an oral glucose load of 1g per kg to rats fasted overnight. C) Insulin sensitivity index (ISI) values were estimated according to formula of Matsuda and Defronzo, where ISI = 100/square root [(mean plasma glucose ! mean plasma insulin) ! (fasting plasma glucose ! fasting plasma insulin)]. All values are expressed as mean ± SEM (n=8). * significantly different from the control groups (P < 0.05). Open bars represent control groups and solid bars represent fructose- fed rats.              ! "#$!                 ! "#"! Figure 5.7 Effect of doxycycline and AG1478 treatment on responses to PE and Ach in SMA isolated from control and fructose-fed rats. A) Concentration response curves to increasing concentrations of PE (10 -9  to 10 -4  M) in SMA. A1 represents the corresponding area under the curve values. B) Concentration response curves to increasing concentration of acetylcholine, Ach (10 -9  to 10 -4  M) in SMA constricted with a concentration that is ED70 of PE. B1 represents the corresponding area under the curve values. All values are expressed as mean ± SEM (n=4-6). * significantly different from control groups (P < 0.05), # different from F group (P<0.05). Open bars represent control groups and solid bars represent fructose-fed rats.              ! "#$!                  ! "#$!      Figure 5.8 Effect of doxycycline and AG1478 on the expression of eNOS in the arteries of control and fructose-fed rats. Representative Western blot showing phospho-eNOS expression, with GAPDH as a loading control. The densitometric values of phospho-eNOS were normalized to their corresponding total eNOS densitometric values and the relative band intensities are expressed as mean ± SEM (n=8). * different from all groups (P < 0.05), # different from the F group (P < 0.05). Open bars represent control groups and solid bars represent fructose-fed rats.   ! "#$! Figure 5.9 Effect of MMP-2 on calcium ionophore stimulated NO release in bovine coronary artery endothelial (BCAE) cells. A) NOx levels in the presence or absence of the eNOS inhibitor, L-NAME (1 mM). Results are expressed as percent control and are means ± SEM (n=4 independent experiments). * different from all other groups (P<0.05). B) NOx levels in the presence of active (striped bars) and inactive MMP-2 (dark bars). Results are expressed as percent control and are means ± SEM (n=4 independent experiments). * different from control and active MMP-2 treated cells (P < 0.05). In all experiments, BCAECs were preincubated with 1 to 8 pM of active (treated with p- aminophenyl mercuric acetate, 1 mM APMA for 2 hours at 37 0 C or inactive human recombinant MMP-2 or its vehicle for 2 hours and stimulated with the calcium ionophore, A-23187 (5µM) for 30 minutes. For, L-NAME experiments, cells were incubated with L-NAME for 2 hours and stimulated with ionophore.             ! "#$!                        ! "#$! Figure 5.10 Effect of MMP and the EGFR inhibition on the maintenance of adrenergic vascular tone induced by PE in superior mesenteric arteries (SMA) isolated from control and fructose-fed rats. A) Percent relaxation to increasing concentrations of GM6001, a broad- spectrum MMP inhibitor or its vehicle in SMA constricted with a ED70 concentration of PE. B) Percent relaxation to increasing concentrations of AG1478 or its vehicle in SMA of control rats constricted with a concentration that is ED70 of PE. All values are expressed as mean ± SEM (n=4-6). C) Percent relaxation to increasing concentrations of AG1478 (10 -7  to 10 -4 M) or its vehicle in SMA obtained from fructose-fed rats and constricted with a concentration that is ED70 of PE. All values are expressed as mean ± SEM (n=4-6). * different from AG1478 treated arteries (P<0.05).              ! "##!                 ! "#$! 5.6 Bibliography 1. Reaven GM. Abnormalities of carbohydrate and lipoprotein metabolism in patients with hypertension. Relationship to obesity. Ann Epidemiol. 1991;1(4):305-311. 2. Collins VR, Dowse GK, Finch CF, Zimmet PZ. An inconsistent relationship between insulin and blood pressure in three Pacific island populations. J Clin Epidemiol. 1990;43(12):1369-1378. 3. Mbanya JC, Thomas TH, Wilkinson R, Alberti KG, Taylor R. Hypertension and hyperinsulinaemia: a relation in diabetes but not essential hypertension. Lancet. 1988;1(8588):733-734. 4. Zavaroni I, Mazza S, Dall'Aglio E, Gasparini P, Passeri M, Reaven GM. 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Trends Endocrinol Metab. 2004;15(6):241- 243.     ! "#$! 6. Agonist induced activation of matrix metalloproteinases promotes increased expression of contractile proteins in insulin resistant vascular smooth muscle cells through the epidermal growth factor receptor pathway 1 .                   1 A version of this chapter will be submitted for publication. Nagareddy PR, Vasudevan H, McClure B, MacLeod KM and McNeill JH. Agonist induced activation of matrix metalloproteinases promotes increased expression of contractile proteins in insulin resistant vascular smooth muscle cells through the epidermal growth factor receptor pathway (2009). ! "#$! 6.1 Introduction The presence of metabolic and hemodynamic abnormalities activates growth-promoting pathways and contributes to the development of hypertension both in insulin resistant and non- insulin resistant states 1 . A growing body of evidence suggests that the characteristically elevated levels of G-protein coupled receptor (GPCR) agonists such as catecholamines, endothelin-1 (ET-1) and angiotensin II (Ang II), promote hypertrophic growth as well contribute to enhanced vascular tone in hypertension 2 . Some of the molecular mechanisms that are associated with these changes in hypertension include upregulation of growth promoting pathways and increased generation of reactive oxygen species 2, 3 . Until recently, the signaling events initiated by GPCR agonists were considered to be rapid, short-lived, and divided into two separate linear but distinct pathways, the vasoconstrictory pathway involving the phospholipase C (PLC)- diacylglycerol (DAG)- Ca 2+ axis and the growth promoting pathway involving receptor tyrosine and mitogen-activated protein (MAP) kinases. However, recent studies have suggested that these signaling pathways do not function independently but are actively engaged in cross talk 4 . The resulting combinatorial signaling events allow GPCRs to take advantage of pathways downstream of growth factor receptors to influence cell function under varying physiological situations 5 . One example of such cross talk connecting GPCR  activation with the MAPK signaling pathway is matrix metalloproteinase (MMP) dependent transactivation of the epidermal growth factor receptor (EGFR) in VSM cells 6-8 . It has been proposed that MMPs transactivate the EGFR in VSM cells in response to stimulation by GPCR agonists such as catecholamines, Ang II and ET-1. Further, overstimulation of the MMP-EGFR pathway leads to enhanced vascular tone, increased oxidative stress and hypertrophic growth in hypertension 9, 10 . Increasing evidence suggest that the EGFR could function as a convergent point for both mitogenic and non-mitogenic signals arising from ! "#$! various stimuli in VSM cells 7 . Studies have demonstrated that inhibition of the activities of MMP or the EGFR promotes vasodilatation 2, 6, 11 , reduces oxidative stress 12, 13 , improves insulin resistance 12, 14 , and reduces elevated blood pressure 6, 12  and cardiovascular hypertrophy 9, 10, 15 . Although it is clear that EGFR activation triggers a series of complex but well-characterized signaling events, how these signaling events are integrated to regulate contractility and influence the expression of contractile proteins is poorly understood. Further, it is not clear if MMP- dependent EGFR signaling is altered in insulin resistance and its role in regulation of contractile protein expression has not been fully defined. One signaling molecule that could play an important role in EGFR signaling and that is of particular importance in insulin resistance is Ras, a small GTP binding protein 1 . Activation of Ras, downstream of the EGFR and other receptor tyrosine kinases, initiates a phosphorylation cascade involving Raf, MAPK kinase (MEK) and extracellular signal-regulated kinases (ERK) I and 2 16, 17 . ERK activation promotes synthesis of contractile proteins by mechanisms involving stimulation of serum response factor (SRF) binding to 2 CC (A/T-rich) GG elements, also referred to as CArG boxes, a DNA sequence within the regulatory regions of smooth muscle genes 18 . Further, it has been reported that nearly all smooth muscle genes contain one or more CArG boxes, underscoring the importance of SRF in regulating gene expression in VSM cells 19 . SRF targets smooth muscle specific genes such as myosin light chain kinase (MLCK). Increased expression and activation of such a gene product could have downstream effects that may result in hypertension 20 . The other targets of SRF may include proteins of the contractile machinery such as the 20-kDa regulatory light chain of myosin-II (MLC20), myosin phosphatase (MYPT), the enzyme involved in dephosphorylation of MLC20, and Rho kinase, an enzyme involved in inactivating myosin phosphatase, all of which are implicated some way in the development of hypertension 20-22 . ! "##! In conditions of insulin resistance, many insulin target tissues, including VSM cells, display a significant defect of phosphatidylinositol 3-kinase (PI3K) signaling, but retain normal sensitivity to insulin via the MAPK signaling pathway 23, 24 . Since insulin resistance is also associated with hyperinsulinemia and elevated levels of various GPCR agonists, we hypothesize that this results in activation of MAPK pathways, promoting increased synthesis of contractile proteins in insulin resistant hypertension via the MMP-dependent transactivation of the EGFR. The main objectives of the present study were 1) to characterize the growth promoting signal pathways that are upregulated in response to GPCR stimulation in insulin resistant VSM cells, 2) to elucidate the mechanisms leading to increased expression of contractile proteins in insulin resistant VSM cells and 3) to study the effect of MMP and the EGFR inhibition on the activation and expression of contractile proteins in VSM cells in vitro and tissues from insulin resistant rats. 6.2 Methods 6.2.1 Cell culture Rat aortic vascular smooth muscle cells (VSMCs) were grown in complete Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen Life Technologies, Carlsbad, CA) with 10% fetal bovine serum (FBS) and 100-units/mL penicillin-streptomycin at 37ºC and 95% O2/5% CO2. Cells from passages 2-6 were used in all experiments. When cells were 80-90% confluent they were made quiescent by incubation in DMEM containing normal glucose (5.5 mM), high glucose (25 mM) or mannitol (5.5 mM glucose +19.5 mM mannitol) and 0.1% calf serum for 72 hours. On the day of experiments, quiescent VSM cells were treated with inhibitors/agonists for the indicated times. In experiments involving inhibitors, drugs were added to the media 1 hour prior to the stimulation by agonists. The media were collected and stored at -70 o C until MMP activities were determined by zymography. At the end of the experiment, cells were washed ! "#$! with ice-cold phosphate-buffered saline (PBS), lysed and the cell lysates collected and stored (at -70 o C) for Western blotting. 6.2.1.1 SiRNA experiments in VSM cells. Small interference RNAs (siRNA) specific to rat EGFR, MMP-2 and MMP-7 were purchased from Santa Cruz Biotech. For optimal siRNA transfection efficiency, the manufacturer’s protocol was followed. Briefly, VSM cells were seeded in a 6 well plate and cultured in 2 ml antibiotic free normal growth medium supplemented with 10% FBS until the cells were 60-80% confluent (~36 hours). On the day of transfection, cells were washed with transfection medium (sc-36868) and incubated with 1 ml of transfection reagent (sc-29528) containing 60 pmoles of either MOCK (scrambled, sc-sc37007) or rat specific EGFR, MMP-2 or MMP-7 siRNA oligonucleotides for 12 hours. After 12 h, the medium was supplemented with fresh DMEM (1ml), containing 2X FBS and antibiotics for another 24 hours. At this point, the cells were washed with warm PBS and incubated with normal DMEM (containing normal growth factors and antibiotics) for an additional 48 hours. When cells were 80-90% confluent they were maintained in DMEM containing normal glucose (5.5 mM), high glucose (25 mM) or mannitol (5.5 mM glucose +19.5 mM mannitol) and 0.1% calf serum for additional 72 hours. On the day of experiment, media were collected for zymography experiments and the serum-starved cells were stimulated with PE for 1 hour. At the end of the experiments, cells were lysed and the lysates collected and stored at -70 o C for Western blotting. 6.2.1.2 Measurement of MMP activity by substrate zymography. MMP-2, MMP-9 and MMP-7 activity in cell culture releasates was measured using gelatin (for MMP-2 and MMP-9) or carboxymethyl-transferrin (CMT) substrate (for MMP-7) zymography assays. The releasates were subjected to SDS PAGE using CMT (2.5 mg/mL) or 2% gelatin in gels. Following electrophoresis, the gels were washed with Triton X-100 (2.5% for 3x20 min) and incubated at ! "#$! 37°C overnight in zymogram development buffer containing 50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 5 mM CaCl2, 0.02% Brij-35Tris. The gels were then stained with coomassie blue and MMPs were detected as transparent bands against the background of coomassie blue-stained undigested CM transferrin. The ratio of pro-MMP to active-MMP was used to measure MMP activity. 6.2.1.3 Western blotting. Cells and tissues were homogenized using a homogenizer or sonicator in modified RIPA buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM PMSF, 1 mM EDTA, 5 !g/ml aprotinin, 5 !g/ml leupeptin, 1% Triton x-100, 1% sodium deoxycholate, 0.1% SDS at 4 °C. The homogenate was centrifuged at 10000"g at 4 °C for 15 min and the protein content of the supernatants was determined by the Bradford protein assay. Equal amounts of protein (15-30µg) from each sample were separated by 8% or 10% SDS PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked for 1 hour in a solution of 5% skim milk (in TBS-T) and then incubated overnight at 4°C with corresponding primary antibodies. Membranes were washed, incubated with their corresponding HRP-conjugated secondary antibodies for 1 hour followed by treatment with chemiluminescence reagents (Amersham Inc, Québec, Canada). Immunoblots were exposed and developed on photographic film. Densitometric analysis was performed using Image J software from the NIH. 6.2.2 Animal Studies Twenty-four male Wistar rats weighing between 280 to 300 g were obtained from Charles River Laboratories Inc., Quebec and allowed to acclimatize to the local vivarium. They were randomly divided into 4 equal groups: control (C), control treated with AG1478 (CA), fructose-fed (F), fructose-fed and treated with AG1478 (FA). Animals were allowed ad libitum access to food and water. Rats in control groups received normal rat chow while the rats in fructose groups ! "#"! were given a diet containing high fructose (60%) for 10 weeks to render them insulin resistant and hypertensive. Following 6 weeks of fructose feeding, rats in control and fructose groups were given vehicle or AG1478, the EGFR tyrosine kinase inhibitor (5mg/kg, daily) 25  by oral gavage for 4 weeks until termination. Basal blood pressure (at week 6) and the final blood pressure (at week 10) were measured in conscious rats using the indirect tail cuff method as described previously 26 . At the end of treatment period an oral glucose tolerance test (OGTT) was performed. At termination, superior mesenteric arteries (SMA) were isolated, cleaned of excess adipose and connective tissues and snap frozen in liquid nitrogen and stored at - 70 0 C for Western blot studies. 6.2.3 Statistical analysis All values are expressed as mean ± SEM. “n” denotes the sample size in each group. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by the Newman-Keuls test for multiple comparisons or unpaired student t test. GraphPad Prism (GraphPad Software, CA) software program were used for statistical analysis. For all results the level of significance was set at P<0.05. 6.3 Results 6.3.1 Modeling insulin resistance in VSM cells As a first step to establish cell culture conditions that mimic insulin resistance, we adapted a previously described procedure by growing VSM cells in normal glucose (NG, 5 mM) or high glucose (HG, 25 mM) for 72 hours 27 . As described, the cells as early as from the explant stage were maintained in normal glucose conditions and switched to low and high glucose conditions before performing experiments. Under these conditions, we found that insulin-dependent phosphorylation of IRS-1 and Akt, the key markers of insulin resistance were altered in VSM cells cultured in HG conditions (Fig.6.1). Specifically, we noticed increased IRS-1 ser307  ! "#$! phosphorylation and decreased Akt phosphorylation in response to insulin in VSM cells incubated in HG conditions. The phosphorylation of Ser 307, which results in insulin resistance was markedly increased in VSM cells cultured in HG and was detected as early as 1 minute after stimulating with insulin 28 . 6.3.2 Effect of PE-stimulation on MMP-EGFR signaling in insulin resistant VSM cells In order to gauge potential differences between the VSM cells grown in normal and high glucose conditions, we performed a survey of signaling pathway activation by vasoactive agonists. Activation of GPCRs such as !1  adrenergic receptors by phenylephrine (PE) and AT I receptors by Ang II has been linked to the activation of MMPs in VSMC 6, 11, 29 . Stimulation of these receptors by their corresponding agonists transactivates the EGFR by an MMP-dependent mechanism. To understand, if the activity and expression of vascular MMPs such as MMP-2, MMP-9 and MMP-7 are altered in response to HG and agonist stimulation, we treated VSM cells cultured in NG and HG conditions with PE for 30 minutes. As shown in the zymograms (Fig.6.2A), MMP-2 activity was significantly elevated in VSM cells grown in HG compared to NG conditions and stimulation with PE increased MMP activity further in HG conditions. The activities of other vascular MMPs such as MMP-9 and MMP-7 were not detected in our experimental settings. However, we found increased expression of all major vascular MMPs in VSM cells grown in HG conditions (data not shown). These data suggest that VSM cells exposed to HG exhibit increased activity and expression of all major vascular MMPs particularly, the MMP-2. Likewise, exposure of VSM cells to HG as well as stimulation with PE significantly increased the levels of phospho-EGFR (Fig.6.2B). Phosphorylation at 1173 on the carboxy-terminal of tyrosine residue, is a major site for autophosphorylation and crucial for the initiation of the downstream signaling and is often used as an index of EGFR activation 6 . These data suggests ! "#$! that VSM cells exhibit increased transactivation of the EGFR in response to HG and or PE stimulation possibly by MMP-dependent mechanisms. 6.3.3 Inhibition of MMPs reduces EGFR phosphorylation in insulin resistant VSM cells To determine whether inhibition of MMP activity or suppression of its expression reduces the EGFR phosphorylation induced by insulin resistance and PE stimulation, we conducted experiments using both pharmacological inhibition and siRNA approaches. As shown (Fig.6.2A), inhibition of MMPs by GM6001, a broad spectrum MMP inhibitor, reduces MMP-2 activity induced by HG as well as PE stimulation. Further, treatment of cells with GM6001 decreased the activation of the EGFR induced by both HG and PE stimulation (Fig.6.2B). On the contrary, GM6001 did not affect the expression of MMP-2, MMP-9 or MMP-7 (data not shown). Similar to pharmacological inhibition of MMPs by GM6001, genetic ablation of MMP- 2 or MMP-7 expression by their corresponding siRNA significantly reduced the phosphorylation of the EGFR induced both by high glucose and PE stimulation (Fig.6.3). Likewise, suppression of the EGFR by EGFR siRNA also decreased both HG and PE-induced increase in the EGFR activity in insulin resistant VSM cells (Fig.6.4). 6.3.4 Effect of PE-stimulation on MAP-kinases in insulin resistant VSM cells Activation of MAP kinase signaling pathways are linked to mitogenic effects including the synthesis of contractile proteins downstream of growth factor receptors such as insulin and the EGFR 18, 27 . We tested the effect of HG and PE stimulation on the classical MAP-kinases such as the ERK, c-Jun-N-terminal kinase (JNK) and the p38MAP kinase. Incubation of VSM cells in HG alone activated only ERK but not other MAP kinases (Fig.6.5). Further, stimulation with PE increased ERK activity as determined by increased phosphorylation of thr 202 and tyr 204 residues on ERK in VSM cells cultured in HG compared to NG conditions (Fig.6.5). PE- stimulation of VSM cells also increased the phosphorylation of p38 MAPK (Fig.6.6A) but not ! "#$! JNK-1 in HG conditions (Fig.6.6B). These data suggest that ERK activation is the most important signaling event in response to HG as well as agonist stimulation in insulin resistant VSM cells possibly to promote increased synthesis of contractile proteins 3, 21 . 6.3.5 Inhibition of MMP-EGFR pathway reduces PE-induced ERK activation in insulin resistant VSM cells Because ERK has been implicated in the synthesis of contractile proteins such as smooth muscle MLCK in response to vasoactive agents 21 , we next wanted to investigate if MMP and EGFR inhibitors can modulate ERK activity. In the present experiment, we stimulated VSM cells cultured in NG and HG with PE (10 µM) for 30 minutes in the presence of GM6001 or AG1478. As described before, stimulation of VSM cells cultured in HG produced a significant elevation in ERK activity as demonstrated by increased expression of phospho-ERK1/2. Prior treatment of cells with either GM6001 or AG1478 significantly reduced the activation of ERK produced by both HG and agonist stimulation (Fig.6.5A). To confirm the involvement of specific MMPs and the EGFR in mediating PE-induced increase in ERK activity, we transfected VSM cells with MMP-2, MMP-7 and EGFR siRNA and studied the effect of HG and PE stimulation. Suppression of the EGFR expression (Fig.6.5B) by the EGFR siRNA significantly decreased ERK activation induced by both HG and PE stimulation. Similarly, suppression of MMP-2 and MMP-7 expression by their corresponding siRNA, significantly decreased ERK activation (Fig.6.7). These data add substantial support to the idea that PE-induced increase in ERK activity is dependent on MMP transactivation of the EGFR in insulin resistant VSM cells. 6.3.6 Effect of PE-stimulation on the expression of contractile proteins in insulin resistant VSM cells We next examined the effects of PE stimulation on signaling pathways that are involved in vascular smooth muscle contraction and regulatory pathways that influence the expression of ! "#$! contractile proteins. The most important proteins involved in smooth muscle contraction are the MLCK, the 20-kDa regulatory light chain (RLC) of myosin-II (MLC20) and MYPT. In smooth muscle, MLCK phosphorylates the RLC of myosin II in the presence of Ca 2+  and calmodulin, which in turn facilitates interaction of myosin with actin filaments. The phosphorylation of the myosin RLC is known to be an obligatory step involved in the initiation of contraction. Therefore, we sought to determine the effect of PE-stimulation on the expression of MLCK and MLC II in VSM cells exposed to normal and high glucose. As shown (Fig.6.8), incubation of VSM cells in HG significantly increased the levels of MLCK as well as the phospho and total- MLC II. Further, stimulation with PE increased the levels of MLC II (both unphosphorylated and total) but not MLCK in insulin resistant VSM cells compared to normal VSM cells. Pharmacological inhibition of MMPs with GM6001 or the EGFR with AG1478 did not change the expression of MLCK or MLC II or phosphorylation of MLC II induced by PE either in control or insulin resistant VSM cells (Fig.6.8A). However, suppression of the expression of MMP-2 (Fig.6.8B) or the EGFR (Fig.6.8C) by their corresponding siRNAs reduced the MLC II levels induced by both insulin resistance and PE-stimulation in VSM cells. 6.3.7 Inhibition of MMP-EGFR pathway reduces SRF activation induced by insulin resistance and PE-stimulation in VSM cells Having found that ERK is activated in response to PE-stimulation and insulin resistance to a greater extent in VSM cells, we next studied the potential mechanisms downstream of ERK involved in increased synthesis of contractile proteins. Exposure of VSM cells to HG alone markedly increased activation of SRF as determined by the increased phosphorylation of ser 103 on SRF (Fig.6.9). Further, stimulation with PE increased the activation of SRF to a greater level in VSM cells exposed to HG compared to NG (Fig.6.9). The ribosomal S6 kinase P90RSK, a growth factor-inducible kinase is known to phosphorylate SRF at serine 103, resulting in the ! "#$! enhancement of the affinity of SRF to its binding site, the serum response element (SRE), within the c-fos promoter 30 . In parallel with increased phosphorylation of SRF, incubation of VSM cells in HG increased the activation of P90RSK as determined by increased levels of ser380 on P90RSK. Further, stimulation with PE increased the levels of p-P90RSK further in VSM cells cultured in HG compared to NG conditions (Fig.6.9A). Inhibition of MMPs by GM6001 and the EGFR tyrosine kinase activity by AG1478 reduced the phosphorylation of SRF induced by both HG and PE stimulation. Similarly, MMP and EGFR inhibitors also inhibited the phosphorylation of P90RSK in VSM cells cultured in HG (Fig.6.9A). Suppression of MMP-2 (Fig.6.9B) or EGFR (Fig.6.9C) expression by their corresponding siRNAs significantly reduced the activation of SRF in VSM cells induced by HG and PE stimulation. These data suggest that MMP-dependent transactivation of the EGFR contributes significantly to increased activation of SRF and P90RSK in VSM cells in response to HG and agonist stimulation. 6.3.8 Treatment of insulin resistant rats with AG1478 decreases the activation of EGFR, ERK, SRF and MLC II and prevents the development of hypertension We previously have shown that inhibition of MMP (by doxycycline) or the EGFR (by AG1478) activity prevented the development of hypertension in high fructose diet fed rats (see previous chapter). In the present study we aimed to understand the importance of ERK, SRF and MLC II in the development of hypertension and to assess the consequences of EGFR inhibition on these markers in vivo. Feeding fructose to rats induced insulin resistance (see results from previous chapter) and made them hypertensive. Treatment of fructose-fed rats with AG1478 significantly prevented the development of hypertension (Fig.6.10A) without any improvement in insulin resistance (chapter-5). However, insulin resistance in these rats was associated with increased EGFR, ERK, SRF and MLC II activity as measured by increased levels of phospho-EGFR, ! "#$! phospho-ERK, phospho-SRF and phospho-MLC II (Fig.6.10B).  Treatment of fructose rats with AG1478 normalized the activities of ERK1/2 (Fig.6.13C), SRF (Fig.6.10D) and MLC II (Fig.6.10E) in fructose-fed rats without any major effects in rats fed with normal chow. 6.4. Discussion We previously have reported elevated MMP and EGFR activities in the arteries of insulin resistant hypertensive rats (see chapter 5). Further, inhibition of either MMPs or the EGFR prevented development of hypertension in high fructose diet-fed rats without any change in insulin resistance. In the present study we examined the role of MMP-EGFR pathway in mediating HG and PE-induced expression of contractile proteins in insulin resistant VSM cells. Because in insulin resistance, VSM cells retain the normal MAP-kinase response to insulin despite defects in PI3K signaling, we reasoned that in such conditions, elevated levels of various vasoactive molecules including insulin could promote transactivation of the EGFR via MMPs and contribute to increased expression of contractile proteins in VSM cells. In support of this idea, in the present study we found that HG and PE stimulation of !1-AR increased MMP- dependent transactivation of the EGFR in VSM cells. Further, we found increased expression of various contractile proteins and their transcriptional activators in insulin resistant VSM cells that was normalized by blockade of the MMP-EGFR pathway. Moreover, inhibiting the EGFR activity prevented the development of hypertension along with normalization of ERK1/2, SRF and MLC II levels in SMA in insulin resistant rats. Hypertension is pathological condition characterized by increased peripheral resistance due to abnormalities in vascular function and underlying structural changes. Functional abnormalities include increased vasoconstriction and/or decreased vasodilatation while the structural changes include reduced lumen diameter and arterial wall thickening as a result of hypertrophic growth and remodeling 3 . Although various mechanisms have been proposed to explain the etiology of ! "#$! hypertension it still remains unclear what causes this condition. Increasing evidence suggest that elevated levels of various GPCR agonists may initiate this condition, wherein increased levels of oxidative stress, enhanced vascular tone and hypertrophic growth of cardiovascular tissues as a result of excessive GPCR stimulation, culminate in the development of hypertension 2 . The paradigm emerging from several recent studies implicates transactivation of the EGFR by MMPs as a converging point in mediating all the above effects of GPCRs agonists in hypertension 2 . The present study explored the mechanisms by which MMP-dependent EGFR transactivation promote increased synthesis of contractile proteins in response to insulin resistance and agonist stimulation in VSM cells. In the present study, we found increased activation of MMP-2 and protein levels of all major MMPs (MMP-2, MMP-9 and MMP-7) in VSM cells exposed to high glucose in vitro. In the vascular tissues, MMP-2 and MMP-9 are produced predominantly by smooth muscle and endothelial cells, whereas MMP-7 is mainly secreted from macrophages and VSM cells 2, 11, 31 . Previous studies have reported increased expression and activity of MMPs in a number of pathophysiological conditions, including insulin resistance 12 , diabetes 32 , hypertension 6, 12, 33  and other cardiac and vascular diseases 2, 31 . The exact nature of signals that contribute to altered activity and expression of MMPs in the present study is unclear. Many agents including growth factors, cytokines and neurohormones have been shown to regulate MMP expression in different cell types 34 . Further, MMP activation is regulated at both transcriptional and post-transcriptional levels including pro-enzyme activation and regulation by TIMPs. In our culture system, we exposed VSM cells to high glucose to induce insulin resistance and therefore hyperglycemia per se might have contributed to increased expression and activity of MMPs 35-37 . Nevertheless, HG exposed VSM cells were also insulin resistant as determined by increased and decreased phosphorylation of ser307 and tyr989 respectively in response to insulin 27 . Further, it has been ! "##! well established both in humans and animal models of diabetes that hyperglycemia is closely associated with insulin resistance 38, 39 . Our cell culture system therefore mimics the conditions of type 2 diabetes wherein insulin resistance and hyperglycemia co-exist and provides a model to examine the role of MMP-EGFR pathway in the synthesis of contractile proteins in response to GPCR agonists. Stimulation of !1-AR with PE increased MMP activity and also promoted EGFR transactivation to greater levels in HG exposed VSM cells. Various mechanisms have been proposed to explain the activation of MMPs in response to agonist stimulation of GPCRs including activation of PKC, Src, release of Ca 2+  and generation of ROS 10 . Once activated, MMPs cleave pro-heparin binding EGF (pro HB-EGF) to HB-EGF. The latter binds to and activates the EGFR leading to the phosphorylation of phosphoinositide-3 kinase (PI3K), Akt and MAP kinases including ERK1/2 and promotes cell growth, proliferation and vasoconstriction 10 . ERK1/2 and Akt are required for the development of hypertrophy, whereas p38 MAPK and Akt (chapter-4) contribute to enhanced vascular tone and hypertension 6 . Although, it is unclear if increased MMP-dependent EGFR transactivation in insulin resistance is due to hyperglycemia or defective PI3K signaling, the fact that EGFR activity is increased underscore the importance of its role in the development of hypertension in insulin resistance. Many animal models of hypertension including the SHR and FHR despite having normal glucose levels also exhibit insulin resistance and therefore increased MMP and EGFR activities could be a common underlying phenomenon in both conditions. Interestingly, MMPs promote insulin resistance by degrading insulin receptors 12  and processing mature TNF-! 14 , an agent that is know to cause insulin resistance, further adding to the complexity of insulin resistance-associated hypertension. VSM cells cultured in HG but not high mannitol (data not shown) showed increased expression of various contractile proteins including the smooth muscle specific MLCK and MLC II. PE- ! "##! stimulation significantly increased the activity and expression of MLC II and MLCK in HG conditions. Our data is consistent with previous studies that have reported increased expression of MLC II and MLCK in VSM cells isolated from the arteries of SHRs 20  and Wistar-Kyoto rats stimulated with Ang II 21 . In the later study MLCK protein expression increased rapidly in concert with increased phosphorylation of MLC II in response to Ang II stimulation. Further, strategies aimed at blocking the AT1 receptor or ERK activation blocked the Ang II–induced increase in MLCK expression, underscoring the importance of ERK pathway in mediating the increase in MLCK expression. We therefore examined the potential role of ERK pathway in mediating increased expression of contractile proteins in insulin-resistant VSM cells. Exposure to HG alone increased ERK activity and agonist stimulation increased the activity to a greater level in VSM cells. A number of studies have previously reported increased ERK activity in response to hyperglycemia and agonist stimulation of ras 16, 21  and receptor tyrosine kinases 10, 40 . Because ERK is a redox sensitive kinase, it is likely activated in response to increased formation of ROS and/ or PKC activation both of which are increased in hyperglycemic conditions (chapter-2) and in response to the GPCR stimulation. Using this model system previous studies have reported increased levels of mitochondrial ROS in response to agonist stimulation of the MMP-EGFR pathway 13 . Nevertheless, pharmacological inhibition of MMPs or the EGFR activity significantly reduced ERK activity in VSM cells exposed to HG. These data suggest that MMP-EGFR pathway modulate ERK activity, possibly to control VSM growth and contraction. ERK is not only critical in the mitogenic response to MAPK activation but also involved in vascular contraction in response to agonist stimulation 40 . Inhibition of MEK, the upstream activator of ERK1/2 by U0126 reduced ERK1/2, MLC II and SRF activation and normalized elevated blood pressure in SHRs 20, 21 . ! "#$! Agonist stimulation of ERK activates the expression of immediate early genes that are linked to cellular growth and muscle differentiation 41 . Such genes are characterized by the presence of serum response elements (SREs) in their promoters, which bind to SRF and recruit ternary complex factors (TCF) such as Elk-1 42 . Elk-1 is phosphorylated by ERK and recruited to the c- fos SRE 43 . However, before recruitment these accessory factors require prior assembly with SRF to form binary complex. SRF is a 508-amino-acid-long protein, consisting of a central core that contains the DNA binding domain, a C-terminal transcriptional activation domain and an N- terminal domain that could be phosphorylated by ribosomal S6 kinase (RSK) 19 . The P90RSK, a growth factor-inducible kinase, phosphorylates SRF at serine 103 and significantly enhances the affinity and rate with which SRF associates with its binding site, the SRE, within the c-fos promoter 30 . Various intracellular signaling pathways, including MAP-kinase cascades, Rho- dependent signaling, and Ca 2+  stimulation regulate SRF complexes. In the present study we found that exposure of VSM cells to HG or agonist stimulation significantly increased the activity and expression of SRF and P90RSK. Further, inhibition of MMP-EGFR activity decreased the phosphorylation of SRF and MLC II. In addition, genetic ablation of MMP and EGFR expression decreased not only MLC II and SRF activity but also their expression in HG treated VSM cells. Taken together, all these data suggest that MMP-EGFR pathway induces the expression of contractile proteins in response to HG and agonist stimulation by regulating ERK and the downstream transcriptional regulators such as SRF and P90RSK. To examine the effect on insulin resistance on the expression of contractile proteins in vivo, we studied FHRs, a model of diet induced systolic hypertension. Previous studies in FHR rats from our lab have reported elevated levels of various GPCR agonists including Ang II 44 , ET-1 45  and TXA2 46  all of which are known to transactivate the EGFR 2, 10 . Further, we also have found increased MMP and EGFR activities in the mesenteric arteries of these rats and that inhibition ! "#"! of MMP by doxycycline improves blood pressure and endothelial function (previous chapter). In the present study we examined the expression of contractile proteins and transcriptional regulators in SMA from FHR. Similar to SHRs, we found increased activities of SRF, P90RSK and MLC II in addition to increased EGFR and ERK activities in FHRs. Treatment of FHR with an EGFR inhibitor prevented the development of hypertension and this was associated with decreased activities of the EGFR, ERK, SRF, P90RSK and MLC-II. Since AG1478 did not improve insulin sensitivity in our model, it is likely that EGFR signaling does not cause insulin resistance in these rats. In summary, our data suggest that vasoactive GPCR stimulation in the presence of insulin resistance or hyperglycemia increases the expression of contractile proteins by mechanisms involving MMP-dependent transactivation of the EGFR. Our work also revealed that activation of ERK and transcriptional activators such as SRF and P90RSK might be necessary to induce the expression of contractile proteins in response to EGFR transactivation. Further, in an insulin resistant animal model, we demonstrated increased expression of ERK, MLC-II and SRF and that blocking EGFR signaling decreased blood pressure by normalizing the expression of above proteins. These results provide new insights into the mechanisms by which GPCR agonists regulate the expression of contractile proteins by involving MMP-EGFR pathways, and thereby influence the hemodynamic outcomes in insulin resistance and possibly in type 2 diabetes. Although, it is unclear if hypertension is the cause or consequence of increased MMP and EGFR activities, the presence of elevated levels of GPCR agonists in both clinical and animal models of hypertension activates this pathway to form a vicious cycle involving enhanced vascular tone, hypertrophic growth and oxidative stress. Because it is virtually impossible to reduce the levels of GPCR agonists an alternative way to break this vicious cycle is to inhibit MMP-EGFR pathway, a potential target for the treatment of hypertension. ! "#$! Limitations The association between increased expression of contractile proteins, hypertrophy and vascular remodeling and its contribution to the development of hypertension is unclear. Although it is generally accepted that increased expression of contractile proteins may indicate hypertrophy in hypertension 20, 47 , it is not clear if that leads to abnormal vascular remodeling 21 . This is because, unlike SHR arteries FHR arteries do not undergo vascular remodeling at the same time they exhibit increased blood pressure 45  despite increases in the expression of contractile proteins. It is likely that increased expression of contractile proteins precedes the development of hypertension and the presence of chronically elevated blood pressure may predispose arteries to abnormal vascular remodeling. Evidence for this idea comes from the discrepancy between SHR and FHR with respect to vascular remodeling wherein both models exhibit increased synthesis of contractile proteins 20, 21  but only in SHR, this leads to abnormal vascular remodeling 21 . Although both models show characteristic symptoms of insulin resistance and hypertension, it is the severity of hypertension that differs between these models wherein FHR are mildly hypertensive compared to SHR. Since the stretch of the vascular wall by intraluminal blood pressure influences vascular remodeling 48  as well as MMP-dependent transactivation of the EGFR 49 , the differences in the magnitude of blood pressure between these two models likely contributes to vascular hypertrophy and remodeling in SHR.  ! "#$! 6.5 Tables and Figures  Figure 6.1 Insulin resistance induced by high glucose in rat aortic VSM cells. Western blot analysis of phospho-IRS-1 and phospho-Akt in response to insulin stimulation (100 nM) in VSM cells cultured in normal glucose (NG, 5.5 mM) or high glucose (HG, 25 mM) conditions. A) Representative Western blot and quantitative analysis of phospho-IRS-1 (Ser 307) showing increased levels in response to insulin (at 1 min) in VSM cells cultured in HG conditions. B) Representative Western and quantitative analysis showing decreased and delayed Akt (Ser 473) phosphorylation in response to insulin (at 1 min) in VSM cells cultured in HG conditions. All values are expressed as mean ± SEM. * different from their NG groups (P<0.05) ! "#$!     Figure 6.2 Effect of GM6001 on phenylephrine-induced activation of MMP-2 and the EGFR in insulin resistant VSM cells. A) Representative zymogram and quantitative analysis of MMP-2 activity as measured by the ratio of active MMP-2 to pro-MMP-2. VSM cells cultured in NG (5.5 mM open bars) or HG (25 mM, solid bars) were treated with GM6001 (25 µM) 1 hour before stimulation with PE (10 µM, 1 hour). All values are expressed as mean ± SEM. * different from their respective control (NG) groups (P<0.05), # different from HG+PE group (P<0.05) B) Representative Western blot and quantitative analysis of EGFR activity as determined by the ratio of phospho-EGFR to total EGFR in VSM cells cultured in NG (5.5 mM, open bars) or HG (25 mM, solid bars) and stimulated with PE (10 µM) in the presence of GM6001 (25 µM). All values are expressed as mean ± SEM. * different from their respective control (NG) groups (P<0.05), # different from HG+PE group (P<0.05). ! "#$!   Figure 6.3 Effect of MMP suppression on phenylephrine-induced EGFR transactivation in insulin resistant VSM cells. Using siRNA techniques, MMP-2 and MMP-7 expressions were suppressed in VSM cells and cultured in NG (5.5 mM) or HG (25 mM). On the day of experiment, cells transfected with either scrambled siRNA (MOCK) or the MMP specific siRNA were stimulated with PE (10 µM) for 1 hour and phosphorylation of the EGFR was determined by Western blotting. A) Representative Western blot and quantitative analysis of phospho-EGFR in VSM cells transfected with MOCK or MMP-2 siRNA and cultured in NG (open bars) or HG (solid bars). All values are expressed as mean ± SEM. * different from their respective control (NG) groups (P<0.05), # different from MOCK siRNA treated groups (P<0.05). B) Representative Western blot and quantitative analysis of phospho-EGFR in VSM cells transfected with MOCK or MMP-7 siRNA and cultured in NG (open bars) or HG (solid bars). All values are expressed as mean ± SEM. * different from their respective control (NG) groups (P<0.05), # different from MOCK siRNA treated groups (P<0.05). ! "#$!           Figure 6.4 Effect of EGFR suppression on phenylephrine-induced EGFR transactivation in insulin resistant VSM cells. Using rat EGFR specific siRNA, the EGFR expression was suppressed in VSM cells and cultured in NG (5.5 mM) or HG (25 mM). On the day of experiment, cells transfected with either scrambled siRNA (MOCK) or the EGFR specific siRNA were stimulated with PE (10 µM) for 1 hour and phosphorylation of the EGFR was determined by Western blotting. A) Representative Western blot and quantitative analysis of phospho-EGFR in VSM cells transfected with MOCK or EGFR siRNA and cultured in NG (open bars) or HG (solid bars). All values are expressed as mean ± SEM. * different from their respective control (NG) groups (P<0.05), # different from MOCK siRNA treated groups (P<0.05). ! "#$!   Figure 6.5 Effect of MMP and EGFR inhibition on phenylephrine-induced ERK activation in insulin resistant VSM cells. A) Representative Western blots and quantitative analysis of ERK activity as determined by the ratio of phospho-ERK1 to total ERK1 in VSM cells cultured in NG (open bars) or HG (solid bars) in the presence of MMP (GM6001) or EGFR (AG1478) inhibitors. All values are expressed as mean ± SEM. * different from their respective control (NG) groups (P<0.05), # different from HG+PE group (P<0.05). B) Representative Western blots and quantitative analysis of ERK activity as determined by the ratio of phospho-ERK1 to total ERK1. VSM cells transfected with either scrambled (MOCK) or EGFR siRNA were cultured in NG (open bars) or HG (solid bars) and stimulated with PE for 1 hour. All values are expressed as mean ± SEM. * different from their respective control (NG) groups (P<0.05), # different from all MOCK siRNA treated groups (P<0.05).  ! "#$!            Figure 6.6 Effect of PE-stimulation on p38MAPK and JNK1 in insulin resistant VSM cells. VSM cells cultured in NG (open bars) or HG (solid bars) were stimulated with phenylephrine (PE, 10 µM) for 1 hour. A) Representative western blot (top panel) and quantitative analysis (bottom panel) showing the expression of phospho and total p38MAPK. B) Representative western blot (top panel) and quantitative analysis (bottom panel) showing the expression of phospho and total JNK1. All values are expressed as mean ± SEM. * different from all other groups (P<0.05).   ! "#$!  Figure 6.7 Effect of MMP suppression on phenylephrine-induced activation of ERK in insulin resistant VSM cells. Using siRNA techniques, MMP-7 and MMP-2 expressions were suppressed in VSM cells and cultured in NG (5.5 mM) or HG (25 mM). On the day of experiment, cells transfected with either scrambled siRNA (MOCK) or the MMP specific siRNAs were stimulated with PE (10 µM) for 1 hour and phosphorylation of the ERK1 was determined by Western blotting. A) Representative Western blot and quantitative analysis of phospho-ERK1 in VSM cells transfected with MOCK or MMP-7 siRNA and cultured in NG (open bars) or HG (solid bars). All values are expressed as mean ± SEM. * different from their respective control (NG) groups (P<0.05), # different from HG and MOCK siRNA treated groups (P<0.05). B) Representative Western blots and quantitative analysis of phospho-ERK1 in VSM cells transfected with MOCK or MMP-2 siRNA and cultured in NG (open bars) or HG (solid bars). All values are expressed as mean ± SEM. * different from their respective control (NG) groups (P<0.05), # different from all MOCK siRNA treated groups (P<0.05). ! "##! Figure 6.8 Effect of MMP and EGFR inhibition on phenylephrine-induced expression of contractile proteins in insulin resistant VSM cells. A) Representative Western blots showing the expression of MLCK and MLC II (phospho and total) in VSM cells cultured in NG (5.5 mM) or HG (25 mM) in the presence of inhibitors of MMPs (GM6001) or the EGFR (AG1478) and stimulated with PE. B) Representative Western blots and quantitative analysis of phospho-MLC II expression in MMP-2 siRNA transfected cells. VSM cells transfected with either scrambled (MOCK) or MMP-2 siRNA were cultured in NG (open bars) or HG (solid bars) and stimulated with PE for 1 hour. Because total MLC II expression was also decreased in MMP-2 siRNA transfected cells, the densitometric values of phospho MLC II were normalized to the densitometric values of GAPDH and expressed as relative band intensity. All values are expressed as mean ± SEM. * different from their respective control (NG) groups (P<0.05), # different from all MOCK siRNA treated groups (P<0.05). C) Representative Western blots and quantitative analysis of phospho-MLC II expression in EGFR siRNA transfected cells. VSM cells transfected with either scrambled (MOCK) or EGFR siRNA were cultured in NG (open bars) or HG (solid bars) and stimulated with PE for 1 hour. Because total MLC II expression was also decreased in EGFR siRNA transfected cells, the densitometric values of phospho MLC II were normalized to the densitometric values of GAPDH and expressed as relative band intensity. All values are expressed as mean ± SEM. * different from their respective control (NG) groups (P<0.05), # different from all HG and MOCK siRNA treated groups (P<0.05).  ! "#"!          ! "#$! Figure 6.9 Effect of MMP and EGFR inhibition on phenylephrine-induced activation of transcriptional activators in insulin resistant VSM cells. A) Representative Western blots showing the expression of phospho-P90RSK and phospho-SRF in VSM cells cultured in NG (5.5 mM) or HG (25 mM) in the presence of inhibitors of MMPs (GM6001) or the EGFR (AG1478) and stimulated with PE. B) Representative Western blots and quantitative analysis of phospho-SRF expression in MMP-2 siRNA transfected cells. VSM cells transfected with either scrambled (MOCK) or MMP-2 siRNA were cultured in NG (open bars) or HG (solid bars) and stimulated with PE for 1 hour. The densitometric values of phospho SRF were normalized to the densitometric values of GAPDH and expressed as relative band intensity. All values are expressed as mean ± SEM. * different from their respective control (NG) groups (P<0.05), # different from HG and MOCK siRNA treated groups (P<0.05). C) Representative Western blots and quantitative analysis of phospho-SRF expression in EGFR siRNA transfected cells. VSM cells transfected with either scrambled (MOCK) or EGFR siRNA were cultured in NG (open bars) or HG (solid bars) and stimulated with PE for 1 hour. The densitometric values of phospho-SRF were normalized to the densitometric values of GAPDH and expressed as relative band intensity. All values are expressed as mean ± SEM. * different from their respective control (NG) groups (P<0.05), # different from all MOCK siRNA treated groups (P<0.05).    ! "#$!            ! "#$! Figure 6.10 In vivo effects of AG1478 on blood pressure and the expression of contractile proteins in the superior mesenteric arteries (SMA) of fructose-hypertensive rats (FHR). A) In vivo effects of inhibiting EGFR activity on systolic blood pressure in FHR. Rats fed on normal chow or high fructose diets were treated orally with vehicle or AG1478 (5mg/kg/day, 4 weeks) from week 6 through week 10. Blood pressure (BP) at week 6 (basal) and after the treatment (final) was measured in all groups by tail cuff method. After treatment, rats were killed and their SMA’s isolated for Western blot analysis of various proteins. Open bars represent basal BP and solid bars represent final BP. All data are expressed as mean ± SEM. * different from all other groups (P<0.05). B) Representative Western blots showing the expression of phospo-EGFR, phospho-ERK, phospho-P90RSK, phospho-SRF and phospho MLC II with GAPDH as loading control in SMAs from control (C), fructose (F), control treated (CA) and fructose treated (FA) with AG1478. C) Quantitative analysis of ERK activity as determined by the ratio of phospho-ERK1 to total ERK1 in SMAs from control (open bars) and fructose (solid bars) rats. All data are expressed as mean ± SEM. * different from all groups (P<0.05), # different from F group (P<0.05). D) Quantitative analysis of phospho-SRF expression. The densitometric values of phospho-SRF were normalized to their corresponding GAPDH densitometric values and expressed as mean ± SEM. * different from all groups (P<0.05), # different from F group (P<0.05). E) Quantitative analysis of phospho-MLC II expression. The densitometric values of phospho-MLC II were normalized to their corresponding GAPDH densitometric values and expressed as mean ± SEM. * different from all groups (P<0.05), # different from F group (P<0.05). ! "#$!            ! "#$! 6.6 Bibliography 1. Kim JA, Montagnani M, Koh KK, Quon MJ. Reciprocal relationships between insulin resistance and endothelial dysfunction: molecular and pathophysiological mechanisms. Circulation. 2006;113(15):1888-1904. 2. Fernandez-Patron C. Therapeutic potential of the epidermal growth factor receptor transactivation in hypertension: a convergent signaling pathway of vascular tone, oxidative stress, and hypertrophic growth downstream of vasoactive G-protein-coupled receptors? 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Ann N Y Acad Sci. 2001;947:42-54; discussion 54-45. 8. Prenzel N, Zwick E, Daub H, Leserer M, Abraham R, Wallasch C, Ullrich A. EGF receptor transactivation by G-protein-coupled receptors requires metalloproteinase cleavage of proHB-EGF. Nature. 1999;402(6764):884-888. 9. Shah BH, Catt KJ. A central role of EGF receptor transactivation in angiotensin II - induced cardiac hypertrophy. Trends Pharmacol Sci. 2003;24(5):239-244. 10. Shah BH, Catt KJ. Matrix metalloproteinase-dependent EGF receptor activation in hypertension and left ventricular hypertrophy. Trends Endocrinol Metab. 2004;15(6):241- 243. 11. Fernandez-Patron C, Radomski MW, Davidge ST. Vascular matrix metalloproteinase-2 cleaves big endothelin-1 yielding a novel vasoconstrictor. Circ Res. 1999;85(10):906-911. ! "#$! 12. DeLano FA, Schmid-Schonbein GW. Proteinase activity and receptor cleavage: mechanism for insulin resistance in the spontaneously hypertensive rat. Hypertension. 2008;52(2):415-423. 13. Hao L, Nishimura T, Wo H, Fernandez-Patron C. Vascular responses to alpha1-adrenergic receptors in small rat mesenteric arteries depend on mitochondrial reactive oxygen species. Arterioscler Thromb Vasc Biol. 2006;26(4):819-825. 14. Morimoto Y, Nishikawa K, Ohashi M. KB-R7785, a novel matrix metalloproteinase inhibitor, exerts its antidiabetic effect by inhibiting tumor necrosis factor-alpha production. Life Sci. 1997;61(8):795-803. 15. Kagiyama S, Qian K, Kagiyama T, Phillips MI. Antisense to epidermal growth factor receptor prevents the development of left ventricular hypertrophy. Hypertension. 2003;41(3 Pt 2):824-829. 16. de Vries-Smits AM, Burgering BM, Leevers SJ, Marshall CJ, Bos JL. Involvement of p21ras in activation of extracellular signal-regulated kinase 2. Nature. 1992;357(6379):602-604. 17. Schauwienold D, Plum C, Helbing T, Voigt P, Bobbert T, Hoffmann D, Paul M, Reusch HP. ERK1/2-dependent contractile protein expression in vascular smooth muscle cells. Hypertension. 2003;41(3):546-552. 18. Treisman R. The serum response element. Trends Biochem Sci. 1992;17(10):423-426. 19. Miano JM. Serum response factor: toggling between disparate programs of gene expression. J Mol Cell Cardiol. 2003;35(6):577-593. 20. Han YJ, Hu WY, Chernaya O, Antic N, Gu L, Gupta M, Piano M, de Lanerolle P. Increased myosin light chain kinase expression in hypertension: Regulation by serum response factor via an insertion mutation in the promoter. Mol Biol Cell. 2006;17(9):4039- 4050. 21. Han YJ, Hu WY, Piano M, de Lanerolle P. Regulation of myosin light chain kinase expression by angiotensin II in hypertension. Am J Hypertens. 2008;21(8):860-865. 22. Seasholtz TM, Zhang T, Morissette MR, Howes AL, Yang AH, Brown JH. Increased expression and activity of RhoA are associated with increased DNA synthesis and reduced p27(Kip1) expression in the vasculature of hypertensive rats. Circ Res. 2001;89(6):488- 495. ! "#$! 23. Cusi K, Maezono K, Osman A, Pendergrass M, Patti ME, Pratipanawatr T, DeFronzo RA, Kahn CR, Mandarino LJ. Insulin resistance differentially affects the PI 3-kinase- and MAP kinase-mediated signaling in human muscle. J Clin Invest. 2000;105(3):311-320. 24. Jiang ZY, Lin YW, Clemont A, Feener EP, Hein KD, Igarashi M, Yamauchi T, White MF, King GL. Characterization of selective resistance to insulin signaling in the vasculature of obese Zucker (fa/fa) rats. J Clin Invest. 1999;104(4):447-457. 25. Ellis AG, Doherty MM, Walker F, Weinstock J, Nerrie M, Vitali A, Murphy R, Johns TG, Scott AM, Levitzki A, McLachlan G, Webster LK, Burgess AW, Nice EC. Preclinical analysis of the analinoquinazoline AG1478, a specific small molecule inhibitor of EGF receptor tyrosine kinase. Biochem Pharmacol. 2006;71(10):1422-1434. 26. Galipeau D, Verma S, McNeill JH. Female rats are protected against fructose-induced changes in metabolism and blood pressure. Am J Physiol Heart Circ Physiol. 2002;283(6):H2478-2484. 27. Patterson C, Mapera S, Li HH, Madamanchi N, Hilliard E, Lineberger R, Herrmann R, Charles P. Comparative effects of paclitaxel and rapamycin on smooth muscle migration and survival: role of AKT-dependent signaling. Arterioscler Thromb Vasc Biol. 2006;26(7):1473-1480. 28. Aguirre V, Werner ED, Giraud J, Lee YH, Shoelson SE, White MF. Phosphorylation of Ser307 in insulin receptor substrate-1 blocks interactions with the insulin receptor and inhibits insulin action. J Biol Chem. 2002;277(2):1531-1537. 29. Fernandez-Patron C, Stewart KG, Zhang Y, Koivunen E, Radomski MW, Davidge ST. Vascular matrix metalloproteinase-2-dependent cleavage of calcitonin gene-related peptide promotes vasoconstriction. Circ Res. 2000;87(8):670-676. 30. Rivera VM, Miranti CK, Misra RP, Ginty DD, Chen RH, Blenis J, Greenberg ME. A growth factor-induced kinase phosphorylates the serum response factor at a site that regulates its DNA-binding activity. 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High glucose enhance expression of matrix metalloproteinase-2 in smooth muscle cells. Acta Pharmacol Sin. 2003;24(6):534-538. 36. Sachidanandam K, Portik-Dobos V, Harris AK, Hutchinson JR, Muller E, Johnson MH, Ergul A. Evidence for vasculoprotective effects of ETB receptors in resistance artery remodeling in diabetes. Diabetes. 2007;56(11):2753-2758. 37. Uemura S, Matsushita H, Li W, Glassford AJ, Asagami T, Lee KH, Harrison DG, Tsao PS. Diabetes mellitus enhances vascular matrix metalloproteinase activity: role of oxidative stress. Circ Res. 2001;88(12):1291-1298. 38. DeFronzo RA. Pathogenesis of type 2 diabetes mellitus. Med Clin North Am. 2004;88(4):787-835, ix. 39. Takada J, Machado MA, Peres SB, Brito LC, Borges-Silva CN, Costa CE, Fonseca-Alaniz MH, Andreotti S, Lima FB. Neonatal streptozotocin-induced diabetes mellitus: a model of insulin resistance associated with loss of adipose mass. Metabolism. 2007;56(7):977-984. 40. Touyz RM, El Mabrouk M, He G, Wu XH, Schiffrin EL. Mitogen-activated protein/extracellular signal-regulated kinase inhibition attenuates angiotensin II-mediated signaling and contraction in spontaneously hypertensive rat vascular smooth muscle cells. Circ Res. 1999;84(5):505-515. 41. Turjanski AG, Vaque JP, Gutkind JS. MAP kinases and the control of nuclear events. Oncogene. 2007;26(22):3240-3253. 42. Shaw PE, Saxton J. Ternary complex factors: prime nuclear targets for mitogen-activated protein kinases. Int J Biochem Cell Biol. 2003;35(8):1210-1226. 43. Zhang HM, Li L, Papadopoulou N, Hodgson G, Evans E, Galbraith M, Dear M, Vougier S, Saxton J, Shaw PE. Mitogen-induced recruitment of ERK and MSK to SRE promoter complexes by ternary complex factor Elk-1. Nucleic Acids Res. 2008;36(8):2594-2607. ! ""#! 44. Tran LT, Macleod KM, McNeill JH. Endothelin-1 modulates angiotensin II in the development of hypertension in fructose-fed rats. Mol Cell Biochem. 2009. 45. Verma S, Skarsgard P, Bhanot S, Yao L, Laher I, McNeill JH. Reactivity of mesenteric arteries from fructose hypertensive rats to endothelin-1. Am J Hypertens. 1997;10(9 Pt 1):1010-1019. 46. Jiang J, Tran L, Vasudevan H, Xia Z, Yuen VG, McNeill JH. Endothelin-1 blockade prevents COX2 induction and TXA2 production in the fructose hypertensive rat. Can J Physiol Pharmacol. 2007;85(3-4):422-429. 47. Owens GK, Schwartz SM. Alterations in vascular smooth muscle mass in the spontaneously hypertensive rat. Role of cellular hypertrophy, hyperploidy, and hyperplasia. Circ Res. 1982;51(3):280-289. 48. Albinsson S, Nordstrom I, Hellstrand P. Stretch of the vascular wall induces smooth muscle differentiation by promoting actin polymerization. J Biol Chem. 2004;279(33):34849-34855. 49. Lucchesi PA, Sabri A, Belmadani S, Matrougui K. Involvement of metalloproteinases 2/9 in epidermal growth factor receptor transactivation in pressure-induced myogenic tone in mouse mesenteric resistance arteries. Circulation. 2004;110(23):3587-3593.    ! """! 7. Summary and conclusions                        ! ""#! 7.1 Summary and conclusions Metabolic disorders such as insulin resistance 1  and diabetes mellitus 2, 3  contribute significantly to morbidity and mortality associated with cardiovascular diseases, although the underlying reasons are not completely understood. However, the hemodynamic changes that occur due to the underlying structural and functional abnormalities in vasculature could contribute significantly to the development of cardiovascular diseases in these patients. The present thesis focused on investigating the mechanisms of vascular dysfunction and its contribution to abnormal hemodynamics at two different stages of diabetes, the pre-diabetic stage of insulin resistance and the late stage of type 1 diabetes. Type 1 diabetes is characterized by an absolute deficiency of insulin attributable to pancreatic insuffiency. Vascular dysfunction in type 1 diabetes is probably the consequence of metabolic abnormalities, in particular hyperglycemia. With age, a number of cardiovascular complications arises in type 1 diabetic patients including retinopathy, nephropathy, peripheral vascular disease and the diabetic foot. However, in animal models of type 1 diabetes such as the streptozotocin (STZ) rat model, probably because of uncontrolled hyperglycemia, in addition to the above complications, various other cardiovascular abnormalities are frequently observed.  Specifically, we reported depressed cardiac function 4 , lower mean arterial blood pressure (MABP) and heart rate (HR) and attenuated pressor responses to vasoactive agents 5, 6 . In our previous studies, we found that hyperglycemia was associated with an increased expression of inducible nitric oxide synthase (iNOS) 7  and nitrotyrosine, and decreased expression of endothelial nitric oxide synthase (eNOS) in the heart and arteries of STZ diabetic rats 8 . In the present thesis, we investigated the mechanisms involved in the cause and consequences of increased expression of iNOS and its role in the development of cardiovascular abnormalities in STZ-diabetic rats. ! ""#! As a first step, towards understanding the molecular mechanisms involved in the cause of iNOS induction in cardiovascular tissues, we examined whether the hyperglycemia-induced increase in the expression of iNOS is protein kinase C !2 (PKC!2)-dependent. The reason that we focused our investigations on PKC!2 was because previous studies from our lab 9, 10  and elsewhere 11-13  have found preferential expression and activation of this kinase in cardiac and vascular tissues of diabetic animals. Further, increased PKC! activity has also been implicated in many of the diabetic secondary complications 14 . To determine whether PKC!2 could increase iNOS expression in cardiovascular tissues we exposed cardiomyocytes and aortic vascular smooth muscle cells to high glucose conditions in the presence of a selective PKC! inhibitor, LY333531. High glucose activated PKC!2 and increased iNOS expression and this was prevented by the PKC! inhibitor. Similarly, treatment of smooth muscle cells with the PKC! inhibitor prevented high glucose-induced activation of nuclear factor kappa B (NF-"B), extracellular signal-regulated kinase (ERK1/2) and iNOS overexpression. Suppression of PKC!2 expression with siRNA also decreased high-glucose–induced NF-"B and ERK1/2 activation and iNOS expression in vascular smooth muscle cells. Taken together, these data suggest that activation of PKC!2 as a result of hyperglycemia induces iNOS in cardiac and vascular tissues by recruiting ERK and the NF-"B pathway 15-17 . As a second step, we examined the consequences of increased expression of iNOS and determined whether selective inhibition of PKC!2 or its downstream target iNOS could improve cardiovascular abnormalities in STZ diabetic rats. We found that increased expression of iNOS in STZ-diabetic rat hearts and arteries was associated with decreased eNOS expression and increased peroxynitrite formation. Further, these rats demonstrated the classical symptoms of cardiovascular depression such as depressed MABP, HR and cardiac function, coupled with endothelial dysfunction and attenuated pressor responses to vasoactive agents. We found that ! ""#! treatment of diabetic rats with the PKC! inhibitor or the iNOS inhibitor led to a significant improvement in cardiovascular function in STZ diabetic rats. Further, increased PKC!2 activation and iNOS expression and elevated nitrotyrosine levels in the heart and arteries of diabetic rats were normalized by PKC! inhibition. From these studies, we conclude that PKC!2 is involved in the increased expression of iNOS in diabetes and that one of the mechanisms by which PKC!2 inhibition improves cardiovascular function is by preventing the upregulation of iNOS and its ability to produce nitrosative stress. Although there is strong evidence that mitochondrial production of superoxide anions is the unifying mechanism and pathological basis of all secondary complications of diabetes 18 , intervention studies with classic antioxidants, such as vitamin E, have failed to demonstrate any beneficial effect in diabetes in humans. The reasons for the apparent non-beneficial effects of antioxidant therapy in clinical settings are not very clear but it has been argued that antioxidants simply work by scavenging already formed reactive oxygen species but do not prevent the damage to DNA and other biomolecules caused by agents such as peroxynitrite 19 . It should be noted that in diabetes, increased levels of oxidative stress are also associated with increased production of nitric oxide from iNOS favoring the formation of peroxynitrite, a strong oxidant and cytotoxic compound. Moreover, and most important of all, peroxynitrite uncouples eNOS, decreases the bioavailability of functional nitric oxide and causes endothelial dysfunction 20 , a vascular abnormality that is implicated in the pathophysiology of many cardiovascular diseases including coronary artery diseases, atherosclerosis and hypertension in diabetes. These findings may explain why classic antioxidants have failed to show beneficial effects on diabetic complications and may suggest new and attractive "causal" antioxidant therapy with agents such PKC! inhibitors, peroxynitrite decomposition catalysts and small molecule antioxidants that scavenge intracellular superoxide anions and improve mitochondrial function 19 . The findings ! ""#! from our studies in the type 1 diabetic rat model supports the idea of causal therapy with agents such as LY333531 or L-NIL to correct hemodynamic abnormalities in diabetes. Among the hemodynamic abnormalities studied, our major focus was on investigating the mechanisms involved in depressed blood pressure and endothelial dysfunction. It has long been agued that diabetes in humans is more commonly associated with hypertension rather than hypotension. Although we do not dispute this claim, it is probably because 90% of diabetic patients have type 2 diabetes and three fourths of these are hypertensive. However, type 1 diabetic patients (10%) are normotensive (many with symptoms of orthostatic hypotension) and not hypertensive unless they suffer from a major renal disease 21 . Although hypotension per se is not a characteristic feature in type 1 diabetic subjects, numerous studies have reported the presence of postural blood pressure changes, particularly orthostatic hypotension in long-term diabetes 22, 23 . Evaluation of diabetic patients participating in the Wisconsin Epidemiologic Study of Diabetic Retinopathy clearly indicated a 16.1% prevalence of orthostatic hypotension (OH), and the risk of OH is higher in diabetic subjects 22 . It is very well established that chronic hyperglycemia causes damage to neural and vascular systems that causes impaired BP responses in people with diabetes 24  similar to the attenuated pressor responses that we demonstrated in STZ diabetic rats. In addition, clinical studies have correlated increased plasma nitrotyrosine /NOx levels with endothelial dysfunction, lower blood pressure and sympathetic nerve dysfunction in type 1 diabetic patients 25, 26 . Although our findings in the STZ diabetic rat model are comparable to many of the cardiovascular anomalies observed in type 1 diabetic patients, the presence of severe uncontrolled hyperglycemia for longer durations represents an exaggerated and extreme condition that is no longer seen in humans. Nevertheless, the mechanisms that were investigated provide vital clues to our understanding of the pathophysiological basis of human diabetic secondary complications. ! ""#! In contrast to type 1 diabetes, insulin resistance, widely believed to be a pre-type 2 diabetic stage, is characterized mainly by impaired sensitivity to the actions of insulin despite high plasma levels of insulin. Over time, because of the progressive dysfunction of the pancreatic !- cells, a loss of both insulin production and function will lead to type 2 diabetes. In contrast, to type 1 diabetes the relationship between vascular dysfunction and cardiovascular abnormalities is much more complex in insulin resistance because of the presence of other factors such as hypertension, dyslipidemia, hyperinsulinemia and glucose intolerance. Although not all people with insulin resistance develop hypertension, the presence of insulin resistance contributes significantly to the impairment of endothelial function and hypertension 27 . Endothelial dysfunction may thus play a primary role in the development of hypertension. Various mechanisms have been proposed to explain the pathophysiology of hypertension both in diabetic and non-diabetic subjects. Among them, the three most commonly accepted mechanisms are enhanced peripheral vascular tone, hypertrophic growth of cardiovascular tissues and endothelial dysfunction due to oxidative stress. Accordingly, various therapeutic agents and regimens are available to treat high blood pressure in humans both in essential and secondary hypertension. However, there is no single and unifying pathway that could be a potential target in the treatment of not only hypertension but also endothelial dysfunction and insulin resistance. Recently, it was proposed that all of the above three mechanisms can be explained by the matrix metalloproteinases (MMP)-dependent transactivation of the epidermal growth factor receptor (EGFR) 28 . We tested the hypothesis that increased MMP activity and EGFR transactivation by vasoconstrictory agonists impair endothelial function and contribute to the development of hypertension in insulin resistance. In collaboration with Dr Fernandez-Patron 28  we investigated the potential role of the MMP-EGFR pathway in the maintenance of enhanced adrenergic vascular tone. Later, we examined the role of MMPs and the EGFR transactivation pathway in ! ""#! the etiology of insulin resistance, endothelial dysfunction and its contribution to the development of hypertension in an animal model of acquired systolic hypertension. Previous studies from Dr Fernandez-Patron’s lab have suggested that agonist-induced stimulation of Gq-protein coupled receptors (such as !1-adrenoceptors and angiotensin receptors) results in the transactivation of growth factor receptors (such as the EGFR) by MMPs 29 . The ensuing signaling events downstream of the EGFR resulted in the activation of PI3K-dependent phosphorylation of protein kinase B (Akt) as well as the generation of reactive oxygen species and the hyperpolarization of mitochondria 30 . These events implied a mechanism of mitochondrial control of agonist-induced vascular tone. Because mitochondrial ATP production is directly coupled to the contractile machinery of the vascular smooth muscle, we examined the hypothesis that agonist-induced MMP transactivation of EGFR maintains vascular tone by modulating the synthesis of ATP. We found that agonist-induced stimulation of GPCRs such as adrenergic receptors causes vasoconstriction possibly as a result of the initial surge in mitochondrial ATP synthesis. GPCR stimulation also resulted in the transactivation of the EGFR via the activation of MMPs. Although we do not have direct evidence, it is likely that activation of the EGFR and PI3-kinase was secondary to initial vascular contraction caused by stimulation of the IP3-Ca 2+ /DAG-PKC  pathway. Once activated, PI3-kinase phosphorylates protein kinase B (Akt) and recruits glucose transporters such as GLUT4, which in turn could provide a continuous supply of substrates for mitochondrial ATP synthesis. Continuous supply of glucose could help sustain the vascular tone initiated by vasoactive agonists for longer periods of time. Alternatively, the ATP synthesized and released into the extracellular milieu in response to vasoactive agents may activate MMPs further to maintain a feed forward cycle of sustained ATP synthesis and hence enhanced vascular tone. These findings suggest a potential role for MMP-EGFR inhibitors in reducing ! ""#! enhanced vascular tone, a characteristic feature in human and other animal models of hypertension. Having determined that MMP-dependent transactivation of the EGFR contributes to enhanced vascular tone in a normal physiological setting, we next investigated the role of this pathway in fructose-fed rats, a model of insulin resistance and acquired systolic hypertension. Unlike STZ- diabetic rats, the FHR animals develop systolic hypertension with abnormalities in vascular and endothelial function. Previous studies in FHRs from our lab have demonstrated various pathological features such as insulin resistance, increased sympathetic nervous system activity and elevated levels of various vasoactive agents including NE, Ang II, ET-1 and TXA2, all of which can cause MMP-transactivation of the EGFR. Our preliminary studies in FHRs found increased activities of all major vascular MMPs, including MMP-2, MMP-9 and MMP-7, in addition to increased EGFR tyrosine kinase activity. Further, inhibition of MMPs with doxycycline improved endothelial function and prevented the development of hypertension. Moreover, in vitro studies in endothelial cells suggested that MMP-2 reduces the ability of eNOS to produce nitric oxide in response to its activation by calcium ionophore. Although we do not have direct evidence, we speculate that MMPs cause endothelial dysfunction by direct degradation of eNOS 31  or its uncoupling 32 . However, further studies are needed to examine this hypothesis. The improvement in blood pressure was not associated with changes in insulin sensitivity in doxycycline treated rats. This was quite surprising because MMPs have been reported to cause insulin resistance either by degradation of insulin receptors 33  or by the processing of mature TNF-! 34 , an agent that is widely believed to cause insulin resistance. Variations in the animal model, subtype of MMP involved and the dose of MMP inhibitor used could explain the observed difference. Similar to doxycycline, inhibition of the EGFR also prevented the development of hypertension however, neither endothelial function nor insulin ! "#$! resistance was improved in these animals. Ex vivo studies in isolated arteries revealed a direct vasorelaxant effect to the EGFR inhibitor, AG1478 possibly by preventing Ca 2+  influx in response to agonist-induced contraction 35 . Having determined the effects of MMP-EGFR inhibitors on vascular function and blood pressure, we next investigated the role of MMPs and the EGFR transactivation pathway in the increased expression and activity of contractile proteins in VSM cells. We found that the presence of insulin resistance and agonist stimulation increases the expression and activity of contractile proteins such as MLCK and MLC II and their transcriptional activators, P90RSK and SRF in VSM cells. Further, we demonstrated that disruption of MMP-EGFR signaling inhibits the increased expression of contractile proteins not only in VSM cells in vitro but also in the arteries of FHRs in vivo. In addition, we found that MMP-EGFR inhibitors prevents the development of hypertension in FHRs. Taken together these data suggest that the MMP-EGFR pathway could be a unifying pathway that connects all the major pathological features of hypertension and hence could be potential strategy in the treatment of hypertension both in insulin resistance and noninsulin resistance conditions (Fig.7.1). The extent to which inhibition of this pathway is clinically applicable to the prevention and treatment of hypertension in human patients remains to be determined. We conclude that mechanisms of vascular dysfunction in type 1 diabetes are inherently different from those in insulin resistance/type 2 diabetes. Chronic hyperglycemia in type 1 diabetes leads to hypotension, whereas in insulin resistance, increased levels of GPCR agonists contribute to the development of hypertension despite impairment of endothelial function in both models.    ! "#$!           Figure 7.1 Schematic summary of the MMP-EGFR signaling in VSM cells investigated in the present thesis. Stimulation of GPCR by agonists such as PE and Ang II leads to the activation of PLC and generation of IP3 and DAG, which in turn causes activation of PKC and release of Ca 2+ from intracellular stores. Intracellular signaling events such as generation of ROS and phosphorylation of Src cause activation of MMPs leading to the enzymatic conversion of pro HB-EGF to HB-EGF. The latter binds and activates the EGFR leading to the phosphorylation of PI3K and ERK1/2 and promoting hypertrophy and vasoconstriction. PI3K- activation of Akt helps to maintain vascular tone by providing continuous supply of substrates via GLUT4 to mitochondrial ATP synthesis. Activation of ERK promotes increased synthesis of contractile proteins and contributes to the development of hypertension. ! "#"! 7.2 Important findings 1. Induction of iNOS in cardiovascular tissues contributes significantly to the depressed cardiac function, mean arterial blood pressure and heart rate, endothelial dysfunction and attenuated pressor responses to vasoactive agents in STZ diabetic rats. 2. Chronic inhibition of iNOS or nitrosative stress prevented the development of the above cardiovascular abnormalities. This was associated with normalization of the expression of vascular eNOS, reduced formation of nitrotyrosine and improved pressor responsiveness to !1-adrenoceptor stimulation. 3. Hyperglycemia-induced activation of PKC"2 is antecedent to increases in superoxide, ERK1/2, NF-#B, and iNOS expression in cardiovascular tissues. Inhibition of this pathway suppresses key signaling events that lead to increased nitrosative stress. Inhibition of PKC"2 may be a useful approach for correcting abnormal hemodynamics in diabetes by preventing iNOS-mediated nitrosative stress. 4. The MMP-EGFR pathway modulates vascular tone, at least in part, via the activation of PI3-kinase and modulation of mitochondrial ATP synthesis. 5. Hypertension in insulin resistance is associated with increased expression and activity of vascular MMPs and the EGFR. Inhibition of MMPs by doxycycline prevents the development of hypertension and impairment of endothelial function without improvement in insulin resistance. Blockade of the EGFR activation by AG1478 prevents the development of hypertension without any change in endothelial function. 6. Sustained agonist stimulation and/or chronic insulin resistance increased the activity and expression of various contractile proteins and their transcriptional regulators in VSM cells that were normalized by blockade of the MMP-EGFR pathway. ! "##! 7. Inhibition of the EGFR activity prevented the development of hypertension along with normalization of ERK1/2, SRF and MLC II activity in the arteries of insulin resistant rats.                      ! "#$! 7.3 Future directions 1. The STZ-diabetic rat is the most widely investigated and closest model that mimics conditions of human type 1 diabetes, including hypoinsulinemia, hyperglycemia and derangements in carbohydrate and lipid metabolism yet it has been criticized with regard to the clinical relevance of the findings in this model. This is because not all characteristics of the cardiovascular changes that occur in STZ-diabetic rats are similar to those in human diabetic patients and moreover blood glucose levels in humans are controlled and less severe. In future, it is important to study the cardiovascular changes under better glycemic control rather than uncontrolled hyperglycemia. 2. We demonstrated quite convincingly that induction of iNOS causes cardiovascular abnormalities in STZ diabetic rats. By conducting studies using iNOS inhibitor, L-NIL, we supported our hypothesis that inhibition of iNOS prevents the development of cardiovascular abnormalities. Conducting studies using iNOS knockout mice and examining the effect of diabetes on cardiovascular function in normal and iNOS knockout mice may strengthen our findings. 3. In the present study, we provided only circumstantial evidence to support our view that changes secondary to increased iNOS activity such as formation of peroxynitrite / nitrotyrosine contributes to cardiovascular abnormalities. Our evidence was based on a positive correlation between increased expression of iNOS and nitrotyrosine levels with depressed blood pressure and pressor responses. Although our data is convincing it still needs to be validated and to demonstrate a “cause and effect” relationship, future studies should focus on using agents such as peroxynitrite decomposition catalysts (e.g FeTMPyP). Further, mechanistic studies should be conducted to elucidate the mechanisms by which peroxynitrite causes cardiovascular abnormalities in diabetes. ! "#$! 4. We demonstrated that PKC!2 is an obligatory mediator of nitrosative stress and that LY333531 significantly improved all but endothelial function in STZ-diabetic rats. Although we could not provide a convincing explanation for the latter observation, it is possible that a compensatory mechanism might have been activated by LY333531, which in turn prevented improvement in endothelial function. We noticed that stimulation of VSM cells with PMA, a non-specific PKC activator produced a 3-fold increase in superoxide anions that was not only not blocked but was potentiated by LY333531 (data not presented in this thesis). These data suggest the possibility that other isoforms of PKC are being unmasked upon PKC stimulation with PMA in the presence of LY333531.  Future studies should focus on investigating if other PKC isoforms are activated when stimulated with PMA in the presence of PKC! inhibitor. 5. We showed that the MMP-EGFR pathway maintains adrenergic vascular tone by PI3- kinase mediated GLUT4 recruitment. We speculated that continuous provision of substrate might be required for constant ATP synthesis in order to maintain enhanced vascular tone. However, we did not study the effect of GLUT4 inhibitors on the maintenance of adrenergic vascular tone, or the role of other transporters in substrate supply. It is therefore important to examine the effect of GLUT4 inhibitors (e.g. indinavir) on vascular responses to adrenergic stimulation and the effect of MMP and EGFR inhibitors on adrenergic vascular tone. Further, studies should be conducted to examine the effects of altered substrate supply and oxygen deprivation on the maintenance of vascular tone and ATP synthesis. 6. Inhibition of MMPs particularly MMP-9 by doxycycline has been shown to improve insulin resistance in the SHR model. However, we did not observe any change in insulin sensitivity. Although, this could be due to differences in the animal model and dosage ! "#$! used, further studies, including a dose-response study should be conducted which may provide explanations for this discrepancy. 7. Doxycycline prevented impairment of endothelial function in FHRs. Although we demonstrated a direct inhibitory effect for MMP-2 on eNOS derived NO production, the mechanisms are still elusive. Using MALDI-TOF analysis we sought to determine if MMPs (MMP-2 and MMP-9) degrade eNOS by acting on vulnerable sites particularly the glycine-leucine bonds on eNOS protein. Unfortunately, we were not successful in our experiments, largely due to the presence of unwanted contaminants in the recombinant eNOS protein preparation. However, using an approach of custom peptide synthesis, synthetic peptides representative of a particular eNOS sequence containing vulnerable sites (e.g MMP sensitive bonds) particularly in the catalytic and regulatory domain of eNOS enzyme should be obtained and studied in future experiments. Further, the effect of MMPs on eNOS uncoupling and/ or eNOS inactivation should be investigated. 8. AG1478 produced a vasorelaxant effect in phenylephrine-constricted arteries although the mechanisms are still unclear. Some studies have suggested that AG1478 prevents the mobilization of intracellular Ca 2+  in response to agonist stimulation. However it is not clear if this is in response to GPCR stimulation or EGFR activation. Future studies should look in to this aspect, as it will reveal the molecular mechanisms involved in the cross talk between GPCRs and the growth factor receptors. 9. Chronic insulin resistance and sustained agonist stimulation in VSM cells increased the expression and activation of contractile proteins, a marker of vascular hypertrophy and this was inhibited by inhibitors of MMP and the EGFR pathway. We also showed increased expression of contractile proteins in FHR arteries. However, it is not clear if ! "#$! FHR exhibit vascular remodeling and if not, the reason why these rats show markers of vascular hypertrophy yet do not undergo vascular remodeling. Long-term studies with chronic fructose feeding or short-term studies with insulin or other GPCR agonist infusions might help accelerate the conditions conducive (e.g high blood pressure) for vascular remodeling in these rats. The effect of MMP-EGFR inhibitors on such markers should provide more clues to our understanding of the relationship between the markers of hypertrophy and vascular remodeling and their overall contribution to the development of hypertension. 10. It has been shown that ROS can activate MMPs and promote EGFR transactivation. Because oxidative stress is a major component in the etiology of vascular complications in STZ diabetic rats, it would be interesting to examine the role of MMPs, particularly their contributions to endothelial function and blood pressure in STZ diabetic rats. We demonstrated that peroxynitrite, a major oxidant in nitrosative stress was associated with cardiovascular abnormalities in STZ-diabetic rats. Peroxynitrite also activates MMPs by acting directly on the inhibitory propetide domain 36 . Further, its action on MMPs appears to be concentration dependent since lower concentrations activate while higher concentrations inhibit MMP activity 37 . In light of such evidence, it is tempting to investigate the role of MMPs in STZ diabetic rats. Does higher nitrosative stress inhibit MMP activity and cause impaired endothelial function, attenuated pressor responses and hypotension?     ! "#$! 7.4 Bibliography 1. Ford ES. Risks for all-cause mortality, cardiovascular disease, and diabetes associated with the metabolic syndrome: a summary of the evidence. Diabetes Care. 2005;28(7):1769-1778. 2. The effect of intensive treatment of diabetes on the development and progression of long- term complications in insulin-dependent diabetes mellitus. The Diabetes Control and Complications Trial Research Group. N Engl J Med. 1993;329(14):977-986. 3. Ritz E, Fliser D, Nowicki M. Hypertension and vascular diseases as complications of diabetes. 2nd edn ed. New York: Raven Press; 1995. 4. Nagareddy PR, Vasudevan H, McNeill JH. Oral administration of sodium tungstate improves cardiac performance in streptozotocin-induced diabetic rats. Can J Physiol Pharmacol. 2005;83(5):405-411. 5. Nagareddy PR, Xia Z, MacLeod KM, McNeill JH. N-acetylcysteine prevents nitrosative stress-associated depression of blood pressure and heart rate in streptozotocin diabetic rats. J Cardiovasc Pharmacol. 2006;47(4):513-520. 6. Nagareddy PR, Xia Z, McNeill JH, MacLeod KM. Increased expression of iNOS is associated with endothelial dysfunction and impaired pressor responsiveness in streptozotocin-induced diabetes. Am J Physiol Heart Circ Physiol. 2005;289(5):H2144- 2152. 7. Bardell AL, MacLeod KM. Evidence for inducible nitric-oxide synthase expression and activity in vascular smooth muscle of streptozotocin-diabetic rats. J Pharmacol Exp Ther. 2001;296(2):252-259. 8. Nagareddy PR, Xia Z, McNeill JH, Macleod KM. Increased expression of iNOS is associated with endothelial dysfunction and impaired pressor responsiveness in streptozotocin-induced diabetes. Am J Physiol Heart Circ Physiol. 2005. 9. Mueed I, Zhang L, Macleod KM. Role of the PKC/CPI-17 pathway in enhanced contractile responses of mesenteric arteries from diabetic rats to alpha-adrenoceptor stimulation. Br J Pharmacol. 2005. 10. Xia Z, Kuo KH, Nagareddy PR, Wang F, Guo Z, Guo T, Jiang J, McNeill JH. N- acetylcysteine attenuates PKCbeta2 overexpression and myocardial hypertrophy in streptozotocin-induced diabetic rats. Cardiovasc Res. 2007;73(4):770-782. ! "#$! 11. King GL, Ishii H, Koya D. Diabetic vascular dysfunctions: a model of excessive activation of protein kinase C. Kidney Int Suppl. 1997;60:S77-85. 12. Koya D, King GL. Protein kinase C activation and the development of diabetic complications. Diabetes. 1998;47(6):859-866. 13. Lee TS, Saltsman KA, Ohashi H, King GL. Activation of protein kinase C by elevation of glucose concentration: proposal for a mechanism in the development of diabetic vascular complications. Proc Natl Acad Sci U S A. 1989;86(13):5141-5145. 14. Ishii H, Jirousek MR, Koya D, Takagi C, Xia P, Clermont A, Bursell SE, Kern TS, Ballas LM, Heath WF, Stramm LE, Feener EP, King GL. Amelioration of vascular dysfunctions in diabetic rats by an oral PKC beta inhibitor. Science. 1996;272(5262):728-731. 15. Jiang B, Brecher P, Cohen RA. Persistent activation of nuclear factor-kappaB by interleukin-1beta and subsequent inducible NO synthase expression requires extracellular signal-regulated kinase. Arterioscler Thromb Vasc Biol. 2001;21(12):1915-1920. 16. Jiang B, Xu S, Hou X, Pimentel DR, Brecher P, Cohen RA. Temporal control of NF- kappaB activation by ERK differentially regulates interleukin-1beta-induced gene expression. J Biol Chem. 2004;279(2):1323-1329. 17. Ramana KV, Friedrich B, Srivastava S, Bhatnagar A, Srivastava SK. Activation of nuclear factor-kappaB by hyperglycemia in vascular smooth muscle cells is regulated by aldose reductase. Diabetes. 2004;53(11):2910-2920. 18. Nishikawa T, Edelstein D, Du XL, Yamagishi S, Matsumura T, Kaneda Y, Yorek MA, Beebe D, Oates PJ, Hammes HP, Giardino I, Brownlee M. Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature. 2000;404(6779):787-790. 19. Ceriello A. New insights on oxidative stress and diabetic complications may lead to a "causal" antioxidant therapy. Diabetes Care. 2003;26(5):1589-1596. 20. Zou MH, Cohen R, Ullrich V. Peroxynitrite and vascular endothelial dysfunction in diabetes mellitus. Endothelium. 2004;11(2):89-97. 21. Mogensen CE. Drug treatment for hypertensive patients in special situations: diabetes and hypertension. Clin Exp Hypertens. 1999;21(5-6):895-906. 22. Hirai FE, Moss SE, Klein BE, Klein R. Postural blood pressure changes and associated factors in long-term Type 1 diabetes: Wisconsin Epidemiologic Study of Diabetic Retinopathy. J Diabetes Complications. 2008. ! "#$! 23. Wu JS, Yang YC, Lu FH, Wu CH, Wang RH, Chang CJ. Population-based Study on the Prevalence and Risk Factors of Orthostatic Hypotension in Subjects with Pre-diabetes and Diabetes Mellitus. Diabetes Care. 2008. 24. Vinik AI, Maser RE, Mitchell BD, Freeman R. Diabetic autonomic neuropathy. Diabetes Care. 2003;26(5):1553-1579. 25. Ceriello A, Piconi L, Esposito K, Giugliano D. Telmisartan shows an equivalent effect of vitamin C in further improving endothelial dysfunction after glycemia normalization in type 1 diabetes. Diabetes Care. 2007;30(7):1694-1698. 26. Hoeldtke RD. Nitrosative stress in early Type 1 diabetes. David H. P. Streeten Memorial Lecture. Clin Auton Res. 2003;13(6):406-421. 27. Kim JA, Montagnani M, Koh KK, Quon MJ. Reciprocal relationships between insulin resistance and endothelial dysfunction: molecular and pathophysiological mechanisms. Circulation. 2006;113(15):1888-1904. 28. Fernandez-Patron C. Therapeutic potential of the epidermal growth factor receptor transactivation in hypertension: a convergent signaling pathway of vascular tone, oxidative stress, and hypertrophic growth downstream of vasoactive G-protein-coupled receptors? Can J Physiol Pharmacol. 2007;85(1):97-104. 29. Hao L, Du M, Lopez-Campistrous A, Fernandez-Patron C. Agonist-induced activation of matrix metalloproteinase-7 promotes vasoconstriction through the epidermal growth factor-receptor pathway. Circ Res. 2004;94(1):68-76. 30. Hao L, Nishimura T, Wo H, Fernandez-Patron C. Vascular responses to alpha1-adrenergic receptors in small rat mesenteric arteries depend on mitochondrial reactive oxygen species. Arterioscler Thromb Vasc Biol. 2006;26(4):819-825. 31. Fernandez-Patron C, Radomski MW, Davidge ST. Vascular matrix metalloproteinase-2 cleaves big endothelin-1 yielding a novel vasoconstrictor. Circ Res. 1999;85(10):906-911. 32. Cheng J, Ou JS, Singh H, Falck JR, Narsimhaswamy D, Pritchard KA, Jr., Schwartzman ML. 20-hydroxyeicosatetraenoic acid causes endothelial dysfunction via eNOS uncoupling. Am J Physiol Heart Circ Physiol. 2008;294(2):H1018-1026. 33. DeLano FA, Schmid-Schonbein GW. Proteinase activity and receptor cleavage: mechanism for insulin resistance in the spontaneously hypertensive rat. Hypertension. 2008;52(2):415-423. ! "#$! 34. Morimoto Y, Nishikawa K, Ohashi M. KB-R7785, a novel matrix metalloproteinase inhibitor, exerts its antidiabetic effect by inhibiting tumor necrosis factor-alpha production. Life Sci. 1997;61(8):795-803. 35. Che Q, Carmines PK. Angiotensin II triggers EGFR tyrosine kinase-dependent Ca2+ influx in afferent arterioles. Hypertension. 2002;40(5):700-706. 36. Okamoto T, Akaike T, Nagano T, Miyajima S, Suga M, Ando M, Ichimori K, Maeda H. Activation of human neutrophil procollagenase by nitrogen dioxide and peroxynitrite: a novel mechanism for procollagenase activation involving nitric oxide. Arch Biochem Biophys. 1997;342(2):261-274. 37. Owens MW, Milligan SA, Jourd'heuil D, Grisham MB. Effects of reactive metabolites of oxygen and nitrogen on gelatinase A activity. Am J Physiol. 1997;273(2 Pt 1):L445-450.   ! "#"! Appendices                          ! "#$! List of publications 1. Nagareddy PR, McNeill JH, Macleod KM. Chronic inhibition of inducible nitric oxide synthase ameliorates cardiovascular abnormalities in streptozotocin diabetic rats. Eur J Pharmacol. 2009 Jun 2;611(1-3):53-9. PubMed PMID: 19344709.  2. Soliman H, Craig GP, Nagareddy PR, Yuen VG, Lin G, Kumar U, McNeill JH, Macleod KM. Role of inducible nitric oxide synthase in induction of RhoA expression in hearts from diabetic rats. Cardiovasc Res. 2008 Jul 15; 79(2):322-30. Epub 2008 Apr 14. PubMed PMID: 18411229.  3. Xia Z, Kuo KH, Nagareddy PR, Wang F, Guo Z, Guo T, Jiang J, McNeill JH. N- acetylcysteine attenuates PKCbeta2 overexpression and myocardial hypertrophy in streptozotocin-induced diabetic rats. Cardiovasc Res. 2007 Mar 1;73(4):770-82. Epub 2006 Nov 30. PubMed PMID: 17250813.  4. Xia Z, Guo Z, Nagareddy PR, Yuen V, Yeung E, McNeill JH. Antioxidant N- acetylcysteine restores myocardial Mn-SOD activity and attenuates myocardial dysfunction in diabetic rats. Eur J Pharmacol. 2006 Aug 21;544(1-3):118-25. Epub 2006 Jun 23. PubMed PMID: 16859669.  5. Vasudevan H, Nagareddy PR, McNeill JH. Gonadectomy prevents endothelial dysfunction in fructose-fed male rats, a factor contributing to the development of hypertension. Am J Physiol Heart Circ Physiol. 2006 Dec;291(6):H3058-64. Epub 2006 Jun 30. PubMed PMID: 16815981.  6. Nagareddy PR, Xia Z, MacLeod KM, McNeill JH. N-acetylcysteine prevents nitrosative stress-associated depression of blood pressure and heart rate in streptozotocin diabetic rats. J Cardiovasc Pharmacol. 2006 Apr;47(4):513-20. PubMed PMID: 16680064.  7. Nagareddy PR, Lakshmana M. Withania somnifera improves bone calcification in calcium-deficient ovariectomized rats. J Pharm Pharmacol. 2006 Apr;58(4):513-9. PubMed PMID: 16597369.  8. Xia Z, Nagareddy PR, Guo Z, Zhang W, McNeill JH. Antioxidant N-acetylcysteine restores systemic nitric oxide availability and corrects depressions in arterial blood pressure and heart rate in diabetic rats. Free Radic Res. 2006 Feb;40(2):175-84. PubMed PMID: 16390827.  9. Nagareddy PR, Xia Z, McNeill JH, MacLeod KM. Increased expression of iNOS is associated with endothelial dysfunction and impaired pressor responsiveness in streptozotocin-induced diabetes. Am J Physiol Heart Circ Physiol. 2005 Nov;289(5):H2144-52. Epub 2005 Jul 8. PubMed PMID: 16006542.  10. Nagareddy PR, Vasudevan H, McNeill JH. Oral administration of sodium tungstate improves cardiac performance in streptozotocin-induced diabetic rats. Can J Physiol Pharmacol. 2005 May;83(5):405-11. PubMed PMID: 15897922  11. Nagareddy PR, Lakshmana M. Withania somnifera improves bone calcification in ! "##! calcium-deficient ovariectomized rats. J Pharm Pharmacol. 2006 Apr;58(4):513-9. PubMed PMID: 16597369.  12. Nagareddy PR, Lakshmana M. Assessment of experimental osteoporosis using CT- scanning, quantitative X-ray analysis and impact test in calcium deficient ovariectomized rats. J Pharmacol Toxicol Methods. 2005 Nov-Dec;52(3):350-5. Epub 2005 Jun 29. PubMed PMID: 15996488.  13. Nagareddy PR, Lakshmana M, Udupa UV. Effect of Praval bhasma (Coral calx), a natural source of rich calcium on bone mineralization in rats. Pharmacol Res. 2003 Dec;48(6):593-9. PubMed PMID: 14527824.  14. Nagareddy PR, Lakshmana M. Prevention of bone loss in calcium deficient ovariectonized rats by OST-6, a herbal preparation. J Ethnopharmacol. 2003 Feb;84(2- 3):259-64. PubMed PMID: 12648824. ! "#$! !

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