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Vascular diseases such as hypertension are marked by changes in calcium (Ca2+) and extracellular signal regulated kinase (ERK) signaling in the arterial wall. The overall goal of this project was to better understand pathways leading to altered gene regulation in cerebral arteries. Two models were tested to determine if, 1) Ca2+/cAMP response element binding protein (CREB) is regulated in intact cerebral arteries by multiple sources of Ca2+, and 2) hypertensive disease causes changes in genes regulated by ERK and CREB. Ca2+-mediated phosphorylation of CREB (P-CREB) was measured by immunofluorescence in both cultured vascular smooth muscle cells (VSMCs) and in intact cerebral arteries. The level of P-CREB was increased by both Ca2+ influx through voltage-dependent calcium channels (VDCCs) and store-operated Ca2+ entry (SOCE) in VSMCs. A similar increase in P-CREB was observed following stimulation of VDCCs and SOCE in intact cerebral arteries. However, unlike the results obtained from VSMCs phosphorylation of CREB following Ca2+ store depletion using thapsigargin, was partially dependent on Ca2+ entry through VDCCs, suggesting that communication between Ca2+ entry pathways in intact arteries may be lost during cell culture. The second model was tested using immunocytochemistry and RNA analysis to measure differences in cerebral artery signal transduction and gene expression caused by chronic hypertension in the Dahl salt sensitive genetic hypertensive rat model. Arteries from hypertensive animals exhibited increased phosphorylation of ERK and expression of Ki-67, a marker of proliferation, when compared to controls. In addition, microarray analysis of arterial RNA revealed overexpression of the matricellular ERK-regulated genes osteopontin (OPN), and plasminogen activator inhibitor 1 (PAI-1), and the activator protein transcription factor (AP-1) member junB in cerebral arteries, with validation using RT qPCR. To elucidate a role for CREB, ERK and JunB in the transcriptional regulation of OPN and PAI-1, VSMCs were treated with angiotensin II (Ang II), a vasoconstrictor linked to hypertension, and confirmed activator of OPN and PAI-1 transcription. Ang II induced an ERK-dependent transient increase in junB mRNA and protein prior to OPN, and PAI-1 induction. Gene silencing experiments indicated that OPN and PAI-1 are reciprocally regulated by junB and CREB, respectively, and that CREB is a negative regulator of OPN. Data from cell culture confirms that the Ang II response in VSMCs is transient, in contrast to the hypertensive in vivo model, suggesting that the CREB and ERK response induces long term changes. Together, these data have revealed mechanisms for regulation of gene expression that are linked to proliferation and remodeling in the arterial wall. Future experiments will explore an in vivo role for Ang II and SOCE in the mediation of ERK- and CREBregulated gene expression. This research has the potential to help in defining therapeutic strategies to prevent arterial remodeling caused by arterial pathologies such as hypertension.

One quarter of any community may have normal (normal/high-normal) as opposed to optimal or hypertensive blood pressures (BP). These individuals may be at risk for a BP-related cardiovascular event, but do not qualify for BP-lowering therapy as those at risk are difficult to identify. In the present thesis I sought to determine whether aspects of non-invasively determined aortic BP may refine BPrelated cardiovascular risk assessment in those with brachial BP values within normotensive ranges. In 1169 participants from a community sample of African ancestry, 319 (27%)

This study investigated the effects of selective estrogen receptor (ER) agonists on constrictor prostanoid (CP) function and on the development of mean arterial pressure (MAP) in aortic coarctation-induced hypertension (ACIH). Female Sprague-Dawley rats were divided randomly into four groups: intact (INT), ovariectomized (OVX), OVX + ERα selective agonist (4, 4’, 4”-(4-Propyl-[1H]-pyrazole-1, 3, 5-triyl)trisphenol; OVX+PPT), or OVX + ERβ selective agonist (2,3-bis(4-Hydroxyphenyl)-propionitrile; OVX+DPN). Rats were then subjected to abdominal aortic coarctation (hypertensive, HT) or sham surgery (normotensive, NT). PPT, DPN or vehicle treatments were given daily as a subcutaneous injection. MAP was measured every other day at 2-14 days after coarctation. Mesenteric arterioles were harvested 12-14 days after coarctation for isometric tension studies to examine concentration-responses to VP. Basal and VP-stimulated prostanoid release and mRNA and protein levels of ERα and ERβ (using real time RT-PCR and immunoblotting) were measured in separate groups of arterioles. MAP was higher in INT-HT, OVX+PPT-HT and OVX+DPN-HT than in OVX-HT after 12 days. Vascular reactivity to VP was greater in OVX+PPT-NT rats than in other groups. There were no significant differences in vascular reactivity to VP in HT groups. Blockade of thromboxane receptor (TP) with SQ 29,548 (TP receptor antagonist) did not have a significant effect in any groups. Inhibition of intracellular calcium release with simvastatin (blocker of IP3 mediated calcium release) was greater in NT than in HT groups, and greater in OVX- and DPN-treated groups than in INT and PPT-treated groups. VP-stimulated release of thromboxane (TXA2) and prostacyclin (PGI2) were highest in INT-HT and OVX+PPT-HT rats. Neither mRNA nor protein expression of ERs changed significantly in response to selective ER agonist treatment or during hypertension. Selective ERα stimulation with PPT during development of ACIH resulted in similar effects to those seen in INT rats for CP release, VP reactivity of mesenteric arterioles and MAP, while selective stimulation of ERβ only increased MAP. While ERα is capable of modulating most of the effects of estrogen on the vasculature, ERβ has stimulatory effects on MAP during the development of ACIH that merit further investigation. Further studies of the vascular actions of ERα and ERβ may lead to better hormonal therapies that successfully prevent and/or treat cardiovascular disease in post-menopausal women.

<p>Glycine, a lipophilic non-essential amino acid biosynthesized from L-serine, L-alanine and L-threonine, plays a role in the biosynthesis of proteins, nucleotides, and glutathione. It is a neurotransmitter in the central nervous system and acts as a co-agonist at the N-methyl-D aspartate (NMDA) receptor. Oral administration of glycine is helpful in the management of schizophrenia. While long-term oral treatment with glycine is considered to exert a cardio-vascular protective role by overcoming endothelial dysfunction and oxidative stress, there are no systematic studies examining the cardiovascular effects of glycine. Recently, we showed that the precursor/metabolite of glycine, L-serine, evoked endothelium-dependent vasodilatation in rat mesenteric arterioles. Acute intravenous administration of L-serine produced a rapid, dose-dependent fall in blood pressure (BP) in both normotensive and hypertensive rats. These responses were abolished in the combined presence of the Ca²⁺ activated small and intermediate conductance K⁺ channel inhibitors, apamin and Tram-34/charybdotoxin. In contrast, intravenous administration of glycine evoked a fall in BP in normotensive Wistar-Kyoto (WKY) rats and an elevation of BP in spontaneously hypertensive rats (SHR), and in WKY rats subjected to chronic nitric oxide (NO) synthase (NOS) inhibition by oral treatment with NOS inhibitor, L-NAME (N^G-nitro-L-arginine-methylester). Therefore, <i>in vivo</i> and <i>in vitro</i> studies were designed to address the mechanisms that contribute to the opposite effects of glycine in normotensive <i>vs</i>. hypertensive rats.</p> <p>Experiments were performed using 14 weeks old male WKY, chronic L-NAME treated WKY and SHR strains. <i>In vivo</i> studies involved examination of changes in systemic hemodynamic parameters such as mean arterial pressure (MAP), heart rate (HR), total peripheral resistance (TPR) and cardiac output (CO) as well as regional hemodynamic parameters of changes in blood flow and vascular resistance in major organs/tissues following acute intravenous administration of glycine using fluorescent microsphere distribution technique. Parallel complementary <i>in vitro</i> studies were conducted to examine the effects of glycine on changes in basal tone and phenylephrine (PE) constricted tone in aortic rings with endothelium-intact and endothelium-denuded states after isolation from WKY and SHR strains. All these studies were conducted in the presence and absence of two NMDA antagonists, MK-801 and memantine.</p> <p>In normotensive WKY rats, glycine (1 mmol/L) administration decreased MAP (P<0.01), TPR (P<0.05) while it increased CO (P<0.01) and blood flow to brain (215%), kidney (190%) and heart (160%). In SHR and L-NAME treated WKY rats, glycine administration elevated MAP and TPR but reduced CO (P<0.01) and blood flow to brain, kidney and heart. These effects were abolished in animals pretreated with either MK-801 or memantine. These data are consistent with the likely expression of vascular NMDA receptors activated by glycine in brain and kidney. Glycine (0.5-3.0 mmol/L) <i>per se</i> increased basal tone (E_max1.4g) in aortic rings from all rats and it was absent following incubation with NMDA antagonist(s). The concentration-dependent vasodilatation (I_max43%) evoked by glycine in PE-constricted rings with intact endothelium of WKY rats was attenuated by either MK-801 or L-NAME. Such vasodilator responses to glycine could be abolished by pretreatment with either NMDA antagonist or L-NAME. In contrast, in PE-constricted rings of SHR strain, addition of glycine enhanced the tone and this was abolished in the presence of NMDA antagonist, MK-801.</p> <p>Taken together, these data suggest that NMDA receptors, likely present on both endothelium and vascular smooth muscle cells, predominantly in brain, kidney and coronary vascular beds as well as the conduit vessel, aorta, could contribute to the systemic and regional hemodynmic effects of glycine. In normotensive WKY rats, addition of glycine promotes endothelium/NO-dependent vasodilatation subsequent to Ca2+ mobilization evoked following endothelial NMDA receptor activation by glycine. Thus, glycine-induced dose-dependent, reversible fall in MAP and TPR in normotensive WKY rats is likely associated with NO-dependent/endothelium-mediated vasodilatation. However, in hypertensive rat models such as in the SHR model with endothelial dysfunction and reduced NO bioavailability or in chronic L-NAME treated rats, glycine administration promotes vascular NMDA receptor activation leading to enhanced vascular tone resulting in increased TPR and MAP.</p> <p>These observations are important and critical in terms of considering glycine as a possible antihypertensive agent as this was proposed by several earlier reports that did not explore the direct vascular effects of glycine in hypertensive animal models.</p>

<p>Glycine, a lipophilic non-essential amino acid biosynthesized from L-serine, L-alanine and L-threonine, plays a role in the biosynthesis of proteins, nucleotides, and glutathione. It is a neurotransmitter in the central nervous system and acts as a co-agonist at the N-methyl-D aspartate (NMDA) receptor. Oral administration of glycine is helpful in the management of schizophrenia. While long-term oral treatment with glycine is considered to exert a cardio-vascular protective role by overcoming endothelial dysfunction and oxidative stress, there are no systematic studies examining the cardiovascular effects of glycine. Recently, we showed that the precursor/metabolite of glycine, L-serine, evoked endothelium-dependent vasodilatation in rat mesenteric arterioles. Acute intravenous administration of L-serine produced a rapid, dose-dependent fall in blood pressure (BP) in both normotensive and hypertensive rats. These responses were abolished in the combined presence of the Ca²⁺ activated small and intermediate conductance K⁺ channel inhibitors, apamin and Tram-34/charybdotoxin. In contrast, intravenous administration of glycine evoked a fall in BP in normotensive Wistar-Kyoto (WKY) rats and an elevation of BP in spontaneously hypertensive rats (SHR), and in WKY rats subjected to chronic nitric oxide (NO) synthase (NOS) inhibition by oral treatment with NOS inhibitor, L-NAME (N^G-nitro-L-arginine-methylester). Therefore, <i>in vivo</i> and <i>in vitro</i> studies were designed to address the mechanisms that contribute to the opposite effects of glycine in normotensive <i>vs</i>. hypertensive rats.</p> <p>Experiments were performed using 14 weeks old male WKY, chronic L-NAME treated WKY and SHR strains. <i>In vivo</i> studies involved examination of changes in systemic hemodynamic parameters such as mean arterial pressure (MAP), heart rate (HR), total peripheral resistance (TPR) and cardiac output (CO) as well as regional hemodynamic parameters of changes in blood flow and vascular resistance in major organs/tissues following acute intravenous administration of glycine using fluorescent microsphere distribution technique. Parallel complementary <i>in vitro</i> studies were conducted to examine the effects of glycine on changes in basal tone and phenylephrine (PE) constricted tone in aortic rings with endothelium-intact and endothelium-denuded states after isolation from WKY and SHR strains. All these studies were conducted in the presence and absence of two NMDA antagonists, MK-801 and memantine.</p> <p>In normotensive WKY rats, glycine (1 mmol/L) administration decreased MAP (P<0.01), TPR (P<0.05) while it increased CO (P<0.01) and blood flow to brain (215%), kidney (190%) and heart (160%). In SHR and L-NAME treated WKY rats, glycine administration elevated MAP and TPR but reduced CO (P<0.01) and blood flow to brain, kidney and heart. These effects were abolished in animals pretreated with either MK-801 or memantine. These data are consistent with the likely expression of vascular NMDA receptors activated by glycine in brain and kidney. Glycine (0.5-3.0 mmol/L) <i>per se</i> increased basal tone (E_max1.4g) in aortic rings from all rats and it was absent following incubation with NMDA antagonist(s). The concentration-dependent vasodilatation (I_max43%) evoked by glycine in PE-constricted rings with intact endothelium of WKY rats was attenuated by either MK-801 or L-NAME. Such vasodilator responses to glycine could be abolished by pretreatment with either NMDA antagonist or L-NAME. In contrast, in PE-constricted rings of SHR strain, addition of glycine enhanced the tone and this was abolished in the presence of NMDA antagonist, MK-801.</p> <p>Taken together, these data suggest that NMDA receptors, likely present on both endothelium and vascular smooth muscle cells, predominantly in brain, kidney and coronary vascular beds as well as the conduit vessel, aorta, could contribute to the systemic and regional hemodynmic effects of glycine. In normotensive WKY rats, addition of glycine promotes endothelium/NO-dependent vasodilatation subsequent to Ca2+ mobilization evoked following endothelial NMDA receptor activation by glycine. Thus, glycine-induced dose-dependent, reversible fall in MAP and TPR in normotensive WKY rats is likely associated with NO-dependent/endothelium-mediated vasodilatation. However, in hypertensive rat models such as in the SHR model with endothelial dysfunction and reduced NO bioavailability or in chronic L-NAME treated rats, glycine administration promotes vascular NMDA receptor activation leading to enhanced vascular tone resulting in increased TPR and MAP.</p> <p>These observations are important and critical in terms of considering glycine as a possible antihypertensive agent as this was proposed by several earlier reports that did not explore the direct vascular effects of glycine in hypertensive animal models.</p>