Background and Purpose: The endothelium is a single-cell layer positioned at the blood-vascular wall interface, where in response to blood-borne signals and hemodynamic forces, endothelial cells act as central regulators of vascular homeostatic processes including vascular tone, growth and remodeling, inflammation and adhesion, and blood fluidity and coagulation. Agonist- or flow-stimulated endothelium-dependent vasorelaxation becomes impaired in states of cardiovascular disease (CVD) risk and has been identified as a possible biomarker of overall endothelial dysfunction leading to vascular dysregulation and disease pathogenesis. Accordingly, it is important to elucidate the mechanisms accounting for this endothelial vasomotor dysfunction. Upon stimulation, endothelial cells can synthesize and release a variety of endothelium-derived relaxing factors (EDRFs), the most prominent of which is nitric oxide (NO) derived from NO synthase (NOS). In addition, under certain CVD risk conditions including hypertension and aging, stimulated endothelial cells can become a prominent source of endothelium-derived contracting factors (EDCFs) produced in a cyclooxygenase (COX)-dependent manner. Consequently, endothelial dysfunction may be caused by under-active EDRF signaling and/or competitive over-active EDCF signaling. Much attention has been given to elucidating the mechanisms of under-active EDRF signaling and its role in causing endothelial dysfunction, wherein excess reactive oxygen species (ROS) accumulation and oxidative stress under CVD risk conditions have been recognized as major factors in reducing NO bioavailability thus causing under-active EDRF signaling and endothelial dysfunction. Less attention however, has been given to elucidating the mechanisms of over-active COX-mediated EDCF signaling and its role in causing endothelial dysfunction. Moreover, while COX-mediated EDCF signaling activity has been investigated in some segments of the vasculature, most notably the aorta, it has not been well-investigated in the common carotid artery (CCA), a highly accessible cerebral blood flow conduit particularly advantageous in exploring the roles of the endothelium in vascular pathogenesis. It was the global purpose of this thesis to gain a better understanding of the cellular-molecular mechanisms accounting for endothelial dysfunction in the CCA of animal models known to exhibit COX-mediated EDCF signaling activity, in particular essential (spontaneous) hypertension and aging. Experimental Objective and Approach: This thesis comprises three studies. Study I and Study II investigated the CCA of young-adult (16-24wk old) normotensive Wistar Kyoto (WKY) and Spontaneously Hypertensive (SHR) rats. Study III investigated the CCA of Adult (25-36wks old) and Aging (60-75wks old) Sprague Dawley (SD) rats treated in vivo (or not; CON) with L-buthionine sulfoximine (BSO) to chronically deplete the cellular anti-oxidant glutathione (GSH) and increase ROS accumulation and oxidative stress. The global objective and approach across these studies was to systematically examine the relative contributions of NOS and COX signaling pathways in mediating the acetylcholine (ACh)-stimulated endothelium-dependent relaxation (EDRF) and contractile (EDCF) activities of isometrically-mounted CCA in tissue baths in vitro, with a particular focus on elucidating the mechanisms of COX-mediated EDCF signaling activity. An added objective was to examine the in vivo hemodynamic characteristics of the CCA in each animal model investigated, serving both to identify the pressure-flow environment that the CCA is exposed to in vivo and to provide assessment of potential hypertension, aging, and oxidative stress effects on large artery hemodynamics. Key Findings: Study I hemodynamic analysis confirmed a hypertensive state in young adult SHR while also exposing a reduction in mean CCA blood flow in SHR compared to WKY accompanied by a multi-faceted pressure-flow interaction across the cardiac cycle relating to flow and pressure augmentation. Study III hemodynamic analysis found that neither aging nor chronic BSO-induced GSH depletion affected CCA blood pressure or blood flow parameters in SD rats. Study I and II demonstrated that a COX-mediated EDCF response impaired ACh-stimulated endothelium-dependent vasorelaxation in pre-contracted CCA from young adult SHR, while EDRF signaling activity, predominantly mediated by NO, remained well-preserved compared to WKY. Examining ACh-stimulated contractile function specifically from a quiescent (non pre-contracted) state revealed that EDCF activity did exist in WKY CCA but could be completely suppressed by NO-mediated EDRF signaling activity, whereas the similarly robust NO-meditated EDRF signaling activity in SHR CCA could not fully suppress its >2-fold augmented EDCF activity vs. WKY CCA. Further pharmaco-dissection of ACh-stimulated contractile function in the SHR-WKY CCA model revealed that the EDCF signaling activity was completely dependent on the COX-1 (but not COX-2) isoform of COX and was almost exclusively mediated by the thromboxane-prostanoid (TP) sub-type of the prostaglandin (PG) G-protein coupled receptor family and by Rho-associated kinase (ROCK), a down-stream effector of the molecular switch RhoA. Furthermore, it was found that while exogenous ROS-stimulated CCA contractile function was similarly >2-fold augmented in SHR vs. WKY and dependent on COX-1 and TP receptor and ROCK effectors, ACh-stimulated CCA EDCF signaling activity was only minimally affected by in-bath ROS manipulating compounds. Additional biochemical and molecular analysis revealed that ACh stimulation was associated with PG over-production from an over-expressed COX-1 in SHR CCA, and with CCA plasma membrane localization and activation of RhoA. Study III demonstrated that a COX-mediated EDCF response impaired ACh-stimulated endothelium-dependent vasorelaxation in pre-contracted CCA from Aging SD rats, while EDRF signaling activity, predominantly mediated by NO, remained well-preserved compared to Adult SD rats. Specific examination of ACh-stimulated contractile function revealed that EDCF activity did exist in Adult CCA but could be completely suppressed by NO-mediated EDRF signaling activity, whereas the similarly robust NO-meditated EDRF signaling activity in Aging CCA could not fully suppress its >3-fold augmented EDCF activity vs. Adult CCA. Further pharmaco-dissection of ACh-stimulated contractile function in the Adult-Aging SD rat CCA model revealed that EDCF signaling activity was completely dependent on COX-1, but while exogenous ROS was able to elicit a COX-dependent CCA contractile response, in-bath ROS manipulating compounds were found to be without effect on ACh-stimulated CCA EDCF signaling activity. Furthermore, biochemical analysis revealed that aging was not associated with a change in tissue (liver and vascular) GSH content or ROS accumulation. Chronic in vivo BSO treatment was effective in depleting tissue GSH content and increasing ROS accumulation, to a similar extent, in both Adult and Aging SD rats. However, regardless of age, neither ACh-stimulated NO-mediated EDRF signaling activity nor COX-mediated EDCF signaling activity were affected by these BSO-induced perturbations. Conclusions and Perspective: In the CCA of animals at the early pathological stages of either essential hypertension (young adult SHR) or normotensive aging (Aging SD rats), endothelial vasomotor dysfunction can be caused solely by over-active EDCF signaling, apparently disconnected from changes in NO bioavailability or oxidative stress. While NO and ROS may act, respectively, as negative and positive modulators of the established COX-PG-TP receptor-RhoA-ROCK cell-signaling axis mediating endothelium-dependent contractile activity, these factors do not appear to be essential to the mechanism(s) underlying the development of over-active EDCF signaling. Further elucidation of the cellular-molecular causes of over-active EDCF signaling, and its patho-biological consequences, in the SHR-WKY and Adult-Aging SD rat CCA models of EDCF activity established and hemodynamically characterized in this thesis, may help to identify new or more effective targets to be used in prevention or treatment strategies to combat the pathogenesis of CVD.

Background and Purpose: The endothelium is a single-cell layer positioned at the blood-vascular wall interface, where in response to blood-borne signals and hemodynamic forces, endothelial cells act as central regulators of vascular homeostatic processes including vascular tone, growth and remodeling, inflammation and adhesion, and blood fluidity and coagulation. Agonist- or flow-stimulated endothelium-dependent vasorelaxation becomes impaired in states of cardiovascular disease (CVD) risk and has been identified as a possible biomarker of overall endothelial dysfunction leading to vascular dysregulation and disease pathogenesis. Accordingly, it is important to elucidate the mechanisms accounting for this endothelial vasomotor dysfunction. Upon stimulation, endothelial cells can synthesize and release a variety of endothelium-derived relaxing factors (EDRFs), the most prominent of which is nitric oxide (NO) derived from NO synthase (NOS). In addition, under certain CVD risk conditions including hypertension and aging, stimulated endothelial cells can become a prominent source of endothelium-derived contracting factors (EDCFs) produced in a cyclooxygenase (COX)-dependent manner. Consequently, endothelial dysfunction may be caused by under-active EDRF signaling and/or competitive over-active EDCF signaling. Much attention has been given to elucidating the mechanisms of under-active EDRF signaling and its role in causing endothelial dysfunction, wherein excess reactive oxygen species (ROS) accumulation and oxidative stress under CVD risk conditions have been recognized as major factors in reducing NO bioavailability thus causing under-active EDRF signaling and endothelial dysfunction. Less attention however, has been given to elucidating the mechanisms of over-active COX-mediated EDCF signaling and its role in causing endothelial dysfunction. Moreover, while COX-mediated EDCF signaling activity has been investigated in some segments of the vasculature, most notably the aorta, it has not been well-investigated in the common carotid artery (CCA), a highly accessible cerebral blood flow conduit particularly advantageous in exploring the roles of the endothelium in vascular pathogenesis. It was the global purpose of this thesis to gain a better understanding of the cellular-molecular mechanisms accounting for endothelial dysfunction in the CCA of animal models known to exhibit COX-mediated EDCF signaling activity, in particular essential (spontaneous) hypertension and aging. Experimental Objective and Approach: This thesis comprises three studies. Study I and Study II investigated the CCA of young-adult (16-24wk old) normotensive Wistar Kyoto (WKY) and Spontaneously Hypertensive (SHR) rats. Study III investigated the CCA of Adult (25-36wks old) and Aging (60-75wks old) Sprague Dawley (SD) rats treated in vivo (or not; CON) with L-buthionine sulfoximine (BSO) to chronically deplete the cellular anti-oxidant glutathione (GSH) and increase ROS accumulation and oxidative stress. The global objective and approach across these studies was to systematically examine the relative contributions of NOS and COX signaling pathways in mediating the acetylcholine (ACh)-stimulated endothelium-dependent relaxation (EDRF) and contractile (EDCF) activities of isometrically-mounted CCA in tissue baths in vitro, with a particular focus on elucidating the mechanisms of COX-mediated EDCF signaling activity. An added objective was to examine the in vivo hemodynamic characteristics of the CCA in each animal model investigated, serving both to identify the pressure-flow environment that the CCA is exposed to in vivo and to provide assessment of potential hypertension, aging, and oxidative stress effects on large artery hemodynamics. Key Findings: Study I hemodynamic analysis confirmed a hypertensive state in young adult SHR while also exposing a reduction in mean CCA blood flow in SHR compared to WKY accompanied by a multi-faceted pressure-flow interaction across the cardiac cycle relating to flow and pressure augmentation. Study III hemodynamic analysis found that neither aging nor chronic BSO-induced GSH depletion affected CCA blood pressure or blood flow parameters in SD rats. Study I and II demonstrated that a COX-mediated EDCF response impaired ACh-stimulated endothelium-dependent vasorelaxation in pre-contracted CCA from young adult SHR, while EDRF signaling activity, predominantly mediated by NO, remained well-preserved compared to WKY. Examining ACh-stimulated contractile function specifically from a quiescent (non pre-contracted) state revealed that EDCF activity did exist in WKY CCA but could be completely suppressed by NO-mediated EDRF signaling activity, whereas the similarly robust NO-meditated EDRF signaling activity in SHR CCA could not fully suppress its >2-fold augmented EDCF activity vs. WKY CCA. Further pharmaco-dissection of ACh-stimulated contractile function in the SHR-WKY CCA model revealed that the EDCF signaling activity was completely dependent on the COX-1 (but not COX-2) isoform of COX and was almost exclusively mediated by the thromboxane-prostanoid (TP) sub-type of the prostaglandin (PG) G-protein coupled receptor family and by Rho-associated kinase (ROCK), a down-stream effector of the molecular switch RhoA. Furthermore, it was found that while exogenous ROS-stimulated CCA contractile function was similarly >2-fold augmented in SHR vs. WKY and dependent on COX-1 and TP receptor and ROCK effectors, ACh-stimulated CCA EDCF signaling activity was only minimally affected by in-bath ROS manipulating compounds. Additional biochemical and molecular analysis revealed that ACh stimulation was associated with PG over-production from an over-expressed COX-1 in SHR CCA, and with CCA plasma membrane localization and activation of RhoA. Study III demonstrated that a COX-mediated EDCF response impaired ACh-stimulated endothelium-dependent vasorelaxation in pre-contracted CCA from Aging SD rats, while EDRF signaling activity, predominantly mediated by NO, remained well-preserved compared to Adult SD rats. Specific examination of ACh-stimulated contractile function revealed that EDCF activity did exist in Adult CCA but could be completely suppressed by NO-mediated EDRF signaling activity, whereas the similarly robust NO-meditated EDRF signaling activity in Aging CCA could not fully suppress its >3-fold augmented EDCF activity vs. Adult CCA. Further pharmaco-dissection of ACh-stimulated contractile function in the Adult-Aging SD rat CCA model revealed that EDCF signaling activity was completely dependent on COX-1, but while exogenous ROS was able to elicit a COX-dependent CCA contractile response, in-bath ROS manipulating compounds were found to be without effect on ACh-stimulated CCA EDCF signaling activity. Furthermore, biochemical analysis revealed that aging was not associated with a change in tissue (liver and vascular) GSH content or ROS accumulation. Chronic in vivo BSO treatment was effective in depleting tissue GSH content and increasing ROS accumulation, to a similar extent, in both Adult and Aging SD rats. However, regardless of age, neither ACh-stimulated NO-mediated EDRF signaling activity nor COX-mediated EDCF signaling activity were affected by these BSO-induced perturbations. Conclusions and Perspective: In the CCA of animals at the early pathological stages of either essential hypertension (young adult SHR) or normotensive aging (Aging SD rats), endothelial vasomotor dysfunction can be caused solely by over-active EDCF signaling, apparently disconnected from changes in NO bioavailability or oxidative stress. While NO and ROS may act, respectively, as negative and positive modulators of the established COX-PG-TP receptor-RhoA-ROCK cell-signaling axis mediating endothelium-dependent contractile activity, these factors do not appear to be essential to the mechanism(s) underlying the development of over-active EDCF signaling. Further elucidation of the cellular-molecular causes of over-active EDCF signaling, and its patho-biological consequences, in the SHR-WKY and Adult-Aging SD rat CCA models of EDCF activity established and hemodynamically characterized in this thesis, may help to identify new or more effective targets to be used in prevention or treatment strategies to combat the pathogenesis of CVD.

Chronic kidney disease (CKD) is defined as glomerular filtration rate (GFR) less than 60 mL/min/1.73 m2 for 3 months and is characterized by progressive loss of renal function. The second leading cause of CKD is hypertension. More than half of CKD patients also suffer from hypertension. Arteries and arterioles adjust to the fluctuations in the systematic blood pressure through a mechanism called autoregulation. In the kidneys, autoregulation protects the delicate glomeruli capillaries from high blood pressure and occurs through myogenic constriction (MC). MC refers to contraction of arterioles in response to an increase in the blood pressure. Chronically hypertensive individuals and animal models have an enhanced MC, leading to minimal renal injury despite their elevated blood pressure. Experimental and clinical evidence point to a role for the MC in the pathogenesis of the CKD, however, the mechanism through which preglomerular arterial MC contributes to CKD has not been fully elucidated. This thesis showed that augmented MC in chronically hypertensive animal models was due to increased thromboxane A2 prostaglandin that was not released from the endothelium (Chapter 2). Nevertheless, inhibiting MC while also reducing the blood pressure prevented salt-induced renal injury even though the blood pressure was still not normalized compared to the normotensive controls (Chapter 3). The resulting improvement in renal structure and function could be attributed to the reduction in the blood pressure, albumin, and uromodulin (UMOD) excretion (Chapter 3). UMOD is a kidney-specific glycoprotein that, based on a genome-wide association study have the strongest association to CKD (Chapter 3). Comparing two CKD hypertensive animal models further revealed that CKD progression was independent of the blood pressure and strongly associated with UMOD excretion levels (Chapter 4). Collectively, the data discussed in this thesis demonstrates potential therapeutic targets in CKD hypertensive animal models. / Dissertation / Doctor of Philosophy (PhD)