Increased peripheral resistance in hypertension may be caused by a vasoconstriction resulting from enhanced transduction of signals promoting contractile protein activation within the vascular myocyte. This research proposal will extend our understanding of the RhoA/Rho-kinase signaling system in vascular smooth muscle from hypertensive mice. The research will be guided by the working hypothesis that augmented vascular reactivity in angiotensin II-induced hypertension occurs by a two-step mechanism to increase the functional activity of the RhoA/Rho-kinase system: 1) during the early phase of blood pressure elevation, angiotensin II stimulates superoxide production leading to transactivation of the epidermal growth factor receptor (EGFR) and subsequent stimulation of RhoA/Rho-kinase to cause vasoconstriction; and 2) during the established phase of blood pressure elevation, angiotensin II increases interleukin-6 (IL-6) in plasma and tissue leading to JAK/STAT activation and increased expression of components of the RhoA/Rho-kinase cascade and maintained vasoconstriction. This working hypothesis will be tested by three specific aims: 1) to test the hypothesis that during the early phase of blood pressure elevation in angiotensin II-induced hypertension, the octapeptide stimulates RhoA/Rho-kinase signaling via superoxide activation of the receptor for epidermal growth factor (EGFR); 2) to test the hypothesis that during the maintained phase of angiotensin II-induced hypertension, the octapeptide and IL-6 increase expression of components of the RhoA/Rho-kinase signaling pathway via activation of JAK/STAT; and 3) to test the hypothesis that augmented RhoA/Rho-kinase signaling is not related to blood pressure per se in angiotensin II-induce hypertension. Mice will be made hypertensive by infusion of angiotensin II and blood pressure will be measured using telemetry. During the initiation and maintenance phases of the hypertensive process, the mice will be killed and blood vessels will be isolated and subjected to functional and biochemical analyses of the RhoA/Rho-kinase and JAK/STAT signal transduction pathways. Superoxide anion and IL-6 levels will be measured. The techniques used to evaluate the contribution of the signaling pathways include: 1) isometric recording of contractile behavior of isolated arteries and measures of pressor activity in perfused mesentery and hindquarters; 2) pharmacological interventions to evaluate components of the signaling cascades; 3) evaluation of pressure-dependent activation of vascular myocytes (protected hindlimb); and 4) measures of protein expression. In a complementary strategy, IL-6 knock-out mice will be used to evaluate the role of this cytokine in the transduction of the angiotensin II signal. We predict that, in vascular myocytes, angiotensin II and IL-6 will lead to an increased activity of regulatory proteins that influence contractile activity.
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