In the previous funding period, we have shown that lipoproteins and the cholesterol lowering agent, HMG-CoA reductase inhibitors or statins, affect the vascular wall through modulation of heterotrimeric and small G-proteins in vascular endothelial cells. In this competitive renewal application, we will expand on this theme and investigate the pathophysiological effects of Rho kinases (ROCKs), the immediate downstream target of the small GTP-binding protein Rho, on endothelial function and lipoprotein-induced vascular disease. Overactivity of ROCKs is observed in cerebral and coronary vasospasm, hypertension, vascular inflammation, and arteriosclerosis. ROCKs, therefore, may be an important and still relatively unexplored therapeutic target in cardiovascular disease. Studies from our laboratory suggest that inhibition of RhoA/ROCK by statins increases eNOS expression via stabilization of eNOS mRNA and stimulates eNOS activity via activation of the phosphatidyinositol (PI)-3 kinase/protein kinase Akt pathway. The proposed studies, therefore, will extend the Pi's long-standing interest in eNOS regulation by statins and lipoproteins to investigations in the Rho/ROCK pathway. The main goal of this project is to determine whether endothelial ROCK isoforms (ROCK1 and ROCK2) contribute to endothelial dysfunction, vascular inflammation, and atherosclerosis. To achieve this, we have developed conditional ROCK1 and ROCK2 KO mice, and using the endothelial-specific Tie2.Cre mice, will attempt to target ROCK deletion to the endothelium for these studies.
Three specific aims are proposed to study these mice and their tissues.
Specific aim 1 will test the hypothesis that endothelial ROCKs are activated following non-endothelial- denuding perivascular cuff-induced injury and that endothelial deletion of ROCKs confer vascular protection.
Specific aim 2 will test the hypothesis that downstream targets of ROCKs regulate endothelial and vascular function, and contribute to some of the beneficial effects of statin therapy in ischemic stroke. The mechanism by which ROCK regulates eNOS mRNA stability will also be explored.
Specific aim 3 will test the hypothesis that endothelial ROCK deletion is protective in a mouse model of atherosclerosis. It is hoped that these studies will provide the mechanistic basis for some of the cholesterol-independent effects of statins and help establish the importance of ROCK as a novel therapeutic target for improving endothelial function and decreasing cardiovascular disease.
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