This competitive renewal application proposes to investigate the downstream mechanisms by which ROCK regulates eNOS mRNA stability and activity. Preliminary studies suggest that phosphorylation of the translational elongation factor-1? (eEF1A) by ROCK2 leads to decreased eNOS mRNA stability and expression. Furthermore, we found that activation of Phosphatase and TENsin homologue (PTEN) by ROCK2 leads to decreased phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P3) levels and inhibition of Akt-mediated eNOS activity. Based upon these pilot studies, two comprehensive specific aims are proposed, which will determine how the regulation of eEF1A and PTEN by ROCK2 could affect eNOS expression and activity, respectively, and whether this contributes importantly to the pathogenesis of endothelial dysfunction and vascular disease. It is hoped that these studies will help establish the importance of ROCK2 as a therapeutic targets for improving endothelial function and decreasing cardiovascular disease.
Specific aim 1 : To determine whether ROCK2 regulates endothelial function in vivo, we will generate endothelial-specific ROCK2Tie2-/- and EC-caROCK mice, and investigate whether eNOS expression, endothelium-dependent vascular relaxation, and atherosclerosis are altered due to the loss- or gain-of-function of endothelial ROCK2. Further studies are proposed to determine how phosphorylation of eEF1A by ROCK2 leads to eNOS mRNA instability and decreased eNOS expression.
Specific aim 2 : To determine how ROCK2 could inhibit the Akt/eNOS pathway, we will investigate how ROCK2 decreases PIP3 levels through inhibition of PI3K activity, stimulation of PTEN activity, or both. We will determine whether ROCK2 can bind to and phosphorylate PTEN, and identify putative ROCK phosphorylation site(s) on PTEN. We will then determine whether phosphorylation of PTEN by ROCK2 increases PTEN activity, leading to decreased PIP3 levels and inhibition of Akt Thr308 phosphorylation and activity. These findings will be correlated with Akt-dependent eNOS Ser1179 phosphorylation and the degree of vascular remodeling.
Cardiovascular disease is a major cause of morbidity and mortality worldwide. The underlying mechanism is partly due to endothelial dysfunction. This research application proposes to investigate the role of an emerging signaling pathway, Rho kinase (ROCK), as an important regulator of endothelial function in vascular disease.
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