The primary goal of this project is to characterize the intracellular signal transduction mechanisms activated by flow in endothelial cells (EC) that confer protection from atherosclerosis. The concept that inflammation plays a key role in the pathogenesis and progression of atherosclerosis has recently received much support. The major hypothesis of this proposal is that steady laminar shear stress limits atherosclerosis by inhibiting the pro-atherogenic program of gene expression induced by inflammatory cytokines. Because the activity of transcription factors responsible for gene expression is controlled by protein phosphorylation our laboratory has focused on protein kinases. We have elucidated mechanisms by which members of the mitogen activated protein kinases (MAPK) are activated by flow and tumor necrosis factor (TNF). MAPK phosphorylate and activate transcription factors that induce expression of both pro- and anti-inflammatory molecules. In particular, c-Jun N-terminal kinase (JNK) is activated by many inflammatory cytokines (e.g., TNF and IL-1). We propose that understanding the mechanisms by which flow regulates LINK activation by cytokines will provide insight into the atheroprotective mechanisms induced by flow. The specific hypothesis to be investigated is that flow regulates several pathways (apoptosis signal regulated kinase (ASK1), Src homology-2 domain containing phosphatase (SHP2), extracellular signal regulated kinases (ERK1/2 and ERK5 or BMK1), and JNK phosphatases) to inhibit JNK activity. Exciting preliminary data indicate that pre-exposure of EC to steady laminar flow prevents TNF-mediated JNK activation, in association with decreases in TNF activation of ASK! and SHP2. This inhibition is not due primarily to effects of nitric oxide (NO), a mechanism that has not been well explored. To prove our hypotheses three specific aims are proposed. 1) Identify signal transduction mechanisms responsible for flow-dependent suppression of JNK activity, focusing on BMK1, ERK1/2, ASK!, SHP2 and LINK phosphatases. 2) Determine the effect of cardiovascular risk factors (oscillatory flow and oxidative stress) on flow-mediated suppression of JNK activity. 3) Characterize the effect of altering JNK activation (by adenoviral and antisense oligonucleotide transfection) on the response of the ex vivo perfused rabbit aorta to TNF. These studies should provide insight into the mechanisms by which flow generates an atheroprotective signal and facilitate development of new therapeutic approaches to limit atherosclerosis.
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