: The end organ sequelae of atherosclerosis and hypertension represent the largest public health problems in industrialized populations. The importance of arachidonic acid (AA) and its metabolites in modulating vascular tone, cell growth, migration, proliferation and gene expression is well known. The rate determining step in the production of eicosanoids is the release of AA by intracellular phospholipases A2 (PLA2). Accordingly, a detailed understanding of the types and modes of regulation of vascular cell intracellular PLA2S are necessary to gain insight into the critical signaling mechanisms, which mediate atherosclerotic, and hypertensive disease states. We have identified the major vascular intracellular phospholipases as iPLA2B and 1PLA2Gamma The structure of iPLA2B is remarkable for: 1) the presence of ankyrin-like repeat domains in its N-terminus which likely modulate protein-protein interactions facilitating its intracellular sorting; and 2) a calmodulin binding domain in its C-terminus which regulates its activity. Accordingly, in Specific Aim 1 we will examine the functional role of the N-terminal ankyrin-like repeat domains in iPLA2B by identifying its protein binding partners. Next, we will explore the role of phosphorylation of the calmodulin binding domain to elucidate the role of protein kinases in regulating iPLA2B activity. Finally, the role of proteolytic activation of iPLA2B activity by caspase-3 and calpain-mediated cleavage will be examined.
In Specific Aim 2, we will generate smooth muscle cell (SMC) specific overexpressors and mice null for IPLA2B and 1PLA2gamma in a SMC specific manner to determine the role of iPLA2s in vascular SMC tone, proliferation and migration.
In Specific Aim 3, we will explore the newly identified role of AA as a modulator of endothelial cell (EC) nitric oxide production and the roles of Akt mediated activation of eNOS and iPLA2B in NO production. Finally, by exploiting chiral mechanism-based inhibitors of iPLA2B and iPLA27 we have developed, we will examine the role of intracellular phospholipases in EC responses to cyclic mechanical force. Alterations in the release of AA, generation of eicosanoid metabolites, lipid composition and cytoskeletal organization during changes in shear stress and mechanical stretch will be examined. Collectively, these studies represent a targeted, multidisciplinary approach to elucidate the roles of intracellular phospholipases in the vascular biology of the hypertensive and atherosclerotic disease states.
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