Activation of smooth muscle cells is thought to be an important pathological event in atherogenesis. Cellular activation promotes release of specific cyclooxygenase and lipoxygenase metabolites which modulate vascular cholesterol homeostasis.To define the molecular basis of this regulation, they investigated the role of the major cyclooxygenase metabolite of smooth muscle cells, namely prostacyclin (PGI2), in the regulation of cholesterol trafficking in arterial smooth muscle cells. They discovered that PGI2 alters cholesterol delivery and hydrolysis by covalent phosphorylation, and cholesterol enrichment in vivo and in vitro reduced PGI2 synthesis and CE hydrolysis. Recently, they and others have reported that PGI2 synthesis is dependent upon the regulation of signal transduction pathways involving cell surface receptors containing intrinsic tryosine kinase domains or cell surface receptors linked to G-proteins. Activated G-proteins stimulate phospholipase A2 and/or phospholipase C/diglyceride lipase activities, resulting in arachidonic acid hydrolysis from cellular phospholipids, and subsequent conversion to PGI2 by cyclooxygenase and PGI2 synthase. Therefore, alterations in the regulation of processes leading to eicosanoid generation may subsequently effect cholesterol trafficking in normal and lipid-laden (foam) cells. Experiments are now designed to test the hypothesis that decreased PGI2 synthesis in CE-enriched cells is due to specific alterations in signal transduction pathways linked to G-protein activation. They will initially focus on the mechanisms by which eicosanoid generation is reduced following cholesterol-enrichment, a logical extension of the previous specific aims. They will determine if cholesterol-enrichment alters transcriptional or translational processes leading to reduction in cyclooxygenase expression. Next, since the mechanism by which G-protein activation stimulates SMC eicosanoid biosynthesis is currently unknown, they will determine the identity of, and the mechanisms by which, G-protein activation stimulates eicosanoid synthesis. To this end, they will define the basis for G-protein- mediation of eicosanoid metabolism, determine which G-proteins mediate eicosanoid synthesis, and determine which the relative role of phospholipase A2 and phospholipase C/diglyceride lipase in mediating eicosanoid biosynthesis following G-protein activation. Finally, they will assess the involvement of G-protein activation and its link to signal transduction pathways leading to eicosanoid synthesis during foam cell development, and examine the role of G- protein activation in cholesterol trafficking (uptake, hydrolysis, esterification, and efflux). These experiments will define the regulatory pathways leading to eicosanoid-mediated regulation of cellular cholesterol trafficking, and provide insights into novel interventions to stimulate cholesterol mobilization under conditions of hypercholesterolemia and arterial accumulation.
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