Arterial prostacyclin synthesis is reduced in human atherosclerotic disease, and in animal models of atherosclerosis. However, the relationship between eicosanoids and vascular cholesterol metabolism is not clear. Experiments proposed are designed to: (i) establish an in vitro model of cholesterol accumulation by exposure of vascular smooth muscle cells to cationized LDL; (ii) determine if eicosanoid metabolism is reduced by arterial smooth muscle cells enriched in cholesterol; (iii) determine the putative mechanism(s) that cholesterol- enrichment alters eicosanoid synthetic capacity. Potential mechanisms to be studied include alterations in cellular fatty acid composition, arachidonic acid availability to cellular phospholipases, or inhibition of phospholipase A2 and C, cyclooxygenase, and/or prostacyclin synthetase. Plasma high density lipoproteins (HDL) promote net cholesterol efflux and stimulate prostacyclin and PGE2 synthesis from vascular smooth muscle cells in vitro. Prostacyclin and its hydrolytic metabolites promote net cholesterol efflux by increasing neutral and acid cholesteryl esterase (NCEH and ACEH) activities in intact smooth muscle cells. Experiments proposed are designed to (i) determine if HDL-induced eicosanoid synthesis in part mediates HDL-induced cholesterol efflux; and (ii) determine if the mechanisms of HDL-induced cholesterol efflux is due to eicosanoid-mediated alterations in ACEH, ACAT and NCEH activities. Endothelial cells (which are a major arterial source of eicosanoids) alter cholesterol metabolism in co-cultured arterial vascular smooth muscle cells. Experiments proposed are designed to (i) determine if endothelial cells modulate cholesterol content and ACEH, ACAT, and NCEH activities in cholesterol-enriched smooth muscle cells, (ii) determine if this effect is dependent upon endothelial-derived eicosanoids; and (iii) determine if HDL enhances endothelial cell-mediated cholesterol efflux from cholesterol-enriched smooth muscle cells via eicosanoid dependent mechanisms. Results from these experiments will further define the cellular mechanisms by which eicosanoids alter cholesterol metabolism in arterial smooth muscle cells.
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