Plasma membrane cholesterol and monocyte /macrophage function

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Early in the pathogenesis of atherosclerosis, aggregated, oxidized lipoprotein particles become attached to proteins in the subendothelial matrix of the affected arteries. Monocytes (Mos) that traverse the overlying endothelium come into contact with these matrix-bound lipoprotein particles, engulf them, and eventually take up large amounts of lipid, developing into lipid-engorged macrophage foam cells, which promote further growth of the atherosclerotic plaque. We hypothesize that alterations in the plasma membrane cholesterol levels in Mos and macrophages caused by interactions with these matrix-bound lipoproteins may be partially responsible for lesion growth. Based on our preliminary studies, we postulate that retained and aggregated lipoproteins alter actin dynamics in Mos and macrophages in the subendothelial space, and therefore inhibit the migration and phagocytic function of these cells. This may promote the retention of cells in the tissue and thus the growth of atherosclerotic plaques. The specific aims of this grant are designed to test this hypothesis. Numerous studies have investigated the effects of reducing membrane cholesterol levels on cellular functions, but far fewer have looked at the effects of the potentially more physiologically significant event of increasing membrane cholesterol levels. Our preliminary data indicate that overloading a Mo's plasma membrane with cholesterol decreases its migration rate in a three dimensional (3D) collagen gel. Similarly, our studies with neutrophils showed that cholesterol depletion inhibited Rac GTPase-mediated actin reorganization and migration. We propose to first quantify the extent to which stimulated actin reorganization in Mos and macrophages is sensitive to raising or lowering membrane cholesterol levels, and then we will study how these changes affect the actin-dependent functions of migration and phagocytosis. Next, we will directly test the idea that effects on migration and phagocytic function can be attributed to disruption of activation and/or targeting of Rho GTPases (i.e. Rho, Rac, and Cdc42). Initially we will use pharmacological means to alter membrane cholesterol, and later we will use progressively more physiological cholesterol modulating scenarios from isolated lipoproteins to matrix-embedded lipoproteins that mimic an atherogenic environment.