(229l) Impact of Membrane Cholesterol on Macrophage Biomechanics

Monocytes/macrophages play a critical role in the pathogenesis of atherosclerosis, and cholesterol has been shown to greatly influence the development and progression of the disease, Although cholesterol is known to affect the various stages of atherosclerotic process from monocyte recruitment to atherosclerotic foci to foam cell formation, the mechanisms are not well understood. Plasma membrane contain majority of the total cellular content, and is the entry point of dietary cholesterol that infiltrate the macrophages. We hypothesized that cellular cholesterol affects macrophage recruitment and transmigration by altering the biophysical properties. To test this hypothesis, we investigated the effect of cholesterol depletion and enrichment on membrane stiffness, lipid raft and receptor clustering, cytoskeletal deformability, and its consequences on monocyte recruitment, transmigration and chemotaxis. We observed that a decrease in cholesterol levels resulted in an increase in membrane fluidity and uniformity in lipid raft distribution but a decrease in overall cellular deformability as measured by atomic force microscopy, and increase in actin polymerization and cell spreading. The changes in the distribution of adhesion receptors CD44 followed the lipid raft distribution. As a consequence, we observed that cholesterol-depleted cells rolled slowly and more uniformly on E-selectin coated surfaces under shear flow. We also observed that a decrease in cholesterol levels resulted in a decrease in the transmigration of monocytes to chemotactic gradient triggered by MCP-1, due to a redistribution of the cognate receptor, CCR2. These effects were reversed when the cells were enriched with cholesterol. Together, our data suggests that the biophysical regulation of monocyte/macrophage behavior by membrane cholesterol levels may influence cellular interactions that lead to atherosclerosis.