OBJECTIVE: During atherogenesis, macrophages migrate into the subendothelial space where they ingest deposited lipoproteins, accumulate lipids, and transform into foam cells. It is unclear why these macrophages do not remove their lipid loads from the region. This study was aimed at testing the hypothesis that macrophage behavior is altered when membrane cholesterol levels are elevated, as might be the case for cells in contact with lipoproteins within atherosclerotic lesions. METHODS AND RESULTS: We examined the effects of elevating membrane cholesterol on macrophage behavior. J774 macrophages were treated with either acetylated low-density lipoprotein (ac-LDL) and ACAT inhibitor or cholesterol-chelated methyl-beta-cyclodextrin (chol-MbetaCD) to increase membrane cholesterol levels. Our results show that elevating the membrane cholesterol of J774 macrophages induced dramatic ruffling, stimulated cell spreading, and affected F-actin organization. Cellular adhesion was required for these effects, and Rac-mediated signaling pathways were involved. Additionally, 3-dimensional transwell chemotaxis assays showed that migration of J774 macrophages was significantly inhibited when membrane cholesterol levels were raised. CONCLUSIONS: These findings indicate that increased membrane cholesterol causes dramatic effects on macrophage cellular functions related to the actin cytoskeleton. They should provide new insights into the early steps of atherogenesis.
