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This investigation has elucidated a mechanism for development of macrophage foam cells when macrophages are incubated with native low density lipoprotein (LDL). LDL is believed to be the main source of cholesterol that accumulates in monocyte-derived macrophages within atherosclerotic plaques, but native LDL has not previously been shown to cause substantial cholesterol accumulation when incubated with macrophages. We have found that activation of human monocyte-derived macrophages with phorbol 12-myristate 13-acetate ( Engorgement of macrophages with cholesterol is the defining pathological characteristic of atherosclerotic plaques, the cause of most heart attacks and strokes. Cholesterol accumulation in macrophages not only contributes to cholesterol retention within the vessel wall, but also alters macrophage biology. Cholesterol-loaded macrophages secrete plaque-disrupting matrix metalloproteinases, and produce tissue factor that promotes thrombosis when plaques rupture (1-3). Thus, how macrophages accumulate cholesterol and become foam cells has been the subject of intense investigation.Low density lipoprotein (LDL), 1 the main carrier of plasma cholesterol, enters the vessel wall and then by some mechanism enters macrophages. Previously, native LDL could not be shown to cause foam cell formation because the cellular receptor that binds LDL is poorly expressed on differentiated macrophages and down-regulates during cholesterol uptake, limiting total cholesterol accumulation (4 -7). Moreover, the LDL receptor is not expressed in human atherosclerotic plaques (8). Thus, most previous studies of macrophage foam cell formation have focused on modifying LDL in some way that increases its binding to macrophages. Increased macrophage binding of LDL has been achieved with chemical modifications to the apoB component of the LDL, aggregation of LDL induced by either vortexing or treatment of LDL with lipases, and complexing of LDL with other molecules, for example, glycosaminoglycans or antibodies, which bind macrophages and promote LDL uptake by piggyback endocytosis (9). Macrophages take up modified LDL by receptormediated endocytosis in pinocytotic vesicles, phagocytic vacuoles, or patocytic surface-connected compartments (9).One popular hypothesis of foam cell formation involves LDL oxidation. LDL oxidation promotes macrophage LDL uptake that is mediated by various macrophage scavenger receptors (10). Although oxidation of LDL has important biological effects that could influence atherosclerotic plaque development (11), oxidation of LDL does not readily explain foam cell formation. Incubation of human monocyte-derived macrophages with oxidized LDL, even strongly oxidized with artificial chemical systems, produces little macrophage cholesterol accumulation (12, 13). Also, oxidized LDL is poorly metabolized within lysosomes of macrophages because of partial inactivation by oxidized LDL of the lysosomal enzymes that degrade LDL (14 -16). This limits the capacity of oxidized LDL to induce acyl-CoA:cholesterol acyltra...

Human monocyte-derived macrophages can efflux accumulated cholesterol without exogenously added cholesterol acceptors (Kruth, H. S., Skarlatos, S. I., Gaynor, P. M., and Gamble, W. (1994) J. Biol. Chem. 269, 24511-24518). Most of the effluxed cholesterol accumulates in the medium as apolipoprotein E-discoidal lipid particles. In the current study, we determined whether and to what degree cholesterol efflux from human monocytemacrophages depended on apolipoprotein E secretion. Unexpectedly, 2-week-old differentiated monocyte-macrophages secreted similar amounts of apolipoprotein E without or with cholesterol enrichment. Apolipoprotein E mRNA levels in these macrophages were not increased by cholesterol enrichment and were comparable with levels in HepG2 cells. Without cholesterol enrichment, monocyte-macrophages secreted lipid-poor apolipoprotein E with a density >1.21 g/ml. By contrast, cholesterol enrichment of monocyte-macrophages induced the association of apoE with phospholipid and cholesterol to form discoidal particles that floated at densities of 1.08 -1.10 g/ml. An anti-apolipoprotein E monoclonal antibody added to the culture medium significantly inhibited cholesterol and phospholipid efflux from the monocytemacrophages. This showed that apolipoprotein E was required for most of the cholesterol efflux, and that apolipoprotein E did not leave macrophages with lipid but rather associated with lipid after it was secreted. Thus, 1) apolipoprotein E was constitutively secreted by differentiated human monocyte-macrophages, 2) apolipoprotein E only formed discoidal particles following macrophage cholesterol enrichment, 3) apolipoprotein E was necessary for cholesterol efflux to occur in the absence of added cholesterol acceptors and, in addition 4) the level of macrophage unesterified cholesterol was not rate-limiting for this cholesterol efflux, and 5) net phospholipid synthesis occurred in macrophages secondary to apoE-mediated loss of macrophage phospholipid. In conclusion, apolipoprotein E functions in an autocrine pathway that mediates cholesterol efflux from human monocyte-derived macrophages. Apolipoprotein (apo)1 E shows an anti-atherogenic effect by facilitating clearance of atherogenic remnant lipoproteins from the plasma (1, 2). Recent studies suggest that apoE may be anti-atherogenic by additional mechanisms. Transgenic expression of apoE restricted to the blood vessel wall (3) or to macrophages (including those in the vessel wall) (4) inhibits development of atherosclerotic lesions in mice, even without altering plasma cholesterol levels. This suggests that expression of apoE locally within the vessel wall is sufficient to produce an anti-atherogenic effect. One mechanism by which locally produced apoE could be anti-atherogenic is by promoting reverse cholesterol transport from the vessel wall. This could occur through the association of apoE with plasma-derived high density lipoprotein (HDL) which increases HDL's capacity to carry cholesterol (5, 6). On the other hand, it is possible that apoE co...