Macrophages incubated with mildly oxidized low density lipoprotein (OxLDL), aggregated low density lipoprotein (AggLDL), or cholesteryl ester-rich lipid dispersions (DISPs) accumulate cholesterol in lysosomes followed by an inhibition of lysosomal cholesteryl ester (CE) hydrolysis.

No abstract

Human macrophages incubated for prolonged periods with mildly oxidized LDL (oxLDL) or cholesteryl ester-rich lipid dispersions (DISP) accumulate free and esterified cholesterol within large, swollen lysosomes similar to those in foam cells of atherosclerosis. The cholesteryl ester (CE) accumulation is, in part, the result of inhibition of lysosomal hydrolysis due to increased lysosomal pH mediated by excessive lysosomal free cholesterol (FC). To determine if the inhibition of hydrolysis was long lived and further define the extent of the lysosomal defect, we incubated THP-1 macrophages with oxLDL or DISP to produce lysosome sterol engorgement and then chased with acetylated LDL (acLDL). Unlike oxLDL or DISP, CE from acLDL normally is hydrolyzed rapidly. Three days of incubation with oxLDL or DISP produced an excess of CE in lipid-engorged lysosomes, indicative of inhibition. After prolonged oxLDL or DISP pretreatment, subsequent hydrolysis of acLDL CE was inhibited. Coincident with the inhibition, the lipid-engorged lysosomes failed to maintain an acidic pH during both the initial pretreatment and subsequent acLDL incubation. This indicates that the alterations in lysosomes were general, long lived, and affected subsequent lipoprotein metabolism. This same phenomenon, occurring within atherosclerotic foam cells, could significantly affect lesion progression.

Mice deficient in scavenger receptor class B type I (SR-BI) and apolipoprotein E (apoE) [double knockout (DKO) mice] develop dyslipidemia, accelerated atherosclerosis, and myocardial infarction, and die prematurely. We examined effects of apoE and SR-BI deficiency on macrophage cholesterol homeostasis. DKO macrophages had increased total cholesterol (TC) stores (220-380 mg/mg protein) compared with apoE 2/2 cells (40 mg/mg), showed significant lysosomal lipid engorgement, and increased their TC by 34% after exposure to HDL. DKO macrophages from apoE 2/2 mice reconstituted with DKO bone marrow showed less cholesterol accumulation (89 mg/mg), suggesting that the dyslipidemia of DKO mice explains part of the cellular cholesterol defect. However, analyses of DKO and apoE 2/2 macrophages from transplanted apoE 2/2 mice revealed a role for macrophage SR-BI, inasmuch as the TC in DKO macrophages increased by 10% in the presence of HDL, whereas apoE 2/2 macrophage TC decreased by 33%. After incubation with HDL, the free cholesterol (FC) increased by 29% in DKO macrophages, and decreased by 8% in apoE 2/2 cells, and only DKO cells had FC in large peri-nuclear pools. Similar trends were observed with apoA-I as an acceptor. Thus, the abnormal cholesterol homeostasis of DKO macrophages is due to the plasma lipid environment of DKO mice and to altered trafficking of macrophage cholesterol. Both factors are likely to contribute to the accelerated atherosclerosis in DKO mice.-

Atherosclerosis is a complex, multifactorial disease. An early event in atherosclerosis is the development of macrophage foam cells. These are primarily formed through the uptake of modifi ed lipoproteins by macrophages within the artery wall ( 1-4 ). In late-stage atherosclerotic lesions, a large amount of both free cholesterol (FC) and cholesteryl ester (CE) accumulates in foam cell lysosomes. The presence of extensive amounts of CE suggests an interruption in the normal lysosomal hydrolysis of lipid particle-derived CE, while the accumulation of FC indicates an interruption in the normal removal of FC from lysosomes to other organelles, primarily the plasma membrane ( 5-7 ). Many studies indicate that lysosomal sequestration of cholesterol can have consequences for atherosclerotic lesion development (8)(9)(10)(11)(12)(13)(14)(15). In addition to a direct effect of accumulating sterol on lysosome function, trapping of sterol in lysosomes can prevent removal of cholesterol by effl ux. In fact, the cholesterol and CE in lysosomes remains trapped in lysosomes, even when further uptake of lipoproteins is halted and acceptor concentrations in the media are increased to levels that effl ux most of the nonlysosomal CE stores ( 16 ). Thus, the sequestration of sterol within lysosomes prevents effl ux and limits the availability of cholesterol to other intracellular processes ( 16 ). Therefore, factors that infl uence the removal of lysosomally sequestered sterol could have profound effects on foam cell biology and atherosclerotic lesion development.Many studies have examined macrophage foam cell metabolism in the presence of various CE-containing particles ( 2-4 ). However, triglyceride-rich particles (TRPs), including VLDL, are also present within the atherosclerotic Abbreviations: aggLDL, aggregated low density lipoprotein; CE, cholesteryl ester; DISP, lipid dispersions; FC, free cholesterol; LAMP-1, lysosomal-associated membrane protein; TG, triglyceride; TRP, triglyceride-rich particle; TPA, phorbol ester.