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.

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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.

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Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.

A hallmark feature of the atherosclerotic lesion is the presence of cholesterol engorged macrophages called foam cells because of the foamy appearance that lipid imparts to their cytoplasm 1 . Cytochemical inspection reveals that much of this cholesterol accumulates within large swollen lysosomes 2 . Although the cholesterol in the atherosclerotic lesion is primarily derived from LDL, it must be modified by the lesion environment before LDL can produce the massive cholesterol accumulation seen in atherosclerosis 3 . Oxidation of LDL is just such a modification and we have mimicked this lysosomal cholesterol accumulation by treating macrophages in culture with mildly oxidized LDL (ox-LDL). The cholesterol in LDL particles is primarily in the form of cholesteryl esters (CE). Following uptake by receptor mediated endocytosis, this CE is delivered to lysosomes/late endosomes for hydrolysis to free cholesterol (FC) and fatty acids 2 . The lysosomal cholesterol accumulation from ox-LDL occurs in two phases, an initial phase where lysosomal hydrolysis occurs normally but the resulting FC is trapped in lysosomes and a second phase where lysosomal CE hydrolysis is inhibited 4 . The result is an accumulation of both FC and CE in lysosomes. This cholesterol is trapped and not available for further metabolism by the cell, presenting grave consequences for the cell and potentially accelerating development of the atherosclerotic lesion.Curiously, another modification of LDL, acetylation, does not produce lysosomal cholesterolengorgement, leading to speculation that it is the oxidized lipids which disrupt lysosomal hydrolysis 5 . Ox-LDL is not the only modified form of cholesterol found in atherosclerotic lesions and so in the present study we investigate uptake and metabolism by macrophages of two non-oxidized modified lipids; small aggregates of LDL (agg-LDL) and CE-rich lipid dispersions (DISP) which were formed from sonicating together phosphatidylcholine, phosphatidylserine, and CE. Cellular cholesterol accumulation was analyzed by biochemical measures of FC and CE mass and the rate of CE hydrolysis and by fluorescence, brightfield, and electron microscopy.Incubation of the human macrophage cell line, THP-1, with either agg-LDL (Figure 1) or DISP produced cellular cholesterol accumulation of greater than 300 ug total cholesterol/mg cell protein as both FC and CE. For the first two days of incubation, cells treated with agg-LDL continued to generate some FC via lysosomal hydrolysis although lysosomal CE also accumulated. However, after the second day, greater than 95% of the cholesterol accumulating in agg-LDL-treated cells occurred as unhydrolyzed lipoprotein in lysosomes. DISP treatment also produced both FC and CE accumulation but the rate of accumulation occurred faster and less FC was generated. The timing of the shutdown of hydrolysis appeared related to the rate of initial free cholesterol accumulation.Measurement of lysosomal pH revealed that the cholesterol engorged lysosomes failed to maintain an acidic pH, sugge...