Uptake Of Native Low Density Lipoprotein Research Articles

Emerging roles of calpain proteolytic systems in macrophage cholesterol handling.

Calpains are Ca2+-dependent intracellular proteases that play central roles in the post-translational processing of functional proteins. In mammals, calpain proteolytic systems comprise the endogenous inhibitor calpastatin as well as 15 homologues of the catalytic subunits and two homologues of the regulatory subunits. Recent pharmacological and gene targeting studies in experimental animal models have revealed the contribution of conventional calpains, which consist of the calpain-1 and -2 isozymes, to atherosclerotic diseases. During atherogenesis, conventional calpains facilitate the CD36-dependent uptake of oxidized low-density lipoprotein (LDL), and block cholesterol efflux through ATP-binding cassette transporters in lesional macrophages, allowing the expansion of lipid-enriched atherosclerotic plaques. In addition, calpain-6, an unconventional non-proteolytic calpain, in macrophages reportedly potentiates pinocytotic uptake of native LDL, and attenuates the efferocytic clearance of apoptotic and necrotic cell corpses from the lesions. Herein, we discuss the recent progress that has been made in our understanding of how calpain contributes to atherosclerosis, in particular focusing on macrophage cholesterol handling.

Imatinib reduces cholesterol uptake and matrix metalloproteinase activity in human THP-1 macrophages.

Imatinib mesylate (Glivec®, formerly STI-571) is a selective tyrosine kinase inhibitor used for the treatment of chronic myeloid leukemia and gastrointestinal stromal tumors. However, there are reports suggesting that imatinib could be atheroprotective by lowering plasma low-density lipoprotein (LDL). To investigate the potential inhibitory effect of imatinib on cholesterol uptake in human macrophages as well as its effect on matrix metalloproteinase (MMP) activity. Uptake of fluorescence-labeled LDL was analyzed using flow cytometry. Macrophages treated with imatinib showed a 23.5%, 27%, and 15% decrease in uptake of native LDL (p<0.05), acetylated LDL (p<0.01), and copper-modified oxidized LDL (p<0.01), respectively. Gel-based zymography showed that secretion and activity of MMP-2 and MMP-9 were inhibited by imatinib. Using GeneChip Whole Transcript Expression array analysis, no obvious gene candidates involved in the mechanisms of cholesterol metabolism or MMP regulation were found to be affected by imatinib. Instead, we found that imatinib up-regulated microRNA 155 (miR155) by 43.8% and down-regulated ADAM metallopeptidase domain 28 (ADAM28) by 41.4%. Both genes could potentially play an atheroprotective role and would be interesting targets in future studies. Our results indicate that imatinib causes post-translational inhibition with respect to cholesterol uptake and regulation of MMP-2 and MMP-9. More research is needed to further evaluate the role of imatinib in the regulation of other genes and processes.

High-capacity selective uptake of cholesteryl ester from native LDL during macrophage foam cell formation

Macrophage foam cells are a defining pathologic feature of atherosclerotic lesions. Recent studies have demonstrated that at high concentrations associated with hypercholesterolemia, native LDL induces macrophage lipid accumulation. LDL particles are taken up by macrophages as part of bulk fluid pinocytosis. However, the uptake and metabolism of cholesterol from native LDL during foam cell formation has not been clearly defined. Previous reports have suggested that selective cholesteryl ester (CE) uptake might contribute to cholesterol uptake from LDL independently of particle endocytosis. In this study we demonstrate that the majority of macrophage LDL-derived cholesterol is acquired by selective CE uptake in excess of LDL pinocytosis and degradation. Macrophage selective CE uptake does not saturate at high LDL concentrations and is not down-regulated during cholesterol accumulation. In contrast to CE uptake, macrophages exhibit little selective uptake of free cholesterol (FC) from LDL. Following selective uptake from LDL, CE is rapidly hydrolyzed by a novel chloroquine-sensitive pathway. FC released from LDL-derived CE hydrolysis is largely effluxed from cells but also is subject to ACAT-mediated reesterification. These results indicate that selective CE uptake plays a major role in macrophage metabolism of LDL.

Native Low-Density Lipoprotein Uptake by Macrophage Colony-Stimulating Factor–Differentiated Human Macrophages Is Mediated by Macropinocytosis and Micropinocytosis

To examine the pinocytotic pathways mediating native low-density lipoprotein (LDL) uptake by human macrophage colony-stimulating factor-differentiated macrophages (the predominant macrophage phenotype in human atherosclerotic plaques). We identified the kinase inhibitor SU6656 and the Rho GTPase inhibitor toxin B as inhibitors of macrophage fluid-phase pinocytosis of LDL. Assessment of macropinocytosis by time-lapse microscopy revealed that both drugs almost completely inhibited macropinocytosis, although LDL uptake and cholesterol accumulation by macrophages were only partially inhibited (approximately 40%) by these agents. Therefore, we investigated the role of micropinocytosis in mediating LDL uptake in macrophages and identified bafilomycin A1 as an additional partial inhibitor (approximately 40%) of macrophage LDL uptake that targeted micropinocytosis. When macrophages were incubated with both bafilomycin A1 and SU6656, inhibition of LDL uptake was additive (reaching 80%), showing that these inhibitors target different pathways. Microscopic analysis of fluid-phase uptake pathways in these macrophages confirmed that LDL uptake occurs through both macropinocytosis and micropinocytosis. Our findings show that human macrophage colony-stimulating factor-differentiated macrophages take up native LDL by macropinocytosis and micropinocytosis, underscoring the importance of both pathways in mediating LDL uptake by these cells.

Native low-density lipoprotein uptake by Macrophage Colony-Stimulating Factor-differentiated human macrophages is mediated by macropinocytosis and micropinocytosis (134.12)

Abstract Human macrophage colony-stimulating factor (M-CSF)-differentiated macrophages—the predominant macrophage phenotype in human atherosclerotic plaques—constitutively take up native low-density lipoprotein (LDL) by fluid-phase pinocytosis, but the pinocytotic pathways mediating LDL uptake by these macrophages have not been thoroughly examined. We identified the kinase inhibitor SU6656 and the Rho GTPase inhibitor toxin B as inhibitors of macrophage fluid-phase pinocytosis of LDL. Assessment of macropinocytosis by time-lapse microscopy revealed that both drugs almost completely inhibited macropinocytosis, although LDL uptake and cholesterol accumulation by macrophages was only partially inhibited (~40%) by these agents. We therefore investigated the role of micropinocytosis in mediating LDL uptake in macrophages and identified bafilomycin A1 as an additional partial inhibitor (~40%) of macrophage LDL uptake that targeted micropinocytosis. When macrophages were incubated with both bafilomycin A1 and SU6656, inhibition of LDL uptake was additive (reaching 80%), showing that these inhibitors target different pathways. Microscopic analysis of fluid-phase uptake pathways in these macrophages confirmed that LDL uptake occurs through both macropinocytosis and micropinocytosis. Our findings show that human M-CSF-differentiated macrophages take up native LDL by macropinocytosis and micropinocytosis, underscoring the importance of both pathways in mediating LDL uptake by these cells.

Native low-density lipoprotein uptake by macrophage colony-stimulating factor differentiated macrophages is mediated by macropinocytosis and micropinocytosis

Native low-density lipoprotein uptake by macrophage colony-stimulating factor differentiated macrophages is mediated by macropinocytosis and micropinocytosis

Endothelial Lipase is Increased by Inflammation and Promotes LDL Uptake in Macrophages

Endothelial lipase (EL) is a member of the lipoprotein lipase family that regulates HDL metabolism. EL is known to act as a bridging molecule for monocytes or lipoproteins in vascular endothelial cells. We investigated the role and regulatory mechanisms of EL expression in macrophages. Macrophages originating from wild-type (EL+/+) and EL-deficient (EL-/-) mice were stimulated with lipopolysaccharide (LPS). The expression of EL mRNA was evaluated by northern blotting. DiI-LDL was used to measure the uptake of native low-density lipoprotein (nLDL). LPS increased EL mRNA levels by increasing intracellular oxidative stress in the macrophages. LPS did not affect EL expression in macrophages derived from Toll-like receptor 4 (TLR4) gene mutant mice, C3H/HeJ. The uptake of nLDL after LPS-treatment was significantly lower in macrophages from EL-/- mice than those from EL+/+ mice. Simvastatin suppressed the LPS-induced upregulation of EL expression and uptake of nLDL. EL expression is upregulated by LPS via TLR4 and promotes the uptake of nLDL by macrophages. Simvastatin inhibits the LPS-induced up-regulation and uptake in macrophages. Thus, our findings provide a novel role for EL in lipoprotein metabolism and would expand the range of anti-atherogenic effects of statins.

Constitutive Receptor-independent Low Density Lipoprotein Uptake and Cholesterol Accumulation by Macrophages Differentiated from Human Monocytes with Macrophage-Colony-stimulating Factor (M-CSF)

Recently, we have shown that macrophage uptake of low density lipoprotein (LDL) and cholesterol accumulation can occur by nonreceptor mediated fluid-phase macropinocytosis when macrophages are differentiated from human monocytes in human serum and the macrophages are activated by stimulation of protein kinase C (Kruth, H. S., Jones, N. L., Huang, W., Zhao, B., Ishii, I., Chang, J., Combs, C. A., Malide, D., and Zhang, W. Y. (2005) J. Biol. Chem. 280, 2352-2360). Differentiation of human monocytes in human serum produces a distinct macrophage phenotype. In this study, we examined the effect on LDL uptake of an alternative macrophage differentiation phenotype. Differentiation of macrophages from human monocytes in fetal bovine serum with macrophage-colony-stimulating factor (M-CSF) produced a macrophage phenotype demonstrating constitutive fluid-phase uptake of native LDL leading to macrophage cholesterol accumulation. Fluid-phase endocytosis of LDL by M-CSF human macrophages showed non-saturable uptake of LDL that did not down-regulate over 48 h. LDL uptake was mediated by continuous actin-dependent macropinocytosis of LDL by these M-CSF-differentiated macrophages. M-CSF is a cytokine present within atherosclerotic lesions. Thus, macropinocytosis of LDL by macrophages differentiated from monocytes under the influence of M-CSF is a plausible mechanism to account for macrophage foam cell formation in atherosclerotic lesions. This mechanism of macrophage foam cell formation does not depend on LDL modification or macrophage receptors.

Endogenously produced lipoprotein lipase enhances the binding and cell association of native, mildly oxidized and moderately oxidized low-density lipoprotein in mouse peritoneal macrophages

It has been well established that purified lipoprotein lipase (LPL) can facilitate the cellular uptake of various native and modified lipoproteins when added exogenously to macrophages. Because activated macrophages express LPL endogenously, it was the aim of this study to investigate the effect of macrophage-produced LPL on the uptake of native low-density lipoprotein (LDL) and LDL that has been modified to various degrees by Cu(2+)-mediated oxidation. Cell binding and uptake of Eu(3+)-labelled native and oxidized LDL was determined in mouse peritoneal macrophages (MPM) from normal mice and induced mutant mice that lack LPL expression in MPM. We found that LPL expressed by MPM was able to increase cell binding and association of native LDL (by 121% and 101% respectively), mildly oxidized LDL (by 47% and 43%) and moderately oxidized LDL (by 30% and 22%). With increased levels of lipoprotein oxidation, the relative proportion of LPL-mediated LDL uptake decreased. This decrease was not due to weakened binding of LPL to oxidized LDL. The drastically increased uptake of highly oxidized LDL in MPM by scavenger-receptor-mediated pathways might dominate the simultaneous exogenous or endogenous LPL-mediated uptake of this lipoprotein. Competition experiments with positively charged poly(amino acids) furthermore suggested that histidine, arginine and lysine residues in LPL are important for the interaction between LDL and LPL. Our results imply that physiological levels of LPL produced by macrophages facilitate the uptake of native LDL as well as mildly and moderately oxidized LDL. This process might, in the micro-environment of arteries, contribute to the accumulation of macrophage lipids and the formation of foam cells.

Induction of macrophage foam cell formation by Chlamydia pneumoniae.

Foam cell formation is the hallmark of early atherosclerosis. It was found that the intracellular bacterium Chlamydia pneumoniae induces foam cell formation by human monocyte-derived macrophages. Exposure of macrophages to C. pneumoniae followed by low-density lipoprotein (LDL) caused a marked increase in the number of foam cells and accumulation of cholesteryl esters. Foam cell formation was not inhibited by the antioxidant butylated hydroxytoluene nor fucoidan, suggesting that lipid accumulation did not involve scavenger receptors. In contrast, addition of heparin, which blocks binding of LDL to the LDL receptor, inhibited C. pneumoniae-induced foam cell formation, suggesting that the pathogen induced lipid accumulation by dysregulating native LDL uptake or metabolism (or both). These data demonstrate that an infectious agent can induce macrophage foam cell formation and implicate C. pneumoniae as a causative factor in atherosclerosis.

Corticotropin increases the receptor-specific uptake of native low-density lipoprotein (LDL)—but not of oxidized LDL and native or oxidized lipoprotein(a) [LP(a)]—in HEPG2 cells: No evidence for Lp(a) catabolism via the LDL-receptor

To understand the interaction of corticotropin (ACTH) and lipid catabolism, we analyzed the influence of ACTH on receptor-mediated lipoprotein uptake and compared the uptake and degradation of human native (N-LDL) and oxidized (Ox-LDL) low-density lipoprotein and native (N-Lp(a)) and oxidized (Ox-Lp(a)) lipoprotein(a) by human hepatoma (HepG2) cells. The receptor affinity of N-LDL, Ox-LDL, N-Lp(a), and Ox-Lp(a) was comparable ( K d , 33, 13, 24, and 13 μg/mL medium), whereas the maximum degradative capacity was 10.5-fold higher in N-LDL (V max, 1,978 ng/mg cell cell protein) compared with Ox-LDL (189 ng/mg). In N-LDL, it was 4.5-fold higher than in N-Lp(a) (442 ng/mg) and eightfold higher than in Ox-Lp(a) (246 ng/mg) ( P < .05). Addition of ACTH to the cell cultures increased receptor-specific degradation of N-LDL by 44% (2,866 v 1,978 ng/mg, P < .05), whereas changes in Ox-LDL, N-Lp(a), and Ox-Lp(a) showed no significant increase. No differences in uptake specificity were observed with or without ACTH. In addition, a 12-hour preincubation of liver cells with LDL increased Lp(a) uptake by 40% to 50% with (411 v 625 ng/mg) and without (393 v 558 ng/mg) ACTH administration. These data indicate that ACTH elevates receptor-specific uptake of N-LDL, but only to a low extent versus Ox-LDL, N-Lp(a), or Ox-Lp(a). These results support the hypothesis that catabolism of oxidized lipoproteins and Lp(a) through the LDL receptor pathway is only a minor route of lipid metabolism, whereas LDL receptor activity itself can be stimulated by ACTH.

Metabolism of Native and Naturally Occurring Multiple Modified Low Density Lipoprotein in Smooth Muscle Cells of Human Aortic Intima

The subfraction of low density lipoprotein (LDL) with low sialic acid content that caused accumulation of cholesterol esters in human aortic smooth muscle cells has been found in the blood of coronary atherosclerosis patients. It was demonstrated that this subfraction consists of LDL with small size, high electronegative charge, reduced lipid content, altered tertiary structure of apolipoprotein B, etc. LDL of this subfraction is naturally occurring multiple-modified LDL (nomLDL). In this study we compared the binding, uptake and proteolytic degradation of native LDL and nomLDL by smooth muscle cells cultured from human grossly normal intima, fatty streaks, and atherosclerotic plaques. Uptake of nomLDL by normal and atherosclerotic cells was 3.5- and 6-fold, respectively, higher than uptake of native LDL. Increased uptake of nomLDL was due to increased binding of this LDL by intimal smooth muscle cells. The enhanced binding is explained by the interaction of nomLDL with cellular receptors other than LDL-receptor. Modified LDL interacted with the scavenger receptor, asialoglycoprotein receptor, and also with cell surface proteoglycans. Rates of degradation of nomLDL were 1.5- and 5-fold lower than degradation of native LDL by normal and atherosclerotic cells, respectively. A low rate of nomLDL degradation was also demonstrated in homogenates of intimal cells. Activities of lysosomal proteinases of atherosclerotic cells were decreased compared with normal cells. Pepstatin A, a cathepsin D inhibitor, completely inhibited lipoprotein degradation, while serine, thiol, or metallo-proteinase inhibitors had partial effect. This fact reveals that cathepsin D is involved in initial stages of apoB degradation by intimal smooth muscle cells. Obtained data show that increased uptake and decreased lysosomal degradation of nomLDL may be the main cause of LDL accumulation in human aortic smooth muscle cells, leading to foam cell formation.

Involvement of the macrophage low density lipoprotein receptor-binding domains in the uptake of oxidized low density lipoprotein.

Macrophages, unlike most other cells, possess both low density lipoprotein (LDL) and scavenger receptors. The scavenger receptor has been shown to mediate the uptake of oxidized LDL (ox-LDL), which ultimately leads to cholesterol loading of the macrophages. The present study was undertaken to define epitopes on ox-LDL that are important for lipoprotein binding to macrophages and to ascertain whether ox-LDL can bind to the LDL receptor. Monoclonal antibodies (Mabs) directed against several epitopes along the apolipoprotein B-100 (apo B-100) molecule were used. LDL (300 micrograms/ml) was oxidized by incubation with 10 microM CuSO4 for 24 hours. Ox-LDL, as opposed to acetylated LDL (ac-LDL), reacted with Mabs directed against the LDL receptor-binding domains (Mabs B1B6 and B1B3). Similarly, uptake of ox-LDL but not ac-LDL by a murine J774 macrophage-like cell line was inhibited by as much as 40% after using Mab B1B6. The anti-LDL receptor antibody IgG-C7 also inhibited 125I-ox-LDL uptake by macrophages by 60%. Chromatography on heparin-Sepharose columns of LDL that was partially oxidized for only 3 hours resulted in two fractions: an unbound fraction with characteristics similar to those of ox-LDL and a bound fraction similar to native LDL. Macrophage degradation of the unbound fraction was inhibited by Mab IgG-C7 and Mab B1B6, which are directed toward the LDL receptor and the LDL receptor-binding domains on apo B-100, respectively. When incubated with three types of macrophages, J774 macrophage cells, mouse peritoneal macrophages, and human monocyte-derived macrophages, excess amounts of unlabeled ox-LDL, like native LDL but unlike ac-LDL, substantially suppressed the uptake and degradation of 125I-labeled LDL. Similar studies with fibroblasts, however, revealed that unlabeled LDL but not unlabeled ox-LDL or ac-LDL competed with 125I-LDL for cellular uptake and degradation. Mab directed against epitopes on the amino terminus domain of apo B-100 (C14) demonstrates a similar immunoreactivity with ox-LDL and native LDL but a much lower reactivity with ac-LDL. Mab C14 inhibited macrophage degradation of ox-LDL by 34% but had no inhibitory effect on the uptake of native LDL or ac-LDL. Thus, the ac-LDL and LDL receptor-binding domains as well as a unique epitope on the amino terminus of apo B-100 may be involved in macrophage binding of ox-LDL.(ABSTRACT TRUNCATED AT 400 WORDS)

In vivo and in vitro catabolism of native and biologically modified LDL

Incubation of human low density lipoprotein (LDL) at 37°C in the presence of human umbilical-vein endothelial cells (EC) causes a time-dependent shift in the charge and density of LDL. After intravenous injection into rats, native LDL is merely cleared from the circulation by Kupffer cells while EC-modified LDL is rapidly cleared by endothelial liver cells. The uptake of native LDL by Kupffer cells and EC-modified LDL by endothelial cells in vivo can be explained by the presence of two different specific receptors on these cell types. It is concluded that the liver endothelial cells form an important protection against a possible atherogenic action of EC-modified LDL.

Nonenzymatic glucosylation of low-density lipoprotein alters its biologic activity.

Human low-density lipoprotein (LDL) was glucosylated by incubation in vitro with glucose (20-80 mM) with or without addition of cyanoborohydride. The incorporation of covalently bound glucose was linear over time, and amino acid analysis showed the presence of glucosyllysine residues. The glucosylated LDL (glc LDL) moved more rapidly than normal LDL on agarose electrophoresis. The rate of degradation of 125I-labeled glucosylated LDL (glc LDL) by cultured human fibroblasts was reduced compared with that of native I-LDL, the difference increasing with extent of glucosylation. Effects were seen with blockage of as few as 6-15% of the LDL lysine residues; high-affinity degradation was completely lost when one-third of the lysine residues were blocked. Conjugation of LDL with glucose-6-phosphate also blocked high-affinity uptake and degradation. Whereas native LDL uptake inhibited the activity of beta-hydroxy-beta-methylglutaryl coenzyme A reductase and stimulated acyl coenzyme A:cholesterol acyltransferase activity, glc LDL had no effects on these enzymes. The fractional catabolic rate of glc LDL in guinea pigs was reduced. Degradation of glc LDL by mouse peritoneal macrophages was not significantly faster than that of native LDL. Finally, the presence of glc LDL in human plasma was demonstrated. Preliminary data show that 1.3% of lysine residues in normal LDL and 2-5.3% of lysines in diabetic LDL were glucosylated. Since, like other plasma proteins, LDL undergoes glucosylation in diabetes, its turnover and sites of catabolism may differ from normal and this may be relevant to the accelerated atherosclerosis of diabetes.

Metabolism of native and of lactosylated human low density lipoprotein: evidence for two pathways for catabolism of exogenous proteins in rat hepatocytes.

Human low density lipoprotein (LDL) covalently conjugated with 200-250 residues of lactose per LDL particle (Lac-LDL) was bound and rapidly taken up by the galactose-specific receptor of rat hepatocytes. Uptake of Lac-LDL was associated with inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase and stimulation of cholesterol esterification. Uptake of native human LDL had no significant effects on these enzyme activities even when the rates of LDL uptake equaled those of Lac-LDL. When injected into rats, Lac-LDL was selectively removed by the liver (98% of injected dose). The hepatic subcellular distribution of simultaneously injected native 125I-labeled LDL and 131I-labeled Lac-LDL differed significantly, Lac-LDL was associated with fractions enriched in lysosomal hydrolases whereas native LDL was found predominantly in the supernatant fraction enriched in lactate dehydrogenase. Chloroquine (0.1 mM) markedly suppressed uptake of Lac-LDL by cultured rat hepatocytes (> 80%) but had only a small effect on uptake of native LDL. Leupeptin (0.625 mM) inhibited degradation of Lac-LDL more than it did degradation of native LDL. Colchicine (0.25 microM) dramatically suppressed uptake of Lac-LDL (> 70%) but did not affect native LDL uptake even at concentrations as high as 10 microM. Uptake of human LDL by rat hepatocytes occurs largely by nonspecific mechanisms, including fluid endocytosis, whereas Lac-LDL, as shown here, is taken up by a specific receptor-mediated mechanism. The results show further that native human LDL, representing an example of a protein taken up nonspecifically, is processed intracellularly by a pathway qualitatively distinct from that for Lac-LDL, an example of a protein taken up by a specific mechanism. Lac-LDL may serve as a vehicle for specifically delivering drugs, hormones, or radioactive compounds to hepatocytes for therapeutic or diagnostic purposes.

Metabolism of cationized lipoproteins by human fibroblasts: biochemical and morphologic correlations

Human plasma low density lipoprotein (LDL) that had been rendered polycationic by coupling with N, N-dimethyl-1, 3-propanediamine (DMPA) was shown by electron microscopy to bind in clusters to the surface of human fibroblasts. The clusters resembled those formed by polycationic ferritin (DMPA-feritin), a visual probe that binds to anionic site on the plasma membrane. Biochemical studies with (125)I-labeled DMPA-LDL showed that the membrane-bound lipoprotein was internalized and hydrolyzed in lysosomes. The turnover time for cell bound (125)I-DMPA-LDL, i.e., the time in which the amount of (125)I-DMPA-LDL degraded was equal to the steady-state cellular content of the lipoprotein, was about 50 h. Because the DMPA-LDL gained access to fibroblasts by binding nonspecifically to anionic sites on the cell surface rather than by binding to the physiologic LDL receptor, its uptake failed to be regulated under conditions in which the uptake of native LDL was reduced by feedback suppression of the LDL receptor. As a result, unlike the case with native LDL, the DMPA-LDL accumulated progressively within the cell, and this led to a massive increase in the cellular content of both free and esterified cholesterol. Studies with (14)C-oleate showed that at least 20 percent of the accumulated cholesteryl esters represented cholesterol that had been esterified within the cell. After 4 days of incubation with 10 mug/ml of DMPA-LDL, fibroblasts had accumulated so much cholesteryl ester that neutral lipid droplets were visible at the light microscope level with Oil Red O staining. By electron microscopy, these intracellular lipid droplets were observed to lack a tripartite limiting membrane. The ability to cause the overaccumulation of cholesteryl esters within cells by using DMPA-LDL provides a model system for study of the pathologic consequences at the cellular level of massive deposition of cholesteryl ester.