Reduced atherosclerosis in hormone-sensitive lipase transgenic mice overexpressing cholesterol acceptors

Macrophage-specific overexpression of cholesteryl ester hydrolysis in hormone-sensitive lipase transgenic (HSL Tg) female mice paradoxically increases cholesterol esterification and cholesteryl ester accumulation in macrophages, and thus susceptibility to diet-induced atherosclerosis compared to nontransgenic C57BL/6 mice. The current studies suggest that whereas increased cholesterol uptake could contribute to transgenic foam cell formation, there are no differences in cholesterol synthesis and the expression of cholesterol efflux mediators (ABCA1, ABCG1, apoE, PPARgamma, and LXRalpha) compared to wild-type macrophages. HSL Tg macrophages exhibit twofold greater efflux of cholesterol to apoA-I in vitro, suggesting the potential rate-limiting role of cholesteryl ester hydrolysis in efflux. However, macrophage cholesteryl ester levels appear to depend on the relative efficacy of alternate pathways for free cholesterol in either efflux or re-esterification. Thus, increased atherosclerosis in HSL Tg mice appears to be due to the coupling of the efficient re-esterification of excess free cholesterol to its limited removal mediated by the cholesterol acceptors in these mice. The overexpression of cholesterol acceptors in HSL-apoA-IV double-transgenic mice increases plasma HDL levels and decreases diet-induced atherosclerosis compared to HSL Tg mice, with aortic lesions reduced to sizes in nontransgenic littermates. The results in vivo are consistent with the effective efflux from HSL Tg macrophages supplemented with HDL and apoA-I in vitro, and highlight the importance of cholesterol acceptors in inhibiting atherosclerosis caused by imbalances in the cholesteryl ester cycle.     

Hormone-sensitive lipase is required for high-density lipoprotein cholesteryl ester-supported adrenal steroidogenesis

Steroid hormones are synthesized using cholesterol as precursor, with a substantial portion supplied by the selective uptake of lipoprotein-derived cholesteryl esters. Adrenals express a high level of neutral cholesteryl ester hydrolase activity, and recently hormone-sensitive lipase (HSL) was shown to be responsible for most adrenal neutral cholesteryl ester hydrolase activity. To determine the functional importance of HSL in adrenal steroidogenesis, adrenal cells were isolated from control and HSL-/- mice, and the in vitro production of corticosterone was quantified. Results show that, even though adrenal cholesteryl ester content was substantially elevated in both male and female HSL-/- mice, basal corticosterone production was reduced approximately 50%. The maximum corticosterone production induced by dibutyryl cAMP, and lipoproteins was approximately 75-85% lower in adrenal cells from HSL-/- mice compared with control. There is no intrinsic defect in the conversion of cholesterol into steroids in HSL-/- mice. Dibutyryl cAMP-stimulated conversion of high-density lipoprotein cholesteryl esters into corticosterone was reduced 97% in HSL-/- mice. An increase in low-density lipoprotein receptor expression appears to be one of the compensatory mechanisms for cholesterol delivery in HSL-/- mice. These findings suggest that HSL is functionally linked to the selective pathway and is critically involved in the intracellular processing and availability of cholesterol for adrenal steroidogenesis.     

A defect in mobilization of cholesteryl esters in rabbit macrophages

Macrophages provide an important way for cholesteryl esters to accumulate in tissues in pathologic amounts. We studied cholesteryl ester metabolism in thioglycollate-induced peritoneal macrophages obtained from normocholesterolemic and hypercholesterolemic rabbits. The macrophage preparations from normocholesterolemic rabbit (MN cells) had 26 nmol esterified cholesterol/mg cellular protein, incorporated 1 nmol of labeled oleate into cholesteryloleate/2 h per mg cellular protein and had an acyl-coenzyme A:cholesterol acyltransferase activity of 22 pmol cholesterylpalmitate formed/min per mg protein in isolated membranes. The macrophage preparations from hypercholesterolemic rabbits (MHC cells) contained a 12-fold greater mass of cholesteryl ester, had an 8-times higher rate of formation of cholesteryloleate, and had 3-times more acyl-coenzyme A:cholesterol acyltransferase activity in the isolated membranes. When a cholesterol acceptor (10% fetal bovine serum or 10 mg of lipid-free fetal bovine serum protein) was added to the culture medium of rabbit MHC cells, the MHC cells retained more than 70% of their cholesteryl esters after 48 h of incubation. In contrast, when a cholesterol acceptor (10% fetal bovine serum) was added to the medium of thioglycollate-induced, cholesterol-enriched macrophages from mice, the mice macrophages retained only 19% of their cholesteryl esters after 48 h of incubation. The limited capacity of rabbit macrophages to release unesterified cholesterol from stored cytoplasmic cholesteryl esters to an exogenous acceptor may be related to the propensity of rabbits to develop atherosclerotic lesions.     

Identification of neutral cholesterol ester hydrolase, a key enzyme removing cholesterol from macrophages

Unstable lipid-rich plaques in atherosclerosis are characterized by the accumulation of macrophage foam cells loaded with cholesterol ester (CE). Although hormone-sensitive lipase and cholesteryl ester hydrolase (CEH) have been proposed to mediate the hydrolysis of CE in macrophages, circumstantial evidence suggests the presence of other enzymes with neutral cholesterol ester hydrolase (nCEH) activity. Here we show that the murine orthologue of KIAA1363, designated as neutral cholesterol ester hydrolase (NCEH), is a microsomal nCEH with high expression in murine and human macrophages. The effect of various concentrations of NaCl on its nCEH activity resembles that on endogenous nCEH activity of macrophages. RNA silencing of NCEH decreases nCEH activity at least by 50%; conversely, its overexpression inhibits the CE formation in macrophages. Immunohistochemistry reveals that NCEH is expressed in macrophage foam cells in atherosclerotic lesions. These data indicate that NCEH is responsible for a major part of nCEH activity in macrophages and may be a potential therapeutic target for the prevention of atherosclerosis.     

The LDL receptor is not necessary for acute adrenal steroidogenesis in mouse adrenocortical cells

Steroid hormones are synthesized using cholesterol as precursor. To determine the functional importance of the low density lipoprotein (LDL) receptor and hormone-sensitive lipase (HSL) in adrenal steroidogenesis, adrenal cells were isolated from control, HSL(-/-), LDLR(-/-), and double LDLR/HSL(-/-) mice. The endocytic and selective uptake of apolipoprotein E-free human high density lipoprotein (HDL)-derived cholesteryl esters did not differ among the mice, with selective uptake accounting for >97% of uptake. In contrast, endocytic uptake of either human LDL- or rat HDL-derived cholesteryl esters was reduced 80-85% in LDLR(-/-) and double-LDLR/HSL(-/-) mice. There were no differences in the selective uptake of either human LDL- or rat HDL-derived cholesteryl esters among the mice. Maximum corticosterone production induced by ACTH or dibutyryl cyclic AMP and lipoproteins was not altered in LDLR(-/-) mice but was reduced 80-90% in HSL(-/-) mice. Maximum corticosterone production was identical in HSL(-/-) and double-LDLR/HSL(-/-) mice. These findings suggest that, although the LDL receptor is responsible for endocytic delivery of cholesteryl esters from LDL and rat HDL to mouse adrenal cells, it appears to play a negligible role in the delivery of cholesterol for acute adrenal steroidogenesis in the mouse. In contrast, HSL occupies a vital role in adrenal steroidogenesis because of its link to utilization of selectively delivered cholesteryl esters from lipoproteins.     

Continuous monitoring of cholesterol oleate hydrolysis by hormone-sensitive lipase and other cholesterol esterases

Hormone-sensitive lipase (HSL) contributes importantly to the hydrolysis of cholesteryl ester in steroidogenic tissues, releasing the cholesterol required for adrenal steroidogenesis. HSL has broad substrate specificity, because it hydrolyzes triacylglycerols (TAGs), diacylglycerols, monoacylglycerols, and cholesteryl esters. In this study, we developed a specific cholesterol esterase assay using cholesterol oleate (CO) dispersed in phosphatidylcholine and gum arabic by sonication. To continuously monitor the hydrolysis of CO by HSL, we used the pH-stat technique. For the sake of comparison, the hydrolysis of CO dispersion was also tested using other cholesteryl ester-hydrolyzing enzymes. The specific activities measured on CO were found to be 18, 100, 27, and 3 micromol/min/mg for HSL, cholesterol esterase from Pseudomonas species, Candida rugosa lipase-3, and cholesterol esterase from bovine pancreas, respectively. The activity of HSL on CO is approximately 4- to 5-fold higher than on long-chain TAGs. In contrast, with all other enzymes tested, the rates of TAG hydrolysis were higher than those of CO hydrolysis. The relatively higher turnover of HSL on CO observed in vitro adds further molecular insight on the physiological importance of HSL in cholesteryl ester catabolism in vivo. Thus, HSL could be considered more as a cholesteryl ester hydrolase than as a TAG lipase.     

Macrophage‐mediated cholesterol handling in atherosclerosis

Formation of foam cells is a hallmark at the initial stages of atherosclerosis. Monocytes attracted by pro‐inflammatory stimuli attach to the inflamed vascular endothelium and penetrate to the arterial intima where they differentiate to macrophages. Intimal macrophages phagocytize oxidized low‐density lipoproteins (oxLDL). Several scavenger receptors (SR), including CD36, SR‐A1 and lectin‐like oxLDL receptor‐1 (LOX‐1), mediate oxLDL uptake. In late endosomes/lysosomes of macrophages, oxLDL are catabolysed. Lysosomal acid lipase (LAL) hydrolyses cholesterol esters that are enriched in LDL to free cholesterol and free fatty acids. In the endoplasmic reticulum (ER), acyl coenzyme A: cholesterol acyltransferase‐1 (ACAT1) in turn catalyses esterification of cholesterol to store cholesterol esters as lipid droplets in the ER of macrophages. Neutral cholesteryl ester hydrolases nCEH and NCEH1 are involved in a secondary hydrolysis of cholesterol esters to liberate free cholesterol that could be then out‐flowed from macrophages by cholesterol ATP‐binding cassette (ABC) transporters ABCA1 and ABCG1 and SR‐BI. In atherosclerosis, disruption of lipid homoeostasis in macrophages leads to cholesterol accumulation and formation of foam cells.     

Reversible accumulation of cholesteryl esters in macrophages incubated with acetylated lipoproteins

Mouse peritoneal macrophages accumulate large amounts of cholesteryl ester when incubated with human low-density lipoprotein that has been modified by chemical acetylation (acetyl-LDL). This accumulation is related to a high-affinity cell surface binding site that mediates the uptake of acetyl-LDL by adsorptive endocytosis and its delivery to lysosomes. The current studies demonstrate that the cholesteryl ester accumulation can be considered in terms of a two-compartment model: (a) the incoming cholesteryl esters of acetyl-LDL are hydrolyzed in lysosomes, and (b) the resultant free cholesterol is re-esterified in the cytosol where the newly formed esters are stored as lipid droplets. The following biochemical and morphologic evidence supports the hydrolysis-re-esterification mechanism: (a) Incubation of macrophages with acetyl-LDL markedly increased the rate of cholesteryl ester synthesis from [14C]oleate, and this was accompanied by an increase in the acyl-CoA:cholesteryl acyltransferase activity of cell-free extracts. (b) When macrophages were incubated with reconstituted acetyl-LDL in which the endogenous cholesterol was replaced with [3H]-cholesteryl linoleate, the [3H]cholesteryl linoleate was hydrolyzed, and at least one-half of the resultant [3H]cholesterol was re-esterified to form [3H]cholesteryl oleate, which accumulated within the cell. The lysosomal enzyme inhibitor chloroquine inhibited the hydrolysis of the [3H]cholesteryl linoleate, thus preventing the formation of [3H]cholesteryl oleate and leading to the accumulation of unhydrolyzed [3H]cholesteryl linoleate within the cells. (c) In the electron microscope, macrophages incubated with acetyl-LDL had numerous cytoplasmic lipid droplets that were not surrounded by a limiting membrane. The time course of droplet accumulation was similar to the time course of cholesteryl ester accumulation as measured biochemically. (d) When acetyl-LDL was removed from the incubation medium, biochemical and morphological studies showed that cytoplasmic cholesteryl esters were rapidly hydrolyzed and that the resultant free cholesterol was excreted from the cell.     

Carboxyl ester lipase expression in macrophages increases cholesteryl ester accumulation and promotes atherosclerosis

Carboxyl ester lipase (CEL, also called cholesterol esterase or bile salt-dependent lipase) is a lipolytic enzyme capable of hydrolyzing cholesteryl esters, triacylglycerols, and phospholipids in a trihydroxy bile salt-dependent manner but hydrolyzes ceramides and lysophospholipids via bile salt-independent mechanisms. Although CEL is synthesized predominantly in the pancreas, a low level of CEL expression was reported in human macrophages. This study used transgenic mice with macrophage CEL expression at levels comparable with that observed in human macrophages to explore the functional role and physiological significance of macrophage CEL expression. Peritoneal macrophages from CEL transgenic mice displayed a 4-fold increase in [(3)H]oleate incorporation into cholesteryl [(3)H]oleate compared with CEL-negative macrophages when the cells were incubated under basal conditions in vitro. When challenged with acetylated low density lipoprotein, cholesteryl ester accumulation was 2.5-fold higher in macrophages expressing the CEL transgene. The differences in cholesteryl ester accumulation were attributed to the lower levels of ceramide and lysophosphatidylcholine in CEL-expressing cells than in CEL-negative cells. CEL transgenic mice bred to an atherosclerosis susceptible apoE(-/-) background displayed an approximate 4-fold higher atherosclerotic lesion area than apoE(-/-) mice without the CEL transgene when both were fed a high fat/cholesterol diet. Plasma level of the atherogenic lysophosphatidylcholine was lower in the CEL transgenic mice, but plasma cholesterol level and lipoprotein profile were similar between the two groups. These studies documented that CEL expression in macrophages is pro-atherogenic and that the mechanism is because of its hydrolysis of ceramide and lysophosphatidylcholine in promoting cholesterol esterification and decreasing cholesterol efflux.     

Phagocytosis of cholesteryl ester is amplified in diabetic mouse macrophages and is largely mediated by CD36 and SR-A

Type 2 diabetes (T2D) is associated with accelerated atherosclerosis, which accounts for approximately 75% of all diabetes-related deaths. Here we investigate the link between diabetes and macrophage cholesteryl ester accumulation. When diabetic (db/db) mice are given cholesteryl ester intraperitoneally (IP), peritoneal macrophages (PerMPhis) recovered from these animals showed a 58% increase in intracellular cholesteryl ester accumulation over PerMPhis from heterozygote control (db/+) mice. Notably, PerMPhi fluid-phase endocytosis and large particle phagocytosis was equivalent in db/+and db/db mice. However, IP administration of CD36 and SR-A blocking antibodies led to 37% and 25% reductions in cholesteryl ester accumulation in PerMPhi. Finally, in order to determine if these scavenger receptors (SRs) were part of the mechanism responsible for the increased accumulation of cholesteryl esters observed in the diabetic mouse macrophages, receptor expression was quantified by flow cytometry. Importantly, db/db PerMPhis showed a 43% increase in CD36 expression and an 80% increase in SR-A expression. Taken together, these data indicate that direct cholesteryl ester accumulation in mouse macrophages is mediated by CD36 and SR-A, and the magnitude of accumulation is increased in db/db macrophages due to increased scavenger receptor expression.     

Morphological characterization of the cholesteryl ester cycle in cultured mouse macrophage foam cells

Mouse peritoneal macrophages can be induced to accumulate cholesteryl esters by incubating them in the presence of acetylated low density lipoprotein. The cholesteryl esters are sequestered in neutral lipid droplets that remain in the cell even when the acetylated low density lipoprotein is removed from the culture media. Previous biochemical studies have determined that the cholesterol component of cholesteryl ester droplets constantly turns over with a half time of 24 h by a cyclic process of de-esterification and re-esterification. We have used morphologic techniques to determine the spatial relationship of cholesteryl ester, free cholesterol, and lipase activity during normal turnover and when turnover is disrupted. Lipid droplets were surrounded by numerous 7.5-10.0-nm filaments; moreover, at focal sites on the margin of each droplet there were whorles of concentrically arranged membrane that penetrated the matrix. Histochemically detectable lipase activity was associated with these stacks of membrane. Using filipin as a light and electron microscopic probe for free cholesterol, we determined that a pool of free cholesterol was associated with each lipid droplet. Following incubation in the presence of the exogenous cholesterol acceptor, high density lipoprotein, the cholesteryl ester droplets disappeared and were replaced with lipid droplets of a different lipid composition. Inhibition of cholesterol esterification caused cholesteryl ester droplets to disappear and free cholesterol to accumulate in numerous myelin-like structures in the body of the cell.     

Relative importance of the LDL receptor and scavenger receptor class B in the beta-VLDL-induced uptake and accumulation of cholesteryl esters by peritoneal macrophages

We investigated the mechanism of beta-very low density lipoprotein (beta-VLDL)-induced foam cell formation derived from peritoneal macrophages from control mice and low density lipoprotein (LDL) receptor-deficient mice to elucidate the role of the LDL receptor in this process. The LDL receptor appeared to be of major importance for beta-VLDL metabolism. Consequently, the accumulation of cholesteryl esters in LDL receptor(-)(/)- macrophages is 2.5-fold lower than in LDL receptor(+)(/)(+) macrophages. In the absence of the LDL receptor, however, beta-VLDL was still able to induce cholesteryl ester accumulation and subsequently we characterized the properties of this residual beta-VLDL recognition site(s) of LDL receptor(-)(/)- macrophages. Although the LDL receptor-related protein is expressed on LDL receptor(-)(/)- macrophages, the cell association of beta-VLDL is not influenced by the receptor-associated protein, and treatment of the macrophages with heparinase and chondroitinase was also ineffective. In contrast, both oxidized LDL (OxLDL) and anionic liposomes were able to inhibit the cell association of (125)I-labeled beta-VLDL in LDL receptor(-)(/)- macrophages by 65%. These properties suggest a role for scavenger receptor class B (SR-B), and indeed, in the LDL receptor(-)(/)- macrophages the selective uptake of cholesteryl esters from beta-VLDL was 2.2-fold higher than that of apolipoproteins, a process that could be inhibited by OxLDL, high density lipoprotein (HDL), and beta-VLDL.In conclusion, the LDL receptor on peritoneal macrophages is directly involved in the metabolism of beta-VLDL and the subsequent foam cell formation. When the LDL receptor is absent, SR-B appears to mediate the remaining metabolism of cholesteryl esters from beta-VLDL.     

Phosphorylation and activation of hormone-sensitive lipase in isolated macrophages.

Hormone-sensitive lipase (HSL) is responsible for the neutral cholesterol ester hydrolase activity in macrophages. Incubation of intact WEHI macrophages or mouse peritoneal macrophages leads to phosphorylation of HSL, which is increased by incubation with either dibutyryl cyclic AMP and 3-isobutyl-1-methylxanthine or okadaic acid. Correspondingly, these agents also activate neutral cholesterol ester hydrolase activity in intact WEHI cells. Regulation of mobilisation of esterified cholesterol in macrophages may be of antiatherogenic value, which this model system now allows us to investigate further.     

Interaction of hormone-sensitive lipase with steroidogenic acute regulatory protein: facilitation of cholesterol transfer in adrenal

Hormone-sensitive lipase (HSL) is responsible for the neutral cholesteryl ester hydrolase activity in steroidogenic tissues. Through its action, HSL is involved in regulating intracellular cholesterol metabolism and making unesterified cholesterol available for steroid hormone production. Steroidogenic acute regulatory protein (StAR) facilitates the movement of cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane and is a critical regulatory step in steroidogenesis. In the current studies we demonstrate a direct interaction of HSL with StAR using in vitro glutathione S-transferase pull-down experiments. The 37-kDa StAR is coimmunoprecipitated with HSL from adrenals of animals treated with ACTH. Deletional mutations show that HSL interacts with the N-terminal as well as a central region of StAR. Coexpression of HSL and StAR in Chinese hamster ovary cells results in higher cholesteryl ester hydrolytic activity of HSL. Transient overexpression of HSL in Y1 adrenocortical cells increases mitochondrial cholesterol content under conditions in which StAR is induced. It is proposed that the interaction of HSL with StAR in cytosol increases the hydrolytic activity of HSL and that together HSL and StAR facilitate cholesterol movement from lipid droplets to mitochondria for steroidogenesis.     

Glucose-dependent regulation of cholesterol ester metabolism in macrophages by insulin and leptin

Insulin resistance, obesity, and diabetes are characterized by hyperglycemia, hyperinsulinemia, and hyperleptinemia and are associated with increased risk of atherosclerosis. In an effort to understand how this occurs, we have investigated whether these factors cause disregulation of cholesterol ester metabolism in J774.2 macrophages. Raising glucose levels alone was sufficient to increase uptake of acetylated low density lipoprotein but did not stimulate synthesis of cholesterol esters. In the presence of high glucose, both insulin and leptin increased the rate of cholesterol ester synthesis, although they did not further increase uptake of acetylated low density lipoprotein. However, in the presence of high glucose both insulin and leptin caused a significant increase in the activity of acyl-CoA: cholesterol O-acyltransferase (ACAT) combined with a significant reduction in the level of hormone-sensitive lipase (HSL). Because ACAT is the main enzyme responsible for cholesterol ester synthesis and HSL contributes significantly to neutral cholesterol ester hydrolase activity, this suggests that glucose primes the J774.2 cells so that in the presence of high insulin or leptin they will store cholesterol esters. This contrasts with 3T3-L1 adipocytes, where HSL activity and expression are increased by insulin in high glucose conditions. These findings may provide an explanation for the observation that in conditions characterized by hyperglycemia, hyperleptinemia, and hyperinsulinemia, triglyceride lipolysis in adipocytes is increased while hydrolysis of cholesterol esters in macrophages is decreased, contributing to foam cell formation.     

Microquantification of cholesterol and cholesteryl esters in rat peritoneal macrophages by reverse-phase high-performance liquid chromatography

A simple and rapid method for the microquantification of cholesterol and cholesteryl esters by reverse-phase high performance liquid chromatography has been established. Comparison of elution patterns of authentic cholesterol and cholesteryl esters revealed that a mu Bondasphere reverse-phase C8 (300-A) column was more suitable than a corresponding reverse-phase C4 or C18 column in terms of rapidity and sensitivity. Recovery of cholesterol and cholesteryl esters from a C8 column was greater than 98% when determined either by radioactive cholesterol and cholesteryl oleate or by cholesteryl heptadecanoate. The sensitivity of the quantification ranged from 5 ng to 50 micrograms for both cholesterol and cholesteryl esters. This method was applied to determination of cellular cholesterol and cholesteryl esters of rat peritoneal macrophages. Lipid extracts of these cells were found to contain 38.01 +/- 2.60 micrograms of cholesterol and 3.18 +/- 0.36 micrograms of cholesteryl esters per milligram of cell protein. When the cells were loaded with cholesteryl esters by incubation for 24 h with various concentrations of acetylated low-density lipoprotein, a cellular level of cholesteryl esters showed a dose-dependent increase and reached a maximal level of 106.60 +/- 3.05 micrograms/mg cell protein. Thus, the present method is useful for the microquantification of cholesterol and cholesteryl esters from lipid extracts of biological samples.     

Induction of macrophage growth by lipids

Lipoproteins from ascitic tumors in mice and lipids extracted from these lipoproteins induced growth of murine peritoneal macrophages in vitro. The lipid components with activity were examined by use of lipid vesicles or liposomes. Liposomes prepared from egg-yolk PC alone did not induce macrophage growth, but those prepared from mixtures of egg-yolk PC and cholesterol or cholesteryl esters other than cholesteryl oleate, or triglycerides other than triolein, enhanced 3H-TdR incorporation into macrophages. The free fatty acids examined had no effect on 3H-TdR incorporation. These results suggest that growth of macrophages is induced by ordinary lipids present in lipoproteins or cell membranes that the macrophages scavenge in the body.     

The interaction of human apoB-containing lipoproteins with mouse peritoneal macrophages: a comparison of Lp(a) with LDL

Cholesteryl ester accumulation in macrophages and foam cell formation is believed to play an important role in atherogenesis. The effect of Lp(a) on the incorporation of [14C]oleate into cholesteryl esters was studied in mouse peritoneal macrophages. In view of the physico-chemical similarities between Lp(a) and LDL, the results were compared with those obtained with LDL. Native Lp(a) and LDL did not stimulate cholesteryl ester formation. Incubation of macrophages with Lp(a)- or LDL-dextran sulfate complexes caused a significant increase in cholesteryl ester formation. A similar effect was observed when Lp(a) or LDL were incubated with macrophages in the presence of antibodies directed against the specific Lp(a) apoprotein or against LpB. Treatment of Lp(a) with acetic anhydride or malondialdehyde (MDA) was followed by precipitation of most of the lipoprotein. Therefore, these modifications were not suitable to study the uptake of modified Lp(a) by macrophages. Studies with acetyl-LDL or MDA-treated LDL caused the well-known stimulation of [14C]oleate incorporation into cholesteryl esters. Thus, the modification of Lp(a) by sulfated polysaccharides or by treatment with antibodies yields similar cholesteryl ester deposition in mouse peritoneal macrophages as observed with modified LDL. This might be one mechanism by which Lp(a) exerts its atherogenicity.