Differences in the metabolism of oxidatively modified low density lipoprotein and acetylated low density lipoprotein by human endothelial cells: inhibition of cholesterol esterification by oxidatively modified low density lipoprotein

The rate of degradation of oxidatively modified low density lipoprotein (Ox-LDL) by human endothelial cells was similar to that of unmodified low density lipoprotein (LDL), and was approximately 2-fold greater than the rate of degradation of acetylated LDL (Ac-LDL). While LDL and Ac-LDL both stimulated cholesterol esterification in endothelial cells, Ox-LDL inhibited cholesterol esterification by 34%, demonstrating a dissociation between the degradation of Ox-LDL and its ability to stimulate cholesterol esterification. Further, while LDL and Ac-LDL resulted in a 5- and 15-fold increase in cholesteryl ester accumulation, respectively, Ox-LDL caused only a 1.3-fold increase in cholesteryl ester mass. These differences could be accounted for, in part, by the reduced cholesteryl ester content of Ox-LDL. However, when endothelial cells were incubated with Ac-LDL in the presence and absence of Ox-LDL, Ox-LDL led to a dose-dependent inhibition of cholesterol esterification without affecting the degradation of Ac-LDL. This inhibitory effect of Ox-LDL on cholesteryl ester synthesis was also manifest in normal human skin fibroblasts incubated with LDL and in LDL-receptor-negative fibroblasts incubated with unesterified cholesterol to stimulate cholesterol esterification. Further, the lipid extract from Ox-LDL inhibited cholesterol esterification in LDL-receptor negative fibroblasts. These findings suggest that the inhibition of cholesterol esterification by oxidized LDL is independent of the LDL and scavenger receptors and may be a result of translocation of a lipid component of oxidatively modified LDL across the cell membrane.     

Metabolism of modified LDL and foam cell formation in murine macrophage-like RAW 264 cells.

The uptake of modified low density lipoprotein (LDL) by arterial macrophages is a key event in the atherogenesis. We studied 1) the uptake and degradation of modified LDL, 2) LDL recognition by specific receptors, and 3) the foam cell formation with murine macrophage-like RAW 264 cells in vitro. The cells took up and degraded effectively 125I-labeled acetylated LDL (Ac-LDL) and aggregated LDL (Aggr-LDL). Also oxidized LDL (Ox-LDL) was taken up but it was degraded poorly. The degradation of 125I-Ac-LDL was efficiently competed by both unlabeled Ac-LDL and Ox-LDL, whereas the degradation of 125I-Ox-LDL was partially competed by unlabeled Ox-LDL and Aggr-LDL but not at all by unlabeled Ac-LDL. The incubation with increasing concentrations of Ac-LDL, Aggr-LDL or Ox-LDL resulted in marked foam cell formation in the RAW 264 cells. Ox-LDL was cytotoxic at 500 to 1000 microg/ml concentrations. The results show that RAW 264 cells have at least two classes of receptors for modified lipoproteins: one that recognizes both Ox-LDL and Ac-LDL, and is similar to the scavenger receptors, and another that recognizes Ox-LDL but not Ac-LDL. RAW 264 cells are a convenient model cell line for examining the metabolism of modified lipoproteins, not only that of Ac-LDL but also that of Ox-LDL and Aggr-LDL, and cellular accumulation of lipids derived from modified LDL.     

Inhibition of acyl coenzyme A:cholesterol acyl transferase in J774 macrophages enhances down-regulation of the low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl-coenzyme A reductase and prevents low density lipoprotein-induced cholesterol accumulation

Cholesteryl ester accumulation in arterial wall macrophages (foam cells) is a prominent feature of atherosclerotic lesions. We have previously shown that J774 macrophages accumulate large amounts of cholesteryl ester when incubated with unmodified low density lipoprotein (LDL) and that this is related to sluggish down-regulation of the J774 LDL receptor and 3-hydroxy-3-methylglutaryl-coenzyme A reductase. To further explore intracellular cholesterol metabolism and regulatory events in J774 macrophages, we studied the effect of inhibitors of acyl-CoA:cholesterol acyl transferase (ACAT) on the cells' ability to accumulate cholesterol and to down-regulate receptor and reductase. Treatment of J774 cells with LDL in the presence of ACAT inhibitor 58-035 (Sandoz) prevented both cholesteryl ester and total cholesterol accumulation. Furthermore, 58-035 markedly enhanced down-regulation of the J774 LDL receptor and 3-hydroxy-3-methylglutaryl-CoA reductase in the presence of LDL. In dose-response studies, down-regulation of the receptor by 58-035 paralleled its inhibition of ACAT activity. Compound 58-035 also increased the down-regulation of the J774 LDL receptor in the presence of 25-hydroxycholesterol and acetyl-LDL but not in the presence of cholesteryl hemisuccinate, which is not an ACAT substrate. The ability of 58-035 to enhance LDL receptor down-regulation was negated when cells were simultaneously incubated with recombinant high density lipoprotein3 discs, which promote cellular cholesterol efflux. In contrast to the findings with J774 macrophages, down-regulation of the human fibroblast LDL receptor was not enhanced by 58-035. These data suggest that in J774 macrophages, but not in fibroblasts, ACAT competes for a regulatory pool of intracellular cholesterol, contributing to diminished receptor and reductase down-regulation, LDL-cholesterol accumulation, and foam cell formation.     

Acyl coenzyme A:cholesterol acyl transferase in macrophages utilizes a cellular pool of cholesterol oxidase-accessible cholesterol as substrate

Cholesterol esterification by acyl CoA:cholesterol acyl transferase (ACAT) in macrophages is a key process in atheroma foam cell formation. However, the process of cholesterol substrate delivery to ACAT is not well defined. In this study, J774 macrophages, which form foam cells with native low density lipoprotein (LDL), were labeled with [3H]cholesterol-containing liposomes. Most (80-90%) of the cholesterol label could be converted by cholesterol oxidase to cholestenone, suggesting plasma membrane localization; only 0.6% of the label was in cholesteryl ester (CE). In cells chased for 6 h in medium lacking LDL, the distribution of label was essentially unchanged, whereas in cells chased with LDL, 28% of the label was incorporated into CE concomitant with a decrease in cholestenone label to 50%. [3H]Cholesterol-labeled mouse peritoneal macrophages incubated with acetyl-LDL, and both J774 and mouse peritoneal macrophages incubated with 25-hydroxy-cholesterol, also showed a shift of label from cholestenone to CE. Similar results were found when cellular cholesterol was biosynthetically labeled with [3H]mevalonate. The percentage of cholesterol substrate for ACAT in LDL-treated J774 macrophages which originates from endogenous cellular pools (versus that originating from LDL itself) is approximately 50%. We conclude that upon activation of ACAT in macrophages, there is a novel process whereby a cholesterol oxidase-accessible pool of cellular cholesterol, presumably plasma membrane cholesterol, is translocated to ACAT in the endoplasmic reticulum.     

Low density lipoprotein modification by cholesterol oxidase induces enhanced uptake and cholesterol accumulation in cells.

Oxidation of low density lipoprotein (LDL) by cells of the arterial wall or in the presence of copper ions was shown to result in the peroxidation of its fatty acids as well as its cholesterol moiety. LDL incubation with cholesterol oxidase (CO) resulted in the conversion of up to 85% of the lipoprotein unesterified cholesterol (cholest-5-en-3-ol) to cholestenone (cholest-4-en-3-one) in a dose- and time-dependent pattern. Plasma very low density lipoprotein (VLDL) and high density lipoprotein (HDL) could be similarly modified by CO. In cholesterol oxidase-modified LDL (CO-LDL), unlike copper ion-induced oxidized LDL (Cu-Ox-LDL), there was no fatty acids peroxidation, and lipoprotein size or charge as well as LDL cholesteryl ester, phospholipids, and triglycerides content were not affected. CO-LDL, however, demonstrated enhanced susceptibility to oxidation by copper ions in comparison to native LDL. Upon incubation of CO-LDL with J-774 A.1 macrophage-like cell line, cellular uptake and degradation of the lipoprotein was increased by up to 62% in comparison to native LDL but was 15% lower than that of Cu-Ox-LDL. Similarly, the binding of CO-LDL to macrophages increased by up to 80%, and cellular cholesterol mass was increased 51% more than the mass obtained with native LDL. Several lines of evidence indicate that CO-LDL was taken up via the LDL receptor: 1) Excess amounts of unlabeled LDL, but not acetyl-LDL (Ac-LDL), effectively competed with 125I-CO-LDL for the uptake by cells. 2) The degradation of CO-LDL by various types of macrophages and by fibroblasts could be dissociated from that of Ac-LDL and was always higher than that of native LDL. 3) A monoclonal antibody to the LDL receptor (IgG-C7) and a monoclonal antibody to the LDL receptor binding domains on apoB-100 (B1B6) inhibited macrophage degradation of CO-LDL. The receptor for Cu-Ox-LDL, which is not shared with Ac-LDL, was also partially involved in macrophage uptake of CO-LDL, since Cu-Ox-LDL demonstrated some competition capability with CO-125I-LDL for its cellular degradation. CO-LDL cellular degradation was inhibited by chloroquine, thus implying lysosomal involvement in the cellular processing of the lipoprotein. Incubation of macrophages with LDL in the presence of increasing concentrations of cholestenone resulted in up to 52% enhanced lipoprotein cellular degradation suggesting that the cholestenone in CO-LDL might be involved in the enhanced cellular uptake of the modified lipoprotein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Uptake of type IV hypertriglyceridemic VLDL by cultured macrophages is enhanced by interferon-gamma.

Hypertriglyceridemic (HTG) very low density lipoproteins (VLDL) from subjects with type IV hyperlipoproteinemia induce both cholesteryl ester (CE) and triglyceride (TG) accumulation in cultured J774 macrophages. We examined whether the cytokine interferon-gamma (IFN-gamma), which is expressed by lymphocytes in atherosclerotic lesions, would modulate macrophage uptake of HTG -VLDL. Incubation of cells with HTG -VLDL alone significantly increased cellular CE and TG mass 17- and 4.3-fold, respectively, while cellular free cholesterol (FC) was unaffected. Pre-incubation of cells with IFN-gamma (50 U/ml) prior to incubation with HTG -VLDL caused a marked enhancement in cellular CE and TG 27- and 6-fold over no additions (controls), respectively, and a 1.5-fold increase in FC. IFN-gamma increased low density lipoprotein (LDL)-induced cellular CE 2-fold compared to LDL alone. IFN-gamma did not enhance the uptake of type III (apoE2/E2) HTG -VLDL or VLDL from apoE knock-out mice. Incubations in the presence of a lipoprotein lipase (LPL) inhibitor or an acylCoA:cholesterol acyltransferase (ACAT) inhibitor demonstrated that the IFN-gamma-enhanced HTG -VLDL uptake was dependent on LPL and ACAT activities. IFN-gamma significantly increased the binding and degradation of 125I-labeled LDL. Binding studies with 125I-labeled alpha2-macroglobulin, a known LDL receptor-related protein (LRP) ligand, and experiments with copper-oxidized LDL indicated that the IFN-gamma-enhanced uptake was not due to increased expression of the LRP or scavenger receptors. Thus, IFN-gamma may promote foam cell formation by accelerating macrophage uptake of native lipoproteins. IFN-gamma-stimulated CE accumulation in the presence of HTG -VLDL occurs via a process that requires receptor binding-competent apoE and active LPL. IFN-gamma-enhanced uptake of both HTG -VLDL and LDL is mediated by the LDL-receptor and requires ACAT-mediated cholesterol esterification.     

Lipoproteins activate acyl-coenzyme A:cholesterol acyltransferase in macrophages only after cellular cholesterol pools are expanded to a critical threshold level

Activation of acyl-CoA:cholesterol actyltransferase (ACAT) in macrophages by lipoproteins is a key event in atheroma foam cell formation. To help elucidate the mechanisms whereby lipoproteins stimulate ACAT, the early cellular events of lipoprotein-induced ACAT stimulation were studied in mouse peritoneal macrophages. As a function of increasing lipoprotein-cholesterol influx to the cell during the first few hours of incubation, ACAT activity was markedly stimulated by beta-very low density lipoprotein (beta-VLDL) and acetyl-low density lipoprotein (acetyl-LDL) only after lipoprotein-cholesterol influx reached a threshold level of approximately 25% above the basal cell cholesterol content. In contrast, LDL stimulated ACAT only minimally at this level of lipoprotein-cholesterol influx. In further experiments, the source of ACAT cholesterol substrate during the initial stimulation of ACAT was shown to be a mixture of cellular (approximately 75%) and lipoprotein-cholesterol (approximately 25%) in proportions that approximated the proportions of originally cellular and lipoprotein-cholesterol in the cell. Thus, lipoprotein-cholesterol rapidly mixed with most or all of cellular cholesterol before ACAT esterification. Additional studies showed that LDL caused significant efflux of cellular cholesterol, thus providing at least a partial explanation for the relatively weak ACAT stimulatory potential of LDL. To support this idea, LDL that was modified to decrease its ability to induce net cellular cholesterol efflux stimulated ACAT 2-fold greater than control LDL when matched for lysosomal LDL-cholesterol influx. Moreover, when the effective efflux potentials of beta-VLDL and acetyl-LDL were increased, ACAT stimulation was markedly decreased despite unchanged lipoprotein-cholesterol influx. Thus, macrophage ACAT is stimulated not directly by the influx of newly hydrolyzed lipoprotein-cholesterol but rather by net expansion of cellular cholesterol pools to a particular threshold level. This scheme has potentially important implications regarding the cellular and molecular mechanisms of foam cell formation.     

Effects of hypoxia on cholesterol metabolism in human monocyte-derived macrophages

We assessed the metabolism of low density lipoprotein (LDL) of human monocyte-derived macrophages under hypoxia. The specific binding and association of 125I-labeled LDL (125I-LDL) were not changed under hypoxia compared to normoxia. However, the degradation of 125I-LDL under hypoxia decreased to 60%. The rate of cholesterol esterification under hypoxia was 2-fold greater on incubation with LDL or 25-hydroxycholesterol. The cellular cholesteryl ester content was also greater under hypoxia on incubation with LDL. Secretion of apolipoprotein E into the medium was not altered under hypoxia, suggesting that apolipoprotein E independent cholesterol efflux may be reduced under hypoxia. Thus, hypoxia affects the intracellular metabolism of LDL, stimulates cholesterol esterification, and enhances cholesteryl ester accumulation in macrophages. Hypoxia is one of the important factors modifying the cellular lipid metabolism in arterial wall.     

Abnormal high density lipoproteins from patients with liver disease regulate cholesterol metabolism in cultured human skin fibroblasts

Apolipoprotein B (apoB) of plasma low density lipoproteins (LDL) binds to high affinity receptors on many cell types. A minor subclass of high density lipoproteins (HDL), termed HDL1, which contains apoE but lacks apoB, binds to the same receptor. Bound lipoproteins are engulfed, degraded, and regulate intracellular cholesterol metabolism and receptor activity. The HDL of many patients with liver disease is rich in apoE. We tested the hypothesis that such patient HDL would reduce LDL binding and would themselves regulate cellular cholesterol metabolism. Normal HDL had little effect on binding, uptake, and degradation of 125I-labeled LDL by cultured human skin fibroblasts. Patient HDL (d 1.063-1.21 g/ml) inhibited these processes, and in 15 of the 25 samples studied there was more than 50% inhibition at 125I-labeled LDL and HDL protein concentrations of 10 micrograms/ml and 25 micrograms/ml, respectively. There was a significant negative correlation between the percentage of 125I-labeled LDL bound and the apoE content of the competing HDL (r = -0.54, P less than 0.01). Patient 125I-labeled HDL was also taken up and degraded by the fibroblasts, apparently through the LDL-receptor pathway, stimulated cellular cholesterol esterification, increased cell cholesteryl ester content, and suppressed cholesterol synthesis and receptor activity. We conclude that LDL catabolism by the receptor-mediated pathway may be impaired in liver disease and that patient HDL may deliver cholesterol to cells.     

High-density lipoprotein particle uptake and selective uptake of high-density lipoprotein-associated cholesteryl esters by J774 macrophages

High-density lipoprotein (HDL) cholesteryl esters are taken up by fibroblasts via HDL particle uptake and via selective uptake, i.e., cholesteryl ester uptake independent of HDL particle uptake. In the present study we investigated HDL selective uptake and HDL particle uptake by J774 macrophages. HDL3 (d = 1.125-1.21 g/ml) was labeled with intracellularly trapped tracers: 125I-labeled N-methyltyramine-cellobiose-apo A-I (125I-NMTC-apo A-I) to trace apolipoprotein A-I (apo A-I) and [3H]cholesteryl oleyl ether to trace cholesteryl esters. J774 macrophages, incubated at 37 degrees C in medium containing doubly labeled HDL3, took up 125I-NMTC-apo A-I, indicating HDL3 particle uptake (102.7 ng HDL3 protein/mg cell protein per 4 h at 20 micrograms/ml HDL3 protein). Apparent HDL3 uptake according to the uptake of [3H]cholesteryl oleyl ether (470.4 ng HDL3 protein/mg cell protein per 4 h at 20 micrograms/ml HDL3 protein) was in significant excess on 125I-NMTC-apo A-I uptake, i.e., J774 macrophages demonstrated selective uptake of HDL3 cholesteryl esters. To investigate regulation of HDL3 uptake, cell cholesterol was modified by preincubation with low-density lipoprotein (LDL) or acetylated LDL (acetyl-LDL). Afterwards, uptake of doubly labeled HDL3, LDL (apo B,E) receptor activity or cholesterol mass were determined. Preincubation with LDL or acetyl-LDL increased cell cholesterol up to approx. 3.5-fold over basal levels. Increased cell cholesterol had no effect on HDL3 particle uptake. In contrast, LDL- and acetyl-LDL-loading decreased selective uptake (apparent uptake 606 vs. 366 ng HDL3 protein/mg cell protein per 4 h in unloaded versus acetyl-LDL-loaded cells at 20 micrograms HDL3 protein/ml). In parallel with decreased selective uptake, specific 125I-LDL degradation was down-regulated. Using heparin as well as excess unlabeled LDL, it was shown that HDL3 uptake is independent of LDL (apo B,E) receptors. In summary, J774 macrophages take up HDL3 particles. In addition, J774 cells also selectively take up HDL3-associated cholesteryl esters. HDL3 selective uptake, but not HDL3 particle uptake, can be regulated.     

The contribution of the macrophage receptor for oxidized LDL to its cellular uptake

Oxidized LDL (Ox-LDL) was shown to be taken up by macrophages via several receptors including the acetyl-LDL(Ac-LDL), the LDL, and the Ox-LDL receptors. Cellular uptake and degradation of Ox-LDL could be dissociated from that of LDL and Ac-LDL as demonstrated by using macrophages that lack the LDL or the Ac-LDL receptors. In J-774 A.1 macrophage-like cell line unlabeled Ox-LDL reduced the 125I-Ox-LDL by up to degradation of 91% whereas unlabeled Ac-LDL and native LDL reduced 125I-Ox-LDL degradation by only 51% and 23%, respectively. Analysis of macrophage degradation of 125I-Ox-LDL in the presence of 30-fold excess concentration of LDL + Ac-LDL (to block uptake of 125I-Ox-LDL via the LDL and the Ac-LDL receptors) revealed that cellular degradation via the Ox-LDL receptor could account for 45% of the macrophage uptake of Ox-LDL.     

Three probe flow cytometry of a human foam-cell forming macrophage.

A human cell line THP-1 was differentiated into macrophages expressing the scavenger receptor for uptake of modified lipoproteins. The cells were exposed to native low-density lipoprotein (n-LDL), acetylated-low-density lipoprotein (Ac-LDL), oxidised-LDL, or 25-OH cholesterol, leading to the accumulation of cholesteryl esters within the cells. Harvested macrophages were studied using three separate probes: 1) 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (diI)-labelled LDL to study lipoprotein uptake; 2) the lipophilic fluorescent dye Nile Red to study cholesteryl ester accumulation within the cells; and 3) the polyene antibiotic Filipin III to study free cholesterol homeostasis. Cells were analysed using fluorescence flow cytometry and the three signals analysed separately. THP-1 macrophages incubated with diI-labelled modified lipoproteins produced a concentration dependent increase in the fluorescence emissions, consistent with accumulation of the labelled particles. Macrophages exposed to unlabelled modified LDLs were demonstrated, by staining with Nile Red, to accumulate cholesteryl esters within their cytoplasm and to alter their cholesterol content as judged by staining with Filipin. The foam-cell forming macrophage and its response to modified lipoproteins is considered a key step in the development of atherosclerosis. The use of these three probes during the formation of foam-cells in vitro offers a way of studying their behaviour at the single cell level.     

Effect of fatty acid supplementation on cholesterol and retinol esterification in J774 macrophages

J774 macrophages exposed to medium containing cholesterol-rich phospholipid dispersions accumulate cholesteryl ester. Supplementing this medium with 100 micrograms oleate/ml increased cellular cholesteryl ester contents 3-fold. Cell retinyl ester contents increased 8-fold when medium containing retinol dispersed in dimethyl sulfoxide was supplemented with oleate. These increases were not the result of increases in total lipid uptake by the cells but rather of redistribution of cholesterol and retinol into their respective ester pools. Effective oleate concentration of 15-30 micrograms/ml increased cellular retinyl and cholesteryl ester contents. The effective oleate concentration was reduced to 5 micrograms/ml when the fatty acid/albumin molar ratio was increased. The oleate-stimulated increase in cholesterol esterification was blocked by incubating cells with Sandoz 58-035, a specific inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT), indicating that the effect of fatty acid exposure is mediated through changes in ACAT activity. When cholesterol or retinol was added to cells which had been exposed to oleate for 24 h to provide a triacylglycerol store, the cellular contents of cholesteryl or retinyl ester were also significantly increased compared to cells not previously exposed to oleate. The oleate-stimulated increase in the esterification of cholesterol and/or retinol was also observed in P388D1 macrophages, human (HepG2) and rat (Fu5AH) hepatomas, human fibroblasts, rabbit aortic smooth muscle cells and MCF-7 breast carcinoma cells. In addition to oleate, a number of other fatty acids increased retinol esterification in J774 macrophages; however, cellular cholesterol esterification in these cells was increased only by unsaturated fatty acids and was inhibited in the presence of saturated fatty acids. Although the cellular uptake of radiolabeled oleate and palmitate was similar, a significant difference in the distribution of these fatty acids among the lipid classes was observed. These data demonstrate that exogenous fatty acids are one factor that regulate cellular cholesteryl and retinyl ester contents in cultured cells.     

Influence of the acyl-CoA: cholesterol O-acyltransferase inhibitor, CL 277082, on cholesteryl ester accumulation in rabbit macrophage-rich granulomas and hepatic tissue

The influence of the acyl-CoA: cholesterol O-acyltransferase (ACAT) inhibitor, CL 277082, on macrophage cholesteryl ester accumulation in a rabbit carrageenan granuloma macrophage-foam cell model was studied. Diets were supplemented with 0.3% cholesterol and 6% peanut oil with or without the inhibitor (0.25%) for 4 weeks prior to granuloma induction, and macrophage-rich granuloma tissue was harvested 14 days after carrageenan injection. Serum cholesterol was monitored biweekly, and plasma lipoproteins were isolated terminally. Total, free and esterified cholesterol contents were measured in hepatic and granuloma tissue. In hepatic tissue, administration of CL 277082 resulted in an 80% reduction in the content of total cholesterol, a 37% decrease in free cholesterol, and a 90% decrease in esterified cholesterol. Similarly, in macrophage-rich granuloma tissue, total cholesterol content was decreased by 44%, and esterified cholesterol content by 61%, with no change in free cholesterol. Additionally, CL 277082 was shown to inhibit granuloma tissue ACAT activity by 45%, VLDL mass was decreased slightly, LDL mass increased 3.4-fold and HDL mass was similar in both the inhibitor-treated and control animals. CL 277082 resulted in a 57% decrease in VLDL cholesteryl ester content and a 4.5-fold increase in triacylglycerol. Cholesteryl ester content in LDL was decreased by 31% and LDL triacylglycerol was increased 5.2-fold, while the only change in HDL composition was a 3.5-fold increase in triacylglycerol. The reductions in both hepatic tissue and macrophage-rich granuloma tissue esterified cholesterol accumulation are considered to be due largely to cellular ACAT inhibition, and the altered distribution and composition of the plasma lipoproteins.     

Pomegranate juice inhibits oxidized LDL uptake and cholesterol biosynthesis in macrophages

Macrophage cholesterol accumulation and foam cell formation are the hallmarks of early atherogenesis. Pomegranate juice (PJ) was shown to inhibit macrophage foam cell formation and development of atherosclerotic lesions. The aim of this study was to elucidate possible mechanisms by which PJ reduces cholesterol accumulation in macrophages. J774.A1 macrophages were preincubated with PJ followed by analysis of cholesterol influx [evaluated as LDL or as oxidized LDL (Ox-LDL) cellular degradation], cholesterol efflux and cholesterol biosynthesis. Preincubation of macrophages with PJ resulted in a significant reduction (P<.01) in Ox-LDL degradation by 40%. On the contrary, PJ had no effect on macrophage degradation of native LDL or on macrophage cholesterol efflux. Macrophage cholesterol biosynthesis was inhibited by 50% (P<.01) after cell incubation with PJ. This inhibition, however, was not mediated at the 3-hydroxy-3 methylglutaryl coenzyme A reductase level along the biosynthetic pathway. We conclude that PJ-mediated suppression of Ox-LDL degradation and of cholesterol biosynthesis in macrophages can lead to reduced cellular cholesterol accumulation and foam cell formation.     

Modified low density lipoproteins activate human macrophages to secrete immunoreactive endothelin

This study attempted to determine if low density lipoproteins (LDL) induce the production of endothelins (ET) by human macrophages. Non-protected LDL from macrophage induced oxidation (n-LDL), copper-oxidized LDL (Ox-LDL), acetylated-LDL (Ac-LDL), butylated hydroxytoluene-LDL (BHT-LDL), BHT-Ac-LDL, polyinosinic acid (PiA, 1.5 micrograms/ml), phorbol myristate acetate (PMA; 0.5 microM) and BHT alone (20 microM) were studied. The different compounds had the following potency to stimulate the ET secretion: PMA greater than Ox-LDL greater than Ac-LDL greater than n-LDL greater than BHT-LDL greater than PiA greater than PiA + Ac-LDL greater than BHT. In conclusion, modified LDL stimulated ET secretion by human macrophages.     

Cellular free cholesterol in Hep G2 cells is only partially available for down-regulation of low-density-lipoprotein receptor activity.

We have previously shown that in Hep G2 cells and human hepatocytes, as compared with fibroblasts, the low-density lipoprotein (LDL) receptor activity is only weakly down-regulated after incubation of the cells with LDL, whereas incubation with high-density lipoproteins (HDL) of density 1.16-1.20 g/ml (heavy HDL) strongly increased the LDL-receptor activity. To elucidate this difference between hepatocytes and fibroblasts, we studied the cellular cholesterol homoeostasis in relation to the LDL-receptor activity in Hep G2 cells. (1) Interrupting the cholesteryl ester cycle by inhibiting acyl-CoA: cholesterol acyltransferase (ACAT) activity with compound 58-035 (Sandoz) resulted in an enhanced LDL-mediated down-regulation of the receptor activity. (2) The stimulation of the receptor activity by incubation of the cells with cholesterol acceptors such as heavy HDL was not affected by ACAT inhibition. (3) Incubation of the Hep G2 cells with LDL, heavy HDL or a combination of both grossly affected LDL-receptor activity, but did not significantly change the intracellular content of free cholesterol, suggesting that in Hep G2 cells the regulatory free cholesterol pool is small as compared with the total free cholesterol mass. (4) We used changes in ACAT activity as a sensitive (indirect) measure for changes in the regulatory free cholesterol pool. (5) Incubation of the cells with compactin (2 microM) without lipoproteins resulted in a 4-fold decrease in ACAT activity, indicating that endogenously synthesized cholesterol is directed to the ACAT-substrate pool. (6) Incubation of the cells with LDL or a combination of LDL and heavy HDL stimulated ACAT activity 3-5 fold, whereas incubation with heavy HDL alone decreased ACAT activity more than 20-fold. Our results suggest that in Hep G2 cells exogenously delivered (LDL)-cholesterol and endogenously synthesized cholesterol are primarily directed to the cholesteryl ester (ACAT-substrate) pool or, if present, to extracellular cholesterol acceptors (heavy HDL) rather than to the free cholesterol pool involved in LDL-receptor regulation.     

Evidence for extralysosomal hydrolysis of high-density lipoprotein cholesteryl esters in rat hepatoma cells (Fu5AH): a model for delivery of high-density lipoprotein cholesterol

Rat hepatoma cells (Fu5AH) were studied as a model for the net delivery of apoE-free high-density lipoprotein (HDL) cholesterol to a cell. Incubating cells with HDL results in 1) a decrease in both media-free cholesterol and cholesteryl ester concentration; 2) decreased cell sterol synthesis; and 3) increased cell cholesteryl ester synthesis. HDL cholesteryl ester uptake is increased when cells are incubated for 18 hr in cholesterol poor media. Coincubation of 3H-cholesteryl ester-labeled low-density lipoprotein (LDL) with 50 microM chloroquine or 25 microM monensin results in a decrease in the cellular free cholesterol/cholesteryl ester (FC/CE) isotope ratio, indicating an inhibition in the conversion of cholesteryl ester to free cholesterol. In contrast, chloroquine and monensin do not alter the cellular FC/CE isotope ratio for 3H-CE HDL. This evidence indicates that acidic lysosomal cholesteryl ester hydrolase does not account for the hydrolysis of HDL-CE. Free cholesterol generated from 3H-cholesteryl ester of both LDL and HDL is reesterified intracellularly. At higher HDL concentrations (above 50 micrograms/ml) HDL cholesteryl ester hydrolysis is sensitive to chloroquine. We propose that an extralysosomal pathway is operating in the metabolism of HDL cholesterol and that at higher HDL concentrations a lysosomal pathway may be functioning in addition to an extralysosomal pathway.     

Regulation of hepatic cholesterol ester hydrolase and acyl-coenzyme A:cholesterol acyltransferase in the rat

Cholesterol exists within the hepatocyte as free cholesterol and cholesteryl ester. The proportion of intrahepatic cholesterol in the free or ester forms is governed in part by the rate of cholesteryl ester formation by acyl-coenzyme A:cholesterol acyltransferase (ACAT) and cholesteryl ester hydrolysis by neutral cholesterol ester (CE) hydrolase. In other cell types both ACAT and CE hydrolase activities are regulated in response to changes in the need for cellular free cholesterol. In rats, we performed a variety of experimental manipulations in order to vary the need for hepatic free cholesterol and to examine what effect, if any, this had on the enzymes that govern cholesteryl ester metabolism. Administration of a 20-mg bolus of lipoprotein cholesterol or a diet supplemented with 2% cholesterol resulted in an increase in microsomal cholesteryl ester content with little change in microsomal free cholesterol. This was accomplished by an increase in cholesteryl esterification as measured by ACAT but no change in CE hydrolase activity. An increased need for hepatic free cholesterol was experimentally induced by intravenous bile salt infusion or cholestyramine (3%) added to the diet. ACAT activity was decreased with both experimental manipulations compared to controls, while CE hydrolase activity did not change. Microsomal cholesteryl ester content decreased significantly with little change in microsomal free cholesterol content. Addition of exogenous liposomal cholesterol to liver microsomes from cholestyramine-fed and control rats resulted in a 784 +/- 38% increase in ACAT activity. Nevertheless, the decrease in ACAT activity with cholestyramine feeding was maintained. These studies allowed us to conclude that changes in hepatic free cholesterol needs are met in part by regulation of the rate of cholesterol esterification by ACAT without a change in the rate of cholesteryl ester hydrolysis by CE hydrolase.     

Regulation of high density lipoprotein receptors in cultured macrophages: role of acyl-CoA:cholesterol acyltransferase

The interaction of human serum high density lipoproteins (HDL) with mouse peritoneal macrophages and human blood monocytes was studied. Saturation curves for binding of apolipoprotein E-free [125I]HDL3 showed at least two components: non-specific binding and specific binding that saturated at approximately 40 micrograms HDL protein/ml. Scatchard analysis of specific binding of apo E-free [125I]-HDL3 to cultured macrophages yielded linear plots indicative of a single class of specific binding sites. Pretreatment of [125I]HDL3 with various apolipoprotein antibodies (anti apo A-I, anti apo A-II, anti apo C-II, anti apo C-III and anti apo E) and preincubation of the cells with anti-idiotype antibodies against apo A-I and apo A-II prior to the HDL binding studies revealed apolipoprotein A-I as the ligand involved in specific binding of HDL. Cellular cholesterol accumulation via incubation with acetylated LDL led to an increase in HDL binding sites as well as an increase in the activity of the cytoplasmic cholesterol esterifying enzyme acyl-CoA:cholesterol acyltransferase (ACAT). Incubation of the cholesterol-loaded cells in the presence of various ACAT inhibitors (Sandoz 58.035, Octimibate-Nattermann, progesterone) revealed a time- and dose-dependent amplification in HDL binding and HDL-mediated cholesterol efflux. It is concluded that the homeostasis of cellular cholesterol in macrophages is regulated in part by the number of HDL binding sites and that ACAT inhibitors enhance HDL-mediated cholesterol efflux from peripheral cells.