Inhibition of the acidification of endosomes and lysosomes by the antibiotic concanamycin B in macrophage J774.

The antibiotic concanamycin B was found to inhibit oxidized-low-density-lipoprotein(LDL)-induced accumulation of lipid droplets in the macrophage J774 at a concentration of 5-10 nM. Concanamycin B inhibited cholesteryl-ester synthesis from [14C]oleate by 50% at 14 nM without affecting the synthesis of triacylglycerol and polar lipids. Degradation of internalized oxidized 125I-LDL was inhibited by about 80% in cells treated with 25 nM concanamycin B, while cell-surface binding of oxidized 125I-LDL at 4 degrees C, uptake of surface-bound oxidized 125I-LDL and microsomal acyl-CoA:cholesterol acyltransferase activity were not significantly affected by the antibiotic at 25 nM. When J774 cells were treated with 25 nM concanamycin B at 37 degrees C for 60 min, there was a reduction of about 50% in the activity of cell-surface receptors. This reduction appeared to be due to partial trapping of the receptors within the cells. Concanamycin B significantly inhibited ATP-dependent acidification of endosomes and lysosomes of the J774 cells at a concentration of 4 nM. Since acidic condition of these organelles is required for receptor recycling and hydrolysis of lipoproteins, the results demonstrate that concanamycin-B inhibition of oxidized-LDL-induced accumulation of lipid droplets and cholesteryl esters in macrophages J774 is associated with reduced ATP-dependent acidification of these organelles.     

Effects of cellular cholesterol loading on macrophage foam cell lysosome acidification

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. The variety of cholesterol-containing particles producing inhibition of hydrolysis suggests that inhibition may relate to general changes in lysosomes. Lysosome pH is a key mediator of activity and thus is a potential mechanism for lipid-induced inhibition. We investigated the effects of cholesterol accumulation on THP-1 macrophage lysosome pH. Treatment with OxLDL, AggLDL, and DISPs resulted in inhibition of the lysosome's ability to maintain an active pH and concomitant decreases in CE hydrolysis. Consistent with an overall disruption of lysosome function, exposure to OxLDL or AggLDL reduced lysosomal apolipoprotein B degradation. The lysosomal cholesterol sequestration and inactivation are not observed in cholesterol-equivalent cells loaded using acetylated low density lipoprotein (AcLDL). However, AcLDL-derived cholesterol in the presence of progesterone (to block cholesterol egression from lysosomes) inhibited lysosome acidification. Lysosome inhibition was not attributable to a decrease in the overall levels of vacuolar ATPase. However, augmentation of membrane cholesterol in isolated lysosomes inhibited vacuolar ATPase-dependent pumping of H+ ions into lysosomes. These data indicate that lysosomal cholesterol accumulation alters lysosomes in ways that could exacerbate foam cell formation and influence atherosclerotic lesion development.     

Triglyceride alters lysosomal cholesterol ester metabolism in cholesteryl ester-laden macrophage foam cells

In late-stage atherosclerosis, much of the cholesterol in macrophage foam cells resides within enlarged lysosomes. Similarly, human macrophages incubated in vitro with modified LDLs contain significant amounts of lysosomal free cholesterol and cholesteryl ester (CE), which disrupts lysosomal function similar to macrophages in atherosclerotic lesions. The lysosomal cholesterol cannot be removed, even in the presence of strong efflux promoters. Thus, efflux of sterol is prevented. In the artery wall, foam cells interact with triglyceride-rich particles (TRPs) in addition to modified LDLs. Little is known about how TRP metabolism affects macrophage cholesterol. Therefore, we explored the effect of TRP on intracellular CE metabolism. Triglyceride (TG), delivered to lysosomes in TRP, reduced CE accumulation by 50%. Increased TG levels within the cell, particularly within lysosomes, correlated with reductions in CE content. The volume of cholesterol-engorged lysosomes decreased after TRP treatment, indicating cholesterol was cleared. Lysosomal TG also reduced the cholesterol-induced inhibition of lysosomal acidification allowing lysosomes to remain active. Enhanced degradation and clearance of CE may be explained by movement of cholesterol out of the lysosome to sites where it is effluxed. Thus, our results show that introduction of TG into CE-laden foam cells influences CE metabolism and, potentially, atherogenesis.—Ullery-Ricewick, J. C., B. E. Cox, E. E. Griffin, and W. G. Jerome. Triglyceride alters lysosomal cholesterol ester metabolism in cholesteryl ester-laden macrophage foam cells.     

Determinants of [Cl-] in recycling and late endosomes and Golgi complex measured using fluorescent ligands

Chloride concentration ([Cl-]) was measured in defined organellar compartments using fluorescently labeled transferrin, alpha2-macroglobulin, and cholera toxin B-subunit conjugated with Cl--sensitive and -insensitive dyes. In pulse-chase experiments, [Cl-] in Tf-labeled early/recycling endosomes in J774 cells was 20 mM just after internalization, increasing to 41 mM over approximately 10 min in parallel to a drop in pH from 6.91 to 6.05. The low [Cl-] just after internalization (compared with 137 mM solution [Cl-]) was prevented by reducing the interior-negative Donnan potential. [Cl-] in alpha2-macroglobulin-labeled endosomes, which enter a late compartment, increased from 28 to 58 mM at 1-45 min after internalization, whereas pH decreased from 6.85 to 5.20. Cl- accumulation was prevented by bafilomycin but restored by valinomycin. A Cl- channel inhibitor slowed endosomal acidification and Cl- accumulation by approximately 2.5-fold. [Cl-] was 49 mM and pH was 6.42 in cholera toxin B subunit-labeled Golgi complex in Vero cells; Golgi compartment Cl- accumulation and acidification were reversed by bafilomycin. Our experiments provide evidence that Cl- is the principal counter ion accompanying endosomal and Golgi compartment acidification, and that an interior-negative Donnan potential is responsible for low endosomal [Cl-] early after internalization. We propose that reduced [Cl-] and volume in early endosomes permits endosomal acidification and [Cl-] accumulation without lysis.     

LDL受体对清道夫受体活性的影响

应用经ＰＭＡ诱导衍生的ＴＨＰ－１巨噬细胞为模型,以单克隆抗体Ｃ７Ｂ封闭ｏｘＬＤＬ上的ＬＤＬ受体结合位点,结果发现,正常细胞在摄取ｏｘＬＤＬ时ＬＤＬ受体与清道夫受体起协同作用;但Ｃ７Ｂ作用于蓄积了脂质的ＴＨＰ－１巨噬细胞时,对细胞脂质蓄积程度无明显影响,清道夫受体活性不但不降低反而有所升高,说明由于脂质蓄积ＬＤＬ受体的作用减弱．     

Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells.

Bafilomycin A1 is known as a strong inhibitor of the vacuolar type H(+)-ATPase in vitro, whereas other type ATPases, e.g. F1,F0-ATPase, are not affected by this antibiotic (Bowman, E.M., Siebers, A., and Altendorf, K. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7972-7976). Effects of this inhibitor on lysosomes of living cultured cells were tested. The acidification of lysosomes revealed by the incubation with acridine orange was completely inhibited when BNL CL.2 and A431 cells were treated with 0.1-1 microM bafilomycin A1. The effect was revealed by washing the cells. Both studies using 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine and fluorescein isothiocyanate-dextran showed that the intralysomal pH of A431 cells increased from about 5.1-5.5 to about 6.3 in the presence of 1 microM bafilomycin A1. The pH increased gradually in about 50 min. In the presence of 1 microM bafilomycin A1, 125I-labeled epidermal growth factor (EGF) bound to the cell surface at 4 degrees C was internalized normally into the cells at 37 degrees C but was not degraded at all, in marked contrast to the rapid degradation of 125I-EGF in the control cells without the drug. Immunogold electron microscopy showed that EGF was transported into lysosomes irrespective of the addition of bafilomycin A1. These results suggest that the vacuolar type H(+)-ATPase plays a pivotal role in acidification and protein degradation in the lysosomes in vivo.     

Defective catabolism of oxidized LDL by J774 murine macrophages.

In J774 murine macrophages, chemically oxidized LDL (OxLDL) and biologically oxidized LDL (BioOxLDL) have similar metabolic fates, characterized by a relatively poor degradation when compared with acetylated LDL (AcLDL), and a modest ability to activate acyl-CoA:cholesterol acyltransferase (ACAT) (850 and 754 pmol [14C]oleate/mg cell protein in OxLDL- and BioOxLDL-incubated cells, versus 425 and 7070 pmol [14C]cholesteryl oleate/mg cell protein in control and AcLDL-incubated cells) with a massive increase of cellular free cholesterol. Therefore, OxLDL were used to investigate the cellular processing of oxidatively modified LDL. Binding and fluorescence microscopy studies demonstrated that OxLDL are effectively bound and internalized by macrophages and accumulate in organelles with density properties similar to those of endo/lysosomes. Although the overall metabolism of OxLDL is modestly affected by 100 microM chloroquine, owing to the poor cellular degradation of the substrate, the drug can further depress OxLDL degradation, indicating that this process takes place in an acidic compartment. Failure to detect products of extensive degradation of OxLDL in the medium is due to their relative resistance to enzymatic hydrolysis, as demonstrated also by in vitro experiments with partially purified lysosomal enzymes, rather than to the intracellular accumulation of degradation products (degraded intracellular protein is, at most, 8.5% of total). This sluggish degradation process is not due to a cytotoxic effect since OxLDL do not affect the intracellular processing of other ligands like AcLDL or IgG. The accumulation of OxLDL-derived products within macrophages may elicit cellular responses, the relevance of which in the atherosclerotic process remains to be addressed.     

Effects of LDL enriched with different dietary fatty acids on cholesteryl ester accumulation and turnover in THP-1 macrophages

LDL enriched with either saturated, monounsaturated, n-6 polyunsaturated, or n-3 polyunsaturated fatty acids were used to study the effects of dietary fatty acids on macrophage cholesteryl ester (CE) accumulation, physical state, hydrolysis, and cholesterol efflux. Incubation of THP-1 macrophages with acetylated LDL (AcLDL) from each of the four diet groups resulted in both CE and triglyceride (TG) accumulation, in addition to alterations of cellular CE, TG, and phospholipid fatty acyl compositions reflective of the individual LDLs. Incubation with monounsaturated LDL resulted in significantly higher total and CE accumulation when compared with the other groups. After TG depletion, intracellular anisotropic lipid droplets were visible in all four groups, with 71% of the cells incubated with monounsaturated AcLDL containing anisotropic lipid droplets, compared with 30% of cells incubated with n-3 AcLDL. These physical state differences translated into higher rates of both CE hydrolysis and cholesterol efflux in the n-3 group. These data suggest that monounsaturated fatty acids may enhance atherosclerosis by increasing both cholesterol delivery to macrophage foam cells and the percentage of anisotropic lipid droplets, while n-3 PUFAs decrease atherosclerosis by creating more fluid cellular CE droplets that accelerate the rate of CE hydrolysis and the efflux of cholesterol from the cell.     

Modulation of endosomal cholesteryl ester metabolism by membrane cholesterol

Cells acquire cholesterol in part by endocytosis of cholesteryl ester containing lipoproteins. In endosomes and lysosomes cholesteryl ester is hydrolyzed by acidic cholesteryl ester hydrolase producing cholesterol and fatty acids. Under certain pathological conditions, however, such as in atherosclerosis, excessive levels of cholesteryl ester accumulate in lysosomes for reasons that are poorly understood. Here, we have studied endosomal and lysosomal cholesteryl ester metabolism in cultured mouse macrophages and with cell-free extracts. We show that net hydrolysis of cholesteryl ester is coupled to the transfer of cholesterol to membranes. When membrane cholesterol levels are low, absorption of cholesterol effectively drives cholesteryl ester hydrolysis. When cholesterol levels in acceptor membranes approach saturation or when cholesterol export is blocked, cholesterol is re-esterified in endosomes. These results reveal a new facet of cellular cholesterol homeostasis and provide a potential explanation for cholesteryl ester accumulation in lysosomes of atherosclerotic cells.     

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.     

Metabolism of cholesteryl ester lipid droplets in a J774 macrophage foam cell model

J774 macrophages rapidly incorporated [3H]cholesteryl oleate droplets by a non-saturable phagocytic process. In less than 2 h, foam cell morphology was acquired. The extent of loading obtained after 2 h was a linear function of the mass of cholesteryl oleate provided to the cells. The cholesteryl oleate incorporated was hydrolyzed in the cells at a linear rate over 24 h and the fractional hydrolysis was constant over a wide range of cellular esterified cholesterol contents. The rate of hydrolysis was influenced by the physical state of the cholesteryl ester; cholesteryl oleate in isotropic droplets was hydrolyzed 2-3-fold more rapidly than cholesteryl oleate in anisotropic droplets. The hydrolysis of both types of droplets was inhibited by lysosomotropic agents, indicating that hydrolysis occurred in the lysosomes. Only a small fraction (less than 10% after 24 h) of the free [3H]cholesterol generated in the lysosomes was esterified by ACAT resulting in a doubling of the cell free cholesterol content. Electron microscopy of cells treated with digitonin revealed the accumulation of free cholesterol in lipid-laden lysosomes. ACAT was active as endogenous free [14C]cholesterol was esterified in a linear manner over 24 h and was responsive to the presence of lysosomally-derived cholesterol, as the extent of esterification of the endogenous pool was directly proportional to the mass of [3H]cholesterol generated in the lysosomes.     

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.     

Characterization of human monocytic cell line, U937, in taking up acetylated low-density lipoprotein and cholesteryl ester accumulation. A flow cytometric and HPLC study

The uptake of LDL and acetylated LDL and the ability of cholesteryl ester accumulation by cells of a human monocytic cell line, U937, has been characterized by flow cytometric assay using a fluorescent probe, DiI, and by high-performance liquid chromatography (HPLC). The increase of mean fluorescence intensity of U937 incubated with DiI-labeled lipoproteins demonstrates that this cell line could incorporate DiI-AcLDL, as well as DiI-labeled LDL. Competition and saturation studies indicate that the manner of taking up DiI-AcLDL is receptor-mediated. While differentiated U937 incubated with 16 nM phorbol myristate acetate for 24 h took up little DiI-AcLDL, HPLC analysis confirmed that intracellular free and esterified cholesterols significantly increase in the U937 cells incubated with AcLDL or LDL. The ability of mouse peritoneal macrophage to abundantly accumulate at least five kinds of cholesteryl ester were also shown in this analysis. In contrast, in U937 cells, free fatty acids are incorporated into various substances rather than into cholesteryl esters (as revealed by HPLC analysis), so that the cholesterol in AcLDL taken up by U937 cells is not synthesized into cholesteryl esters to any great extent.     

Direct observation of rapid internalization and intracellular transport of sterol by macrophage foam cells

Transport of the fluorescent cholesterol analog dehydroergosterol (DHE) from the plasma membrane was studied in J774 macrophages (Mphis) with normal and elevated cholesterol content. Cells were labeled with DHE bound to methyl-beta-cyclodextrin. In J774, Mphis with normal cholesterol, intracellular DHE became enriched in recycling endosomes, but was not highly concentrated in the trans-Golgi network or late endosomes and lysosomes. After raising cellular cholesterol by incubation with acetylated low-density lipoprotein (AcLDL), DHE was transported to lipid droplets, and less sterol was found in recycling endosomes. Transport of DHE to droplets was very rapid (t1/2 = 1.5 min after photobleaching) and did not require metabolic energy. In cholesterol-loaded J774 Mphis, the initial fraction of DHE in the plasma membrane was reduced, and rapid DHE efflux from the plasma membrane to intracellular organelles was observed. This rapid sterol transport was not related to plasma membrane vesiculation, as DHE did not become enriched in endocytic vesicles formed after sphingomyelinase C treatment of cells. When cells were incubated with DHE ester incorporated into AcLDL, fluorescence of the sterol was first found in punctate endosomes. After a chase, this DHE colocalized with transferrin in a distribution similar to cells labeled with DHE delivered by methyl-beta-cyclodextrin. Our results indicate that elevation of sterol levels in Mphis enhances transport of sterol from the plasma membrane by a non-vesicular pathway.     

Essential involvement of 12-lipoxygenase in regiospecific andstereospecific oxidation of low density lipoprotein by macrophages.

To establish a role of the 12-lipoxygenase on the generation of oxidized low density lipoprotein (LDL) in macrophages that leads to foam cell formation in atherosclerosis, we overexpressed 12-lipoxygenases in a macrophage-like cell line, J774A.1, that does not show intrinsic enzyme activity. When the 12-lipoxygenase-expressing cells were incubated with 400 microg.mL-1 LDL in Dulbecco's modified Eagle's medium at 37 degrees C for 12 h, LDL oxidation, as determined by thiobarbituric acid reactive substance, was markedly increased compared with the mock-transfected cells. Oxygenated products in the modified LDL were examined by HPLC before and after alkaline hydrolysis. Most of the oxygenated derivatives were of an esterified form, and the major product was identified as 13S-hydroxyoctadeca-9Z,11E-dienoic acid. These results clearly demonstrate that esterified fatty acids in LDL are oxygenated by the 12-lipoxygenases expressed in the J774A.1 cells. Furthermore, the oxidized LDL generated by intracellular 12-lipoxygenases was recognized by a scavenger receptor as assessed by macrophage degradation assay.     

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.     

Alpha-tocopherol decreases CD36 expression in human monocyte-derived macrophages

Cholesterol-laden macrophages are the hallmark of atherogenesis. The class B scavenger receptor, CD36, binds oxidized low density lipoprotein (OxLDL), is found in atherosclerotic lesions, and is upregulated by OxLDL. We tested the effects of alpha-tocopherol (AT) enrichment of human monocyte-derived macrophages on CD36 expression and cholesteryl ester accumulation. Monocytes isolated from normal volunteers were cultured into macrophages. Macrophages were enriched overnight with various doses of AT (25, 50, and 100 microM). LDL from normal volunteers was oxidized or acetylated (AcLDL) and incubated with macrophages for 48 h at a concentration of 50 or 100 microg/ml. CD36 expression was assessed by flow cytometry. Quantitative analysis of scavenger receptor class A (SR-A) activity was performed with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanide perchlorate (DiI)-labeled LDL. CD36 expression was maximal after 8;-10 days of culture. AT (> or =50 microM) significantly decreased CD36 expression upregulated by OxLDL and AcLDL (P < 0.01). Other antioxidants (beta- or gamma-tocopherol) or protein kinase C inhibitors failed to decrease CD36 expression. Concomitantly, DiI-AcLDL and DiI-OxLDL uptake was significantly decreased after AT treatment (P < 0.001). Cholesteryl ester accumulation was significantly decreased after AT enrichment (AcLDL + AT, 77% inhibition; OxLDL + AT, 42% inhibition). In conclusion, AT decreases both CD36 and SR-A expression and cholesteryl ester accumulation in human macrophages. This provides additional scientific support for the antiatherogenic properties of AT.     

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.