Cholesteryl Ester Accumulation In Macrophages Research Articles

Superoxide-mediated modification of low density lipoprotein by arterial smooth muscle cells.

Extracellular superoxide was detected in cultures of monkey and human arterial smooth muscle cells as indicated by superoxide dismutase inhibitable reduction of cytochrome c. Superoxide production by these cells in the presence of Fe or Cu resulted in modification of low density lipoprotein (LDL). The degree of LDL modification was directly proportional to the rate of superoxide production by cells. Superoxide dismutase (100 micrograms/ml), and the general free radical scavengers butylated hydroxytoluene and butylated hydroxyanisole (50 microM), inhibited Fe- and Cu-mediated modification of LDL by monkey smooth muscle cells, while catalase (100 micrograms/ml) and mannitol (25 mM) had no effect. The chelators desferrioxamine and diethylenetriamine pentaacetic acid completely inhibited Fe- and Cu-promoted modification of LDL, while EGTA had no inhibitory effect. EDTA stimulated Fe-promoted modification in the 1-100 microM range while inhibiting Cu-mediated modification of LDL. LDL modified by smooth muscle cells in the presence of 10 microM Fe or Cu stimulated [14C]oleate incorporation into cholesteryl ester by human macrophages and murine J774 cells to a degree comparable to that produced by acetylated LDL. LDL incubated with smooth muscle cells and metal ions in the presence of superoxide dismutase failed to enhance macrophage cholesteryl ester accumulation. Thus, arterial smooth muscle cells in culture generate superoxide and modify LDL by a superoxide-dependent, Fe or Cu catalyzed free radical process, resulting in enhanced uptake of the modified LDL by macrophages. Neither hydroxyl radicals nor H2O2 are likely to be involved. Superoxide-dependent lipid peroxidation may contribute to biological modification of LDL, resulting in foam cell formation and atherogenesis.

Complexes of low-density lipoproteins and arterial proteoglycan aggregates promote cholesteryl ester accumulation in mouse macrophages

We studied the effect of complexes of low-density lipoproteins (LDL) and different proteoglycan preparations from bovine aorta on LDL degradation and cholesteryl ester accumulation in mouse peritoneal macrophages. Native proteoglycan aggregate containing proteoglycan monomers, hyaluronic acid and link protein was isolated by associative extraction of aortic tissue, while proteoglycan monomer was obtained by dissociative isopycnic centrifugation of the native proteoglycan aggregate. In vitro proteoglycan aggregates were prepared by reaction of the proteoglycan monomer with exogenous hyaluronic acid. 125I-labeled LDL-proteoglycan complexes were formed in the presence of 30 mM Ca 2+ and incubated with macrophages. At equivalent uronic acid levels in the proteoglycans the degradation of 125I-labeled LDL contained in the native proteoglycan aggregate complex was 3.7–7.5-fold greater than the degradation of the lipoprotein in the proteoglycan monomer complex. Degradation of 125I-LDL in the in vitro aggregate complex, while higher than that in the monomer complex, was markedly less than that in the native aggregate complex. The larger size and the greater complex-forming ability of the native proteoglycan aggregate might account for the greater capacity of the aggregate to promote LDL degradation in macrophages. The proteoglycan-stimulated degradation of LDL produced a marked increase in cholesteryl ester synthesis and content in macrophages. The LDL-proteoglycan complex was degraded with saturation kinetics, suggesting that these complexes are internalized through high-affinity receptors. Degradation was inhibited by the lysosomotropic agent, chloroquine. Acetyl-LDL, but not native LDL, competitively inhibited the degradation of the 125I-LDL component of the complex. Polyanionic compounds such as polyinosinic acid and fucoidin, while completely blocking the acetyl-LDL-stimulated cholesteryl ester formation, had no effect on the proteoglycan aggregate-stimulated cholesterol esterification. This suggests that LDL-proteoglycan complex and acetyl-LDL are not entering the cells through the same receptor pathway. These results demonstrate that the interaction of LDL with arterial wall proteoglycan aggregates results in marked cholesteryl ester accumulation in macrophages, a process likely to favor foam cell formation. A role for arterial proteoglycans in atherosclerosis is obvious.

Characterization of very low density lipoproteins and intermediate density lipoproteins of normo- and hyperlipidemic apolipoprotein E-2 homozygotes.

Triglyceride-rich lipoproteins derived from ten normo- and hyperlipidemic apoE-2 homozygotes were analyzed for their composition, beta-VLDL content, and their ability to induce cholesteryl ester storage in macrophages. In six of these probands apoE sequence analysis revealed that the cysteine residues were at positions 112 and 158 of the amino acid sequence (Rall et al. 1983. J. Clin. Invest. 71: 1023-1031). ApoE-2 of these six and the other four patients was further analyzed by SDS electrophoresis to exclude the presence of apoE-2* (Rall et al. 1982. Proc. Natl. Acad. Sci. USA. 79: 4696-4700). The relative serum concentrations of free and esterified cholesterol transported in the d less than 1.006 g/ml and d 1.006-1.019 g/ml lipoproteins of the apoE-2 homozygotes was significantly higher as compared to controls. Compositional analysis of these lipoproteins revealed a relative reduction of triglycerides and a relative increase of cholesteryl esters as compared to controls. In most patients, with increasing serum triglyceride levels the cholesteryl ester concentration increased in d less than 1.006 g/ml and d 1.006-1.019 g/ml lipoproteins. However, in three patients with a low content of beta-VLDL, the increase in the d less than 1.006 g/ml fraction cholesterol was mostly due to free cholesterol and not due to cholesteryl esters. The degree of the macrophage cholesteryl ester accumulation induced by d less than 1.006 g/ml lipoproteins was mostly dependent on the concentration of the beta-migrating fraction (beta-VLDL). The amount of beta-VLDL and pre-beta-VLDL contained in the d less than 1.006 g/ml fraction was determined densitometrically after electrophoretic separation. It could be demonstrated that the beta-VLDL content in the d less than 1.006 g/ml fraction of the apoE-2 homozygous patients was largely independent of serum triglyceride and serum cholesterol levels. When macrophages were incubated with the IDL fraction (d 1.006-1.019 g/ml) from the apoE-2 patients, no significant increase in cellular cholesteryl esters above control levels was observed. Studies with purified lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) clearly revealed that both enzymes interacted with apoE-2 VLDL (binding, hydrolysis) to a lesser degree compared to control preparations. However, the apoE-2 VLDL preparations containing a low content of beta-VLDL were better substrates for LPL and HTGL than those containing a high beta-VLDL content. It is concluded from our studies that the plasma beta-VLDL content in apoE-2 homozygotes is a major determinant for cholesteryl ester accumulation in macrophages.(ABSTRACT TRUNCATED AT 400 WORDS)

Lipoprotein-heparin-fibronectin-denatured collagen complexes enhance cholesteryl ester accumulation in macrophages.

The sequestration of low-density lipoprotein (LDL) by components of the vascular extracellular matrix has long been recognized as a contributing factor to lipid accumulation during atherogenesis. The effects, however, that components of the extracellular matrix might have on LDL catabolism by scavenger cells have been little investigated. For these purposes we have prepared insoluble complexes of LDL, heparin, fibronectin, and denatured collagen (gelatin) and examined their effects on lipid accumulation, LDL uptake and degradation, and cholesteryl ester synthesis in mouse peritoneal macrophages. The results of these experiments have demonstrated that the cholesteryl ester content of macrophages incubated with a particular suspension of LDL, heparin, fibronectin, and collagen complexes is four- to fivefold that of cells incubated with LDL alone. The uptake of complexes containing 125I-LDL is rapid; however, in contrast to either endocytosed 125I-LDL or 125I-acetyl LDL, the degradation of complex-derived LDL is impaired. In addition, the uptake of complex-derived LDL stimulates the incorporation of [14C]oleic acid into cholesteryl oleate, however, the stimulation was a small fraction of that observed in cells incubated with acetyl LDL. Ultrastructurally, macrophages incubated with LDL, heparin, fibronectin, and collagen complexes did not contain many lipid droplets, but rather their cytoplasm is filled with phagosomes containing material similar in appearance to LDL-matrix complexes. These results indicate that components of the extracellular matrix can alter the catabolism of LDL by scavenger cells, suggesting that they may play a role in cellular lipid accumulation in the atherosclerotic lesion.

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.

Cholesteryl ester accumulation in mouse peritoneal macrophages induced by beta-migrating very low density lipoproteins from patients with atypical dysbetalipoproteinemia.

The d < 1.006 lipoproteins of patients in a kindred with atypical dysbetalipoproteinemia induced marked cholesteryl ester accumulation in mouse peritoneal macrophages. The affected family members had severe hypercholesterolemia and hypertriglyceridemia, xanthomatosis, premature vascular disease, the apo-E3/3 phenotype, and a predominance of cholesterol-rich beta-very low density lipoproteins (beta-VLDL) in the d < 1.006 fraction. When incubated with mouse peritoneal macrophages, the d < 1.006 lipoproteins or beta-VLDL from the affected family members stimulated cholesteryl [(14)C]oleate synthesis 15- to 30-fold above that caused by normal, control d < 1.006 lipoproteins (VLDL). The ability of the beta-VLDL to stimulate macrophage cholesteryl ester accumulation was greatly reduced as a consequence of treatment with hypolipidemic agents, which specifically reduced the concentration of beta-VLDL. Two important differences were noted in a comparison of the beta-VLDL from these atypical dysbetalipoproteinemic subjects with that of classic E2/2 dysbetalipoproteinemics: (a) the beta-VLDL from the atypical subjects were severalfold more active in stimulating cholesteryl ester accumulation in macrophages, and (b) both the intestinal and hepatic beta-VLDL from the atypical subjects were active. The triglyceriderich, alpha(2)-migrating VLDL from the affected family members constituted <10% of the d < 1.006 fraction and were similar to normal VLDL in that they did not stimulate cholesteryl ester synthesis in the macrophages. Several lines of evidence indicate that the macrophage accumulation of cholesteryl esters was induced by a receptor-mediated uptake process and that the beta-VLDL were bound by a specific beta-VLDL receptor. First, the uptake and degradation of the lipoproteins and the induction of cholesteryl ester formation displayed qualities of high affinity, saturable kinetics. Second, the uptake and degradation process was inhibited when the lysyl residues of the beta-VLDL apoproteins were modified by reductive methylation. Third, the beta-VLDL from the affected subjects competed with diet-induced canine (125)I-beta-VLDL for the same cell surface receptors, but did not compete with chemically modified low density lipoproteins. Finally, the receptor-mediated uptake of these beta-VLDL resulted in lysosomal degradation of the lipoproteins, which could be prevented by incubating the cells with chloroquine. Normal, triglyceride-rich VLDL were also degraded when incubated with the macrophages, but they were not degraded by the same receptor-mediated process responsible for the degradation of the beta-VLDL of the patients. The degradation of the VLDL was not abolished by reductive methylation of the lipoproteins or by treatment of the cells with choloroquine. These studies demonstrate that the beta-VLDL from subjects with atypical dysbetalipoproteinemia are taken up by macrophages via the same receptor-mediated process responsible for the uptake of diet induced beta-VLDL. The accelerated vascular disease seen in these patients may be the result of high concentrations of beta-VLDL capable of binding to and delivering large quantities of cholesterol to macrophages and converting them into cells resembling the foam cells of atherosclerotic lesions.

Cholesteryl ester accumulation in macrophages resulting from receptor-mediated uptake and degradation of hypercholesterolemic canine beta-very low density lipoproteins.

The synthesis and accumulation of cholesteryl esters by monolayers of mouse peritoneal macrophages was stimulated 20- to 160-fold by incubation with beta-migrating very low density lipoproteins (beta-VLDL, density less than 1.006 g/ml) isolated from the plasma of cholesterol-fed dogs. Three other cholesterol-rich lipoprotein fractions obtained from the plasma of the same hypercholesterolemic dogs, including low density lipoprotein (LDL), cholesterol-induced high density lipoprotein (HDLc), and apo-E HDLc, had little to no stimulatory effect. Plasma VLDL (density less than 1.006 g/ml) from normal dogs did not increase cholesteryl ester formation in macrophages. The enhancement in cholesteryl ester synthesis and accumulation by hypercholesterolemic canine beta-VLDL was due to the presence of a high affinity binding site on the macrophage cell surface that mediated the uptake and lysosomal degradation of the beta-VLDL. Competition studies with fucoidin and dextran sulfate indicated that the receptor for canine beta-VLDL was different from that previously described for human acetylated low density lipoprotein (acetyl-LDL). Prior incubation of macrophage monolayers with either unlabeled canine beta-VLDL or human acetyl-LDL, both of which raised the cellular content of cholesteryl esters, reduced the ability of the cells to degrade 125I-labeled beta-VLDL, suggesting that the receptor for beta-VLDL is subject to regulation. The current findings indicate: 1) that macrophages possess a high affinity receptor that recognizes one of the four cholesterol-rich lipoproteins present in the plasma of cholesterol-fed dogs, beta-VLDL, and 2) that the receptor-mediated ingestion of beta-VLDL leads to cholesteryl ester deposition in these cells.