Stimulation Of Cholesteryl Ester Synthesis Research Articles

Type III hyperlipoproteinema with apolipoprotein E2/2 genotype in Japan

Limited information is available concerning type III hyperlipoproteinemia (HLP) in the Asian population. Therefore, clinical and biochemical characteristics of type III HLP were examined in 16 Japanese patients. Mean plasma triglyceride (TG) and total cholesterol (chol) levels were 381 mg/dl and 253 mg/dl, respectively, and the mean very low density lipoprotein (VLDL)-chol/plasma TG ratio was 0.27, which were lower than those reported in Western countries. Eighty percent of the patients had high plasma remnant-like particles (RLP)-chol levels above 50 mg/dl and a high RLP-chol/plasma TG ratio above 0.1. Twelve patients (75.0%) were obese. Seven patients (43.8%) had type 2 diabetes mellitus and four patients (25.0%) had impaired glucose tolerance. Six patients (37.5%) had coronary heart disease (CHD), but none had peripheral vascular disease or xanthomas. TG-rich lipoproteins from type III HLP patients with diabetes mellitus stimulated cholesteryl ester synthesis by human macrophages significantly (p < 0.001) more than those from type III HLP patients without diabetes mellitus. In conclusion, the Japanese type III HLP patients had lower plasma TG and total chol levels and a lower VLDL-chol/plasma TG ratio, but CHD was more common. The patients were characterized by a high frequency of obesity and/or glucose intolerance. The TG-rich lipoproteins from type III HLP patients with diabetes mellitus were more atherogenic.

The Hypocholesterolemic Agent LY295427 Reverses Suppression of Sterol Regulatory Element-binding Protein Processing Mediated by Oxysterols

The sterol LY295427 reduces plasma cholesterol levels in animals by increasing the expression of hepatic low density lipoprotein (LDL) receptors. Here we trace the hypocholesterolemic activity of LY295427 to an ability to reverse oxysterol-mediated suppression of sterol regulatory element-binding protein (SREBP) processing. Micromolar concentrations of LY295427 induced the metabolism of LDL in oxysterol-treated cultured cells and inhibited the stimulation of cholesteryl ester synthesis mediated by oxysterols. cDNA microarray and RNA blotting experiments revealed that LY295427 increased levels of the LDL receptor mRNA and those of other SREBP target genes. The compound stimulated the accumulation of SREBPs in the nuclei of cells grown in the presence of oxysterols within 4-6 h of addition to the medium. Induction required components of the normal SREBP-processing pathway, including the SREBP cleavage-activating protein and the Site 1 protease. LY295427 overcame the suppression of SREBP processing mediated by several oxysterols but not by LDL-derived cholesterol. We conclude that LY295427 achieves a therapeutically desirable end point by an unique mechanism of action.

Triglyceride-rich lipoproteins from apolipoprotein E3/2 subjects with hypertriglyceridemia enhance cholesteryl ester synthesis in human macrophages

The ability of triglyceride (TG)-rich lipoproteins from apolipoprotein (apo) E2 heterozygote, apo E3/2 which is relatively common, to stimulate cholesteryl ester synthesis in human monocyte-derived macrophages was studied to clarify the atherogenicity of apo E3/2. Twenty-three subjects were randomly sampled from our hospitals, and divided into the following 4 groups: 7 apo E3/3 subjects with normolipidemia, 6 apo E3/3 subjects with hypertriglyceridemia (HTG), 5 apo E3/2 subjects with normolipidemia and 5 apo E3/2 subjects with HTG. Plasma levels of TG and total cholesterol (chol) were significantly ( P<0.001) higher in apo E3/3 and apo E3/2 subjects with HTG than in apo E3/3 and apo E3/2 subjects with normolipidemia, but plasma levels of TG and total chol were not significantly different between apo E3/3 and apo E3/2 subjects with HTG. [ 14C]oleate incorporation into cholesteryl esters in macrophages was significantly ( P<0.001) higher in apo E3/2 subjects with HTG (0.661 nmol/mg cell protein) than in apo E3/3 subjects with normolipidemia (0.228) and with HTG (0.325) and in apo E3/2 subjects with normolipidemia (0.257). It is concluded that TG-rich lipoproteins from apolipoprotein E3/2 subjects with HTG enhance cholesteryl ester synthesis in human macrophages.

Isolation and characterization of a proteoglycan variant from human aorta exhibiting a marked affinity for low density lipoprotein and demonstration of its enhanced expression in atherosclerotic plaques

Proteoglycans (PG) are implicated in the pathophysiology of atherosclerosis due to their ability to complex with plasma low density lipoproteins (LDL). Studies were conducted to determine whether human aorta contains PG subclasses that exhibit enhanced LDL binding ability. PG were isolated from normal and atherosclerotic aortas by a combination of dissociative extraction and ion-exchange chromatography. The PG were further subfractionated on an LDL affinity column based on their binding affinity to LDL. Two PG fractions exhibiting high-affinity binding to LDL, as evidenced by their elution at 1.0 and 1.5 M NaCl, respectively, were isolated from both normal and atherosclerotic tissue. Compared with normal tissue, atherosclerotic tissue showed a twofold increase in the high-affinity PG that eluted at 1.5 M NaCl. Gel filtration of the high-affinity PG from normal tissue yielded two peaks (nPG2 and nPG3), while the high-affinity PG from plaque tissue was resolved into three peaks (pPG1, pPG2, and pPG3). pPG1 eluted at the void volume of the column, indicating that it was of very large molecular size. The hydrodynamic size of pPG2 was larger than that of the corresponding nPG2 ( K av = 0.44 versus 0.51), while pPG3 had the same hydrodynamic size as nPG3 ( K av = 0.86). The high-affinity PG subfractions from normal aorta contained varying proportions of chondroitin sulfates, dermatan sulfates, and heparan sulfate. In contrast, the PG subfractions from plaque tissue contained predominantly chondroitin sulfates and heparan sulfate. In vitro complexes of LDL and the high-affinity PG fractions from normal aorta and plaque tissue stimulated cholesteryl ester synthesis in human monocyte-derived macrophages. However, the LDL-plaque PG complex was significantly more potent than the LDL-normal aorta PG complex in this respect. These results indicate that PG subclasses with enhanced binding affinity to LDL occur in the normal human aorta and that their concentration increases significantly in atherosclerotic lesions. In addition, the high-affinity PG in plaque tissue have altered characteristics and increased ability to stimulate LDL-mediated cholesterol ester synthesis in macrophages. This could lead to increased lipid deposition during atherogenesis.

Macrophages stimulate cholesteryl ester accumulation in cocultured smooth muscle cells incubated with lipoprotein-proteoglycan complex.

Foam cells of atherosclerotic lesions originate from both macrophages and smooth muscle cells (SMCs). We explored the mechanism by which SMCs may become lipid laden. Confluent bovine aortic SMCs were cocultured with P388D, macrophages, and the cocultures were incubated for various times with low-density lipoprotein (LDL), acetyl-LDL, or lipoprotein-proteoglycan (PG) complex isolated from human atherosclerotic lesions. Macrophages were then removed from the SMCs and the cholesteryl ester (CE) content of the SMCs was quantitated. Lipoprotein-PG complex but not LDL or acetyl-LDL produced a 6-fold to 9-fold stimulation of CE synthesis and a 4.4-fold increase in cellular CE mass in cocultured SMCs relative to control SMCs. In similar studies with human aortic SMC-macrophage cocultures, macrophages stimulated lipoprotein-PG complex-mediated CE synthesis 7-fold to 13-fold and CE mass 7.8-fold in cocultured SMCs compared with SMCs cultured alone. CE synthesis that was mediated by lipoprotein-PG complex was dose dependent and increased linearly with time. Incubation of lipoprotein-PG complex with SMC-macrophage cocultures but not with SMCs or macrophages alone resulted in aggregation of the complex and stimulation of cholesterol esterification in SMCs by the conditioned media containing the aggregated complex. Cytochalasin D, an inhibitor of phagocytosis, inhibited CE synthesis mediated by lipoprotein-PG complex by 73%, whereas polyinosinic acid, an inhibitor of the scavenger receptor, had no effect. Upregulation or downregulation of apolipoprotein B,E receptors did not affect the lipoprotein-PG complex-mediated CE synthesis by cocultured SMCs. Lipoprotein-PG complex did not stimulate CE synthesis in SMCs cocultured with aortic endothelial cells or macrophages cocultured with SMCs. These results indicate that macrophages can stimulate CE synthesis and accumulation in cocultured SMCs when incubated with lipoprotein-PG complexes isolated from atherosclerotic lesions. This could be a potential mechanism for myocyte foam cell formation.

Low-density lipoprotein binding affinity of arterial chondroitin sulfate proteoglycan variants modulates cholesteryl ester accumulation in macrophages

Proteoglycans are considered to facilitate lipid accumulation in the arterial wall, as part of the injury and repair process in atherogenesis. The present study determined (1) characteristics of arterial tissue chondroitin sulfate proteoglycan (CS-PG) monomers of versican type that vary in binding affinity to low-density lipoproteins (LDL), and (2) the ability of these variants to modulate LDL metabolism by macrophages. A large CS-PG devoid of dermatan sulfate (DS) was isolated and purified from bovine aorta intima-media under dissociative conditions. The proteoglycan was further subfractionated by LDL affinity chromatography into CS-PGI and CS-PGII variants, the former eluting at 0.1 M NaCl and the latter at 1.0 M NaCl. The core protein of both variants had a similar molecular mass (1.7 · 10 5). However, CS-PGII contained more glycosarninoglycan (GAG) chains (30 vs. 25) with higher average molecular mass (4.2 · 10 4 vs. 3.8 · 10 4) than CS-PGI. Furthermore, CS-PGII contained a relatively higher proportion of CS6-sulfate to CS4-sulfate (65:35 vs. 52:48). Sulfate-to-hexosamine molar ratio of GAG measured approximately 1 in both variants. In terms of metabolism by macrophages, when compared to complex of LDL and CS-PGI, complex of LDL and CS-PGII produced consistent increase in degradation (10.3-fold vs. 8.4-fold over native LDL) and cell association (16.3-fold vs. 10.2-fold over native LDL) of the ligand, and stimulation of cholesteryl ester synthesis (8.4-fold vs. 6.4-fold over native LDL). CS-PGII was as potent as native CS/DS-PG aggregate, which is a complex made of proteoglycan monomers, hyaluronate, and link protein(s), in stimulating the above activities in macrophages. Thus, variations in LDL-binding affinity of CS-PG can potentially modulate the lipid accumulation in atherogenesis.

Neutral sphingomyelinase action stimulates signal transduction of tumor necrosis factor-alpha in the synthesis of cholesteryl esters in human fibroblasts.

We have investigated biochemical mechanisms of tumor necrosis factor (TNF)-alpha signaling in cultured human skin fibroblasts. We found that TNF-alpha signaling may involve activation of a cell membrane neutral sphingomyelinase (N-SMase) in that within 2.5-5 min of treatment of cells with TNF-alpha there was a 2-fold increase in the activity of N-SMase compared to control. This reaction led to the hydrolysis of sphingomyelin as evidenced by a decrease in sphingomyelin mass and in the radioactivity associated with [14C]choline-labeled sphingomyelin. This was accompanied by a 4-fold increase in the formation of cholesteryl [14C]oleate within 2.5 min of incubation with TNF-alpha. This reaction also stimulated the mobilization of cell surface-associated [3H]cholesterol and its utilization in the synthesis of [3H]cholesteryl esters via acyl coenzyme-A cholesterol acyltransferase (ACAT). Gas chromatographic analysis revealed that the cellular level of cholesteryl esters increased about 2.5-3-fold following treatment with TNF-alpha compared to control. Cholesteryl ester synthesis was compromised upon incubation of cells with antibody against N-SMase and remained unaltered with TNF-beta and fibroblast growth factor. Furthermore, TNF-alpha-mediated stimulation of cholesteryl ester synthesis was compromised by incubation of cells with an inhibitor of ACAT. These findings suggest a possible biological role of N-SMase in the signal transduction of TNF-alpha in the synthesis of cholesteryl esters in human fibroblasts.

Lipoprotein-proteoglycan complexes induce continued cholesteryl ester accumulation in foam cells from rabbit atherosclerotic lesions.

We studied the metabolism of lipoprotein-proteoglycan complexes by macrophage-derived foam cells. Foam cells were isolated from atherosclerotic rabbit aortas. ApoB-lipoprotein-proteoglycan complex was isolated from human aorta fibrous plaque lesions and LDL-proteoglycan complex was formed in vitro. Both in vitro and in vivo complexes stimulated cholesteryl ester synthesis in foam cells by a dose-dependent, saturable process that resulted in the intracellular accumulation of cholesteryl ester. Stimulation of cholesteryl ester synthesis was linear with time over a 32-h period. Polyinosinic acid inhibited the stimulation of cholesteryl ester synthesis by the complexes by 32-37%, whereas cytochalasin D only produced a 6-16% inhibition. Foam cells degraded 125I-LDL-proteoglycan complex and 125I-acetyl LDL in a saturable, dose-dependent manner. Excess unlabeled acetyl-LDL inhibited the degradation of 125I-LDL-proteoglycan complex by 52%, while LDL had no effect. Similarly, excess unlabeled complex suppressed the degradation of 125I-acetyl-LDL by 48%. Foam cells degraded 125I-methyl-LDL-proteoglycan complex to the same extent as 125I-LDL-proteoglycan complex. These results show that foam cells from atherosclerotic lesions metabolize lipoprotein-proteoglycan complexes predominantly via receptor-mediated endocytosis and consequently continue to accumulate intracellular cholesteryl ester.

Human monocyte-derived macrophages bind low-density-lipoprotein-proteoglycan complexes by a receptor different from the low-density-lipoprotein receptor

We have shown recently that lipoprotein-proteoglycan complexes isolated from human atherosclerotic lesions stimulated cholesteryl ester synthesis in human monocyte-derived macrophages [Vijayagopal, Srinivasan, Radhakrishnamurthy and Berenson (1992) Arterioscler. Thromb. 12, 237-249]. The present study was conducted to determine the mechanism of cellular uptake of the complexes. A chondroitin sulphate-dermatan sulphate proteoglycan was isolated from normal human aorta and complexed to 125I-labelled human low-density lipoprotein (LDL). The binding and degradation of 125I-LDL-proteoglycan complex were then studied in human monocyte-derived macrophages. The specific binding and degradation of the complex showed saturability and concentration-dependency. The Kd for binding was 1.5 x 10(-8) M, which was greater than that reported for LDL in monocyte-derived macrophages. Binding of the complex was not subject to down-regulation. Chloroquine inhibited degradation of the complex and the resultant stimulation of cholesteryl ester synthesis. Limited treatment of macrophages with proteolytic enzymes abolished binding and degradation of the complex significantly. Macrophages bound 125I-methyl-LDL-proteoglycan complex to the same extent as 125I-LDL-proteoglycan complex. Excess LDL and proteoglycan did not compete against the binding of the complex; however, excess acetyl-LDL competed for 61% of the binding. Likewise, excess LDL-proteoglycan complex inhibited the binding of 125I-acetyl-LDL by 64%. Polyinosinic acid and cytochalasin D inhibited the binding of 125I-LDL-proteoglycan complex by 60% and 36% respectively. Compared with that of acetyl-LDL, the degradation of LDL-proteoglycan complex was retarded in human macrophages. The results indicate that the uptake of LDL-proteoglycan complex in human monocyte-derived macrophages is not mediated through binding to the LDL receptor; but occurs predominantly via the scavenger receptor, with phagocytosis playing a minor role in the process.

Neutral sphingomyelinase increases the binding, internalization, and degradation of low density lipoproteins and synthesis of cholesteryl ester in cultured human fibroblasts.

I have investigated the effects of human urinary neutral sphingomyelinase (N-SMase) (Chatterjee, S., and Ghosh, N. (1989) J. Biol. Chem. 264, 12554-12561) on the cell-surface binding, internalization, and degradation of 125I-low density lipoprotein (LDL) and on cholesteryl ester synthesis in cultured human fibroblasts. N-SMase exerted a concentration-dependent continuous stimulation of 125I-LDL cell-surface binding, internalization, and degradation in normal human fibroblasts. A 3-fold increase in binding, internalization, and degradation was observed at the maximum amount (600 units of N-SMase/ml) examined. This phenomenon was accompanied by a continuous stimulation of cholesteryl ester synthesis. A 5-fold increase in cholesteryl ester synthesis was observed after incubation for 4 h with N-SMase. Antibody against N-SMase and heat inactivation of N-SMase compromised the stimulatory effects of N-SMase on 125I-LDL metabolism and cholesteryl ester synthesis in these cells. Incubation of cells with phospholipase D and phospholipase C did not alter 125I-LDL binding, internalization, or degradation. This finding suggests that the stimulatory effects of N-SMase on LDL metabolism and on cholesteryl ester synthesis in fibroblasts is specific. Moreover, unlabeled LDL competitively displaced 125I-LDL from binding to N-SMase-treated cells. None of the precursors of sphingomyelin could mimic the stimulatory effects of N-SMase on 125I-LDL metabolism in these cells. Taken together, these studies suggest that one of the biological roles of N-SMase involves modulating LDL metabolism and cholesterol metabolism in fibroblasts.

Low density lipoprotein receptor-related protein and gp330 bind similar ligands, including plasminogen activator-inhibitor complexes and lactoferrin, an inhibitor of chylomicron remnant clearance.

The low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP) and gp330, two members of the low density lipoprotein receptor gene family, share a multitude of cysteine-rich repeats. LRP has been shown to act as an endocytosis-mediating receptor for several ligands, including protease-antiprotease complexes and plasma lipoproteins. The former include alpha 2-macroglobulin-protease complexes and plasminogen activator inhibitor-activator complexes. The latter include chylomicron remnant-like particles designated beta-very low density lipoproteins (beta-VLDL) complexed with apoprotein E or lipoprotein lipase. The binding specificity of gp330 is unknown. In the current studies we show that gp330 from rat kidney membranes binds several of these ligands on nitrocellulose blots. We also show that both LRP and gp330 bind an additional ligand, bovine lactoferrin, which is known to inhibit the hepatic clearance of chylomicron remnants. Lactoferrin blocked the LRP-dependent stimulation of cholesteryl ester synthesis in cultured human fibroblasts elicited by apoprotein E-beta-VLDL or lipoprotein lipase-beta-VLDL complexes. Cross-competition experiments in fibroblasts showed that the multiple ligands recognize at least three distinct, but partially overlapping sites on the LRP molecule. Binding of all ligands to LRP and gp330 was inhibited by the 39-kDa protein, which co-purifies with the two receptors, suggesting that the 39-kDa protein is a universal regulator of ligand binding to both receptors. The correlation of the inhibitory effects of lactoferrin in vivo and in vitro support the notion that LRP functions as a chylomicron remnant receptor in liver. LRP and gp330 share a multiplicity of binding sites, and both may function as endocytosis-mediating receptors for a large number of ligands in different organs.

Studies on the mechanism of uptake of low density lipoprotein-proteoglycan complex in macrophages

Earlier, we (Vijayagopal, P. et al. (1988) Biochim. Biophys. Acta 960, 210) showed that mouse peritoneal macrophages metabolize low density lipoprotein (LDL)-proteoglycan complex by a receptor pathway distinct from the acetyl-LDL receptor. Further studies were conducted to probe further into the mechanism of LDL-proteoglycan complex uptake by macrophages. Both 125I-methyl-LDL-proteoglycan complex and 125I-LDL-proteoglycan complex were taken up and degraded by the cells to the same extent. Similarly, the ability of these ligands to stimulate cholesteryl ester synthesis was also indistinguishable. These results rule out the possibility of apoB,E receptor involvement in the uptake of LDL-proteoglycan complex in macrophages. Sodium fluoride, cytochalasin D and aggregated LDL inhibited degradation of the complex by 24%, 26% and 28%, respectively, indicating that phagocytosis is only a minor pathway for the uptake. Both binding and degradation of the complex were not inhibited by excess hyaluronic acid suggesting that ligand recognition was not through hyaluronic acid binding sites. As compared to acetyl-LDL, the cellular degradation of LDL-proteoglycan complex was retarded. Macrophages exhibited a rapid stimulation of [ 3H]inositol trisphosphate (IP 3) release and diacylglycerol production when incubated with LDL-proteoglycan complex. Furthermore, pertussis toxin produced a 62% inhibition of LDL-proteoglycan complex mediated IP 3 release, suggesting that LDL-proteoglycan complex metabolism in macrophages is dependent upon the G-protein coupled signal transduction mechanism. These results show that receptor mediated endocytosis plays a major role in the metabolism of LDL-proteoglycan complex in macrophages.

Interaction of a high-affinity heparin subfraction with low-density lipoprotein stimulates cholesteryl ester accumulation in mouse macrophages

A high-affinity heparin subfraction accounting for 8% of whole heparin from bovine lung was isolated by low-density lipoprotein (LDL)-affinity chromatography. When compared to whole heparin, the high-affinity subfraction was relatively higher in molecular weight (11 000 vs. 17 000) and contained more iduronyl sulfate as hexuronic acid (76% vs. 86%), N-sulfate ester (0.75 vs. 0.96 mol/mol hexosamine), and O-sulfate ester (1.51 vs. 1.68 mol/mol hexosamine). Although both heparin preparations formed insoluble complexes with LDL quantitatively in the presence of 30 mM Ca 2+, the concentrations of NaCl required for 50% reduction in maximal insoluble complex formation was markedly higher with high-affinity subfraction (0.55 M vs. 0.04 M). When compared to complex of 125I-LDL and whole heparin (H- 125I-LDL), complex of 125I-LDL and high-affinity heparin subfraction (HAH- 125I-LDL) produced marked increase in the degradation of lipoproteins by macrophages (7-fold vs. 1.4-fold over native LDL, after 5 h incubation) as well as cellular cholesteryl ester synthesis (16.7-fold vs. 2.2-fold over native LDL, after 18 h incubation) and content (36-fold vs. 2.7-fold over native LDL, after 48 h incubation). After a 5 h incubation, macrophages accumulated 2,3-fold more cell-associated radioactivity from HAH- 125I-LDL complex than from [ 125I]acetyl-LDL. While unlabeled HAH-LDL complex produced a dose-dependent inhibition of the degradation of labeled complex, native unlabeled LDL did not elicit any effect even at a 20-fold excess concentration. Unlabeled particulate LDL aggregate completed for 33% of degradation of labeled complex; however, cytochalasin D, known inhibitor of phagocytosis, did not effectively inhibit the degradation of labeled complex. Unlabeled acetyl-LDL produced a partial (33%) inhibition of the degradation of labeled complex. These results indicate that (1) the interaction of high-affinity heparin subfraction with LDL leads to scavenger receptor mediated endocytosis of the lipoprotein, and stimulation of cholesteryl ester synthesis and accumulation in the macrophages; and (2) with respect to macrophage recognition and uptake, HAH-LDL complex was similar but not identical to acetyl-LDL. These observations may have implications for atherogenesis, because both mast cells and endothelial cells can synthesize heparin in the arterial wall.

Opposing effects of apolipoproteins E and C on lipoprotein binding to low density lipoprotein receptor-related protein.

The low density lipoprotein receptor-related protein (LRP) from rat liver membranes binds apoprotein E (apoE)-enriched rabbit beta-migrating very low density lipoproteins (beta-VLDL) in a ligand blotting assay on nitrocellulose membranes. Binding was markedly activated when the beta-VLDL was preincubated with recombinant human apoE-3, native human apoE-3 or E-4, or native rabbit apoE. Human apoE-2, which binds poorly (1-2% of apo E-3 binding) to low density lipoprotein receptors, was approximately 40% as effective as apoE-3 or apoE-4 in binding to LRP. Stimulation of apoE-dependent binding to LRP was blocked by the inclusion of a mixture of human apoC proteins, but not apoA-I or A-II, in the preincubation reaction. High concentrations of apoE did not overcome the apoC inhibition. The effects of apoE and apoC on the ligand blotting assay were paralleled by similar effects in the ability of beta-VLDL to stimulate cholesteryl ester synthesis in mutant human fibroblasts that lack low density lipoprotein receptors. These properties of LRP are consistent with the known effects of apoE and apoC on uptake of chylomicron and very low density lipoprotein remnants in the liver and raise the possibility that LRP functions as a receptor for apoE-enriched forms of these lipoproteins in intact animals.

Interaction of very-low-density lipoprotein isolated from type I (insulin-dependent) diabetic subjects with human monocyte-derived macrophages

Very-low-density lipoproteins (VLDL) (density < 1.006 g/mL) were isolated from type I (insulin-dependent) diabetic patients in good to fair glycemic control and from age-, sex-, and race-matched, nondiabetic, control subjects. VLDL were incubated with human, monocyte-derived macrophages obtained from nondiabetic donors, and the rates of cellular cholesteryl ester synthesis and cholesterol accumulation were determined. VLDL isolated from diabetic patients stimulated significantly more cholesteryl ester synthesis than did VLDL isolated from control subjects (4.04 ± 1.01 v 1.99 ± 0.39 nmol 14C-cholesteryl oleate synthetized/mg cell protein/20 h; mean ± SEM, P < .05). The stimulation of cholesteryl ester synthesis in macrophages incubated with VLDL isolated from diabetic patients was paralleled by a significant increase in intracellular cholesteryl ester accumulation ( P < .05). The increases in cholesteryl ester synthesis and accumulation in macrophages were mediated by a significant increase in the receptor mediated, high affinity degradation (2.55 ± 0.23 v 2.12 ± 0.20 μg degraded/mg cell protein/20 h) and accumulation (283 ± 35 v 242 ± 33 ng/mg cell protein/20 h) of 125I-VLDL isolated from diabetic patients compared with VLDL from control subjects. To determine if changes in VLDL apoprotein composition were responsible for the observed changes in cellular rates of cholesteryl ester synthesis and accumulation, we also examined the apoprotein composition of the VLDL from both groups. There were no significant differences between the apoproteins B, E, and C content of VLDL from both groups. We also determined the chemical composition of VLDL isolated from both groups of subjects. The free cholesterol content of VLDL isolated from diabetic patients increased significantly and, thus, also contributed to the increased rates of cellular cholesteryl ester synthesis and accumulation. In conclusion, we have shown that VLDL isolated from Type I diabetic patients interact abnormally with human macrophages, and this altered interaction may contribute to the increased prevalence of atherosclerosis in diabetes.

Oestrogen‐induced changes in lipoprotein metabolism: role in prevention of atherosclerosis in the cholesterol‐fed rabbit

Administration of moderate pharmacological doses of oestrogen to cholesterol-fed rabbits for 12 weeks resulted in a dramatically retarded development of arterial lesions as compared to non-oestrogen-treated, cholesterol-fed rabbits (7% vs. 47% aortal involvement). Oestrogen treatment was associated with a retarded increase in plasma cholesterol, and five times higher high density lipoprotein (HDL) to very low density lipoprotein (VLDL) cholesterol ratio. Expression of hepatic lipoprotein receptor activity, as detected by heparin-releasable binding of 125I-hypercholesterolaemic VLDL, was heavily suppressed by cholesterol feeding. Administration of oestrogen modulated this response and resulted in higher receptor expression. In accordance, oestrogen treatment resulted in a less prominent reduction of 125I-hypercholesterolaemic VLDL clearance in the cholesterol-fed rabbits. VLDL from both groups of cholesterol-fed animals stimulated cholesteryl ester synthesis in cultured macrophages to the same extent. Thus, in rabbits under a dietary cholesterol load, oestrogen counteracted hepatic lipoprotein receptor suppression, reduced plasma VLDL- and increased plasma HDL-cholesterol levels, and to a large extent abolished the development of atherosclerosis.

β-VLDL and acetylated-LDL binding to pigeon monocyte macrophages

Blood-derived monocytes are an important source of foam cells in atherosclerotic lesions of White Carneau pigeons. Based upon studies with cultured blood monocytes (monocyte macrophages) and peritoneal macrophages from a variety of mammalian species, it has been proposed that these cells become loaded with cholesteryl esters through the uptake of lipoproteins including, β-migrating very low density lipoproteins (β-VLDL) and low density lipoproteins that have been chemically modified in a manner analogous to experimental acetylation (Ac-LDL). The purpose of this study was to determine whether similar mechanisms functioned in pigeon monocyte macrophages. Radioiodinated pigeon β-VLDL and Ac-LDL were incubated with White Carneau pigeon monocyte macrophages that had been maintained in culture for 7 days. Scatchard analysis of the specific binding data revealed the presence of specific and saturable receptors for both β-VLDL and Ac-LDL. β-VLDL receptors had both low and high affinity binding components, whereas Ac-LDL receptors displayed a single class of high affinity binding sites. β-VLDL binding remained relatively constant from 3 to 10 days in culture while Ac-LDL binding increased with time in culture. Competition studies demonstrated a high degree of binding specificity for 125I-Ac-LDL, but less for 125I-β-VLDL. Binding of 125I-β-VLDL was not competed for by Ac-LDL, but was by β-VLDL and by low density lipoproteins from both normal and hypercholesterolemic pigeons. Following binding of β-VLDL and Ac-LDL, the lipoproteins were rapidly internalized and degraded. Although the majority of degradation was secondary to internalization by the monocyte macrophages, approx. 5% of the degradation resulted from enzymatic activity in the culture medium, presumably due to secretion of proteolytic enzymes by the cells. As measured by esterification of [ 14C]oleate to cholesterol, it was shown that the cholesterol liberated from the degradation of both β-VLDL and Ac-LDL stimulated cholesteryl ester synthesis in pigeon monocyte macrophages. These studies confirm the existence of specific β-VLDL and Ac-LDL receptors on the surface of pigeon monocyte macrophages which facilitate both internalization of the lipoproteins and subsequent stimulation of cholesteryl ester synthesis. This is the first demonstration of β-VLDL and Ac-LDL receptors on monocyte macrophages from an avian species, and the findings support the potential role for the receptor-mediated uptake of a variety of abnormal lipoproteins in the formation of monocyte-derived foam cells in the arterial wall of White Carneau pigeons during the development of atherosclerosis.

Characterization of very low density lipoprotein from Watanabe heritable hyperlipidemic rabbits.

We investigated the properties of very low density lipoprotein (VLDL) from two types of Watanabe heritable hyperlipidemic (WHHL) rabbits: one with a high incidence of coronary atherosclerosis (type 1), and the other with a low incidence (type 2). When incubated with mouse peritoneal macrophages, VLDL from type 1 WHHL rabbit (type 1-VLDL) stimulated cholesteryl ester synthesis 10.5-fold more than VLDL from the type 2 WHHL rabbit (type 2-VLDL) did. Moreover, a similar difference was seen in the stimulation of cholesteryl ester synthesis in peritoneal macrophages isolated from the WHHL rabbits. The mass ratios of cholesterol to protein in type 1- and type 2-VLDL were 5.69 and 2.05, respectively. Agarose gel electrophoresis of type 1-VLDL showed beta mobility, and that of type 2-VLDL showed pre-beta mobility. No difference was seen between the sizes of VLDL particles of the two types. The amount of apolipoprotein E in type 1-VLDL was greater than that in type 2-VLDL. In conclusion, the difference between type 1 and type 2 WHHL rabbits is at least partly due to the presence in type 1 animals of VLDL particles rich in cholesteryl esters and apolipoprotein E, particles which are very similar to beta-VLDL in conformation.

Fibronectin stimulates macrophage uptake of low density lipoprotein-heparin-collagen complexes.

In this study, we investigated whether fibronectin will enhance macrophage uptake of particulate complexes of low density lipoproteins (LDL), heparin, and fibrillar collagen and whether fibronectin's opsonic effect could be modulated by the heparin component in these model matrices. We isolated a heparin fraction (HepFn) based on its affinity to fibronectin. HepFn appeared more charged than unfractionated heparin, as evidenced by enhanced electrophoretic mobility and ability to effect a cathodic shift in the electrophoretic migration of fibronectin. HepFn lacked the smaller molecular weight species present in unfractionated heparin. Macrophage endocytosis of LDL-heparin-collagen complexes, as evidenced by the intracellular accumulation of LDL-derived cholesteryl esters and endogenously synthesized cholesteryl esters, was enhanced by fibronectin. When LDL matrix complexes were prepared with HepFn, fibronectin's opsonic properties were significantly enhanced. F(ab)2 fragments of anti-fibronectin, capable of inhibiting fibronectin's opsonization of gelatin-derivatized latex particles, inhibited the fibronectin-dependent stimulation of cholesteryl ester synthesis by macrophages exposed to LDL-HepFn-collagen complexes. Thus, fibronectin stimulates macrophage endocytosis of LDL matrix complexes. The affinity of the constituent glycosaminoglycan for fibronectin is important in the regulation of this phenomenon.

Metabolism of low-density lipoprotein-proteoglycan complex by macrophages: further evidence for a receptor pathway

Earlier, we (Vijayagopal, P., et al. (1985) Biochim. Biophys. Acta 837-251) have shown that complexes of plasma low-density lipoproteins (LDL) and arterial chondroitin sulfate-dermatan sulfate proteoglycan aggregate promote LDL degradation and cholesteryl ester accumulation in mouse peritoneal macrophages. Further studies were conducted to determine whether LDL-proteoglycan complex is metabolized by a receptor-mediated process. Native proteoglycan aggregate was isolated from bovine aorta by associative CsCI isopycnic centrifugation. Complex of 125I-labeled LDL and proteoglycan aggregate formed in the presence of 30 mM Ca 2+ was incubated with macrophages, and the binding at 4°C and degradation at 37°C of 125I-labeled LDL in the complex was monitored. Both binding and degradation of the complex were specific and saturable, suggesting that the processes are receptor mediated. The K d for binding was 23μg LDL protein per ml in the complex. Degradation of 125I-labeled LDL-proteoglycan complex was not suppressed by preincubation of macrophages with excess unlabeled complex, suggesting that the receptor for the complex is not subject to down regulation. Both binding and degradation of the complex and the resultant stimulation of cholesteryl ester synthesis were inhibited by limited treatment of cells with low doses of trypsin and pronase, indicating that the binding sites are protein or glycoprotein in nature. Binding was not inhibited by an excess of native LDL and β-VLDL and exhibited only partial competition by excess unlabeled acetyl-LDL; however, polyinosinic acid, fucoidin and dextran sulfate, known inhibitors of acetyl-LDL binding and degradation in macrophages, did not affect LDL-proteoglycan complex binding and degradation. Similarly, excess unlabeled LDL-proteoglycan complex produced only partial inhibition of the binding and degradation of 125I-labeled acetyl-LDL by macrophages, suggesting that the binding sites for acetyl-LDL and LDL-proteoglycan complex are probably not identical. These studies provide evidence for a receptor-mediated pathway for the metabolism of LDL-proteoglycan complex in macrophages.