Distinct murine macrophage receptor pathway for human triglyceride-rich lipoproteins.

Murine P388D1 macrophages have a receptor pathway that binds human hypertriglyceridemic very low density lipoproteins (HTG-VLDL) that is fundamentally distinct from the LDL receptor pathway. Trypsin-treated HTG-VLDL (tryp-VLDL), devoid of apolipoprotein (apo)-E, fail to bind to the LDL receptor, yet tryp-VLDL and HTG-VLDL cross-compete for binding to P388D1 macrophage receptors, indicating that these lipoproteins bind to the same sites. The specific, high affinity binding of tryp-VLDL and HTG-VLDL to macrophages at 4 degrees C is equivalent and at 37 degrees C both produce rapid, massive, curvilinear (receptor-mediated) triglyceride accumulation in macrophages. Ligand blots show that P388D1 macrophages express a membrane protein of approximately 190 kD (MBP190) that binds both tryp-VLDL and HTG-VLDL; this binding is competed by HTG-VLDL, trypsinized HTG-VLDL, and trypsinized normal VLDL but not by normal VLDL or LDL. The macrophage LDL receptor (approximately 130 kD) and cellular uptake of beta-VLDL, but not MBP 190 nor uptake of tryp-VLDL, are induced when cells are exposed to lipoprotein-deficient medium and decreased when cells are cholesterol loaded. Unlike the macrophage LDL receptor, MBP 190 partitions into the aqueous phase after phase separation of Triton X-114 extracts. An anti-LDL receptor polyclonal antibody blocks binding of HTG-VLDL to the LDL receptor and blocks receptor-mediated uptake of beta-VLDL by P388D1 cells but fails to inhibit specific cellular uptake of tryp-VLDL or to block binding of tryp-VLDL to MBP 190. Human monocytes, but not human fibroblasts, also express a binding protein for HTG-VLDL and tryp-VLDL similar to MBP 190. We conclude that macrophages possess receptors for abnormal human triglyceride-rich lipoproteins that are distinct from LDL receptors in ligand specificity, regulation, immunological characteristics, and cellular distribution. MBP 190 shares these properties and is a likely receptor candidate for the high affinity uptake of TG-rich lipoproteins by macrophages.     

Phospholipid liposomes acquire apolipoprotein E in atherogenic plasma and block cholesterol loading of cultured macrophages.

A single infusion of phospholipid liposomes promptly and persistently abolished the ability of hypercholesterolemic rabbit plasma to cause cholesteryl ester loading in cultured macrophages. This phospholipid enrichment of plasma caused moderate stimulation of cellular cholesterol efflux and, unexpectedly, almost complete inhibition of cellular uptake of beta-very low density lipoprotein (beta-VLDL), the major cholesteryl ester-rich particle in hypercholesterolemic rabbit plasma. Cell viability and LDL receptor activity were unaffected. Incubation of liposomes with beta-VLDL resulted in transfer of apolipoprotein-E (apoE) to the liposomes; reisolated apoE-phospholipid liposomes then competed efficiently for cellular apoprotein receptors. Thus, a major mechanism by which phospholipid infusions result in diminished accumulation of cholesteryl ester in cultured macrophages is by blocking cellular uptake of beta-VLDL. The liposomes deplete beta-VLDL of apoE, then compete for receptor-mediated uptake. These results suggest a novel mechanism contributing to the known antiatherogenic effect of phospholipid infusions: infused liposomes acquire apoE, then block uptake of atherogenic lipoproteins by arterial wall macrophages.     

High receptor binding affinity of lipoproteins in atypical dysbetalipoproteinemia (type III hyperlipoproteinemia).

Familial dysbetalipoproteinemia (or type III hyperlipoproteinemia) is characterized by the presence of abnormal, cholesteryl ester-rich beta-very low density lipoproteins (beta-VLDL) in the plasma. Subjects with typical dysbetalipoproteinemia are homozygous for an amino acid substitution in apolipoprotein (apo-) E at residue 158 and have defective apo-E-mediated binding of both pre-beta-VLDL and beta-VLDL to apo-B,E(LDL) (or LDL) receptors (1988. Chappell, D.A., J. Clin. Invest. 82:628-639). To understand the effect of substitutions in apo-E at sites other than residue 158, nine dysbetalipoproteinemic (dys-beta) subjects who were either homozygous or heterozygous for substitutions in apo-E at atypical sites were studied. These substitutions occurred at residue 142 (n = 6), 145 (n = 2), or 146 (n = 1) and are known to cause less defective binding than does the 158 substitution. The chemical composition and electrophoretic mobility of pre-beta-VLDL and beta-VLDL from atypical and typical dys-beta subjects were indistinguishable. However, lipoproteins from atypical and typical dys-beta subjects differed in their affinity for the apo-B,E(LDL) receptor on cultured human fibroblasts. The pre-beta-VLDL and beta-VLDL from atypical dys-beta subjects had 640- or 17-fold higher affinity, respectively, than did corresponding lipoproteins from typical dys-beta subjects. The higher binding affinity of lipoproteins from atypical dys-beta subjects was associated with a higher ratio of apo-E to total apo-C. Since higher binding affinity should cause more rapid receptor-mediated clearance of beta-VLDL in atypical than in typical dys-beta subjects in vivo, the mechanism of beta-VLDL accumulation may differ in these two groups.     

Stimulation of cholesteryl ester synthesis in mouse peritoneal macrophages by cholesterol-rich very low density lipoproteins from the Watanabe heritable hyperlipidemic rabbit, an animal model of familial hypercholesterolemia.

Cholesterol-rich very low density lipoproteins (VLDL) from the homozygous Watanabe heritable hyperlipidemic (WHHL) rabbit induced marked cholesteryl ester accumulation in mouse peritoneal macrophages. This WHHL rabbit, an animal model of human familial hypercholesterolemia, has severe hypercholesterolemia, cutaneous xanthomas, and fulminant atherosclerosis due to the deficiency of the low density lipoprotein (LDL) receptor. When incubated with mouse peritoneal macrophages, the VLDL from WHHL rabbit (WHHL-VLDL) stimulated cholesteryl [14C]oleate synthesis 124-fold more than did VLDL from the normal Japanese White rabbit (control-VLDL). The enhancement in cholesteryl ester synthesis and accumulation of WHHL-VLDL was due to the presence of a high affinity binding receptor site on the macrophage cell surface that mediated the uptake and lysosomal degradation of WHHL-VLDL. Competition studies showed that the uptake and degradation of 125I-WHHL-VLDL was inhibited by unlabeled excess WHHL-VLDL and beta-migrating VLDL (beta-VLDL), but not LDL. Furthermore, the degradation of WHHL-VLDL was not blocked by either fucoidin, polyinosinic acid, or polyguanylic acid, potent inhibitors of the acetylated (acetyl)-LDL binding site, or by acetyl-LDL. These results suggest that macrophages possess a high affinity receptor that recognizes the cholesterol-rich VLDL present in the plasma of the WHHL rabbit and that the receptor which mediates ingestion of WHHL-VLDL seems to be the same as that for beta-VLDL and leads to cholesteryl ester deposition within macrophages. Thus the uptake of the cholesterol-rich VLDL from the WHHL rabbit by macrophages in vivo may play a significant role in the pathogenesis of atherosclerosis in the WHHL rabbit.     

Lipoprotein lipase enhances binding of lipoproteins to heparan sulfate on cell surfaces and extracellular matrix.

Lipoprotein lipase enhances binding at 4 degrees C of human plasma lipoproteins (chylomicrons, VLDL, intermediate density lipoprotein, LDL, and HDL3) to cultured fibroblasts and hepG-2 cells and to extracellular matrix. Heparinase treatment of cells and matrix reduces the lipoprotein lipase enhanced binding by 90-95%. Lipoprotein lipase causes only a minimal effect on the binding of lipoproteins to heparan sulfate deficient mutant Chinese hamster ovary cells while it promotes binding to wild type cells that is abolished after heparinase treatment. With 125I-LDL, lipoprotein lipase also enhances uptake and proteolytic degradation at 37 degrees C by normal human skin fibroblasts but has no effect in heparinase-treated normal cells or in LDL receptor-negative fibroblasts. These observations prove that lipoprotein lipase causes, predominantly, binding of lipoproteins to heparan sulfate at cell surfaces and in extracellular matrix rather than to receptors. This interaction brings the lipoproteins into close proximity with cell surfaces and may promote metabolic events that occur at the cell surface, including facilitated transfer to cellular receptors.     

Hypertriglyceridemic Very Low Density Lipoproteins Induce Triglyceride Synthesis and Accumulation in Mouse Peritoneal Macrophages

Triglyceride-rich lipoproteins may be responsible for the lipid accumulation in macrophages that can occur in hypertriglyceridemia. Chylomicrons and very low density lipoproteins (VLDL, total and with flotation constant [S(f)] 100-400) from fasting hypertriglyceridemic subjects induced a massive accumulation of oil red O-positive inclusions in unstimulated peritoneal macrophages. Cell viability was not affected. The predominant lipid that accumulated in cells exposed to hypertriglyceridemic VLDL was triglyceride. Hypertriglyceridemic VLDL stimulated the incorporation of [(14)C]oleate into cellular triglyceride up to ninefold in 16 h, but not into cholesteryl esters. Mass increase in cellular triglyceride was 38-fold. The stimulation of cellular triglyceride formation was dependent on time, temperature, and concentration of hypertriglyceridemic VLDL. By contrast, VLDL, low density, and high density lipoproteins from fasting normolipemic subjects had no significant effect on oleate incorporation into neutral lipids or on visible lipid accumulation.(125)I-Hypertriglyceridemic VLDL (S(f) 100-400) were degraded by macrophages in a dose-dependent manner, with 50 and 100% saturation observed at 3 and 24 mug protein/ml (2.5 and 20 nM), respectively. Hypertriglyceridemic VLDL inhibited the internalization and degradation of (125)I-hypertriglyceridemic VLDL (4 nM) by 50% at 3 nM. Cholesteryl ester-rich VLDL from cholesterol-fed rabbits gave 50% inhibition at 5 nM. Low density lipoproteins (LDL) inhibited by 10% at 5 nM and 40% at 47 nM. Acetyl LDL at 130 nM had no effect. We conclude that the massive triglyceride accumulation produced in macrophages by hypertriglyceridemic VLDL is a direct consequence of uptake via specific receptors that also recognize cholesteryl ester-rich VLDL and LDL but are distinct from the acetyl LDL receptor. Uptake of these triglyceride-rich lipoproteins by monocyte-macrophages in vivo may play a significant role in the pathophysiology of atherosclerosis.     

Lipoprotein lipase-mediated uptake and degradation of low density lipoproteins by fibroblasts and macrophages.

Lipoprotein lipase (LPL), the rate limiting enzyme for hydrolysis of lipoprotein triglyceride, also mediates nonenzymatic interactions between lipoproteins and heparan sulfate proteoglycans. To determine whether cell surface LPL increases LDL binding to cells, bovine milk LPL was added to upregulated and nonupregulated human fibroblasts along with media containing LDL. LDL binding to cells was increased 2-10-fold, in a dose-dependent manner, by the addition of 0.5-10 micrograms/ml of LPL. The amount of LDL bound to the cells in the presence of LPL far exceeded the capacity for LDL binding via the LDL receptor. Treatment of fibroblasts with heparinase and heparitinase resulted in a 64% decrease in LPL-mediated LDL binding. Compared to studies performed without LPL, more LDL was internalized and degraded in the presence of LPL, but the time course was slower than that of classical lipoprotein receptor mediated pathways. In LDL receptor negative fibroblasts, LPL increased surface bound LDL > 140-fold, intracellular LDL > 40-fold, and LDL degradation > 6-fold. These effects were almost completely inhibited by heparin and anti-LPL monoclonal antibody. LPL also increased the binding and uptake by fibroblasts of apolipoprotein-free triglyceride emulsions; binding was increased > 8-fold and cellular uptake was increased > 40-fold with LPL. LPL increased LDL binding to THP-1 monocytes, and increased LDL uptake (4.5-fold) and LDL degradation (2.5-fold) by THP-1 macrophages. In the absence of added LPL, heparin and anti-LPL monoclonal antibodies decreased LDL degradation by > 40%, and triglyceride emulsion uptake by > 50%, suggesting that endogenously produced LPL mediated lipid particle uptake and degradation. We conclude that LPL increases lipid and lipoprotein uptake by cells via a pathway not involving the LDL receptor. This pathway may be important for lipid accumulation in LPL synthesizing cells.     

Regulation of low density lipoprotein receptor activity in freshly isolated human lymphocytes.

Circulating human lymphocytes freshly isolated from venous blood of 15 normal subjects exhibited a low capacity to bind, take up, and degrade 125I-labeled low density lipoprotein (LDL). However, when these cells were incubated for 72 h in the absence of lipoproteins, they gradually acquired in increased number of high affinity cell surface receptors for LDL. The increase in the number of LDL receptors was associated with a 16-fold increase in the rate at which the cells were able to take up and degrade the lipoprotein. The LDL binding and degradation processes that developed in normal lymphocytes exhibited the following characteristics; (a) high affinity (saturation was achieved at LDL concentrations below 50 mug protein/ml); (b) specificity (unlabeled LDL was much more effective than human high density lipoprotein or other plasma proteins in competing with 125I-LDL for binding to the LDL receptor); and(c) feedback regulation (the increase in the number of LDL receptors that appeared after incubation of freshly isolated lymphocytes in lipoprotein-deficient medium was prevented by exposure of the cells to either LDL or a mixture of 25-hydroxycholesterol plus cholesterol but not to HDL). Freshly isolated lymphocytes obtaine from three subjects with the homozygous form of familial hypercholesterolemia failed to develop normal amounts of LDL receptor activity when incubated in medium devoid of lipoproteins. The current data indicate: (a) that the LDL receptors that appear on the surface of cholesterol-deprived, normal human lymphocytes are genetically identical to the previously characterized LDL receptors of cultured human fibroblasts and long-term lymphoid cells and (b) that at least one cell type in the human body, the circulating human lymphocyte, has the capacity to produce a high affinity LDL receptor that mediates the cellular uptake and degradation of plasma LDL.     

The atherogenic lipoprotein Lp(a) is internalized and degraded in a process mediated by the VLDL receptor.

Lp(a) is a major inherited risk factor associated with premature heart disease and stroke. The mechanism of Lp(a) atherogenicity has not been elucidated, but likely involves both its ability to influence plasminogen activation as well as its atherogenic potential as a lipoprotein particle after receptor-mediated uptake. We demonstrate that fibroblasts expressing the human VLDL receptor can mediate endocytosis of Lp(a), leading to its degradation within lysosomes. In contrast, fibroblasts deficient in this receptor are not effective in catabolizing Lp(a). Lp(a) degradation was prevented by antibodies against the VLDL receptor, and by RAP, an antagonist of ligand binding to the VLDL receptor. Catabolism of Lp(a) was inhibited by apolipoprotein(a), but not by LDL or by monoclonal antibodies against apoB100 that block LDL binding to the LDL receptor, indicating that apolipoprotein(a) mediates Lp(a) binding to this receptor. Removal of Lp(a) antigen from the mouse circulation was delayed in mice deficient in the VLDL receptor when compared with control mice, indicating that the VLDL receptor may play an important role in Lp(a) catabolism in vivo. We also demonstrate the expression of the VLDL receptor in macrophages present in human atherosclerotic lesions. The ability of the VLDL receptor to mediate endocytosis of Lp(a) could lead to cellular accumulation of lipid within macrophages, and may represent a molecular basis for the atherogenic effects of Lp(a).     

Specific high-affinity binding of high density lipoproteins to cultured human skin fibroblasts and arterial smooth muscle cells

Binding of human high density lipoproteins (HDL, d = 1.063-1.21) to cultured human fibroblasts and human arterial smooth muscle cells was studied using HDL subjected to heparin-agarose affinity chromatography to remove apoprotein (apo) E and B. Saturation curves for binding of apo E-free 125I-HDL showed at least two components: low-affinity nonsaturable binding and high-affinity binding that saturated at approximately 20 micrograms HDL protein/ml. Scatchard analysis of high-affinity binding of apo E-free 125I-HDL to normal fibroblasts yielded plots that were significantly linear, indicative of a single class of binding sites. Saturation curves for binding of both 125I-HDL3 (d = 1.125-1.21) and apo E-free 125I-HDL to low density lipoprotein (LDL) receptor-negative fibroblasts also showed high-affinity binding that yielded linear Scatchard plots. On a total protein basis, HDL2 (d = 1.063-1.10), HDL3 and very high density lipoproteins (VHDL, d = 1.21-1.25) competed as effectively as apo E-free HDL for binding of apo E-free 125I-HDL to normal fibroblasts. Also, HDL2, HDL3, and VHDL competed similarly for binding of 125I-HDL3 to LDL receptor-negative fibroblasts. In contrast, LDL was a weak competitor for HDL binding. These results indicate that both human fibroblasts and arterial smooth muscle cells possess specific high affinity HDL binding sites. As indicated by enhanced LDL binding and degradation and increased sterol synthesis, apo E-free HDL3 promoted cholesterol efflux from fibroblasts. These effects also saturated at HDL3 concentrations of 20 micrograms/ml, suggesting that promotion of cholesterol efflux by HDL is mediated by binding to the high-affinity cell surface sites.     

Roles of apolipoproteins B and E in the cellular binding of very low density lipoproteins.

Apoproteins B and E both interact with cellular low density lipoprotein (LDL) apolipoprotein B and E (apo B,E)-receptors, and very low density lipoproteins (VLDL) contain both apo B and apo E. Our aim was to study the relative importance of apo B and apo E in the binding of VLDL subfractions to cells. Two monoclonal anti-LDL-apo B antibodies (464B1B3 and 464B1B6, 2a and 2b, respectively) and two anti-apo E antibodies (1506 A1.4 and 1907 F6.4) were used to inhibit lipoprotein-cell interactions. In confirmation of previous findings, the binding and degradation of 125I-LDL by human fibroblasts were inhibited approximately 90% by antibodies 2a or 2b or the antigen-binding fragments of 2a, whereas the cellular processing of 125I-VLDL3 (Sf20-60), 125I-VLDL2 (Sf60-120), and 125I-VLDL1 (Sf greater than 120) were inhibited by only approximately 50%, approximately 25%, and less than 10%, respectively. The VLDL1-3 and LDL-dependent intracellular esterification of cholesterol with [3H]oleate were inhibited to a similar extent. Other monoclonal anti-human apo B antibodies inhibited lipoprotein-cell interactions much less effectively and nonimmune IgG isolated from mouse serum did not inhibit at all. 20-fold excesses of LDL produced about the same patterns of inhibition of degradation of 125I-VLDL1-3 and LDL by cells as did antibodies 2a and 2b, whereas homologous unlabeled VLDL1-3 in like amounts inhibited the matched 125I-VLDL subfraction more effectively. Two anti-apo E monoclonal antibodies and a polyclonal anti-apo E antibody inhibited cell-mediated degradation of and lipoprotein-dependent cholesterol esterification by VLDL1 but not VLDL3 or LDL. The results suggest that receptor recognition sites on apo E in preference to sites on apo B mediate the cellular binding of hypertriglyceridemic VLDL1. However, the proportion of particles bound via apo B seems to increase as VLDL decreases in size toward LDL, and virtually all of LDL binding is mediated by apo B.     

An apolipoprotein E synthetic peptide targets to lipoproteins in plasma and mediates both cellular lipoprotein interactions in vitro and acute clearance of cholesterol-rich lipoproteins in vivo.

Apolipoprotein (apo) E mediates lipoprotein binding to cellular lipoprotein receptors. Previously we reported that a synthetic peptide representing a linear dimeric repeat of amino acids 141-155 binds cellular LDL receptors. To prepare an apoE peptide that bound to both cholesterol-rich lipoproteins and lipoprotein receptors, an NH2-terminal acetylated apoE dimer peptide was synthesized. This acetylated peptide preferentially associated with lipoproteins in plasma, whereas nonacylated peptides were poor lipid binders. Acetylated peptide/LDL complexes (molar ratios of 4-5:1) enhanced the interaction of LDL with cultured human fibroblasts by 7-12-fold. Participation by both receptors and cell surface heparin sulfate proteoglycans was observed. When a preformed peptide/125I-LDL complex was injected intravenously into C57BL/6J apoE-deficient mice, its rate of removal was threefold higher than that of 125I-LDL alone. The liver and the spleen were major tissue distribution sites. Intravenous administration of free acetylated peptide resulted in a 30% reduction in total plasma cholesterol within 3-30 min, which reflected a 40-50% and 20-26% reduction in very low density lipoproteins and intermediate density lipoproteins, respectively. Therefore, this peptide selectively associated with cholesterol-rich lipoproteins and mediated their acute clearance in vivo.     

Familial dysbetalipoproteinemia. Abnormal binding of mutant apoprotein E to low density lipoprotein receptors of human fibroblasts and membranes from liver and adrenal of rats, rabbits, and cows.

Patients with familial dysbetalipoproteinemia (F. Dys.), also called familial type 3 hyperlipoproteinemia, are homozygous for a mutant allele, Ed, that specifies an abnormal form of apoprotein (apo) E, a prominent constituent of remnant lipoproteins derived from very low density lipoproteins (VLDL) and chylomicrons. Apo E is thought to mediate the removal of remnant lipoproteins from the plasma by virtue of its ability to bind to hepatic lipoprotein receptors. In F. Dys. patients, remnant-like lipoproteins accumulate, apparently because of delayed clearance by the liver. In the current studies, we show that the abnormal protein specified by the Ed allele (apo E-D) from some, but not all, patients with F. Dys. has a markedly deficient ability to bind to low density lipoprotein (LDL) receptors. Apo E was isolated from eight control subjects and nine patients with F. Dys. and incorporated into phospholipid complexes. The complexes were tested for their ability to compete with human 125I-LDL or rabbit 125I-beta-VLDL fo binding to LDL receptors in four assay systems: cultured human fibroblasts, solubilized receptors from bovine adrenal cortex, liver membranes from rats treated with 17 alpha-ethinyl estradiol, and liver membranes from normal rabbits. The apo E-D from six of the nine patients with F. Dys. showed binding affinities for LDL receptors that were reduced by greater than 98% in all receptor assays (group 1 patients). All of these group 1 patients were unequivocally of phenotype apo E-D/D by the criterion of isoelectric focussing. The apo E from the three other F. Dys. patients showed a near normal binding ability in all four of the receptor assays (group 2 patients). One of these group 2 patients appeared to have the apo E-D/D phenotype by isoelectric focussing. In the other two patients in group 2, apo E-D was the predominant protein (phenotype, apo E-D/D), but traces of protein in the region corresponding to normal apo E (apo E-N) were also present. The difference between group 1 and group 2 patients was also apparent when the apo E was iodinated and tested directly for binding to liver membranes from rats treated with 17 alpha-ethinyl estradiol. The 125I-labeled apo E from a group 2 patient, but not a group 1 patient, showed enhanced uptake when perfused through the liver of an estradiol-treated rate, indicating that the receptor binding ability of apo E correlated with uptake in the intact liver. The current studies allow the subdivision of patients with F. Dys. into two groups. In group 1, the elevated plasma level of remnants appears to be due to a diminished receptor binding activity of the abnormal protein specified by the Ed allele; in group 2 patients, the cause of the elevated plasma level of remnants remains to be explained.     

Hydrocortisone decreases the internalization of low density lipoprotein in cultured human fibroblasts and arterial smooth muscle cells

The effect of hydrocortisone on the cellular low density lipoprotein (LDL) pathway was studied in cultured human skin fibroblasts and arterial smooth muscle cells. Hydrocortisone decreased both uptake and degradation of 125I-LDL, LDL binding, measured at 4 C, was not affected by the hormone. Physiological concentrations of hydrocortisone (4 . 1 X 10(-8) mol/l) resulted in a 30% reduction of LDL uptake and degradation which could not be accounted for by an effect of the hormone on macromolecular synthesis, cell protein or cell number. To test whether the decrease in uptake and degradation of 125I-LDL was due to reduced internalization, the effect of hydrocortisone on the internalization of prebound LDL was determined and found to be decreased. Also, preincubation with unlabelled LDL in the presence of hydrocortisone resulted in less down regulation of LDL receptor activity than when no hydrocortisone was present. An effect of the hormone on bulk phase endocytosis has been excluded, since hydrocortisone did not affect either LDL degradation by receptor negative cells or endocytosis of 14C-sucrose by normal skin fibroblasts. Thus, hydrocortisone impairs LDL catabolism by decreasing the internalization of LDL normally bound to its cell surface receptor. These results may be relevant to the pathogenesis of atherosclerosis in conditions associated with reduced cellular LDL catabolism.     

Stimulated arachidonate metabolism during foam cell transformation of mouse peritoneal macrophages with oxidized low density lipoprotein.

Changes in arachidonate metabolism were examined in mouse peritoneal macrophages incubated with various types of lipoproteins. Oxidized low density lipoprotein (LDL) was incorporated by macrophages and stimulated macrophage prostaglandin E2 (PGE2) and leukotriene C4 syntheses, respectively, 10.8- and 10.7-fold higher than by the control. Production of 6-keto-PGF1 alpha, a stable metabolite of prostacyclin, was also stimulated. No stimulation was found with native LDL, which was minimally incorporated by the cells. Acetylated LDL and beta-migrating very low density lipoprotein (beta-VLDL), though incorporated more efficiently than oxidized LDL, also had no stimulatory effect. When oxidized LDL was separated into the lipoprotein-lipid peroxide complex and free lipid peroxides, most of the stimulatory activity was found in the former fraction, indicating that stimulation of arachidonate metabolism in the cell is associated with uptake of the lipoprotein-lipid peroxide complex. These results suggest that peroxidative modification of LDL could contribute to the progression of atheroma by stimulating arachidonate metabolism during incorporation into macrophages.     

Stimulation with a monoclonal antibody (mAb4E4) of scavenger receptor-mediated uptake of chemically modified low density lipoproteins by THP-1-derived macrophages enhances foam cell generation.

mAb4E4, a murine monoclonal antibody that is specific for acetylated LDL and malondialdehyde-treated LDL, binds specifically to modified LDL present in human atherosclerotic lesions. It is directed against an epitope that is poorly exposed in delipidated and solubilized apolipoprotein B-100 from modified LDL. mAb4E4, as well as its F(ab')2 and Fab fragments, enhanced the uptake of both acetylated LDL and malondialdehyde-treated LDL by THP-1-derived macrophages resulting in a sixfold increase of cytoplasmic cholesteryl ester levels. The increased uptake of modified LDL/mAb4E4 complexes did not occur via the Fc receptor and did not depend on aggregation of modified LDL particles. However, their uptake was inhibited by blocking the scavenger receptors with fucoidin or by downregulation of receptor expression with endotoxins or interferon-gamma, indicating that their uptake is mediated via these receptors. Thus, generation of autoimmune antibodies against modified LDL and subsequent endocytosis of soluble modified LDL/antibody complexes via scavenger receptors may enhance foam cell generation. This mechanism may contribute to the progression of atherosclerotic lesions.     

Altered Metabolism (In Vivo and In Vitro) of Plasma Lipoproteins after Selective Chemical Modification of Lysine Residues of the Apoproteins

Chemical modification of lysine residues by acetoacetylation of the apoproteins of iodinated canine and human low density lipoproteins (LDL) and canine high density lipoproteins (HDL) resulted in a marked acceleration in the rate of removal of these lipoproteins from the plasma after intravenous injection into dogs. Clearance of the lipoproteins from the plasma correlated with their rapid appearance in the liver. Acetoacetylated canine (125)I-LDL (30-60% of the lysine residues modified) were essentially completely removed from the plasma within an hour, and > 75% of the activity cleared within 5 min. Reversal of the acetoacetylation of the lysine residues of the LDL restored to these lipoproteins a rate of clearance essentially identical to that of control LDL. Identical results were obtained with modified human LDL injected into dogs. At 10 min, when congruent with 90% of the acetoacetylated human (125)I-LDL had been removed from the plasma, 90% of the total injected activity could be accounted for in the liver. Furthermore, it was possible to demonstrate an enhancement in uptake and degradation of acetoacetylated LDL by canine peritoneal macrophages in vitro. The mechanism(s) responsible for the enhanced removal of the LDL and HDL in vivo and in vitro remains to be determined. By contrast, however, acetoacetylation of canine (125)I-apoE HDL(c) did not accelerate their rate of removal from the plasma but, in fact, retarded their clearance. Control (native) apoE HDL(c) were removed from the plasma (64% within 20 min) and rapidly appeared in the liver (39% at 20 min). At the same time point, only 45% of the acetoacetylated apoE HDL(c) were cleared from the plasma and <10% appeared in the liver. Acetoacetylation of the apoE HDL(c) did not enhance their uptake or degradation by macrophages. The rapid clearance from the plasma of the native apoE HDL(c) in normal and hypercholesterolemic dogs suggests that the liver may be a normal site for the removal of the cholesteryl ester-rich apoE HDL(c). The retardation in removal after acetoacetylation of apoE HDL(c) indicates that the uptake process may be mediated by a lysine-dependent recognition system.     

Lipoprotein binding to cultured human hepatoma cells.

Binding of various 125I-lipoproteins to hepatic receptors was studied on cultured human hepatoma cells (Hep G2). Chylomicrons, isolated from a chylothorax, chylomicron remnants, hypertriglyceridemic very low-density lipoproteins, normotriglyceridemic very low-density lipoproteins (NTG-VLDL), their remnants, low-density lipoproteins (LDL), and HDL-E (an Apo E-rich high-density lipoprotein isolated from the plasma of a patient with primary biliary cirrhosis) were bound by high-affinity receptors. Chylomicron remnants and HDL-E were bound with the highest affinity. The results, obtained from competitive binding experiments, are consistent with the existence of two distinct receptors on Hep G2 cells: (a) a remnant receptor capable of high-affinity binding of triglyceride-rich lipoproteins and HDL-E, but not of Apo E free LDL, and (b) a LDL receptor capable of high-affinity binding of LDL, NTG-VLDL, and HDL-E. Specific binding of Apo E-free LDL was completely abolished in the presence of 3 mM EDTA, indicating that binding to the LDL receptor is calcium dependent. Specific binding of chylomicron remnants was not inhibited by the presence of even 10 mM EDTA. Preincubation of the Hep G2 cells in lipoprotein-containing medium resulted in complete suppression of LDL receptors but did not affect the remnant receptors. Hep G2 cells seem to be a suitable model for the study of hepatic receptors for lipoprotein in man.     

Role of Microtubules in Low Density Lipoprotein Processing by Cultured Cells

The effect of the microtubule inhibitor colchicine on the metabolism of (125)I-low density lipoprotein (LDL) by cultured human skin fibroblasts and aortic medial cells was studied in vitro. Colchicine did not alter the binding of LDL to cell surface receptors. However, the rate of LDL endocytosis was reduced to 58% of that expected. Despite diminished endocytosis, LDL was found to accumulate within the cells to 165% of that expected, whereas the release of LDL protein degradation products into the medium was reduced to 34% of control, findings consistent with a reduced rate of intracellular LDL breakdown. Colchicine did not alter cell content of the acid protease which degrades LDL, nor did [(3)H]colchicine accumulate in lysosomal fractions. However, colchicine did alter the intracellular distribution of both fibroblast lysosomes and endosomes. After colchicine, lysosomes tended to accumulate in the perinuclear region, whereas endosomes were found at the cell periphery. These findings are consistent with the hypothesis that ingested LDL is less available to lysosomal enzymes in the presence of colchicine. The actions of colchicine appear to be a result of destruction of cell microtubules. Lumicolchicine, a mixture of colchicine isomers which (unlike the parent compound) does not bind to the subunit of microtubules, was without effect.The uptake and degradation of LDL by cultured cells consists of both a receptor-specific component and nonspecific pinocytosis. Important differences must exist between these processes because even large amounts of LDL taken up and degraded by the nonspecific route fail to regulate key aspects of intracellular cholesterol metabolism. Colchicine selectively inhibited receptor-mediated LDL degradation. No effect was demonstrable on the nonspecific degradation of LDL by familial hypercholesterolemia fibroblasts grown in medium containing serum and added sterols. The degradation of bovine albumin by normal cells was also unaffected. Colchicine sensitivity appears to be a biochemical marker for the LDL receptor-specific metabolic pathway.Cytochalasins inhibit crosslinking and polymerization of cell microfilaments (although other important cell effects also occur). Cytochalasin D reduced LDL degradation to 44% of that expected. This result and the actions of colchicine suggest that cytoskeletal components such as microtubules and possibly microfilaments facilitate normal LDL metabolism.     

Characterization of high density lipoprotein binding to human adipocyte plasma membranes.

Freshly isolated human adipocytes showed specific uptake of 125I-labeled human high density lipoprotein (HDL2 and HDL3), a portion of which could be released by subsequent incubation with excess unlabeled ligand. To study the mechanism of HDL binding, sucrose gradient-purified adipocyte plasma membranes were incubated with radioiodinated lipoprotein particles under equilibrium conditions in the absence (total binding) or presence (nonspecific binding) of 100-fold excess unlabeled ligand. Specific binding of HDL2 and HDL3, calculated by subtracting nonspecific from total binding, was Ca++ independent, unaffected by EDTA, and not abolished by pronase treatment of the membranes. Modification of HDL3 by reductive methylation or cyclohexanedione treatment also failed to affect its binding to adipocyte plasma membranes. High salt concentration (200 mM NaCl) inhibited specific binding of HDL2 and HDL3 but had no effect on LDL binding. A significant portion of 125I-HDL2 or 125I-HDL3 binding was consistently inhibited by adding excess unlabeled LDL, but this inhibition was incomplete as compared with a similar molar excess of unlabeled HDL2 or HDL3. The role of apoproteins (apo) in HDL binding to adipocyte membranes was examined by comparing binding of HDL2 and HDL3 isolated from normal, abetalipoproteinemic (abeta) and apo E-deficient (apo E0) plasma. Specific binding was observed with all normal and mutant HDL particles. Furthermore, a significant portion (61-78%) of abeta-HDL2, apo E0-HDL2, and apo E0-HDL3 binding was inhibited by adding 100-fold excess of unlabeled low density lipoproteins (LDL). The cross-competition of LDL and HDL binding was confirmed by the ability of normal, abeta, and apo E0-HDL2 to completely inhibit 125I-LDL binding. These data suggest that HDL binding is independent of apo E and that the responsible apoprotein(s) of HDL complete with LDL-apo B for binding to the same or closely related site in the adipocyte plasma membrane. Normal and apo E0-HDL3 binding was also completely inhibited by normal HDL2, which suggested that HDL2 and HDL3 probably bind to the same site. Scatchard analysis of normal HDL2, normal HDL3, and apo E0-HDL3 binding data best fitted a one-component binding profile with similar equilibrium dissociation constants (40-96 nM). HDL3 binding was found to be effectively inhibited by anti-human apo AI or anti-human apo AII, but not by anti-human apo B antisera. This binding was also unaffected by monoclonal anti-human apo B or E antibodies known to inhibit binding of apo B or apo E containing lipoprotein to the LDL receptor of cultured fibroblasts. These findings, taken together, suggest that human fat cells possess HDL binding sites with apo AI and /or apo AII specificity. The significant but partial inhibition of HDL2 and HDL3 binding by LDL along with the complete inhibition of LDL binding by HDL2 and HDL3 tends to exclude a single binding site that interacts both lipoproteins and favors the interpretation that LDL and HDL particles bind to multiple recognition sites or to different conformation of the same lipoprotein binding domain on the human fat cell.