Macrophage proteases can modify low density lipoproteins to increase their uptake by macrophages

When low density lipoprotein (LDL) was incubated with sonicated macrophages at acidic pH, its protein moiety was partially degraded by cathepsins B and D. The reisolated LDL was taken up by intact macrophages up to about 20 times as fast as control LDL. LDL proteolysis and its enhanced uptake could be inhibited almost entirely by the selective protease inhibitors leupeptin and pepstatin. If macrophages in atherosclerotic lesions were to release acidic proteases (either by exocytosis or following cell death) and these were to modify LDL, this may help to explain why so much cholesteryl ester accumulates in these cells.     

Binding of 5-methoxypsoralen to human serum low density lipoproteins

5-methoxypsoralen (5-MOP) binds to human serum low density lipoproteins (LDL) according to a two-step process. Scatchard analysis of the first step yields K = 1.4 × 10⁵ M^?1 and 4 binding sites. It involves the LDL apoprotein. The second step corresponds to a solubilization, in the lipidic core, of ? 45 molecules of 5MOP per LDL molecule. It is accompanied by a large blue shift of the 5MOP fluorescence. The ability of LDL to bind 5MOP and to carry it into various cells may explain some biological effects sometimes encountered during PUVA therapy.     

Increased resistance to oxidation of betalain-enriched human low density lipoproteins

Betalains are natural pigments recently considered as compounds with potential antioxidative properties. In this work, ex vivo plasma spiking of pure either betanin or indicaxanthin, followed by isolation of low density lipoprotein (LDL), and measurement of its resistance to copper-induced oxidation, has been used to research if these betalains can bind to LDL and prevent oxidation of LDL lipids. When pooled human plasma from 10 healthy volunteers was incubated in the presence of 25-100 μM either betanin or indicaxanthin, incorporation of both compounds in LDL was observed, with a maximum binding of 0.52±0.08, and 0.51±0.06 nmoles of indicaxanthin and betanin, respectively, per mg LDL protein. Indicaxanthin-enriched and betanin-enriched LDL were more resistant than homologous native LDL to copper-induced oxidation, as assessed by the elongation of the induction period. The incorporated indicaxanthin, however, appeared twice as effective as betanin in increasing the length of the lag phase, while both compounds did not affect the propagation rate. Both betalains were consumed during the inhibition period of lipid oxidation, and delayed consumption of LDL-beta carotene. Indicaxanthin, but not betanin, prevented vitamin E consumption at the beginning of LDL oxidation, and prolonged the time of its utilization. The resistance of LDL to oxidation when vitamin E and indicaxanthin acted separately in a sequence, was lower than that measured when they were allowed to act in combination, indicating some synergistic interaction between the two molecules. No prooxidant effect over a large concentration range of either betanin or indicaxanthin was observed, when either betalain was added to the LDL system undergoing a copper-induced oxidation.

These results show than indicaxanthin and betanin may bind to LDL, and are highly effective in preventing copper-induced lipid oxidation. Interaction with vitamin E appears to add a remarkable potential to indicaxanthin in the protection of LDL. Although molecular mechanisms remain uncompletely understood, various aspects of the action of betanin and indicaxanthin in preventing LDL lipid oxidation are discussed.     

Modification of low density lipoproteins by polymorphonuclear cell elastase leads to enhanced uptake by human monocyte-derived macrophages via the low density lipoprotein receptor pathway

In previous studies we reported that polymorphonuclear cell (PMN) elastase cleaves apoB-100 of human plasma low density lipoprotein (LDL) into seven or eight large Mr fragments (1, Polacek, D., R.E. Byrne, G.M. Fless, and A.M. Scanu. 1986. J. Biol. Chem. 261: 2057-2063). In the present studies we examined the interaction of native and elastase-digested LDL (ED-LDL) with primary cultures of human monocyte-derived macrophages (HMD-M). For this purpose LDL was digested with purified PMN elastase, re-isolated by ultracentrifugation at d 1.063 g/ml to remove the enzyme, and radiolabeled with 125I. At all LDL concentrations in the medium, the degradation of 125I-labeled ED-LDL was 1.5- to 2.5-fold greater than that of 125I-labeled native LDL, and for both lipoproteins species it was further enhanced by prior incubation of the cells in autologous lipoprotein-deficient serum (ALPDS). ED-LDL incubated with HMD-M in a medium containing [14C]oleate stimulated cholesteryl [14C]oleate formation 2- to 3-fold more than native LDL. In competitive degradation experiments, unlabeled ED-LDL did not inhibit the degradation of 125I-labeled acetylated LDL, whereas it caused a 90% inhibition of the degradation of 125I-labeled native LDL. At 4 degrees C, the binding of both 125I-labeled native and 125I-labeled ED-LDL was specific and of a high affinity. At saturation (Bmax), the binding of 125I-labeled ED-LDL was 2-fold higher (68 ng/mg cell protein) than that of 125I-labeled native LDL (31 ng/mg), with Kd values of 6.5 x 10(-8) M and 2.1 x 10(-8) M, respectively. A possible explanation of the binding data was provided by electrophoretic analyses suggesting that ED-LDL was twice the size of native LDL and thus potentially capable of delivering proportionately more cholesterol to the cells. Taken together, the results indicate that 1) digestion of LDL by purified PMN elastase results in a greater mass of ED-LDL (relative to native LDL) being degraded per unit time by HMD-M; 2) uptake of ED-LDL occurs via the LDL receptor; and 3) LDL digested by PMN elastase undergoes a physical change that may be responsible for its unique interactions with HMD-M. We speculate that if this process were to occur in vivo during an inflammatory process, macrophages could acquire excess cholesterol and be transformed into foam cells which are considered to be precursors of the atherosclerotic process.     

Binding of lipopolysaccharide and complexes of lipopolysaccharide to serum low density lipoproteins to liver macrophages

Binding of [³H]-lipopolysaccharide toxin (LPS) and complexes of LPS with serum [¹²⁵I]-labeled low density lipoproteins (LDL) to primary culture of rat liver macrophages (Kupffer cells) has been studied. Total, specific and nonspecific binding was determined. The receptor interaction was shown to dominate for both LPS and LDL-LPS complexes, representing 70–77% and 80–85%, respectively. The Scatchard plot was essentially non-linear for LPS binding but linear for the LDL-LPS complexes. At the Scatchard graph of LPS binding, however, two regions approximately fitting the linear regression could be identified. These regions correspond to two different types of specific binding sites: the first is for lower toxin concentrations of 0.25–0.50 μg/ml with K _d = 0.75 μg/ml; while the second is for higher LPS concentrations of 7.5–15 μg/ml with K _d = 5.39 μg/ml. For LDL-LPS complexes only K _d of 2.80 μg/ml was obtained. The LDL-LPS complexes significantly blocked the LPS binding (?40%) while acetylated or oxidized LDLs exerted a less pronounced effect. LPS inhibited binding of LDL-LPS complexes (?60%), while acetylated or oxidized LDLs suppressed interaction of LDL-LPS complexes with Kupffer cells insignificantly. It is suggested that, while binding to the Kupffer cell surface, a substantial portion of both LPS and LDL-LPS complexes share the same scavenger receptors with which, however, modified LDLs interact weakly. The LDL-LPS complexes can interact, apart from receptors common with LPS, with other receptors exhibiting similar binding parameters, with the apo-B/E receptors playing an inessential role.     

Multiple receptors for modified low density lipoproteins in mouse peritoneal macrophages: different uptake mechanisms for acetylated and oxidized low density lipoproteins

Receptor-mediated incorporations of two modified low density lipoproteins (LDL), acetylated LDL (acetyl-LDL) and oxidized LDL were compared in vitro in mouse peritoneal macrophages by cross-competition experiments. Excess amount of oxidized LDL inhibits the binding of [125I]acetyl-LDL only partially, and excess amount of acetyl-LDL inhibits that of [125I]oxidized LDL also only partially, suggesting that the uptake of the two LDL by macrophages is mediated by partially overlapped yet different mechanisms. Scatchard analysis of [125I]acetyl-LDL binding showed a linear plot and addition of excess amount of oxidized LDL partially displaced the binding sites without changing the affinity, suggesting that there are two classes of receptors with similar affinity; one is specific for acetyl-LDL and the other is common. And the plot of [125I]oxidized LDL binding showed a curvilinear plot and excess amount of acetyl-LDL partially displaced the binding sites of the low affinity, suggesting that there are two classes of binding sites with different affinities and the low affinity one is shared with acetyl-LDL. These results indicate that macrophage receptors for modified LDL consist of at least three receptors, two of which are specific for each LDL and the rest is a common receptor.     

ApoE-3-specific metabolism of human plasma very low density lipoproteins in cultured J-774 macrophages

The effect of apoprotein E on the cellular metabolism of very low density lipoproteins (VLDL) was studied using the J-774 macrophage-like cell line as a foam cell model. Exogenous (plasmatic and recombinant) apoE-3 caused a marked enhancement of the cellular binding, association, and degradation of VLDL fractions I, II, and III from both normolipidemic and hypertriglyceridemic subjects. ApoE-3 did not affect the cellular metabolism of low density lipoproteins (LDL). The stimulatory effect of apoE-3 was specific and was not observed with E-2. ApoE-mediated enhancement of VLDL metabolism was markedly suppressed by competition with LDL or by down-regulation of the LDL receptor while the basal cellular metabolism of VLDL was not. The macrophage, however, appears also to exhibit a second apoE-3-dependent pathway for VLDL metabolism which is discerned from the LDL and scavenger receptors and is relatively resistant to cholesterol in the culture medium. This pathway is responsible for the basal and perhaps a small fraction of the apoE-3-stimulated metabolism of VLDL in the macrophage. Such activity may play a role in promoting foam cell formation by triglyceride-rich lipoproteins.     

Chemically modified low density lipoproteins as inducers of enzyme release from macrophages

Macrophages carry receptors on their surface for acetylated low density lipoprotein (ac-LDL). Receptor-mediated endocytosis of ac-LDL is followed by intracellular cholesterol accumulation. We investigated whether occupation of these binding sites evokes the release of hydrolytic enzymes from mouse peritoneal macrophages cultured for up to 48 h. ac-LDL at concentrations ranging from 25-250 micrograms protein/ml was noted to promote in a dose-dependent fashion secretion of the neutral proteinase elastase (EC 3.4.21.37) and the lysosomal acid hydrolases N-acetyl-beta-glucosaminidase (EC 3.2.1.30), beta-glucuronidase (EC 3.2.1.31), beta-galactosidase (EC 3.2.1.23), alpha-mannosidase (EC 3.2.1.24) and cathepsin D (EC 3.4.23.5). This stimulatory effect was non-cytotoxic. LDL modified by treatment with malondialdehyde was also capable of augmenting enzyme liberation into culture supernates. These findings may have implications for some aspects of the atherosclerotic process.     

Unesterified fatty acids inhibit the binding of low density lipoproteins to the human fibroblast low density lipoprotein receptor

Micromolar concentrations of oleate were found to inhibit reversibly the binding of low density lipoprotein (LDL) to the human fibroblast LDL receptor. The decrease in LDL binding caused a parallel reduction of both 125I-LDL uptake and degradation at 37 degrees C. At 4 degrees C, oleate was also found to displace 125I-LDL already bound to the LDL receptor. The effect of oleate was rapid, reaching 70-80% of maximum displacement with 5-10 min of incubation, and was closely correlated to oleate-albumin molar ratios. Partition analysis of unesterified fatty acids between cells and LDL showed that the inhibitory effect of oleate resulted mainly from an interaction of unesterified fatty acids with the cell surface rather than with the LDL particles. Using different unesterified fatty acids and fatty acid analogs, we found that the inhibitory effect was modulated by both the length and the conformation of the monomeric carbon chain and was directly dependent on the presence of a negative charge on the carboxylic group. At 4 degrees C, the inhibitory effect of oleate never exceeded half of maximum binding capacity. This limitation was associated with the ability of oleate to interact only with part of the population of LDL receptors which spontaneously recycles in the absence of ligand, as demonstrated by the fact that oleate did not induce any reduction of LDL binding after cell treatment with monensin in the absence of LDL. Our results indicate that unesterified fatty acids could participate in the control of LDL catabolism in vivo by direct modulation of the ability of LDL receptor to bind LDL.     

Uptake of canine beta-very low density lipoproteins by mouse peritoneal macrophages is mediated by a low density lipoprotein receptor

The receptor on mouse peritoneal macrophages that mediates the uptake of canine beta-very low density lipoproteins (beta-VLDL) has been identified in this study as an unusual apolipoprotein (apo-) B,E(LDL) receptor. Ligand blots of Triton X-100 extracts of mouse peritoneal macrophages using 125I-beta-VLDL identified a single protein. This protein cross-reacted with antibodies against bovine apo-B,E(LDL) receptors, but its apparent Mr was approximately 5,000 less than that of the human apo-B,E(LDL) receptor. Binding studies at 4 degrees C demonstrated specific and saturable binding of low density lipoproteins (LDL), beta-VLDL, and cholesterol-induced high density lipoproteins in plasma that contain apo-E as their only protein constituent (apo-E HDLc) to mouse macrophages. Apolipoprotein E-containing lipoproteins (beta-VLDL and apo-E HDLc) bound to mouse macrophages and human fibroblasts with the same high affinity. However, LDL bound to mouse macrophages with an 18-fold lower affinity than to human fibroblasts. Mouse fibroblasts also bound LDL with a similar low affinity. Compared with the apo-B,E(LDL) receptors on human fibroblasts, the apo-B,E(LDL) receptors on mouse macrophages were resistant to down-regulation by incubation of the cells with LDL or beta-VLDL. There are three lines of evidence that an unusual apo-B,E(LDL) receptor on mouse peritoneal macrophages mediates the binding and uptake of beta-VLDL: LDL with residual apo-E removed displaced completely the 125I-beta-VLDL binding to mouse macrophages, preincubation of the mouse macrophages with apo-B,E(LDL) receptor antibody inhibited both the binding of beta-VLDL and LDL to the cells and the formation of beta-VLDL- and LDL-induced cholesteryl esters, and binding of 125I-beta-VLDL to the cells after down-regulation correlated directly with the amount of mouse macrophage apo-B,E(LDL) receptor as determined on immunoblots. This unusual receptor binds LDL poorly, but binds apo-E-containing lipoproteins with normal very high affinity and is resistant to down-regulation by extracellular cholesterol.     

Methylamine-treated low density lipoproteins elicit different responses in HepG2 cells and macrophages

Recent results from this laboratory have demonstrated the existence of labile thiolester bonds in apolipoprotein B (ApoB). Thiolester bonds can be cleaved with nucleophiles such as methylamine, resulting in conformational change. The purpose of this study was to explore whether the cellular interactions would be altered after methylamine treatment of low density lipoproteins (LDL). Human hepatoma cells, HepG2, and human monocyte derived macrophages were used for these studies. Fresh LDL were incubated with methylamine under mild alkaline conditions under N₂ and with preservatives for 24 h. The methylamine-treated LDL showed particle size and net charge identical to fresh native LDL. In addition, no oxidative modification of LDL occurred under the experimental conditions. The methylamine-treated LDL were indistinguishable from native LDL in HepG2 cells as judged by binding, degradation, cholesterol accumulation andde novo sterol synthesis. However, methylamine-treated LDL caused an increased accumulation of cholesteryl esters in macrophages which was comparable to the accumulation caused by acetylated LDL. Dual color digital imaging fluorescence microscopy revealed no competition between acetylated and methylamine-treated LDL, suggesting that the excessive uptake of methylamine-treated LDL was not mediated by the scavenger receptor. The increased accumulation of cholesteryl ester in macrophages also did not appear to stem from the classical LDL receptor. These results suggest that a new receptor binding domain is exposed due to the conformational change upon treatment of LDL with methylamine. (Mol Cell Biochem124: 67–79, 1993)Abbreviations LDL low density lipoproteins (d 1.032–1.043 g/ml for this study) - ApoB apolipoprotein B - MA methylamine - TBAR thiobarbituric acid reactive - HepG2 human hepatoma cell line - HMG-CoA reductase, -hydroxy--methylglutaryl CoA reductase - DIFM digital imaging fluorescence microscopy - FITC fluorescence isothiocyanate - ²M ²M-macroglobulin - BSA bovine serum albumin - PBS phosphate buffered saline - ACA -amino caproic acid - SDS-PAGE polyacrylamide gel electrophoresis containing SDS - TCA trichloroacetic acid - LRP lipoprotein receptor-related protein     

Gradient acrylamide/agarose gels for electrophoretic separation of intact human very low density lipoproteins, intermediate density lipoproteins, lipoprotein a, and low density lipoproteins

An exponential gradient gel with 0-10% acrylamide and 0.5% agarose was developed for electrophoresis of intact high molecular weight lipoproteins. This system resolves very low density lipoproteins, intermediate density lipoproteins, lipoprotein a, and low density lipoproteins in a size-dependent fashion. The characteristic relative mobility of these species can be determined in relation to protein and colloidal gold reference materials. Electron microscopy of selected lipoprotein fractions confirmed that relative mobility was related to apparent lipoprotein diameter. The composite gel medium can be used with prestained lipoproteins and permits immunoelectroblotting for qualitative analysis of apolipoprotein constituents.     

Interactions of high density lipoproteins with very low and low density lipoproteins during lipolysis

Interactions of high density lipoproteins (HDL) with very low (VLDL) and low (LDL) density lipoproteins were investigated during in vitro lipolysis in the presence of limited free fatty acid acceptor. Previous studies had shown that lipid products accumulating on lipoproteins under these conditions promote the formation of physical complexes between apolipoprotein B-containing particles (Biochim. Biophys. Acta, 1987. 919: 97-110). The presence of increasing concentrations of HDL or delipidated HDL progressively diminished VLDL-LDL complex formation. At the same time, association of HDL-derived apolipoprotein (apo) A-I with both VLDL and LDL could be demonstrated by autoradiography of gradient gel electrophoretic blots, immunoblotting, and apolipoprotein analyses of reisolated lipoproteins. The LDL increased in buoyancy and particle diameter, and became enriched in glycerides relative to cholesterol. Both HDL2 and HDL3 increased in particle diameter, buoyancy, and relative glyceride content, and small amounts of apoA-I appeared in newly formed particles of less than 75 A diameter. Association of apoA-I with VLDL or LDL could be reproduced by addition of lipid extracts of lipolyzed VLDL or purified free fatty acids in the absence of lipolysis, and was progressively inhibited by the presence of increasing amounts of albumin. We conclude that lipolysis products promote multiple interactions at the surface of triglyceride-rich lipoproteins undergoing lipolysis, including physical complex formation with other lipoprotein particles and transfers of lipids and apolipoproteins. These processes may facilitate remodeling of lipoproteins in the course of their intravascular metabolism.     

Presence of immunoreactive endothelin in human plasma

A highly specific and sensitive radioimmunoassay has been established for measurement of human endothelin (hET) in human plasma. After extraction of plasma with an octyl-silica column, this assay allowed for detection of immunoreactive (IR) hET as low as 0.2 fmol/ml. In 16 healthy subjects, the mean concentration of plasma IR-hET was 0.6 fmol/ml. Reverse-phase HPLC coupled with radioimmunoassay revealed two major IR-hET components, one corresponding to authentic hET(1-21) and another with more hydrophilicity than hET(1-21). These data indicate that ET is a circulating vasoconstrictor hormone in man.     

Presence of immunoreactive endothelin in human milk

Endothelin-like immunoreactivity was detected in human milk at a concentration of 6.8 +/- 1.6 pmol/l (mean +/- SEM; n = 16) using a highly sensitive radioimmunoassay. Gel filtration and fast protein liquid chromatography (FPLC) verified the identity of the endothelin. FPLC revealed 4 peaks, one eluting just after the void volume, and the other three in the positions of endothelin-1, -2, and -3, respectively.     

The influence of oxidatively modified low density lipoproteins on expression of platelet-derived growth factor by human monocyte-derived macrophages

Platelet-derived growth factor (PDGF) is secreted by several cells that participate in the process of atherogenesis, including arterial wall monocyte-derived macrophages. Macrophages in human and non-human primate lesions have recently been demonstrated to contain PDGF-B chain protein in situ. In developing lesions of atherosclerosis, macrophages take up and metabolize modified lipoproteins, leading to lipid accumulation and foam cell formation. Oxidatively modified low density lipoproteins (LDL) have been implicated in atherogenesis and have been demonstrated in atherosclerotic lesions. The effects of the uptake of various forms of modified LDL on PDGF gene expression, synthesis, and secretion in adherent cultures of human blood monocyte-derived macrophages were examined. LDL oxidized in a cell-free system in the presence of air and copper inhibited the constitutive expression of PDGF-B mRNA and secretion of PDGF in a dose-dependent fashion. Oxidatively modified LDL also attenuated lipopolysaccharide-induced PDGF-B mRNA expression. These changes were unrelated to the mechanism of lipid uptake and the degree of lipid loading and were detectable within 2 h of exposure to oxidized LDL. The degree of inhibition of both basal and lipopolysaccharide-induced PDGF-B-chain expression increased with the extent of LDL oxidation. Monocyte-derived macrophages exposed to acetylated LDL or LDL aggregates accumulated more cholesterol than cells treated with oxidized LDL, but PDGF expression was not consistently altered. Thus, uptake of a product or products of LDL oxidation modulates the expression and secretion of one of the principal macrophage-derived growth factors, PDGF. This modulation may influence chemotaxis and mitogenesis of smooth muscle cells locally in the artery wall during atherogenesis.     

High density lipoproteins reduce the uptake of low density lipoproteins by human endothelial cells in culture.

Endothelial cells, explanted from human umbilical veins and cultured, maintained morphological characteristics of vascular endothelium. When exposed to human serum lipoproteins, the cells bound and took up low density lipoproteins in preference to high density lipoproteins. High density lipoproteins reduced markedly the uptake of low density lipoproteins and affected surface binding to a lesser extent. These data suggest that the different levels of high density lipoprotein encountered in normal plasma of males and females could modulate differently the transendothelial transport of low density lipoproteins and provide a possible explanation for the lesser severity of atheromatosis in the aortic intima of premenopausal females.     

Binding of plasma low density lipoproteins to erythrocytes

Low density lipoproteins (LDL) containing apolipoprotein B bind to intact, freshly isolated erythrocytes. The LDL-erythrocyte interaction is of low affinity, with a Kd of 1.1 x 10(-6) M. Binding is noncooperative. There are about 200 binding sites per cell and, within the limits of experimental uncertainty, these sites comprise a homogeneous class. Binding of LDL is a temperature-independent process. The maximum amount of LDL blood increases following proteolytic digestion of the cells with trypsin or chymotrypsin. The specificity of the binding sites for LDL is not absolute: high density lipoproteins and lipid vesicles composed of phosphatidylcholine or phosphatidylcholine/cholesterol (equimolar) complete with LDL for occupancy of 60% of the binding sites. Modification of 5--6 of the 9 apolipoprotein B arginine residues with 1,2-cyclohexanedione/borate or of 10--15 of the 20 lysine residues by reductive methylation does not alter the ability of LDL to bind to erythrocytes. Native LDL and methylated-LDL alter erythrocyte morphology. However, LDL in which the arginine residues are derivatized with 1,2-cyclohexanedione/borate do not induce the discocyte leads to echinocyte transformation. Chemically modified and native LDL exchange cholesterol with erythrocytes at equal rates and to nearly equal extents. Taken together, the data suggest that the binding sites for LDL on the erythrocyte membrane are distinct from the LDL receptors at the surface of other cells--e.g., fibroblasts and lymphocytes--which do not bind HDL and which do not recognize LDL with derivatized arginine or lysine residues. It is proposed that the biological function of the erythrocyte binding sites is to mediate the exchange of cholesterol between the cell membrane and lipoproteins.