A macrophage receptor that recognizes oxidized low density lipoprotein but not acetylated low density lipoprotein

The formation of cholesterol-loaded macrophage foam cells in arterial tissue may occur by the uptake of modified lipoproteins via the scavenger receptor pathway. The macrophage scavenger receptor, also called the acetylated low density lipoprotein (Ac-LDL) receptor, has been reported to recognize Ac-LDL as well as oxidized LDL species such as endothelial cell-modified LDL (EC-LDL). We now report that there is another class of macrophage receptors that recognizes EC-LDL but not Ac-LDL. We performed assays of 0 degrees C binding and 37 degrees C degradation of 125I-Ac-LDL and 125I-EC-LDL by mouse peritoneal macrophages. Competition studies showed that unlabeled Ac-LDL could compete for only 25% of the binding and only 50% of the degradation of 125I-EC-LDL. Unlabeled EC-LDL, however, competed for greater than 90% of 125I-EC-LDL binding and degradation. Unlabeled Ac-LDL was greater than 90% effective against 125I-Ac-LDL; EC-LDL competed for about 80% of 125I-Ac-LDL binding and degradation. Copper-oxidized LDL behaved the same as EC-LDL in all the competition studies. Copper-mediated oxidation of Ac-LDL produced a superior competitor which could now displace 90% of 125I-EC-LDL binding. After 5 h at 37 degrees C in the presence of ligand, macrophages accumulated six times more cell-associated radioactivity from 125I-EC-LDL than from 125I-Ac-LDL, despite approximately equal amounts of degradation to trichloroacetic acid-soluble products, which may imply different intracellular processing of the two lipoproteins. Our results suggest that 1) there is more than one macrophage "scavenger receptor" for modified lipoproteins; and 2) oxidized LDL and Ac-LDL are not identical ligands with respect to macrophage recognition and uptake.     

The contribution of the macrophage receptor for oxidized LDL to its cellular uptake

Oxidized LDL (Ox-LDL) was shown to be taken up by macrophages via several receptors including the acetyl-LDL(Ac-LDL), the LDL, and the Ox-LDL receptors. Cellular uptake and degradation of Ox-LDL could be dissociated from that of LDL and Ac-LDL as demonstrated by using macrophages that lack the LDL or the Ac-LDL receptors. In J-774 A.1 macrophage-like cell line unlabeled Ox-LDL reduced the 125I-Ox-LDL by up to degradation of 91% whereas unlabeled Ac-LDL and native LDL reduced 125I-Ox-LDL degradation by only 51% and 23%, respectively. Analysis of macrophage degradation of 125I-Ox-LDL in the presence of 30-fold excess concentration of LDL + Ac-LDL (to block uptake of 125I-Ox-LDL via the LDL and the Ac-LDL receptors) revealed that cellular degradation via the Ox-LDL receptor could account for 45% of the macrophage uptake of Ox-LDL.     

Low density lipoprotein modification by cholesterol oxidase induces enhanced uptake and cholesterol accumulation in cells.

Oxidation of low density lipoprotein (LDL) by cells of the arterial wall or in the presence of copper ions was shown to result in the peroxidation of its fatty acids as well as its cholesterol moiety. LDL incubation with cholesterol oxidase (CO) resulted in the conversion of up to 85% of the lipoprotein unesterified cholesterol (cholest-5-en-3-ol) to cholestenone (cholest-4-en-3-one) in a dose- and time-dependent pattern. Plasma very low density lipoprotein (VLDL) and high density lipoprotein (HDL) could be similarly modified by CO. In cholesterol oxidase-modified LDL (CO-LDL), unlike copper ion-induced oxidized LDL (Cu-Ox-LDL), there was no fatty acids peroxidation, and lipoprotein size or charge as well as LDL cholesteryl ester, phospholipids, and triglycerides content were not affected. CO-LDL, however, demonstrated enhanced susceptibility to oxidation by copper ions in comparison to native LDL. Upon incubation of CO-LDL with J-774 A.1 macrophage-like cell line, cellular uptake and degradation of the lipoprotein was increased by up to 62% in comparison to native LDL but was 15% lower than that of Cu-Ox-LDL. Similarly, the binding of CO-LDL to macrophages increased by up to 80%, and cellular cholesterol mass was increased 51% more than the mass obtained with native LDL. Several lines of evidence indicate that CO-LDL was taken up via the LDL receptor: 1) Excess amounts of unlabeled LDL, but not acetyl-LDL (Ac-LDL), effectively competed with 125I-CO-LDL for the uptake by cells. 2) The degradation of CO-LDL by various types of macrophages and by fibroblasts could be dissociated from that of Ac-LDL and was always higher than that of native LDL. 3) A monoclonal antibody to the LDL receptor (IgG-C7) and a monoclonal antibody to the LDL receptor binding domains on apoB-100 (B1B6) inhibited macrophage degradation of CO-LDL. The receptor for Cu-Ox-LDL, which is not shared with Ac-LDL, was also partially involved in macrophage uptake of CO-LDL, since Cu-Ox-LDL demonstrated some competition capability with CO-125I-LDL for its cellular degradation. CO-LDL cellular degradation was inhibited by chloroquine, thus implying lysosomal involvement in the cellular processing of the lipoprotein. Incubation of macrophages with LDL in the presence of increasing concentrations of cholestenone resulted in up to 52% enhanced lipoprotein cellular degradation suggesting that the cholestenone in CO-LDL might be involved in the enhanced cellular uptake of the modified lipoprotein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modification of low density lipoprotein by lipoprotein lipase or hepatic lipase induces enhanced uptake and cholesterol accumulation in cells

Incubation of low density lipoprotein(s) (LDL) with either lipoprotein lipase or hepatic lipase led to modification of the core lipid composition of LDL. Both lipases modified LDL by substantially reducing core triglyceride content without producing marked differences in size, charge, or lipid peroxide content in comparison to native LDL. The triglyceride-depleted forms of LDL that result from treatment with these two enzymes were degraded at approximately twice the rate of native LDL by human monocyte-derived macrophages (HMDM). Lipase-modified LDL degradation was inhibited by chloroquine, suggesting lysosomal involvement in LDL cellular processing. The increased degradation by macrophages of the LDL modified by these lipases was accompanied by enhanced cholesterol esterification rates, as well as by an increase in cellular free and esterified cholesterol content. In a patient with hepatic triglyceride lipase deficiency, degradation of the triglyceride-rich LDL by HMDM was approximately half that of normal LDL. Following in vitro incubation of LDL from this patient with either lipoprotein or hepatic lipase, lipoprotein degradation increased to normal. Several lines of evidence indicate that LDL modified by both lipases were taken up by the LDL receptor and not by the scavenger receptor. 1) The degradation of lipase-modified LDL in nonphagocytic cells (human skin fibroblast and arterial smooth muscle cells) as well as in phagocytic cells (HMDM, J-774, HL-60, and U-937 cell lines) could be dissociated from that of acetylated LDL and was always higher than that of native LDL. A similar pattern was found for cellular cholesterol esterification and cholesterol mass. 2) LDL receptor-negative fibroblasts did not degrade lipase-modified LDL. 3) A monoclonal antibody to the LDL receptor inhibited macrophage degradation of the lipase-modified LDL. 4) Excess amounts of unlabeled LDL competed substantially with 125I-labeled lipase-modified LDL for degradation by both macrophages and fibroblasts. Thus, lipase-modified LDL can cause significant cholesterol accumulation in macrophages even though it is taken up by LDL and not by the scavenger receptor. This effect could possibly be related to the reduced triglyceride content in the core of LDL, which may alter presentation of the LDL receptor-binding domain of apolipoprotein B on the particle surface, thereby leading to increased recognition and cellular uptake via the LDL receptor pathway.     

Metabolism of modified LDL and foam cell formation in murine macrophage-like RAW 264 cells.

The uptake of modified low density lipoprotein (LDL) by arterial macrophages is a key event in the atherogenesis. We studied 1) the uptake and degradation of modified LDL, 2) LDL recognition by specific receptors, and 3) the foam cell formation with murine macrophage-like RAW 264 cells in vitro. The cells took up and degraded effectively 125I-labeled acetylated LDL (Ac-LDL) and aggregated LDL (Aggr-LDL). Also oxidized LDL (Ox-LDL) was taken up but it was degraded poorly. The degradation of 125I-Ac-LDL was efficiently competed by both unlabeled Ac-LDL and Ox-LDL, whereas the degradation of 125I-Ox-LDL was partially competed by unlabeled Ox-LDL and Aggr-LDL but not at all by unlabeled Ac-LDL. The incubation with increasing concentrations of Ac-LDL, Aggr-LDL or Ox-LDL resulted in marked foam cell formation in the RAW 264 cells. Ox-LDL was cytotoxic at 500 to 1000 microg/ml concentrations. The results show that RAW 264 cells have at least two classes of receptors for modified lipoproteins: one that recognizes both Ox-LDL and Ac-LDL, and is similar to the scavenger receptors, and another that recognizes Ox-LDL but not Ac-LDL. RAW 264 cells are a convenient model cell line for examining the metabolism of modified lipoproteins, not only that of Ac-LDL but also that of Ox-LDL and Aggr-LDL, and cellular accumulation of lipids derived from modified LDL.     

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.     

Sialic acid content of human low density lipoproteins affects their interaction with cell receptors and intracellular lipid accumulation.

Low density lipoproteins (LDL) isolated from the plasma of patients with angiographically demonstrable coronary heart disease (CHD) induced accumulation of triglycerides, free cholesterol, and cholesteryl esters in cultured macrophages, smooth muscle cells, and endothelial cells derived from uninvolved intima of human aorta, but not in skin fibroblasts or hepatoma cells. The sialic acid content of LDL from CHD patients was 40-75% lower than that from healthy donors. There was a negative correlation between LDL sialic acid content and the LDL-induced accumulation of total intracellular cholesterol. Neuraminidase treatment of LDL from normal healthy donors produced sialic acid-depleted LDL (Ds-LDL) which was able to stimulate intracellular lipid accumulation. Neuraminidase treatment of LDL from CHD patients further increased its capacity to induce intracellular lipid accumulation. Sialic acid-poor LDL isolated by affinity chromatography of LDL from CHD patients induced a 2- to 4-fold increase of free and esterified cholesterol in human intimal smooth muscle cells. Binding, uptake, and degradation of 125I-labeled Ds-LDL by macrophages and endothelial cells were 1.5- to 2-fold higher than for native LDL. Binding and uptake of Ds-LDL was inhibited 64-93% by the addition of 20-fold excess acetylated LDL (Ac-LDL); in the inverse experiment, the level of inhibition was 35-54%. These data indicate that a sialic acid-poor form of LDL isolated from CHD patients can interact with both native and scavenger LDL receptors. A sialic acid-poor form of LDL may be a naturally occurring ligand that interacts with the scavenger receptor(s) on macrophages and endothelial cells.     

Differential uptake of proteoglycan-selected subfractions of low density lipoprotein by human macrophages

Macrophages and arterial chondroitin sulfate proteoglycans (CSPG) are probably associated with extracellular and intracellular lipoprotein deposition during atherogenesis. We found that human arterial CSPG can be used to select subclasses from low density lipoprotein (LDL) with different structural properties and capacities to interact with human monocyte-derived macrophages (HMDM). Four subclasses, LDL(PG)1 to LDL(PG)4, in order of decreasing CSPG-complexing capacity, were prepared and characterized in terms of their ability to interact with HMDM. The LDL subclasses with highest avidity for CSPG, LDL(PG)1 and LDL(PG)2, were bound, internalized, and degraded more efficiently than those of lower avidity for CSPG. From LDL(PG)1 to LDL(PG)4, the gradual decrease in uptake by HMDM and decreasing avidity for CSPG were associated with a gradual decrease in isoelectric point (from 5.93 to 5.68) and an augmented ratio of surface polar lipid to core nonpolar components (from 0.35 to 0.54). Competition experiments indicated that the proteoglycan-selected subfractions shared the binding sites and uptake mechanisms of native LDL. The results suggest the existence of a structurally related gradation in the avidity of LDL subpopulations for cells and matrix components. The presence within LDL subpopulations of a differential capacity to interact with intimal extracellular and cellular elements could be associated with a similar heterogeneity in their atherogenic potential.     

Glycosylation of LDL decreases its ability to interact with high-affinity receptors of human fibroblasts in vitro and decreases its clearance from rabbit plasma in vivo

Incubation of human LDL in vitro at 37 degrees C for 48 h with [14C]glucose at concentrations from 5 to 200 mM resulted in a glycosylated LDL, containing 0.4-20 mol of glucose incorporated per apolipoprotein B of 250 000 daltons. The extent of glucose incorporated was proportional to the time of incubation and concentration of glucose. Glycosylation of LDL abolished its uptake and degradation by the high-affinity process for LDL in normal human skin fibroblasts. 125I-labeled glycosylated LDL was bound, internalized and degraded by the fibroblasts via a nonspecific low-affinity process. The 125I-labeled glycosylated LDL and 125I-labeled LDL were taken up and degraded at similar rates in a non-saturable, low-affinity process by peritoneal macrophages isolated from mice. When 125I-labeled glycosylated LDL or 125I-labeled LDL were injected into rabbits, the glycosylated LDL had a delayed plasma clearance in comparison to the LDL. The mean fractional catabolic rates were 0.67 day-1 and 1.70 day-1 for 125I-labeled glycosylated LDL and 125I-labeled LDL, respectively. The uptake and degradation of 125I-labeled LDL by human skin fibroblasts was decreased as the concentration of free carbohydrate, glucose, sucrose or sorbitol, in the medium was increased from 10 mM to 1 M. It is speculated that pathologic levels of plasma glucose in vivo could result in a decrease in LDL uptake as a result of glycosylation of LDL. A decrease in uptake of native or modified LDL in vivo could contribute to hypercholesterolemia and its pathophysiology.     

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 CsCl isopycnic centrifugation. Complex of 125I-labeled LDL and proteoglycan aggregate formed in the presence of 30 mM Ca2+ was incubated with macrophages, and the binding at 4 degrees C and degradation at 37 degrees 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 Kd for binding was 23 micrograms 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 beta-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.     

Receptors for modified low-density lipoproteins on human endothelial cells: different recognition for acetylated low-density lipoprotein and oxidized low-density lipoprotein

We examined the uptake pathway of acetylated low-density lipoprotein and oxidatively modified LDL (oxidized LDL) in human umbilical vein endothelial cells in culture. Proteolytic degradation of 125I-labeled Ac-LDL or Ox-LDL in the confluent monolayer of human endothelial cells was time-dependent and showed saturation kinetics in the dose-response relationship, which suggests that their incorporation is receptor-mediated. Cross-competition studies between acetylated LDL and oxidized LDL showed that the degradation of 125I-labeled acetylated LDL was almost completely inhibited by excess amount of unlabeled acetylated LDL, while only partially inhibited by excess unlabeled oxidized LDL. On the other hand, the degradation of 125I-labeled oxidized LDL was equally inhibited by excess amount of either acetylated or oxidized LDL. Cross-competition results of the cell-association assay paralleled the results shown in the degradation assay. These data indicate that human endothelial cells do not have any additional receptors specific only for oxidized LDL. On the contrary, they may have additional receptors, as we previously indicated on mouse macrophages, which recognize acetylated LDL, but not oxidized LDL.     

Catabolism of human low density lipoproteins by human hepatoma cell line HepG2

The mechanism of hepatic catabolism of human low density lipoproteins (LDL) by human-derived hepatoma cell line HepG2 was studied. The binding of 125I-labeled LDL to HepG2 cells at 4 degrees C was time dependent and inhibited by excess unlabeled LDL. The specific binding was predominant at low concentrations of 125I-labeled LDL (less than 50 micrograms protein/ml), whereas the nonsaturable binding prevailed at higher concentrations of substrate. The cellular uptake and degradation of 125I-labeled LDL were curvilinear functions of substrate concentration. Preincubation of HepG2 cells with unlabeled LDL caused a 56% inhibition in the degradation of 125I-labeled LDL. Reductive methylation of unlabeled LDL abolished its ability to compete with 125I-labeled LDL for uptake and degradation. Chloroquine (50 microM) and colchicine (1 microM) inhibited the degradation of 125I-labeled LDL by 64% and 30%, respectively. The LDL catabolism by HepG2 cells suppressed de novo synthesis of cholesterol and enhanced cholesterol esterification; this stimulation was abolished by chloroquine. When tested at a similar content of apolipoprotein B, very low density lipoproteins (VLDL), LDL and high density lipoproteins (HDL) inhibited the catabolism of 125I-labeled LDL to the same degree, indicating that in HepG2 cells normal LDL are most probably recognized by the receptor via apolipoprotein B. The current study thus demonstrates that the catabolism of human LDL by HepG2 cells proceeds in part through a receptor-mediated mechanism.     

Losartan inhibits cellular uptake of oxidized LDL by monocyte-macrophages from hypercholesterolemic patients

Angiotensin II (Ang II) and oxidized LDL (Ox-LDL) are risk factors for atherosclerosis, and both of them contribute to macrophage cholesterol accumulation, the hallmark of early atherosclerosis. As Ang II was shown to increase macrophage uptake of Ox-LDL, we investigated the effect of losartan, an Ang II receptor antagonist with antiatherogenic properties, on the cellular uptake of Ox-LDL by human monocyte-derived macrophages (HMDM) from hypercholesterolemic patients. Eight normotensive hypercholesterolemic patients were treated with losartan (50 mg/day) for a period of 4 weeks. Losartan therapy did not significantly affect the degradation of native LDL by the patients' HMDM. However, losartan therapy significantly reduced HMDM uptake of Ox-LDL as shown by a 78% reduction in Ox-LDL cell-association and a 21% reduction in Ox-LDL degradation. CD36 (an Ox-LDL receptor) mRNA expression in HMDM obtained after losartan treatment was decreased by 54% compared to HMDM obtained before treatment. The ability of losartan to inhibit HMDM CD36 mRNA expression and, hence, Ox-LDL uptake and macrophage foam cell formation is probably related to the blockage of Ang II binding to the cell surface and thus to the prevention of Ang II atherogenic effects.     

The effect of felodipine on the uptake and degradation of acetylated LDL in mouse peritoneal cells and on the distribution of acetylated LDL in macrophage-rich organs of the rat

The effect of felodipine on lipoprotein metabolism ex vivo and in vivo was investigated. In the ex vivo studies mice were given felodipine (40–125 μ mol/kg body weight) or vehicle for one week. Peritoneal macrophages from these animals and controls were isolated and used in binding and degradation studies with human iodinated acetylated LDL (Ac-LDL). Macrophages from felodipine-treated mice showed a significant decrease of binding and degradation of Ac-LDL compared to macrophages from control animals (P<0.05). The in vivo studies were performed in rats pretreated with felodipine or vehicle. To determine the distribution and plasma turnover of LDL and Ac-LDL, ¹²⁵I-tyramine cellobiose labelled LDL or Ac-LDL were given i.v. No differences in the removal rate of Ac-LDL or LDL were observed between felodipine-treated or untreated rats. However, an increased uptake of Ac-LDL could be seen in the liver of the felodipine-treated rats. This increased uptake could be ascribed to the parenchymal cells because no differences in uptake could be seen in the liver endothelial cells. However, a significant decreased uptake was seen in the Kuppfer cells and in the spleen, a macrophage-rich organ, of the felodipine-treated rats. The present study suggests a possible mechanism behind the antiatherogenic effects of calcium antagonists, a decreased uptake of atherogenic modified lipoproteins by peripheral macrophages and an increased uptake by the liver.     

Lipoproteins of the extravascular space: enhanced macrophage degradation of low density lipoproteins from interstitial inflammatory fluid

Current evidence has demonstrated that cholesteryl ester-loaded macrophages are important components of the atherosclerotic lesion. Additional studies have implicated low density lipoproteins (LDL) and circulating monocytes as central to the origin of lipid-laden foam cells found in the arterial wall. This is a result of the finding of accelerated macrophage uptake of LDL chemically modified by reaction with malondialdehyde (MDA-LDL), acetic anhydride (Ac-LDL), or incubation with arterial cells in vitro. In concert with these chemical modifications, we have previously demonstrated selective in vivo modification of LDL isolated from interstitial inflammatory fluid (IF) of the rabbit. Utilizing the polyvinyl sponge implant model, we reported that IF-LDL had an altered chemical composition, electrophoretic mobility, and particle size distribution when compared to LDL isolated from homologous plasma (WP-LDL). In this study reported herein, we examined the metabolism of IF-LDL by resident mouse peritoneal macrophages (MPM) in culture. IF-LDL was degraded substantially faster by MPM, and resulted in a substantial increase in cellular cholesteryl ester when compared to cells incubated with WP-LDL. IF-LDL binding to MPM was inhibited by Ac-LDL derived from WP-LDL, but only minimally by unmodified WP-LDL. Transmission electron microscopy of MPM revealed extensive lipid deposition in cells incubated with Ac-LDL and IF-LDL. These results implicate LDL from interstitial inflammatory fluid as an in vivo modified lipoprotein that can enhance uptake via the acetyl LDL receptor pathway in resident macrophages.     

Regulation of the uptake and degradation of beta-very low density lipoprotein in human monocyte macrophages

In normal human monocyte macrophages 125I-labeled beta-migrating very low density lipoproteins (125I-beta-VLDL), isolated from the plasma of cholesterol-fed rabbits, and 125I-human low density lipoprotein (LDL) were degraded at similar rates at protein concentrations up to 50 micrograms/ml. The high affinity degradation of 125I-labeled human LDL saturated at approximately 50 micrograms/ml; however, 125I-labeled rabbit beta-VLDL high affinity degradation saturated at 100-120 micrograms/ml. The activity of the beta-VLDL receptor was 3-fold higher than LDL receptor activity on freshly isolated normal monocyte macrophages, but with time-in-culture both receptor activities decreased and were similar after several days. The degradations of both beta-VLDL and LDL were Ca2+ sensitive, were markedly down regulated by sterols, and were up regulated by preincubation of the cells in a lipoprotein-free medium. The beta-VLDL receptor is genetically distinct from the LDL receptor as indicated by its presence on monocyte macrophages from a familial hypercholesterolemic homozygote. Human thoracic duct lymph chylomicrons as well as lipoproteins of Sf 20-5000 from fat-fed normal subjects inhibited the degradation of 125I-labeled rabbit beta-VLDL as effectively as nonradioactive rabbit beta-VLDL. We conclude: 1) the beta-VLDL receptor is genetically distinct from the LDL receptor, and 2) intestinally derived human lipoproteins are recognized by the beta-VLDL receptor on macrophages.     

Regulation of low density lipoprotein receptor activity by cultured human arterial smooth muscle cells.

The ability of cultured human arterial smooth muscle cells to regulate low density lipoprotein (LDL) receptor activity was tested. In contrast to human skin fibroblasts incubated with lipoprotein deficient medium under identical conditions, smooth muscle cells showed significantly reduced enhancement of 125I-labeled LDL and 125I-labeled VLDL (very low density lipoprotein) binding. Smooth muscle cells also failed to suppress LDL receptor activity during incubation with either LDL or cholesterol added to the medium, while fibroblasts shoed an active regulatory response. Thus, in comparison with the brisk LDL receptor regulation characteristic of skin fibroblasts, arterial smooth muscle cells have and attenuated capacity to regulate their LDL receptor activity. These results may be relevant to the propensity of these cells to accumulate LDL and cholesterol and form "foam cells" in the arterial wall in vivo, a process associated with atherogenesis.     

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

We studied the effect of complexes of low-density lipoproteins (LDL) and different proteoglycan preparations from bovine aorta on LDL degradation and cholesteryl ester accumulation in mouse peritoneal macrophages. Native proteoglycan aggregate containing proteoglycan monomers, hyaluronic acid and link protein was isolated by associative extraction of aortic tissue, while proteoglycan monomer was obtained by dissociative isopycnic centrifugation of the native proteoglycan aggregate. In vitro proteoglycan aggregates were prepared by reaction of the proteoglycan monomer with exogenous hyaluronic acid. 125I-labeled LDL-proteoglycan complexes were formed in the presence of 30 mM Ca2+ and incubated with macrophages. At equivalent uronic acid levels in the proteoglycans the degradation of 125I-labeled LDL contained in the native proteoglycan aggregate complex was 3.7-7.5-fold greater than the degradation of the lipoprotein in the proteoglycan monomer complex. Degradation of 125I-LDL in the in vitro aggregate complex, while higher than that in the monomer complex, was markedly less than that in the native aggregate complex. The larger size and the greater complex-forming ability of the native proteoglycan aggregate might account for the greater capacity of the aggregate to promote LDL degradation in macrophages. The proteoglycan-stimulated degradation of LDL produced a marked increase in cholesteryl ester synthesis and content in macrophages. The LDL-proteoglycan complex was degraded with saturation kinetics, suggesting that these complexes are internalized through high-affinity receptors. Degradation was inhibited by the lysosomotropic agent, chloroquine. Acetyl-LDL, but not native LDL, competitively inhibited the degradation of the 125I-LDL component of the complex. Polyanionic compounds such as polyinosinic acid and fucoidin, while completely blocking the acetyl-LDL-stimulated cholesteryl ester formation, had no effect on the proteoglycan aggregate-stimulated cholesterol esterification. This suggests that LDL-proteoglycan complex and acetyl-LDL are not entering the cells through the same receptor pathway. These results demonstrate that the interaction of LDL with arterial wall proteoglycan aggregates results in marked cholesteryl ester accumulation in macrophages, a process likely to favor foam cell formation. A role for arterial proteoglycans in atherosclerosis is obvious.     

Oxidative stress impairs endocytosis of the scavenger receptor class A

We report the characterization of a cell system employing Chinese hamster ovary (CHO) cells and CHO cells transfected with the scavenger receptor class A (CHO-SRA) using extracellularly produced reactive oxygen species (ROS) in order to study the endocytic function of the scavenger receptor. The oxidative environment was produced using tert-butyl hydroperoxide (TBH) and characterized by flow cytometry and cell viability. Once an adequate oxidative environment was established, binding and internalization studies of radiolabeled acetylated LDL particles (125I-labeled Ac-LDL) with CHO-SRA cells were carried out. RT-PCR analysis using total RNAs from CHO-SRA cells revealed that oxidative stress does not alter the expression of the scavenger receptor. However, internalization of 125I-labeled Ac-LDL through this receptor carried out by these cells was completely abolished under extracellularly oxidative conditions. Together, these results support the idea that an oxidative stress produced extracellularly, inhibiting the endocytosis of the scavenger receptor, could help to understand and explain the mechanisms by which several physiologically important ligands are accumulated in the extracellular space with its consequent cell damage.     

Uptake of chylomicron remnants by the native LDL receptor in human monocyte-derived macrophages

Human chylomicron remnants were taken up by cultured human monocyte-derived macrophages. Competition studies using 125I-labeled and unlabeled lipoproteins demonstrated that the remnant particles were not taken up by the modified LDL (acetyl LDL) receptor in these cells, which also contain a receptor for native LDL. The data thus suggest that the apolipoprotein E- and B-containing remnant particles are mainly taken up through an extra-hepatic E and B receptor (the classical LDL receptor pathway) in macrophages as is the case in cultured human skin fibroblasts.