Receptor-dependent uptake of human chylomicron remnants by cultured skin fibroblasts

Human chylomicrons were isolated from plasma from a subject with familial hypertriglyceridemia and converted to chylomicron remnants by incubation with postheparin plasma. The interaction of these apolipoprotein E-containing, cholesterol-rich human chylomicron remnants with cultured skin fibroblasts was studied. Chylomicron remnants were internalized by skin fibroblasts as a unit, mainly via the low density lipoprotein (LDL)-receptor pathway, resulting in increased cell cholesterol content. After entering the fibroblast, chylomicron remnants stimulated cholesterol esterification, suppressed 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, and down-regulated LDL receptor activity similar to the action of LDL. As a function of increasing lipolysis, remnant particles were progressively more effectively taken up by skin fibroblasts, despite a decrease in the apolipoprotein E content per lipoprotein particle. Remnant particles produced after hydrolysis of 70 to 80% of chylomicron triglyceride increased cell cholesterol content to an amount nearly identical to that observed with LDL when the two lipoproteins were incubated at an equal cholesterol concentration. However, when incubated on the basis of equal particle number, chylomicron remnants were 2 to 3 times more effective than LDL in delivering cholesterol to the cells. These results suggest that chylomicron remnants play a role in the regulation of postabsorptive cholesterol homeostasis in nonhepatic cells, and possibly in the pathogenesis of atherosclerosis.     

Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in mouse peritoneal macrophages.

The lipoprotein-mediated regulation of 3-hydroxy-3-methylglutaryl-(HMG-) CoA reductase in cultured mouse peritoneal macrophages has been investigated. In contrast to what has been reported for other cells, HMG-CoA reductase activity is not suppressed by normal serum or by normal low density lipoproteins (LDL) from humans or dogs. Suppression of reductase activity occurred when cells were cultured in the presence of beta-migrating very low density lipoproteins (beta-VLDL) or LDL from hypercholesterolaemic dogs, or LDL modified by acetoacetylation. Human beta-VLDL from an atypical type III hyperlipoproteinaemic patient was also effective, as was apolipoprotein (apo) E-containing high density lipoproteins (HDL) from cholesterol-fed dogs (apo-E HDLc). The results indicate that cholesterol biosynthesis in mouse peritoneal macrophages is regulated by lipoprotein cholesterol entering via receptor-mediated endocytosis. Normal LDL were not effective because of the poor binding and uptake of these lipoproteins by the apo-B, E (LDL) receptor. Only beta-VLDL, apo-E HDLc, and hypercholesterolaemic LDL were avidly taken up by this receptor and were able to suppress HMG-CoA reductase. Acetoacetylated LDL were internalized via the acetyl-LDL (scavenger) receptor. Thus, mouse macrophages differ from human fibroblasts and smooth muscle cells in their physiological regulation of cholesterogenesis.     

Characterization of the estrogen-induced lipoprotein receptor of rat liver

The ethinyl estradiol-induced lipoprotein receptor of rat liver was purified and characterized. Liver membranes were prepared from ethinyl estradiol-treated rats, solubilized, and subjected to DEAE chromatography. A fraction with a high specific activity for low density lipoprotein (LDL) binding was isolated and used to immunize mice. Hybridomas were prepared from their spleen cells, and a clone that secreted an IgG antibody, which cross-reacted with an ethinyl estradiol-induced protein of the same molecular weight as the bovine adrenal LDL receptor, was expanded. This antibody, designated P1B3, immunoprecipitated the induced lipoprotein receptor. P1B3 was used to purify the receptor, and a polyclonal antibody was raised against the pure protein. This antibody recognized a protein of similar molecular weight in rat liver, adult dog liver, and human skin fibroblasts, thus demonstrating that the induced rat lipoprotein receptor was related to the LDL receptor of other species. This receptor is present in normal rat liver, and its content is reduced by feeding an atherogenic diet, but not by feeding a diet containing 0.5% cholesterol. Moreover, cholestyramine supplementation of the diet did not induce the receptor on liver membranes. The polyclonal antibody could prevent the binding of LDL to liver membranes from control or ethinyl estradiol-treated rats. It decreased chylomicron remnant binding to membranes from ethinyl estradiol-treated membranes, but did not affect chylomicron remnant binding to liver membranes of untreated rats, a result compatible with the existence of a distinct receptor for these latter particles. The amount of LDL receptor-independent, specific remnant binding was the same in both control and ethinyl estradiol-treated rats. This is consistent with the concept that the remnant receptor is not regulated by this treatment. Based on the above, we conclude that the ethinyl estradiol-induced lipoprotein receptor of rat liver is biochemically and immunologically similar to the LDL receptor of other species. It is present on the liver of normal adult rats and could account for LDL as well as beta VLDL and HDLc removal. Although it may contribute to chylomicron remnant removal, there appears to be a second unrelated receptor or process which recognizes this lipoprotein.     

The effect of concanavalin A-stimulated mononuclear cells on low density lipoprotein receptor activity of cultured fibroblasts

Secretory products of freshly isolated human circulating blood cells such as platelets, monocytes, and B lymphocytes, but not T lymphocytes, have previously been shown to enhance low density lipoprotein (LDL) metabolism by arterial wall cells. This study was undertaken to evaluate how secretory factor(s) from mononuclear cells that had been stimulated by concanavalin A (Con A) alters LDL receptor activity by cultured human skin fibroblasts. Conditioned medium from Con A-stimulated mononuclear cells produced an increase of 125I-LDL degradation accompanied by increased thymidine incorporation into DNA. The effect of conditioned medium from the Con A-stimulated mononuclear cells was mediated by the LDL receptor pathway. Degradation of HDL and methylated LDL, neither of which is taken up by the classical LDL receptor pathway, was not affected. The conditioned medium from these Con A-stimulated cells also failed to stimulate fluid pinocytosis, as measured by the uptake of [14C]sucrose. Some strains of fibroblasts, deficient in LDL receptors, responded to the conditioned medium from the Con A-stimulated mononuclear cells by increasing the very small amounts of LDL degraded by these cells. Fibroblasts from other homozygous familial hypercholesterolemic cell strains were unresponsive, however. The effect on LDL receptors was characterized by an increase in LDL receptor number without a change in the affinity of LDL for its receptor. Thus stimulated mononuclear cells secrete mitogens that also stimulate LDL receptor activity in human skin fibroblasts.     

SR-BI-mediated selective lipid uptake segregates apoA-I and apoA-II catabolism

The HDL receptor scavenger receptor class B type I (SR-BI) binds HDL and mediates the selective uptake of cholesteryl ester. We previously showed that remnants, produced when human HDL(2) is catabolized in mice overexpressing SR-BI, become incrementally smaller, ultimately consisting of small alpha-migrating particles, distinct from pre-beta HDL. When mixed with mouse plasma, some remnant particles rapidly increase in size by associating with HDL without the mediation of cholesteryl ester transfer protein, LCAT, or phospholipid transfer protein. Here, we show that processing of HDL(2) by SR-BI-overexpressing mice resulted in the preferential loss of apolipoprotein A-II (apoA-II). Short-term processing generated two distinct, small alpha-migrating particles. One particle (8.0 nm diameter) contained apoA-I and apoA-II; the other particle (7.7 nm diameter) contained only apoA-I. With extensive SR-BI processing, only the 7.7 nm particle remained. Only the 8.0 nm remnants were able to associate with HDL. Compared with HDL(2), this remnant was more readily taken up by the liver than by the kidney. We conclude that SR-BI-generated HDL remnants consist of particles with or without apoA-II and that only those containing apoA-II associate with HDL in an enzyme-independent manner. Extensive SR-BI processing generates small apoA-II-depleted particles unable to reassociate with HDL and readily taken up by the liver. This represents a pathway by which apoA-I and apoA-II catabolism are segregated.     

Scavenger receptor BI plays a role in facilitating chylomicron metabolism

The function of scavenger receptor class B type I (SR-BI) in mediating the selective uptake of high density lipoprotein (HDL) cholesterol esters is well established. However, the potential role of SR-BI in chylomicron and chylomicron remnant metabolism is largely unknown. In the present investigation, we report that the cell association of 160 nm-sized triglyceride-rich chylomicron-like emulsion particles to freshly isolated hepatocytes from SR-BI-deficient mice is greatly reduced (>70%), as compared with wild-type littermate mice. Competition experiments show that the association of emulsion particles with isolated hepatocytes is efficiently competed for (>70%) by the well established SR-BI ligands, HDL and oxidized low density lipoprotein (LDL), whereas LDL is ineffective. Upon injection into SR-BI-deficient mice the hepatic association of emulsion particles is markedly decreased ( approximately 80%) as compared with wild-type mice. The relevance of these findings for in vivo chylomicron (remnant) metabolism was further evaluated by studying the effect of SR-BI deficiency on the intragastric fat load-induced postprandial triglyceride response. The postprandial triglyceride response is 2-fold higher in SR-BI-deficient mice as compared with wild-type littermates (area-under-the-curve 39.6 +/- 1.2 versus 21.1 +/- 3.6; p < 0.005), with a 4-fold increased accumulation of chylomicron (remnant)-associated triglycerides in plasma at 6 h after intragastric fat load. We conclude that SR-BI is important in facilitating chylomicron (remnant) metabolism and might function as an initial recognition site for chylomicron remnants whereby the subsequent internalization can be exerted by additional receptor systems like the LDL receptor and LDL receptor-related protein.     

Low-density lipoproteins modified by lipid transfer protein have altered biological activity

Low-density lipoproteins (LDL) were modified by incubation with very-low-density lipoproteins (VLDL) and lipid transfer protein(s) to yield LDL particles that were enriched in triacylglycerol, depleted in cholesteryl esters, and contained apolipoprotein C. The uptake and degradation of these 125I-labeled modified LDL particles by cultured skin fibroblasts was reduced by approx. 30% when compared with LDL that had not been exposed to lipid transfer protein. Incubation of fibroblasts for 24 h in the presence of modified LDL resulted in less inhibition of LDL receptor activity and sterol synthesis than did incubation with control LDL. Both the degradation of 125I-labeled modified LDL and the effect of unlabeled modified LDL on the regulation of LDL binding and sterol synthesis were progressively decreased as the extent of modification of the LDL was increased. Even when identical amounts of modified LDL or control LDL protein were degraded, less inhibition of LDL receptor activity and sterol synthesis was observed with modified LDL than with control LDL, suggesting that the effects of modified LDL on these regulatory events are related to both the reduced degradation of the modified lipoprotein particles and to the alteration in its chemical composition. Uptake and degradation of modified LDL by human monocyte-derived macrophages in culture was reduced in a manner similar to that observed in the cultured fibroblasts, and was considerably less than that observed with acetylated LDL. No differences were observed between modified LDL prepared by exposure to lipid transfer activity in the lipoprotein deficient fraction of serum or when partially purified lipid transfer was used. Modified LDL, with similar composition to that used in the experiments, has been observed in certain diabetic and non-diabetic hypertriglyceridemic states. Thus, it is possible that the cellular metabolism of LDL in vivo might be altered in the presence of hypertriglyceridemia.     

Plasmin-mediated macrophage reversal of low density lipoprotein aggregation

Evidence suggests that aggregated low density lipoprotein (AgLDL) accumulates in atherosclerotic lesions. Previously, we showed that AgLDL induces and enters surface-connected compartments (SCC) in human monocyte-derived macrophages by a process we have named patocytosis. Most AgLDL taken up by these macrophages in the absence of serum is stored in SCC and remains undegraded. We now show that macrophages released AgLDL (prepared by vortexing or treatment with phospholipase C or sphingomyelinase) from their SCC when exposed to 10% human lipoprotein-deficient serum (LPDS). Macrophages also took up AgLDL in the presence of LPDS, but subsequently released it. In both cases, the released AgLDL was disaggregated. Although the AgLDL that macrophages took up could not pass through a 0.45-micrometer filter, >60% of AgLDL could pass this filter after release from the macrophages. Disaggregation of AgLDL was verified by gel-filtration chromatography and electron microscopy that also showed particles larger than LDL, reflecting fusion of LDL that aggregates. The factor in serum that mediated AgLDL release and disaggregation was plasmin generated from plasminogen by macrophage urokinase plasminogen activator. AgLDL release was decreased >90% by inhibitors of plasmin (epsilon-amino caproic acid and anti-plasminogen mAb), and also by inhibitors of urokinase plasminogen activator (plasminogen activator inhibitor-1 and anti-urokinase plasminogen activator mAb). Moreover, plasminogen could substitute for LPDS and produce similar macrophage release and disaggregation of AgLDL. Because only plasmin bound to the macrophage surface is protected from serum plasmin inhibitors, interaction of AgLDL with macrophages was necessary for reversal of its aggregation by LPDS. The released disaggregated LDL particles were competent to stimulate LDL receptor-mediated endocytosis in cultured fibroblasts. Macrophage-mediated disaggregation of aggregated and fused LDL is a mechanism for transforming LDL into lipoprotein structures size-consistent with lipid particles found in atherosclerotic lesions.     

Remnant lipoproteins induced proliferation of human prostate cancer cell,PC-3 but not LNCaP,via low density lipoprotein receptor

Background: Hypertriglyceridemia has been shown to be one of the risk factors for prostate cancer. In this study, we investigated the effect of remnant lipoproteins on cell growth in prostate cancer cell lines. Methods: Remnant lipoproteins were isolated as remnant like particles (RLP) from human plasma. We used RLP for TG-rich lipoproteins and low density lipoproteins (LDL) for cholesterol-rich lipoproteins respectively and examined the effect of lipoproteins on proliferation of PC-3 and LNCaP cells using MTS assays. Moreover, we studied the effect of RLP and LDL treatment on the regulation of lipoprotein receptors in prostate cancer cells to investigate the relationship between lipoprotein-induced cell proliferation and lipoprotein receptor expression using real-time PCR, Western blotting assays and siRNA. Results: RLP effectively induced PC-3 cell proliferation more than LDL, whereas both RLP and LDL could not induce LNCaP cell proliferation except at a higher concentration of RLP. LDL receptor (LDLr) was expressed in both prostate cancer cells but there was a sharp difference of sterol regulation between two cells. In PC-3 cells, LDL decreased the LDLr expression in some degree, but RLP did not. Meanwhile LDLr expression in LNCaP was easily downregulated by RLP and LDL. Blocking LDLr function significantly inhibited both RLP- and LDL-induced PC-3 cell proliferation. Conclusions: This study demonstrated that RLP-induced PC-3 cell proliferation more than LDL; however, both RLP and LDL hardly induced LNCaP cell proliferation. The differences of proliferation by lipoproteins might be involved in the regulation of LDLr expression.     

Binding of rat chylomicrons and their remnants to the hepatic low-density-lipoprotein receptor and its role in remnant removal.

Binding and uptake of rat chylomicrons of different metabolic stages by the hepatic low-density-lipoprotein (LDL) receptor were studied. Pure chylomicrons, characterized by apolipoprotein B-48 devoid of contaminating B-100, were labelled in their cholesteryl esters. Lymph chylomicrons and serum chylomicrons, enriched in apolipoprotein E and the C-apolipoproteins, bound poorly to rat hepatic membranes. In contrast, chylomicron remnants, containing the apolipoproteins B-48 and E, bound with high affinity. Specific binding of remnants was virtually completely competed for by LDL free of apolipoprotein E. In addition, in ligand blots both remnants and LDL associated with the same protein with an Mr characteristic of the LDL receptor. Uptake of remnants during a single pass through isolated perfused rat livers was decreased to about 50% by an excess of LDL. It is concluded that rat chylomicron remnants are a ligand of the hepatic LDL receptor. The much higher affinity as compared with LDL is mediated by apolipoprotein E but not B-48, and is inhibited by the C-apolipoproteins. This explains why serum chylomicrons are not taken up by the liver, whereas remnants are rapidly removed from the circulation. Results from experiments in vivo suggest that the LDL receptor makes an important contribution to the hepatic uptake of remnants and may be the principal binding site of the liver responsible for remnant removal.     

Role of low density lipoprotein receptor-dependent and -independent sites in binding and uptake of chylomicron remnants in rat liver

The role of the low density lipoprotein (LDL) receptor in the binding of chylomicron remnants to liver membranes and in their uptake by hepatocytes was assessed using a monospecific polyclonal antibody to the LDL receptor of the rat liver. The anti-LDL receptor antibody inhibited the binding and uptake of chylomicron remnants and LDL by the poorly differentiated rat hepatoma cell HTC 7288C as completely as did unlabeled lipoproteins. The antireceptor antibody, however, decreased binding of chylomicron remnants to liver membranes from normal rats by only about 10%. This was true for intact membranes and for solubilized reconstituted membranes and with both a crude membrane fraction as well as with purified sinusoidal membranes. Further, complete removal of the LDL receptor from solubilized membranes by immunoprecipitation with antireceptor antibody only decreased remnant binding to the reconstituted supernatant by 10% compared to solubilized, nonimmunoprecipitated membranes. Treatment of rats with ethinyl estradiol induced an increase in remnant binding by liver membranes. All of the increased binding could be inhibited by the antireceptor antibody. The LDL receptor-independent remnant binding site was not EDTA sensitive and was not affected by ethinyl estradiol treatment. LDL receptor-independent remnant binding was competed for by beta-VLDL = HDLc greater than rat LDL greater than human LDL (where VLDL is very low density lipoprotein, and HDL is high density lipoprotein). There was weak and incomplete competition by apoE-free HDL, probably due to removal of apoE from the remnant. The LDL receptor-independent remnant-binding site was also present in membranes prepared from isolated hepatocytes and had the same characteristics as the site on membranes prepared from whole liver. In contrast, when chylomicron remnants were incubated with a primary culture of rat hepatocytes, the anti-LDL receptor antibody prevented specific cell association by 84% and degradation of chylomicron remnants completely. Based on these studies, we conclude that although binding of chylomicron remnants to liver cell membranes is not dependent on the LDL receptor, their intact uptake by hepatocytes is.     

Incorporation of lycopene into chylomicron remnant-like particles inhibits their uptake by HepG2 cells

The influence of the incorporation of the antioxidant tomato pigment, lycopene, into chylomicron remnant-like particles (CRLPs) on their uptake by the liver cells was investigated. CRLPs or CRLPs containing lycopene (lycCRLPs) radiolabelled with [³H]triacylglycerol were incubated with cells of the human liver hepatoma cell line, HepG2, and the radioactivity taken up by the cells was determined. LycCRLPs were taken up significantly more slowly than CRLPs over a concentration range of 5-60 μg cholesterol/ml and a time course of 2-6 h. Pre-incubation of the hepatocytes with an excess of low density lipoprotein (LDL) inhibited the uptake of CRLPs by about 50%, but had no effect on the uptake of lycCRLPs, and under these conditions the CRLPs and lycCRLPs were taken up at similar rates. In HepG2 cells pre-treated with suramin, which inhibits uptake via the LDL receptor-related protein (LRP), the uptake of CRLPs was also inhibited (− 37%) to a greater extent than that of lycCRLPs (− 24%), so that the values for the two types of particle were no longer significantly different. Heparinase increased the uptake of lycCRLPs (about 2 fold), but not CRLPs, bringing it to a level equivalent to that seen with the control particles. These findings demonstrate that the incorporation of lycopene into CRLPs decreases their uptake by HepG2 cells and suggest that this effect is due to differential interaction with the LDL receptor and the LRP-receptor-mediated pathways, and may also involve binding of the particles to HSPG.     

The hepatic uptake of VLDL in lrp-ldlr-/-vldlr-/- mice is regulated by LPL activity and involves proteoglycans and SR-BI

LPL activity plays an important role in preceding the VLDL remnant clearance via the three major apolipoprotein E (apoE)-recognizing receptors: the LDL receptor (LDLr), LDL receptor-related protein (LRP), and VLDL receptor (VLDLr). The aim of this study was to determine whether LPL activity is also important for VLDL remnant clearance irrespective of these receptors and to determine the mechanisms involved in the hepatic remnant uptake. Administration of an adenovirus expressing LPL (AdLPL) into lrp(-)ldlr(-/-)vldlr(-/-) mice reduced both VLDL-triglyceride (TG) and VLDL-total cholesterol (TC) levels. Conversely, inhibition of LPL by AdAPOC1 increased plasma VLDL-TG and VLDL-TC levels. Metabolic studies with radiolabeled VLDL-like emulsion particles showed that the clearance and hepatic association of their remnants positively correlated with LPL activity. This hepatic association was independent of the bridging function of LPL and HL, since heparin did not reduce the liver association. In vitro studies demonstrated that VLDL-like emulsion particles avidly bound to the cell surface of primary hepatocytes from lrp(-)ldlr(-/-)vldlr(-/-) mice, followed by slow internalization, and involved heparin-releaseable cell surface proteins as well as scavenger receptor class B type I (SR-BI). Collectively, we conclude that hepatic VLDL remnant uptake in the absence of the three classical apoE-recognizing receptors is regulated by LPL activity and involves heparan sulfate proteoglycans and SR-BI.     

Receptor binding activity of lipid recombinants of apolipoprotein B-100 thrombolytic fragments

Apolipoprotein (apo) B-100, the protein constituent of low density lipoproteins (LDL), is the determinant responsible for LDL binding to the apoB,E(LDL) receptor on cells. The current study was designed to identify the region(s) of apoB-100 that interact with the apoB,E(LDL) receptor. Apolipoprotein B-100 was fragmented by thrombin digestion, and the isolated fragments (T2, T3, T4) were recombined with cholesterol-induced canine high density lipoproteins (HDLc). Before the recombination, the receptor binding activity of apoE of the HDLc was abolished by reductive methylation and extensive trypsin treatment. This treatment permitted almost complete replacement of the small residual apoE fragments by the large apoB fragments. Recombinant apoB particles were isolated by ultracentrifugation and tested for binding to receptors on cultured human fibroblasts. The recombinant particles had chemical and physical properties similar to those of native HDLc. Recombinants of both the whole thrombolytic digest and of isolated fragments displayed specific binding to the apoB,E (LDL) receptor. Anti-apoB,E(LDL) receptor antibodies abolished 90% of the binding, and there was almost no specific binding to receptor-negative fibroblasts or to cells in which the receptors had been down-regulated. The binding of apoB-100 recombinants to the receptor also demonstrated calcium dependency; in addition, the surface binding of the recombinants was released by polyanionic compounds. All these recombinants had binding affinities comparable to one another but less than that of native LDL. Although T2, T3 and T4 recombinants can all bind specifically to the apoB,E(LDL) receptor, it remains to be established whether their activity represents physiologically relevant binding. Nevertheless, the present findings illustrate the potential of the recombinant method using HDLc lipids to reconstitute biological activity.     

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.     

Sphingomyelinase enhances low density lipoprotein uptake and ability to induce cholesteryl ester accumulation in macrophages.

Cholesteryl ester-loaded macrophages, or foam cells, are a prominent feature of atherosclerotic lesions. Low density lipoprotein (LDL) receptor-mediated endocytosis of native LDL is a relatively poor inducer of macrophage cholesteryl ester accumulation. However, the data herein show that in the presence of a very small amount of sphingomyelinase, LDL receptor-mediated endocytosis of 125I-LDL was enhanced and led to a 2-6-fold increase in 125I-LDL degradation and up to a 10-fold increase in cholesteryl ester accumulation in macrophages. The enhanced lipoprotein uptake and cholesterol esterification was seen after only approximately 12% hydrolysis of LDL phospholipids, was specific for sphingomyelin hydrolysis, and appeared to be related to the formation of fused or aggregated spherical particles up to 100 nm in diameter. Sphingomyelinase-treated LDL was bound by the macrophage LDL receptor. However, when unlabeled acetyl-LDL, a scavenger receptor ligand, was present during or after sphingomyelinase treatment of 125I-LDL, 125I-LDL binding and degradation were enhanced further through the formation of LDL-acetyl-LDL mixed aggregates. Experiments with cytochalasin D suggested that endocytosis, not phagocytosis, was involved in internalization of sphingomyelinase-treated LDL. Nonetheless, the sphingomyelinase effect on LDL uptake was macrophage-specific. These data illustrate that LDL receptor-mediated endocytosis of fused LDL particles can lead to foam cell formation in cultured macrophages. Furthermore, since both LDL and sphingomyelinase are present in atherosclerotic lesions and since some lesion LDL probably is fused or aggregated, there is a possibility that sphingomyelinase-treated LDL is a physiologically important atherogenic lipoprotein.     

LDL uptake by Leishmania amazonensis: involvement of membrane lipid microdomains

Leishmania amazonensis lacks a de novo mechanism for cholesterol synthesis and therefore must scavenge this lipid from the host environment. In this study we show that the L. amazonensis takes up and metabolizes human LDL(1) particles in both a time and dose-dependent manner. This mechanism implies the presence of a true LDL receptor because the uptake is blocked by both low temperature and by the excess of non-labelled LDL. This receptor is probably associated with specific microdomains in the membrane of the parasite, such as rafts, because this process is blocked by methyl-β-cyclodextrin (MCBD). Cholesteryl ester fluorescently-labeled LDL (BODIPY-cholesteryl-LDL) was used to follow the intracellular distribution of this lipid. After uptake it was localized in large compartments along the parasite body. The accumulation of LDL was analyzed by flow cytometry using FITC-labeled LDL particles. Together these data show for the first time that L. amazonensis is able to compensate for its lack of lipid synthesis through the use of a lipid importing machinery largely based on the uptake of LDL particles from the host. Understanding the details of the molecular events involved in this mechanism may lead to the identification of novel targets to block Leishmania infection in human hosts.     

The beta-VLDL receptor pathway of murine P388D1 macrophages

Very low density lipoproteins Sf 100-400 (VLDL1) from hypertriglyceridemic (HTG) subjects and chylomicrons cause receptor-mediated lipid engorgement in unstimulated macrophages in vitro via the beta-VLDL receptor pathway. We now report that the murine macrophage P388D1 cell line possesses the characteristics of the beta-VLDL receptor pathway observed previously in freshly isolated resident murine peritoneal macrophages or human monocyte-macrophages. HTG-VLDL1 isolated from the plasma of subjects with hypertriglyceridemia types 3, 4, and 5 interact with P388D1 macrophages in a high-affinity, curvilinear manner. beta-VLDL, HTG-VLDL1, chylomicrons, and thrombin-treated HTG-VLDL1 (which do not bind to the LDL receptor) compete efficiently and similarly for the uptake and degradation of HTG-VLDL1. LDL and acetyl LDL do not compete, indicating that uptake of HTG-VLDL1 is via neither the LDL receptor nor the acetyl LDL receptor. Binding of thrombin-treated HTG-VLDL1 to the beta-VLDL receptor indicates that the thrombin-accessible apoE, which is absolutely required for interaction of HTG-VLDL Sf greater than 60 with the LDL receptor, is not required for binding to the beta-VLDL receptor. The uptake and degradation of 125I-labeled HTG-VLDL1 is suppressed up to 80-90% by preincubation of the cells with sterols, acetyl LDL, or beta-VLDL, indicating that this process is not via the irrepressible chylomicron remnant (apoE) receptor. Chylomicrons, HTG-VLDL1, and thrombin-treated HTG-VLDL1-but not normal VLDL1, beta-VLDL, LDL, or acetyl LDL-produce massive triglyceride accumulation (10-20-fold mass increases in 4 hr) in P388D1 macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphological studies on the binding of low-density lipoproteins and acetylated low-density lipoproteins to the plasma membrane of cultured monocytes

Binding of low density lipoproteins (LDL) and acetyl-LDL to the plasma membrane of cultured swine monocytes was investigated by immunofluorescent and immunoelectron microscopy. Binding sites for native LDL, visualized on both the light microscopical and the ultrastructural level, were found to be comparable to those of cultured human fibroblasts. These techniques, however, failed to reveal binding of acetyl-LDL to the cell surface. Biochemical experiments showed that both LDL and acetyl-LDL have specific receptors, the acetyl-LDL receptor being distinctly different from the LDL receptor. It is concluded that there are morphological differences in the binding of LDL and acetyl-LDL to cultured monocytes. These differences are supported by biochemical data.