Clearance of chylomicron remnants by the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor.

The involvement of the low density lipoprotein receptor-related protein (LRP) in chylomicron remnant (CR) catabolism was investigated. Ligand blot analyses demonstrated that beta-very low density lipoproteins (beta-VLDL) incubated with apolipoprotein E (beta-VLDL+E) bound to the LRP and low density lipoprotein receptors, whereas active (receptor-binding) alpha 2-macroglobulin (alpha 2M) bound only to LRP partially purified from rat liver membranes. Iodinated beta-VLDL+E and active alpha 2M showed high affinity binding to the LRP/alpha 2M receptor of low density lipoprotein receptor-negative fibroblasts. The binding and degradation of radiolabeled alpha 2M by these cells were partially inhibited by beta-VLDL+E. Furthermore, alpha 2M interfered with the internalization of beta-VLDL+E and subsequent induction in the cholesterol esterification by these cells. These studies suggested that remnant lipoproteins and active alpha 2M compete for binding to the LRP/alpha 2M receptor. Next, we examined whether the LRP/alpha 2M receptor plays a role, in the presence of low density lipoprotein receptors, in the in vivo catabolism of CR in mice. In vivo studies demonstrated that the unlabeled active, but not the native, alpha 2M partially inhibited the plasma clearance and hepatic uptake of radiolabeled CR or apoE-enriched radiolabled CR. Likewise, apoE-enriched CR retarded the plasma clearance and hepatic uptake of radiolabeled active alpha 2M. These studies provide physiological evidence that the LRP/alpha 2M receptor may function as a CR receptor that removes CR from the plasma.     

Elimination of cholesterol ester from macrophage foam cells by adenovirus-mediated gene transfer of hormone-sensitive lipase

Cholesterol ester (CE)-laden foam cells are a hallmark of atherosclerosis. To determine whether stimulation of the hydrolysis of cytosolic CE can be used as a novel therapeutic modality of atherosclerosis, we overexpressed hormone-sensitive lipase (HSL) in THP-1 macrophage-like cells by adenovirus-mediated gene delivery, and we examined its effects on the cellular cholesterol trafficking. We show here that the overexpression of HSL robustly increased neutral CE hydrolase activity and completely eliminated CE in the cells that had been preloaded with CE by incubation with acetylated low density lipoprotein. In these cells, cholesterol efflux was stimulated in the absence or presence of high density lipoproteins, which might be at least partially explained by the increase in the expression of ABCA1. Importantly, these effects were achieved without the addition of acyl-CoA:cholesterol acyltransferase inhibitor, cAMP, or even high density lipoproteins. Furthermore, the uptake and degradation of acetylated low density lipoprotein was significantly reduced probably by decreased expression of scavenger receptor A and CD36. Notably, the cells with stimulated CE hydrolysis did not exhibit either buildup of free cholesterol or cytotoxicity. In conclusion, increased hydrolysis of CE by the overexpression of HSL leads to complete elimination of CE from THP-1 foam cells not only by increasing efflux but also by decreasing influx of cholesterol.     

Characteristics of chylomicron binding and lipid uptake by endothelial cells in culture.

Bovine vascular endothelial cells bind chylomicrons via a high affinity membrane receptor site. Subsequent to binding, the chylomicron apoprotein was neither internalized nor degraded by either sparse or confluent (contact-inhibited) cells. However, the adsorption of chylomicrons was associated with interiorization of chylomicron cholesteryl ester and triglyceride and the hydrolysis of these lipids to free cholesterol and unesterified fatty acids by a lysosome-dependent pathway. This pathway was active in both subconfluent and contact-inhibited cells. The chylomicron free cholesterol so produced inhibited endogeneous cholesterol synthesis measured in terms of the incorporation of [1-14C]-acetate into sterol. An excess of high density lipoprotein was 2- to 3-fold more effective in reducing both binding of chylomicrons and interiorization of chylomicron lipid than was low density lipoprotein. Chylomicron binding was not "down-regulated" by preincubation of the cells with low density lipoprotein or chylomicrons. The results are discussed in the context of cholesterol sources for contact-inhibited endothelial cells which do not interiorize low density lipoprotein cholesterol.     

Macrophage colony-stimulating factor rapidly enhances beta-migrating very low density lipoprotein metabolism in macrophages through activation of a Gi/o protein signaling pathway

Previous studies have examined lipoprotein metabolism by macrophages following prolonged exposure (>24 h) to macrophage colony-stimulating factor (M-CSF). Because M-CSF activates several signaling pathways that could rapidly affect lipoprotein metabolism, we examined whether acute exposure of macrophages to M-CSF alters the metabolism of either native or modified lipoproteins. Acute incubation of cultured J774 macrophages and resident mouse peritoneal macrophages with M-CSF markedly enhanced low density lipoproteins (LDL) and beta-migrating very low density lipoproteins (beta-VLDL) stimulated cholesteryl [(3)H]oleate deposition. In parallel, M-CSF treatment increased the association and degradation of (125)I-labeled LDL or beta-VLDL without altering the amount of lipoprotein bound to the cell surface. The increase in LDL and beta-VLDL metabolism did not reflect a generalized effect on lipoprotein endocytosis and metabolism because M-CSF did not alter cholesterol deposition during incubation with acetylated LDL. Moreover, M-CSF did not augment beta-VLDL cholesterol deposition in macrophages from LDL receptor (-/-) mice, indicating that the effect of M-CSF was mediated by the LDL receptor. Incubation of macrophages with pertussis toxin, a specific inhibitor of G(i/o) protein signaling, had no effect on cholesterol deposition during incubation with beta-VLDL alone, but completely blocked the augmented response promoted by M-CSF. In addition, incubation of macrophages with the direct G(i/o) protein activator, mastoparan, mimicked the effect of M-CSF by enhancing cholesterol deposition in cells incubated with beta-VLDL, but not acetylated LDL. In summary, M-CSF rapidly enhances LDL receptor-mediated metabolism of native lipoproteins by macrophages through activation of a G(i/o) protein signaling pathway. Together, these findings describe a novel pathway for regulating lipoprotein metabolism.     

Identification and characterization of a high density lipoprotein-binding protein in cell membranes by ligand blotting

Cholesterol efflux from cultured cells can be mediated through binding of high density lipoprotein (HDL) to a cell-surface site which shows many characteristics of a biological receptor. To determine whether a specific protein forms a component of this site, cell membrane proteins were analyzed by ligand blotting using 125I-HDL3. Results demonstrated that membranes from a number of cell types possess a protein with an apparent molecular mass of 110 kDa that binds HDL and apoA-I and apoA-II proteoliposomes, but not low density lipoprotein, acetylated low density lipoprotein, or apoE proteoliposomes. The binding activity of this protein was increased by loading cells with cholesterol and was abolished by trypsin treatment of intact cell monolayers. These results suggest that HDL binds with specificity to a cell-surface protein which is regulated by intracellular cholesterol levels. Since HDL binding to intact cell monolayers shows the same characteristics, the 110-kDa binding protein may represent the proposed HDL receptor that functions to facilitate transport of cholesterol from cells to HDL particles.     

A prospective mechanism and source of cholesterol uptake by Plasmodium falciparum-infected erythrocytes co-cultured with HepG2 cells

Plasmodium falciparum (P. falciparum) parasites still cause lethal infections worldwide, especially in Africa (https://www.who.int/publications/i/item/world-malaria-report-2019). During P. falciparum blood-stage infections in humans, low-density lipoprotein, high-density lipoprotein and cholesterol levels in the blood become low. Because P. falciparum lacks a de novo cholesterol synthesis pathway, it must import cholesterol from the surrounding environment. However, the origin of the cholesterol and how it is taken up by the parasite across the multiple membranes that surround it is not fully understood. To answer this, we used a cholesterol synthesis inhibiter (simvastatin), a cholesterol transport inhibitor (ezetimibe), and an activating ligand of the peroxisome proliferator-activated receptor α, called ciprofibrate, to investigate the effects of these agents on the intraerythrocytic growth of P. falciparum, both with and without HepG2 cells as the lipoprotein feeders. P. falciparum growth was inhibited in the presence of ezetimibe, but ezetimibe was not very effective at inhibiting P. falciparum growth when used in the co-culture system, unlike simvastatin, which strongly promoted parasite growth in this system. Ezetimibe is known to inhibit cholesterol absorption by blocking the activity of Niemann-Pick C1 like 1 (NPC1L1) protein, and simvastatin is known to enhance NPC1L1 expression in the human body's small intestine. Collectively, our results support the possibility that cholesterol import by P. falciparum involves hepatocytes, and cholesterol uptake into the parasite occurs via NPC1L1 protein or an NPC1L1 homolog during the erythrocytic stages of the P. falciparum lifecycle.     

Macrophages take up triacylglycerol-rich emulsions at a faster rate upon co-incubation with native and modified LDL: An investigation on the role of natural chylomicrons in atherosclerosis.

Chylomicrons play a role in atherosclerosis, however, because the mechanisms involved in the cell uptake of these particles are not fully understood, investigations were carried out using a radioactively labeled protein-free triacylglycerol-rich emulsion incubated with peritoneal macrophages obtained from normal and apoE-knockout mice. Experiments were done in the presence of substances that inhibit several endocytic processes: EDTA for low density lipoprotein receptor, fucoidan for scavenger receptor, cytochalasin B for phagocytosis, and a lipopolysaccharide for lipoprotein lipase. In addition, triacylglycerol-rich emulsions were also prepared in the presence of native or modified radioactively labeled low density lipoprotein particles that are known to accumulate in the arterial intima. Probucol was also used to prevent the possible role played by an antioxidant in triacylglycerol-rich emulsion uptake. We have shown that triacylglycerol-rich emulsion alone is taken up by a coated-pit-dependent mechanism, mediated by macrophage secretion of apolipoprotein E. Furthermore, native, aggregated, acetylated, and moderately macrophage-oxidized low density lipoprotein stimulate the uptake of a triacylglycerol-rich emulsion through several mechanisms such as an actin-dependent pathway, scavenger receptors, and lipolysis mediated by lipoprotein lipase. On the other hand, in spite of the interaction of low density lipoprotein forms with a triacylglycerol-rich emulsion, the cellular triacylglycerol-rich emulsion uptake is impaired by copper-oxidized low density lipoprotein, possibly due to its diminished affinity towards lipoprotein lipase. We have also shown that macrophages take up aggregated low density lipoprotein better than the acetylated or oxidized forms of low density lipoprotein.     

Murine macrophage tumors are a source of a 260,000-dalton acetyl-low density lipoprotein receptor

Subcutaneous injection of murine macrophage cell line P388D1 into syngeneic DBA/2 produced tumors, which upon solubilization with 40 mM octyl glucoside contained acetylated low density lipoprotein binding activity. The tumor-derived receptor specifically bound acetylated low density lipoprotein with an affinity of approximately 3 X 10(-8) M but did not bind low density lipoprotein or high density lipoprotein. It was identical in binding specificity, affinity, and Pronase sensitivity to the receptor in intact cells or that obtained from solubilized cultured cell membranes. Partial purification of the receptor was achieved by solubilizing tumors with 1% Triton X-100 followed by chromatography on polyethyleneimine cellulose. After elution with a NaCl gradient in the presence of octyl glucoside and association with liposomes, a 287-fold purification of the receptor was achieved. The receptor was identified by specific ligand blotting as a 260,000-dalton protein having a pI of approximately 6.0. Binding to the receptor by acetylated low density lipoprotein, malondialdehyde-modified low density lipoprotein, and maleic anhydride-modified serum albumin was demonstrated by ligand blotting. A single receptor protein can, therefore, account for the binding of multiple types of charge-modified lipoprotein and nonlipoprotein ligands to the macrophage cell surface.     

Macrophage free cholesterol content regulates apolipoprotein E synthesis

The relationship between macrophage cholesterol content and apolipoprotein E (apoE) synthesis was studied in mouse peritoneal macrophages. Incubations in acetylated low density lipoprotein led to a concentration-dependent increase in macrophage free and esterified cholesterol content and apoE synthesis. Enhanced apoE production reflected increased apoE mRNA abundance in cholesterol-enriched cells. Including an inhibitor of acyl-CoA:cholesterol acyltransferase in incubations with acetylated low density lipoprotein did not diminish the apoE response, suggesting that increased macrophage free cholesterol content was responsible for enhancing apoE production. Incubations in 25-OH cholesterol also produced a dose-dependent stimulation of macrophage apoE synthesis. Removing free cholesterol from cells using high density lipoprotein returned apoE synthetic rates toward base line. Macrophage lysate apoE and medium apoE levels changed in parallel during cholesterol loading and efflux indicating that regulation of apoE by free cholesterol was not primarily at the level of secretion. It is concluded that (a) cholesterol enrichment of macrophages increases apoE mRNA abundance and stimulates apoE synthesis and secretion; (b) neither cholesterol esterification nor cholesteryl ester accumulation are required for increased apoE production.     

Provision of cholesterol to lymphocytes by high density and low density lipoproteins. Requirement for low density lipoprotein receptors

The capacity of lipoprotein fractions to provide cholesterol necessary for human lymphocyte proliferation was examined. When endogenous synthesis of cholesterol was blocked, proliferation of mitogen-stimulated normal human lymphocytes was markedly inhibited unless an exogenous source of sterol was supplied. All lipoprotein fractions with the exception of high density lipoprotein subclass 3 were able to provide cholesterol for lymphocyte proliferation. Each of the lipoprotein subfractions capable of providing cholesterol was also able to regulate endogenous sterol synthesis in cultured human lymphocytes. Provision of cholesterol by lipoproteins required the interaction of apolipoprotein B or apolipoprotein E with specific receptors on normal lymphocytes. Apolipoprotein modification by acetylation or methylation, which markedly reduced the ability to regulate sterol biosynthesis, also diminished the capacity of lipoproteins to provide cholesterol. In addition, depletion of apolipoprotein B- and apolipoprotein E-containing particles from high density lipoprotein decreased its ability to suppress cholesterol synthesis and prevented it from providing cholesterol to proliferating lymphocytes. Monoclonal antibodies directed against the receptor-recognition sites on apolipoprotein B and apolipoprotein E were used to define the specific apolipoproteins required for the provision of cholesterol to lymphocytes by the various lipoprotein fractions. The antibody to apolipoprotein B inhibited cholesterol provision by both low density lipoprotein (LDL) and other lipoprotein fractions. The antibody to apolipoprotein E did not decrease provision of cholesterol by LDL but did inhibit the capacity of other fractions to provide cholesterol. In addition, a monoclonal antibody against the ligand binding site on the LDL receptor inhibited provision of cholesterol to normal lymphocytes by all lipoproteins. Finally, lymphocytes lacking LDL receptors were unable to obtain cholesterol from any lipoprotein fraction. These studies demonstrate that LDL receptor-mediated interaction with apolipoprotein B or apolipoprotein E is essential for the provision of cholesterol to normal human lymphocytes from all lipoprotein sources.     

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.     

The apolipoprotein E-dependent low density lipoprotein cholesteryl ester selective uptake pathway in murine adrenocortical cells involves chondroitin sulfate proteoglycans and an alpha 2-macroglobulin receptor

Cells acquire lipoprotein cholesterol by receptor-mediated endocytosis and selective uptake pathways. In the latter case, lipoprotein cholesteryl ester (CE) is transferred to the plasma membrane without endocytosis and degradation of the lipoprotein particle. Previous studies with Y1/E/tet/2/3 murine adrenocortical cells that were engineered to express apolipoprotein (apo) E demonstrated that apoE expression enhances low density lipoprotein (LDL) CE uptake by both selective and endocytic pathways. The present experiments test the hypothesis that apoE-dependent LDL CE selective uptake is mediated by scavenger receptor, class B, type I (SR-BI). Surprisingly, SR-BI expression was not detected in the Y1/E/tet/2/3 clone of Y1 adrenocortical cells, indicating the presence of a distinct apoE-dependent pathway for LDL CE selective uptake. ApoE-dependent LDL CE selective uptake in Y1/E/tet/2/3 cells was inhibited by receptor-associated protein and by activated alpha(2)-macroglobulin (alpha(2)M), suggesting the participation of the LDL receptor-related protein/alpha(2)M receptor. Reagents that inhibited proteoglycan synthesis or removed cell surface chondroitin sulfate proteoglycan completely blocked apoE-dependent LDL CE selective uptake. None of these reagents inhibited SR-BI-mediated LDL CE selective uptake in the Y1-BS1 clone of Y1 cells in which LDL CE selective uptake is mediated by SR-BI. We conclude that LDL CE selective uptake in adrenocortical cells occurs via SR-BI-independent and SR-BI-dependent pathways. The SR-BI-independent pathway is an apoE-dependent process that involves both chondroitin sulfate proteoglycans and an alpha(2)M receptor.     

Scavenger receptor class B type I-mediated reverse cholesterol transport is inhibited by advanced glycation end products

Cellular interactions of advanced glycation end products (AGE) are mediated by AGE receptors. We demonstrated previously that class A scavenger receptor types I and II (SR-A) and CD36, a member of class B scavenger receptor family, serve as the AGE receptors. In this study, we investigated whether scavenger receptor class B type I (SR-BI), another receptor belonging to class B scavenger receptor family, was also an AGE receptor. We used Chinese hamster ovary (CHO) cells overexpressed hamster SR-BI (CHO-SR-BI cells). (125)I-AGE-bovine serum albumin (AGE-BSA) was endocytosed in a dose-dependent fashion and underwent lysosomal degradation by CHO-SR-BI cells. (125)I-AGE-BSA exhibited saturable binding to CHO-SR-BI cells (K(d) = 8.3 microg/ml). Endocytic uptake of (125)I-AGE-BSA by CHO-SR-BI cells was completely inhibited by oxidized low density lipoprotein (LDL) and acetylated LDL, whereas LDL exerted only a weak inhibitory effect (<20%). Cross-competition experiments showed that AGE-BSA had no effect on HDL binding to these cells and vice versa. Interestingly, however, SR-BI-mediated selective uptake of HDL-CE was completely inhibited by AGE-BSA in a dose-dependent manner (IC(50) <10 microg/ml). Furthermore, AGE-BSA partially inhibited (by <30%) the selective uptake of HDL-CE in human hepatocarcinoma HepG2 cells (IC(50) <30 microg/ml). In addition, [(3)H]cholesterol efflux from CHO-SR-BI cells to HDL was significantly inhibited by AGE-BSA in a dose-dependent manner (IC(50) <30 microg/ml). Our results indicate that AGE proteins, as ligands for SR-BI, effectively inhibit both SR-BI-mediated selective uptake of HDL-CE and cholesterol efflux from peripheral cells to HDL, suggesting that AGE proteins might modulate SR-BI-mediated cholesterol metabolism in vivo.     

Regulation of sphingomyelin long chain base synthesis in human fibroblasts in culture. Role of lipoproteins and the low density lipoprotein receptor

We have studied regulation of synthesis of long chain bases in human fibroblasts using 3 different radioactive precursors and 2 different hydrolysis and separation procedures. Serum and low density lipoproteins inhibited synthesis. Inhibition of long chain base synthesis by various concentrations of low density lipoproteins paralleled inhibition of cholesterol synthesis. This inhibition was dependent on the low density lipoprotein receptor pathway since fibroblasts from homozygous familial hypercholesterolemic patients did not show the inhibition observed with normal fibroblasts. Incorporation of precursor palmitate into free or total long chain bases was inhibited by low density lipoproteins to the same extent as incorporation into sphingomyelin long chain bases. We thus propose that an enzyme in the pathway leading to sphinganine synthesis, probably palmitoyl-CoA:L-serine C-palmitoyltransferase (decarboxylating) EC 2.3.1.50, is regulated by low density lipoproteins.     

Effects of cellular cholesterol loading on macrophage foam cell lysosome acidification

Macrophages incubated with mildly oxidized low density lipoprotein (OxLDL), aggregated low density lipoprotein (AggLDL), or cholesteryl ester-rich lipid dispersions (DISPs) accumulate cholesterol in lysosomes followed by an inhibition of lysosomal cholesteryl ester (CE) hydrolysis. The variety of cholesterol-containing particles producing inhibition of hydrolysis suggests that inhibition may relate to general changes in lysosomes. Lysosome pH is a key mediator of activity and thus is a potential mechanism for lipid-induced inhibition. We investigated the effects of cholesterol accumulation on THP-1 macrophage lysosome pH. Treatment with OxLDL, AggLDL, and DISPs resulted in inhibition of the lysosome's ability to maintain an active pH and concomitant decreases in CE hydrolysis. Consistent with an overall disruption of lysosome function, exposure to OxLDL or AggLDL reduced lysosomal apolipoprotein B degradation. The lysosomal cholesterol sequestration and inactivation are not observed in cholesterol-equivalent cells loaded using acetylated low density lipoprotein (AcLDL). However, AcLDL-derived cholesterol in the presence of progesterone (to block cholesterol egression from lysosomes) inhibited lysosome acidification. Lysosome inhibition was not attributable to a decrease in the overall levels of vacuolar ATPase. However, augmentation of membrane cholesterol in isolated lysosomes inhibited vacuolar ATPase-dependent pumping of H+ ions into lysosomes. These data indicate that lysosomal cholesterol accumulation alters lysosomes in ways that could exacerbate foam cell formation and influence atherosclerotic lesion development.     

Plasma lipoprotein cholesterol concentrations in the golden-mantled ground squirrel (Spermophilus lateralis): a comparison between pre-hibernators and hibernators.

1. The concentrations of total cholesterol (free cholesterol plus cholesteryl ester) in the plasma and in two lipoprotein fractions of golden-mantled ground squirrels (Spermophilus lateralis) were measured during pre-hibernation and compared to those values measured during hibernation. 2. Hibernating ground squirrels had significantly higher (P less than 0.005) very low density lipoprotein plus low density lipoprotein cholesterol (VLDL + LDL-C) concentrations than did pre-hibernating ground squirrels. 3. Hibernating squirrels additionally exhibited significantly higher (P less than 0.005) total plasma cholesterol concentration per high density lipoprotein cholesterol concentration (TPC/HDL-C) ratios than did pre-hibernating squirrels. 4. The significant differences in the lipoprotein cholesterol concentrations observed in this study suggest that lipoprotein metabolism in pre-hibernators was significantly different from that in hibernators and was a reflection of the marked biochemical and physiological adjustments these animals must undergo during their transition from pre-hibernation to hibernation.     

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)     

Human platelets exclusively bind oxidized low density lipoprotein showing no specificity for acetylated low density lipoprotein.

The widely studied macrophage scavenger receptor system is known to bind both acetylated low density lipoprotein and oxidized low density lipoprotein. Although only the latter ligand has been shown to occur in vivo, acetylated low density lipoprotein is often used to evaluate the contribution of scavenger receptors to different (patho)physiologic processes, assuming that all existing subtypes of scavenger receptors recognise both lipoproteins. In the present work, we identify human platelets as the first natural cell type to bind oxidized low density lipoprotein without showing specificity for acetylated low density lipoprotein. Consequently, platelets possess exclusive receptor(s) for oxidized low density lipoprotein distinct from the 'classical' scavenger receptor AI/AII. From the data presented in this work, we conclude that the class B scavenger receptor CD36 (GPIV) is responsible for this exclusive oxidized low density lipoprotein binding.     

Cholesterol delivered to macrophages by oxidized low density lipoprotein is sequestered in lysosomes and fails to efflux normally

Oxidized low density lipoprotein (LDL) has been found to exhibit numerous potentially atherogenic properties, including transformation of macrophages to foam cells. It is believed that high density lipoprotein (HDL) protects against atherosclerosis by removing excess cholesterol from cells of the artery wall, thereby retarding lipid accumulation by macrophages. In the present study, the relative rates of HDL-mediated cholesterol efflux were measured in murine resident peritoneal macrophages that had been loaded with acetylated LDL or oxidized LDL. Total cholesterol content of macrophages incubated for 24 h with either oxidized LDL or acetylated LDL was increased by 3-fold. However, there was no release of cholesterol to HDL from cells loaded with oxidized LDL under conditions in which cells loaded with acetylated LDL released about one-third of their total cholesterol to HDL. Even mild degrees of oxidation were associated with impairment of cholesterol efflux. Macrophages incubated with vortex-aggregated LDL also displayed impaired cholesterol efflux, but aggregation could not account for the entire effect of oxidized LDL. Resistance of apolipoprotein B (apoB) in oxidized LDL to lysosomal hydrolases and inactivation of hydrolases by aldehydes in oxidized LDL were also implicated. The subcellular distribution of cholesterol in oxidized LDL-loaded cells and acetylated LDL-loaded cells was investigated by density gradient fractionation, and this indicated that cholesterol derived from oxidized LDL accumulates within lysosomes. Thus impairment of cholesterol efflux in oxidized LDL-loaded macrophages appears to be due to lysosomal accumulation of oxidized LDL rather than to impaired transport of cholesterol from a cytosolic compartment to the plasma membrane.