Binding and uptake of differently oxidized low density lipoprotein in mouse peritoneal macrophages and THP-1 macrophages: involvement of negative charges as well as oxidation-specific epitopes

Oxidatively modified low-density lipoprotein (LDL) has been found in vivo, and oxidized LDL (oxLDL) could bind to scavenger receptors, leading to foam cell formation. Macrophages bear a number of different scavenger receptors for oxLDL, and macrophages of different origins may have a different scavenger receptor repertoire. In addition, LDL oxidized to different degrees may differ in the ability to bind macrophage scavenger receptors. In this study, we characterized the patterns of the binding and uptake of differently oxidized LDL in mouse peritoneal macrophages (MPM) and human THP-1 macrophages, and the influence of negative charge and oxidation-specific epitopes in oxLDL on these processes. Thresholds of increased binding and uptake in MPM were found when LDL was oxidized to the degrees with a relative electrophoretic mobility (REM) of 2.6 (minor threshold) and 3.0 (major threshold), corresponding to 49 and 57%, respectively, of the loss of free amino groups in these oxLDL. There was no threshold for the binding of oxLDL to THP-1 macrophages, while for uptake, a major threshold with REM of 3.0 (57% free amino groups lost) was found. The presence of the F(ab')(2) fragments of the monoclonal antibody OB/04, which was raised against copper-oxidized LDL, led to the reduction of the binding and uptake, respectively, of Eu(3+)-oxLDL (REM:3.6) in MPM by 31 and 29%, and by 19 and 22% in THP-1 macrophages. It is concluded that LDL oxidized to different degrees binds differently to macrophages, and the patterns of binding and uptake are different for MPM and human THP-1 macrophages. Both, the negative charge and the oxidation-specific epitopes of oxLDL are involved in these processes.     

Oxidation of low density lipoprotein leads to particle aggregation and altered macrophage recognition.

Oxidized (ox-) low density lipoproteins (LDL) is characterized by the formation of lipid peroxides and their decomposition to reactive aldehydes which covalently link to apoB in LDL. These chemical changes are believed to be responsible for the enhanced recognition of ox-LDL by receptors on macrophages in culture. When oxidation is extensive, particle aggregation also occurs. The aim of this study was to characterize aggregation formation and how this influences the interaction of ox-LDL with macrophages in culture. When LDL was oxidized by incubating at 500 micrograms of protein/ml with 10 microM Cu2+ at 20 degrees C for up to 25 h, time-dependent increases in thiobarbituric acid reactive substances, conjugated diene content, electrophoretic mobility, and fluorescence at 360 excitation/430 emission were found. Particle aggregation increased in parallel with several parameters of oxidation and increased with increasing incubation temperatures and LDL concentrations used. When evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, apoB fragments of reproducible sizes and higher molecular weight species appeared after mild oxidation of LDL. The percent of total apoB remaining aggregated in sodium dodecyl sulfate was 50-80% at high degrees of oxidation, whereas it was far less in LDL that had been aggregated without chemical modification. This suggested that intermolecular cross-linking of apoB had occurred during oxidation of LDL at high concentrations. Degradation of ox-LDL in mouse peritoneal macrophages (MPM) increased in parallel with the degree of oxidation and with particle aggregation but reached a plateau after 12 h. Results from cross-competition studies in MPM with soluble and insoluble portions of extensively ox-LDL and with acetyl-LDL were consistent with uptake of soluble ox-LDL via both the scavenger receptor and another receptor on MPM, and uptake of the insoluble ox-LDL by an alternative mechanism.     

Scavenger receptors class A-I/II and CD36 are the principal receptors responsible for the uptake of modified low density lipoprotein leading to lipid loading in macrophages

Modification of low density lipoprotein (LDL) can result in the avid uptake of these lipoproteins via a family of macrophage transmembrane proteins referred to as scavenger receptors (SRs). The genetic inactivation of either of two SR family members, SR-A or CD36, has been shown previously to reduce oxidized LDL uptake in vitro and atherosclerotic lesions in mice. Several other SRs are reported to bind modified LDL, but their contribution to macrophage lipid accumulation is uncertain. We generated mice lacking both SR-A and CD36 to determine their combined impact on macrophage lipid uptake and to assess the contribution of other SRs to this process. We show that SR-A and CD36 account for 75-90% of degradation of LDL modified by acetylation or oxidation. Cholesteryl ester derived from modified lipoproteins fails to accumulate in macrophages taken from the double null mice, as assessed by histochemistry and gas chromatography-mass spectrometry. These results demonstrate that SR-A and CD36 are responsible for the preponderance of modified LDL uptake in macrophages and that other scavenger receptors do not compensate for their absence.     

Scavenger receptor-mediated uptake and metabolism of lipid vesicles containing acidic phospholipids by mouse peritoneal macrophages

We studied the mechanism of uptake and metabolism of exogenous phospholipids in mouse peritoneal macrophages using vesicles composed of various phospholipids and cholesterol. Macrophages in culture were found to actively incorporate and metabolize phosphatidylcholine/cholesterol vesicles containing small amounts of acidic phospholipids such as phosphatidylserine, phosphatidylinositol, or phosphatidic acid and to store the fatty acyl chains and cholesterol in triacylglycerol and cholesteryl ester form in their cytosol. These cells exhibited massive amounts of oil red O-positive lipid droplets, a typical feature of foam cells. The metabolism of exogenous phospholipid vesicles was completely inhibited by chloroquine and cytochalasin B, suggesting that vesicle uptake occurs by endocytosis. A similar type of metabolism was observed in guinea pig peritoneal macrophages, macrophage cell line J774.1, but not in Swiss 3T3 fibroblasts. Competition studies using various ligands for the scavenger receptor showed that acetylated low density lipoprotein (acetyl-LDL), dextran sulfate, or fucoidan was able to compete for up to 60% of the binding of phosphatidylserine-containing vesicles, and that copper-oxidized LDL (oxidized LDL) competed for more than 90% of the vesicle binding. On the other hand, phosphatidylserine-containing vesicles was able to compete for more than 90% of the binding of acetyl-LDL. These results indicate that acidic phospholipids are recognized by the scavenger receptors on the surface of macrophages and that more than one scavenger receptor exists on mouse peritoneal macrophages, i.e. one capable of recognizing acetyl-LDL, oxidized LDL, and an array of acidic phospholipids on membranes, and the other recognizing both acidic phospholipids and oxidized LDL but not acetyl-LDL.     

Implication of lipoprotein associated phospholipase A2 activity in oxLDL uptake by macrophages

Recognition and uptake of oxidized LDL (oxLDL) by scavenger receptors of macrophages and foam cell formation are mediated by the oxidatively modified apolipoprotein B (ApoB) and lipid moiety of oxLDL. A great amount of oxidized phosphatidylcholine (oxPC) of oxLDL is hydrolyzed at the sn-2 position by lipoprotein associated phospholipase A₂ (Lp-PLA₂) to lysophosphatidylcholine and small oxidation products. This study examines the involvement of Lp-PLA₂ in the uptake of oxLDL by mouse peritoneal macrophages. LDL with intact Lp-PLA₂ activity [LDL (+)] and LDL with completely inhibited Lp-PLA₂ activity [LDL (-)] were subjected to oxidation with 5 μM CuSO₄ for 6 h [moderately oxLDL (MoxLDL)], or 24 h [heavily oxLDL (HoxLDL)] and peritoneal macrophages were incubated with these preparations. The uptake of MoxLDL(-) was about 30% increased compared with that of MoxLDL(+), and HoxLDL(-) uptake was about 20% increased compared with that of HoxLDL(+). Inhibition of Lp-PLA₂ activity had no effect on the uptake of ApoB-liposomes conjugates with ApoB isolated from MoxLDL(-), MoxLDL(+), HoxLDL(-), and HoxLDL(+). Liposomes prepared from the lipid extract of MoxLDL(-), MoxLDL(+), HoxLDL(-), and HoxLDL(+) exhibited a similar pattern to that observed in the uptake of the corresponding intact lipoproteins. This study suggests that the progressive inactivation of Lp-PLA₂ during LDL oxidation leads to an increased uptake of oxLDL by macrophages, which could be primarily attributed to the increased uptake of the oxidized phospholipids enriched lipid moiety of oxLDL.     

Phospholipids in oxidized LDL not adducted to apoB are recognized by the CD36 scavenger receptor

Previous studies have shown that oxidation of low-density lipoprotein (oxLDL) results in its recognition by scavenger receptors on macrophages. Whereas blockage of lysyl residues on apoB-100 of oxLDL by lipid peroxidation products appears to be critical for recognition by the scavenger receptor class A (SR-A), modification of the lipid moiety has been suggested to be responsible for recognition by the scavenger class B receptor, CD36. We studied the recognition by scavenger receptors of oxidized LDL in which lysyl residues are blocked prior to oxidation through methylation [ox(m)LDL]. This permits us to minimize any contribution of modified apoB-100 to the recognition of oxLDL, but does not disrupt the native configuration of lipids in the particle. We found that ox(m)LDL was recognized by receptors on mouse peritoneal macrophages (MPM) almost as well as oxLDL. Ox(m)LDL was recognized by CD36-transfected cells but not by SR-A-transfected cells. Oxidized phospholipids (oxPC) transferred from oxLDL or directly from oxPC to LDL, conveyed recognition by CD36-transfected cells, confirming that CD36 recognized unbound oxidized phospholipids in ox(m)LDL. Collectively, these results suggest that oxPC not adducted to apoB within the intact oxLDL particle are recognized by the macrophage scavenger receptor CD36, that these lipids are not recognized by SR-A, and that they can transfer from oxidized to unoxidized LDL and induce CD36 recognition.     

Macrophages can decrease the level of cholesteryl ester hydroperoxides in low density lipoprotein

Murine and human macrophages rapidly decreased the level of cholesteryl ester hydroperoxides in low density lipoprotein (LDL) when cultured in media non-permissive for LDL oxidation. This process was proportional to cell number but could not be attributed to the net lipoprotein uptake. Macrophage-mediated loss of lipid hydroperoxides in LDL appears to be metal ion-independent. Degradation of cholesteryl linoleate hydroperoxides was accompanied by accumulation of the corresponding hydroxide as the major product and cholesteryl keto-octadecadienoate as a minor product, although taken together these products could not completely account for the hydroperoxide consumption. Cell-conditioned medium possessed a similar capacity to remove lipid hydroperoxides as seen with cellular monolayers, suggesting that the activity is not an integral component of the cell but is secreted from it. The activity of cell-conditioned medium to lower the level of LDL lipid hydroperoxides is associated with its high molecular weight fraction and is modulated by the availability of free thiol groups. Cell-mediated loss of LDL cholesteryl ester hydroperoxides is facilitated by the presence of alpha-tocopherol in the lipoprotein. Together with our earlier reports on the ability of macrophages to remove peroxides rapidly from oxidized amino acids, peptides, and proteins as well as to clear selectively cholesterol 7-beta-hydroperoxide, results presented in this paper provide evidence of a potential protective activity of the cell against further LDL oxidation by removing reactive peroxide groups in the lipoprotein.     

Macrophage receptors responsible for distinct recognition of low density lipoprotein containing pyrrole or pyridinium adducts: models of oxidized low density lipoprotein

Oxidation of low density lipoproteins (LDL) induced by incubation with Cu(2+) ions results in the formation of a heterogeneous group of aldehydic adducts on lysyl residues (Lys) of apolipoprotein B (apoB) that are thought to be responsible for the uptake of oxidized LDL (oxLDL) by macrophages. To define the structural and chemical criteria governing such cell recognition, we induced two modifications of lysines in LDL that mimic prototypic adducts present in oxLDL; namely, epsilon-amino charge-neutralizing pyrrolation by treatment with 2,5-hexanedione (hdLDL), and epsilon-amino charge-retaining pyridinium formation via treatment with 2,4,6-trimethylpyrylium (tmpLDL). Both modifications led to recognition by receptors on mouse peritoneal macrophages (MPM). To assess whether the murine scavenger receptor class A-I (mSR-A) was responsible for recognition of hdLDL or tmpLDL in MPM, we measured binding at 4 degrees C and degradation at 37 degrees C of these modified forms of (125)I-labeled LDL by mSR-A-transfected CHO cells. Although uptake and degradation of hdLDL by mSR-A-transfected CHO cells was quantitatively similar to that of the positive control, acLDL, tmpLDL was not recognized by these cells. However, both tmpLDL and hdLDL were recognized by 293 cells that had been transfected with CD36. In the human monocytic cell line THP-1 that had been activated with PMA, uptake of tmpLDL was significantly inhibited by blocking monoclonal antibodies to CD36, further suggesting recognition of tmpLDL by this receptor. Macrophage uptake and degradation of LDL oxidized by brief exposure to Cu(2+) was inhibited more effectively by excess tmpLDL and hdLDL than was more extensively oxidized LDL, consistent with the recognition of the former by CD36 and the latter primarily by SR-A.Collectively, these studies suggest that formation of specific pyrrole adducts on LDL leads to recognition by both the mSR-A and mouse homolog of CD36 expressed on MPM, while formation of specific pyridinium adducts on LDL leads to recognition by the mouse homolog of CD 36 but not by mSR-A. As such, these two modifications of LDL may represent useful models for dissecting the relative contributions of specific modifications on LDL produced during oxidation, to the cellular uptake of this heterogeneous ligand.     

CD36 is a receptor for oxidized high density lipoprotein: implications for the development of atherosclerosis

Atherosclerotic plaques result from the excessive deposition of cholesterol esters derived from lipoproteins and lipoprotein fragments. Tissue macrophage within the intimal space of major arterial vessels have been shown to play an important role in this process. We demonstrate in a transfection system using two human cell lines that the macrophage scavenger receptor CD36 selectively elicited lipid uptake from Cu(2+)-oxidized high density lipoprotein (HDL) but not from native HDL or low density lipoprotein (LDL). The uptake of oxHDL displayed morphological and biochemical similarities with the CD36-dependent uptake of oxidized LDL. CD36-mediated uptake of oxidized HDL by macrophage may therefore contribute to atheroma formation.     

Modification of human serum low density lipoprotein by oxidation--characterization and pathophysiological implications

Plasma low density lipoprotein (LDL) can undergo free radical oxidation either catalyzed by divalent cations, such as Cu2+ or Fe2+ or promoted by incubation with cultured cells such as endothelial cells, smooth muscle cells and monocytes. The content of vitamin E, beta-carotene and unsaturated fatty acids is decreased in oxidized LDL. A breakdown of apolipoprotein-B (apoB), hydrolysis of the phospholipids, an increase of thiobarbituric acid reactive substances and the generation of aldehydes also occur. Changes in the ratio of lipid to protein, the electrophoretic mobility and the fluorescent properties have also been reported to accompany oxidation of this lipoprotein. The functional changes of oxidized LDL include its recognition by the scavenger receptor on macrophages, its cytotoxicity especially to proliferating cells, its chemotactic properties with respect to monocyte-macrophages and its regulation of platelet-derived growth factor-like protein (PDGFc) production by endothelial cells. In this article we summarize some of the contributions to this topic and present speculations relating oxidized LDL to pathological conditions such as atherosclerosis.     

Recognition of oxidized low density lipoprotein by the scavenger receptor of macrophages results from derivatization of apolipoprotein B by products of fatty acid peroxidation

Uptake of cholesterol-containing lipoproteins by macrophages in the arterial intima is believed to be an important step in the pathogenesis of atherosclerosis. There are a number of possible mechanisms by which macrophages might accumulate cholesterol, and one that has attracted much interest recently involves the uptake of oxidatively modified low density lipoprotein (LDL) via a specific cell surface receptor, termed the scavenger or acetyl-LDL receptor. Previous studies have shown that chemical derivatization of LDL with reagents that result in neutralization of the charge of lysine amino groups also allows recognition by this receptor. As well, it has been shown that oxidation of LDL is accompanied by a decrease in free lysine groups and binding of lipid products to apolipoprotein B. The present studies were done to further characterize the receptor-binding domain on oxidized LDL. It was found that LDL could be modified by incubation with water-soluble products derived from autoxidized unsaturated fatty acids under conditions that inhibited oxidation of the LDL itself. The LDL modified in this way had increased electrophoretic mobility but showed no evidence of the oxidative damage that typifies LDL oxidized by exposure to metal ions. Furthermore, the oxidation product-modified LDL was rapidly degraded by cultured macrophages through the scavenger receptor pathway. Bovine albumin modified by oxidation products also showed greatly accelerated degradation by macrophages. When analyzed by reverse-phase high pressure liquid chromatography, the reactive oxidation products appeared less polar than fatty acids or simple medium-chain aldehydes. When treated with the carbonyl reagent 2,4-dinitrophenylhydrazine, the reactive fractions yielded derivatives, some of which were identified by mass spectrometry as hydrazones of nonenal, heptenal, pentenal, and crotonaldehyde. A series of 2-unsaturated aldehydes (acrolein to 2-nonenal) were all found to modify LDL, but none of these aldehyde-modified LDLs were recognized by the scavenger receptor of macrophages and all were degraded much more slowly by these cells than LDL modified with oxidation products. Furthermore, copper-oxidized LDL had only very slight immunoreactivity toward a panel of antibodies specific for adducts of simple 2-unsaturated aldehydes. Analysis of underivatized autoxidized fatty acids by coupled liquid chromatography/thermospray mass spectrometry revealed compounds with m/z corresponding to M+17, M+31, and 2M+31 in fractions that were capable of modifying LDL. The unoxidized fatty acids showed a dominant peak at M-1. These results indicate that the scavenger receptor of macrophages can recogn     

Suppression of endothelial or lipoprotein lipase in THP-1 macrophages attenuates proinflammatory cytokine secretion

LPL and endothelial lipase (EL) are associated with macrophages in human atherosclerotic lesions, and overexpression of LPL in mouse macrophages is associated with a greater extent of atherosclerosis. To investigate potential mechanisms by which macrophage-derived lipase expression may mediate proatherogenic effects, we used lentivirus-mediated RNA interference to suppress the expression of either LPL or EL within THP-1 macrophages. After suppression of either LPL or EL, significant decreases in the concentration of interleukin-1beta, interleukin-6, monocyte chemoattractant protein-1, and tumor necrosis factor-alpha were observed. Incubation of THP-1 macrophages with either mildly or extensively oxidized LDL consistently decreased cytokine expression, which was additive to that contributed by lipase suppression. Decreased lipase expression was also associated with an altered lipid composition, with reduced percentages of cholesterol (unesterified and esterified), triglycerides, and lysophosphatidylcholine. Microarray data indicated a decreased expression of proinflammatory genes, growth factors, and antiapoptotic genes. By contrast, there was an increased expression of lipoprotein receptors (scavenger receptor 1, low density lipoprotein receptor, scavenger receptor class B type I, and CD36). Thus, we conclude that the suppression of either LPL or EL decreases proinflammatory cytokine expression and influences the lipid composition of THP-1 macrophages. These results provide further insight into the specific metabolic and potential pathological roles of LPL and EL in human macrophages.     

Increased uptake of alpha-hydroxy aldehyde-modified low density lipoprotein by macrophage scavenger receptors

Reactive aldehydes can be formed during the oxidation of lipids, glucose, and amino acids and during the nonenzymatic glycation of proteins. Low density lipoprotein (LDL) modified with malondialdehyde are taken up by scavenger receptors on macrophages. In the current studies we determined whether alpha-hydroxy aldehydes also modify LDL to a form recognized by macrophage scavenger receptors. LDL modified by incubation with glycolaldehyde, glyceraldehyde, erythrose, arabinose, or glucose (alpha-hydroxy aldehydes that possess two, three, four, five, and six carbon atoms, respectively) exhibited decreased free amino groups and increased mobility on agarose gel electrophoresis. The lower the molecular weight of the aldehyde used for LDL modification, the more rapid and extensive was the derivatization of free amino groups. Approximately 50-75% of free lysine groups in LDL were modified after incubation with glyceraldehyde, glycolaldehyde, or erythrose for 24-48 h. Less extensive reductions in free amino groups were observed when LDL was incubated with arabinose or glucose, even at high concentration for up to 5 days. LDL modified with glycolaldehyde and glyceraldehyde labeled with (125)I was degraded more extensively by human monocyte-derived macrophages than was (125)I-labeled native LDL. Conversely, LDL modified with (125)I-labeled erythrose, arabinose, or glucose was degraded less rapidly than (125)I-labeled native LDL. Competition for the degradation of LDL modified with (125)I-labeled glyceraldehyde was nearly complete with acetyl-, glycolaldehyde-, and glyceraldehyde-modified LDL, fucoidin, and advanced glycation end product-modified bovine serum albumin, and absent with unlabeled native LDL.These results suggest that short-chain alpha-hydroxy aldehydes react with amino groups on LDL to yield moieties that are important determinants of recognition by macrophage scavenger receptors.     

Different apolipoprotein B breakdown patterns in models of oxidized low density lipoprotein.

Low density lipoprotein (LDL) oxidation is characterized by alterations in biological properties and structure of the lipoprotein particles, including breakdown and modification of apolipoprotein B (apoB). We compared apoB breakdown patterns in different models of minimally and extensively oxidized LDL using Western blotting techniques and several monoclonal and polyclonal antibodies. It was found that copper and endothelial cell-mediated oxidation produced a relatively similar apoB banding pattern with progressive fragmentation of apoB during LDL oxidation, whereas malondialdehyde (MDA)- and hydroxynonenal (HNE) -modified LDL produced an aggregated apoB. It is conceivable that apoB fragments present in copper and endothelial cell oxidized LDL lead to the exposure on the lipoprotein surface of different protein epitopes than in aggregated MDA-LDL and HNE-LDL. Although all models of extensively oxidized LDL led to increased lipid uptake in macrophages, mild degrees of oxidation interfered with LDL uptake in fibroblasts and extensively oxidized LDL impaired degradation of native LDL in fibroblasts. We suggest that in order to improve interpretation and comparison of results, data obtained with various models of oxidized LDL should be compared to the simpliest and most reproducible models of 3 h and 18 h copper-oxidized LDL (apoB breakdown) and MDA-LDL (apoB aggregation) since different models of oxidized LDL have significant differences in apoB breakdown and aggregation patterns which may affect immunological and biological properties of oxidized LDL.     

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.     

Oxidized low density lipoprotein is resistant to cathepsins and accumulates within macrophages

The rate of uptake of oxidized low density lipoprotein (LDL) by mouse peritoneal macrophages is similar to that of acetyl LDL; but only approximately 50% of the internalized oxidized LDL is ultimately degraded, in contrast to the near-complete degradation seen with acetyl LDL. The objectives of this study were to determine if this was due to increased surface binding of oxidized LDL, different uptake pathways for oxidized LDL and acetyl LDL, lysosomal dysfunction caused by oxidized LDL, or resistance of oxidized LDL to hydrolysis by lysosomal proteinases. LDL binding studies at 4 degrees C showed that the increased cell association with oxidized LDL could not be explained by differences in cell-surface binding. Immunofluorescence microscopy confirmed intracellular accumulation of apoB-immunoreactive material in macrophages incubated with oxidized LDL, but not with acetyl LDL. The scavenger receptor ligand polyinosinic acid inhibited both the cell association and degradation of oxidized LDL in macrophages by greater than 75%, suggesting a common uptake pathway for degraded LDL and nondegraded LDL. Studies in THP-1 cells also did not reveal more than one specific uptake pathway for oxidized LDL. LDL derivatized by incubation with oxidized arachidonic acid (under conditions that prevented oxidation of the LDL itself) showed inefficient degradation, similar to oxidized LDL. When macrophages were incubated with oxidized LDL together with acetyl 125I-LDL, the acetyl LDL was degraded normally, excluding lysosomal dysfunction as the explanation for the accumulation of oxidized LDL. Generation of trichloroacetic acid-soluble products from oxidized 125I-LDL by exposure to cathepsins B and D was less than that observed with native 125I-LDL. LDL modified by exposure to reactive products derived from oxidized arachidonic acid was also degraded more slowly than native 125I-LDL by cathepsins. In contrast, acetyl 125I-LDL was degraded more rapidly by cathepsins than native 125I-LDL, and aggregated LDL and malondialdehyde-modified LDL were degraded at the same rate as native 125I-LDL. It is concluded that the intracellular accumulation of oxidized LDL in macrophages can be explained at least in part by the resistance of oxidatively modified apolipoprotein B to cathepsins. This resistance to cathepsins does not appear to be due to aggregation of oxidized LDL, but may be a consequence of modification of apolipoprotein B by lipid peroxidation products.     

The role of sulfur-containing amino acids in superoxide production and modification of low density lipoprotein by arterial smooth muscle cells

Extracellular superoxide (O2-.) was detected in cultures of monkey arterial smooth muscle cells as measured by the superoxide dismutase-inhibitable reduction of cytochrome c and acetylated cytochrome c. Reduction of cytochrome c by these cells required L-cystine in the incubation medium. A variety of other sulfur-containing amino acids, including D-cystine, L-cystathionine, L-methionine, and djenkolic acid did not support O2-. generation when present at concentrations equimolar to L-cystine. At millimolar concentrations, the chelators EDTA and diethylene triamine penta-acetic acid inhibited O2-. production by smooth muscle cells. This effect was maximal when the chelator was present at the same concentration as the sum of the Ca2+ and Mg2+ in the medium, suggesting a role for these cations in O2-. generation by cells. Modification of low density lipoprotein (LDL) by arterial smooth muscle cells, as assessed by changes in lipid peroxide content, mobility on agarose gel electrophoresis, and apoprotein B fragmentation, was also L-cystine-dependent. LDL modification also required micromolar concentrations of the transition metal ion Cu(II) or Fe(III) and was inhibited by superoxide dismutase. LDL modified by smooth muscle cells in the presence of L-cystine and Cu(II) was taken up and degraded less well than native LDL by human skin fibroblasts, suggesting that recognition by the LDL receptor was lost. In contrast, LDL modified by smooth muscle cells was taken up and degraded to a greater degree than native LDL by mouse peritoneal macrophages, consistent with recognition by the scavenger receptor. These results indicate that monkey arterial smooth muscle cells produce O2-. and modify LDL by an L-cystine-dependent process. This may involve reduction of cystine to a thiol, possibly cysteine or a cysteine-containing peptide such as glutathione. Sulfur-containing amino acids may play a role in atherogenesis by supporting cell-mediated generation of reactive oxygen species and modification of lipoprotein to a form recognized by the scavenger receptor.     

TGF-beta1 downregulates CD36 and scavenger receptor A but upregulates LOX-1 in human macrophages

Transforming growth factor-beta1 (TGF-beta1), a key cytokine for control of cell growth, extracellular matrix formation, and inflammation control, is secreted by many cells present in the arteriosclerotic plaque. Lipid accumulation in the vessel wall is regarded as an early step in atherogenesis and depends on uptake of modified low-density lipoprotein (LDL) by macrophages through scavenger receptors and their transformation into foam cells. Prominent members of the scavenger receptor family are the class A type I and II receptors (ScR-A), the class B receptor CD36, and the recently detected lectin-like oxidized LDL receptor-1 (LOX-1), which, unlike the native LDL receptor (LDL-R), are not feedback controlled. CD36 is responsible for >50% of modified LDL uptake into human monocyte-derived macrophages. We therefore studied whether TGF-beta1 influences expression and function of ScR-A, CD36, and LOX-1 in monocytes using RT-PCR and flow cytometry. Total uptake of oxidized LDL by monocytoid cells, reflecting the combined function of all scavenger receptors, was significantly reduced by TGF-beta1. At initially low picomolar concentrations, TGF-beta1 decreased CD36 mRNA and protein surface expression and ScR-A mRNA levels in the human monocytic cell line THP-1 and in freshly isolated and cultivated human monocytes, whereas LOX-1 mRNA was increased. Expression of LDL-R and beta-actin was not affected by TGF-beta1. In conclusion, depression of scavenger receptor function in monocytes by TGF-beta1 in low concentrations reduces foam cell formation. Together with matrix control by TGF-beta1, this may be important for atherogenesis and plaque stabilization.