Oxidation of human low density lipoprotein results in derivatization of lysine residues of apolipoprotein B by lipid peroxide decomposition products

Modification of low density lipoproteins (LDL) by oxidation has been shown to permit recognition by the acetyl-LDL receptor of macrophages. The extensive oxidation of LDL that is required before interaction occurs with this receptor produces major alterations in both the lipid and protein components of LDL. Several chemical modifications of LDL also lead to recognition by this receptor; all of these involve derivatization of lysine residues of apolipoprotein B by adducts that neutralize the positively charged epsilon-amino group. The present studies show that oxidation also results in derivatization of LDL lysine residues. Analysis of amino acid composition indicated that 32% of lysine residues were modified after oxidation of LDL by exposure to 5 microM CuSO4 for 20 h. About one-half of the derivatized lysines were labile under the conditions of acid hydrolysis. Fluorescence of LDL protein was greatly increased by oxidation, with excitation maximum at 350 nm and emission maximum at 433 nm. When LDL containing phosphatidylcholine with isotopically labeled arachidonic acid in the sn-2 position was oxidized, there was a 5-fold increase in radioactivity bound to protein compared to nonoxidized LDL or oxidized LDL labeled with 2-[1-14C]palmitoyl phosphatidylcholine. Prior methylation of LDL prevented the rapid uptake and degradation by macrophages that normally accompanies oxidation. These findings suggest that oxidation of LDL is accompanied by derivatization of lysine epsilon-amino groups by lipid products and that these adducts may be important in the interaction of oxidized LDL with the acetyl-LDL receptor.     

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.     

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

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

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.     

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.     

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.     

Hypochlorous acid-modified low-density lipoprotein inactivates the lysosomal protease cathepsin B: protection by ascorbic and lipoic acids

Unregulated uptake of oxidized LDL by the scavenger receptor(s) of macrophages is thought to be an early event in atherosclerotic lesion development. Accumulation of oxidized LDL within macrophages may result from resistance of the modified LDL to enzymatic hydrolysis or from direct inactivation of lysosomal enzymes by reactive LDL-associated moieties. Since HOCl-modified LDL has been detected in vivo, the effects of HOCI-modified LDL on the activities of the cysteine protease cathepsin B and the aspartyl protease cathepsin D were investigated. LDL (0.5 mg protein/ml), which had been exposed to HOCl (25-200 microM), caused rapid dose-dependent inactivation of cathepsin B, but not of cathepsin D. Exposure of LDL to HOCl results primarily in the formation of LDL-associated chloramines, and the model chloramine N(alpha)-acetyl-lysine chloramine also caused dose-dependent inactivation of cathepsin B. Incubation of HOCl-modified LDL with ascorbic and lipoic acids (25-200 microM) resulted in dose-dependent reduction of LDL-associated chloramines and concomitant protection against cathepsin B inactivation. Thus, the data indicate that HOCl-modified LDL inactivates cathepsin B by a chloramine-dependent mechanism, most likely via oxidation of the enzyme's critical cysteine residue. Furthermore, small molecule antioxidants, such as ascorbic and lipoic acids, may be able to inhibit this potentially pro-atherogenic process by scavenging LDL-associated chloramines.     

Minimally oxidized LDL inhibits macrophage selective cholesteryl ester uptake and native LDL-induced foam cell formation

Scavenger receptor-mediated uptake of oxidized LDL (oxLDL) is thought to be the major mechanism of foam cell generation in atherosclerotic lesions. Recent data has indicated that native LDL is also capable of contributing to foam cell formation via low-affinity receptor-independent LDL particle pinocytosis and selective cholesteryl ester (CE) uptake. In the current investigation, Cu²⁺-induced LDL oxidation was found to inhibit macrophage selective CE uptake. Impairment of selective CE uptake was significant with LDL oxidized for as little as 30 min and correlated with oxidative fragmentation of apoB. In contrast, LDL aggregation, LDL CE oxidation, and the enhancement of scavenger receptor-mediated LDL particle uptake required at least 3 h of oxidation. Selective CE uptake did not require expression of the LDL receptor (LDL-R) and was inhibited similarly by LDL oxidation in LDL-R^−/− versus WT macrophages. Inhibition of selective uptake was also observed when cells were pretreated or cotreated with minimally oxidized LDL, indicating a direct inhibitory effect of this oxLDL on macrophages. Consistent with the effect on LDL CE uptake, minimal LDL oxidation almost completely prevented LDL-induced foam cell formation. These data demonstrate a novel inhibitory effect of mildly oxidized LDL that may reduce foam cell formation in atherosclerosis.     

Hypochlorous acid-modified low-density lipoprotein inactivates the lysosomal protease cathepsin B: protection by ascorbic and lipoic acids

Abstract

Unregulated uptake of oxidized LDL by the scavenger receptor (s) of macrophages is thought to be an early event in atherosclerotic lesion development. Accumulation of oxidized LDL within macrophages may result from resistance of the modified LDL to enzymatic hydrolysis or from direct inactivation of lysosomal enzymes by reactive LDL-associated moieties. Since HOCl-modified LDL has been detected in vivo, the effects of HOCl-modified LDL on the activities of the cysteine protease cathepsin B and the aspartyl protease cathepsin D were investigated. LDL (0.5 mg protein/ml), which had been exposed to HOCl (25–200 µM), caused rapid dose-dependent inactivation of cathepsin B, but not of cathepsin D. Exposure of LDL to HOCl results primarily in the formation of LDL-associated chloramines, and the model chloramine N^-acetyl-lysine chloramine also caused dose-dependent inactivation of cathepsin B. Incubation of HOCl-modified LDL with ascorbic and lipoic acids (25–200 µM) resulted in dose-dependent reduction of LDL-associated chloramines and concomitant protection against cathepsin B inactivation. Thus, the data indicate that HOCl-modified LDL inactivates cathepsin B by a chloramine-dependent mechanism, most likely via oxidation of the enzyme's critical cysteine residue. Furthermore, small molecule antioxidants, such as ascorbic and lipoic acids, may be able to inhibit this potentially proatherogenic process by scavenging LDL-associated chloramines.     

The role of lipid peroxidation and antioxidants in oxidative modification of LDL.

The purpose of this study is to provide a comprehensive survey on the compositional properties of LDL (e.g., lipid classes, fatty acids, antioxidants) relevant for its susceptibility to oxidation, on the mechanism and kinetics of LDL oxidation, and on the chemical and physico-chemical properties of LDL oxidized by exposure to copper ions. Studies on the occurrence of oxidized LDL in plasma, arteries, and plaques of humans and experimental animals are discussed with particular focus on the use of poly- and monoclonal antibodies for immunochemical demonstration of apolipoprotein B modifications characteristic for lipid peroxidation. Apart from uptake of oxidized LDL by macrophages, studies describing biological effects of heavily or minimally oxidized LDL are only briefly addressed, since several reviews dealing with this subject were recently published. This article is concluded with a section on the role of natural and synthetic antioxidants in protecting LDL against oxidation, as well as some previously unpublished material from our laboratories.