Oxidative Modification Of Lipoproteins Research Articles

Oxidatively modified LDL contains phospholipids with platelet-activating factor-like activity and stimulates the growth of smooth muscle cells.

Oxidative modification of lipoproteins is believed to be important in the genesis of atherosclerosis. We established cultures of smooth muscle cells (SMC) and exposed them to native LDL or oxidized LDL. Oxidized LDL, but not native LDL, was mitogenic as measured by incorporation of [3H]-thymidine into DNA. This effect was concentration dependent, averaged 288% of control, and was blocked by a platelet-activating factor (PAF) receptor antagonist. We hypothesized that phospholipids with PAF-like activity were generated during the oxidation of LDL. To test this hypothesis we extracted phospholipids from copper-oxidized LDL and assayed for PAF-like activity. Phospholipids extracted from oxidized LDL and purified by HPLC induced neutrophil adhesion equivalent to PAF (10 nM) and were mitogenic for smooth muscle cells. These effects were not seen with phospholipids extracted from native LDL and were blocked by two structurally different, competitive antagonists of the PAF receptor. The effects of these lipids were also abolished by pretreating them with PAF acetylhydrolase. Finally, we used Chinese hamster ovary cells that had seen stably transfected with a cDNA for the PAF receptor to confirm that phospholipids from oxidized LDL act via this receptor. We found that PAF (control) and the oxidized phospholipids each induced release of arachidonic acid from the transfected cells, but had no effect on wildtype Chinese hamster ovary cells, which lack the PAF receptor. This effect was also blocked by a PAF receptor antagonist. Thus, phospholipids generated during oxidative modification of LDL may participate in atherosclerosis by stimulating SMC proliferation and leukocyte activation.

Effect of the Antioxidant N,N′ -Diphenyl 1,4-Phenylenediamine (DPPD) on Atherosclerosis in ApoE-Deficient Mice

Apolipoprotein (apo) E-deficient mice develop atherosclerotic lesions that contain epitopes formed during the oxidative modification of lipoproteins, and they demonstrate high titers of circulating autoantibodies against such epitopes, suggesting that this murine strain may provide a model to investigate the atherogenic mechanisms of oxidized lipoproteins (Palinski et al, Arterioscler Thromb. 1994; 14:605-616). To test the hypothesis that lipoprotein oxidation contributes to lesion formation in apoE-deficient mice, we studied the effect of the antioxidant N,N'-diphenyl 1,4-phenylenediamine (DPPD) in mice fed a high-fat diet containing 0.15% cholesterol. Animals were divided into two subgroups matched for sex and plasma cholesterol levels, and DPPD (0.5% wt/wt) was added to the diet of one subgroup. Throughout the 6 months of intervention, DPPD treatment had no significant effect on plasma cholesterol. Plasma levels of DPPD at the end of the experiment were 33.1 mumol/L. As judged by resistance to loss of polyunsaturated fatty acids, lipoproteins (d < 1.019 g/mL) from DPPD-treated animals showed greater resistance to copper-induced oxidation than lipoproteins from control animals. In addition, there was a greater than twofold prolongation of the lag time in the formation of conjugated dienes in the LDL and IDL fractions of DPPD-treated mice. Atherosclerosis was significantly reduced, by 36% in the DPPD-treated mice (14.0 +/- 4.53% of aortic surface area versus 21.9 +/- 11.6%; n = 32; P < .02). These results are consistent with the hypothesis that lipoprotein oxidation contributes to atherogenesis in apoE-deficient mice. However, further studies with other antioxidants are needed to validate this hypothesis.

917-100 8-Epi-Prostaglandin F2α(8-Isoprostane), a Novel Marker of Lipid Peroxidation, is Elevated and Inversely Correlated to Serum Antioxidant Vitamins in Smokers

Oxidative modification of lipoproteins is thought to be an essential event in atherosclerosis. Current methods of quantifying lipid peroxidation in vivo are technically difficult and/or nonspecific. A simple, clinically reliable test is needed to assess an individual's degree of lipid peroxidation which could be a powerful predictor of cardiovascular disease risk. 8-lsoprostane is a chemically stable prostanoid resulting from free radical catalyzed peroxidation of arachidonic acid. This molecule is produced by oxidation of low density lipoprotein and is present in both plasma and urine. To evaluate this novel marker of in vivo lipid peroxidation, we compared 24 hour urinary levels of 8-isoprostane in healthy smokers (n = 15) and nonsmokers (n = 9) with a competitive enzyme immunoassay. Serum was also obtained for analysis of lipid and antioxidant vitamin levels. Smokers were found to have higher levels of urinary 8-isoprostane than nonsmokers and these levels were inversely correlated with serum beta carotene (P = 0.04, r = -0.44) and vitamin C (P = 0.01, r = -0.52). No significant differences were detected in lipid profiles between smokers and nonsmokers.8-lsoprostaneMean ± SEMP-ValueNonsmokers1.55 ± 0.22P = 0.002Smokers2.77 ± 0.21Values = μg/24 hours Values = μg/24 hours Urinary 8-isoprostane is elevated in smokers and may provide a sensitive, specific and noninvasive method for assessment of in vivo lipid peroxidation. Further studies are indicated to determine whether elevated 8-isoprostane identifies individuals at risk for developing atherosclerosis.

Effect of Dietary Supplementation of Different β-Carotene Isomers on Lipoprotein Oxidative Modification

Atherogenesis involves oxidative modification of low-density lipoprotein (LDL), which is associated with the depletion of the LDL endogenous anti-oxidants. Thus, enrichment of LDL with the anti-oxidant β-carotene has the potential of reducing the susceptibility of LDL to lipid peroxidation. We compared a pure synthetic all-trans β-carotene isomer with a stereoisomeric mixture of 9-cis and all-trans β-carotene derived from the alga Dunaliella bardawil. Ingestion of β-carotene either in a single dose (300 mg) or for 14 days (180 mg/day) by healthy subjects resulted in a significant elevation in the plasma and the LDL content of the pro-vitamin. However, there was substantially less elevation in β-carotene in response to the Dunaliella β-carotene, with a 1.8-fold elevation in plasma in comparison to a 3.6-fold elevation upon synthetic β-carotene supplementation. Oxidative modification of LDL, measured both after 14 days and a single-dose intake, was similarly reduced, as measured by the thiobarbituric acid-reactive substances (TBARS) generation (which indicated LDL susceptibility to in vitro lipid perodixation, in the presence of copper ions), by 40% or 49%, after 14-day supplementation of the synthetic or the Dunaliella fi-carotene, respectively. The present study thus demonstrates an inhibitory effect by both isomers of β-carotene on the susceptibility of LDL to oxidative modification. Supplementation with fi-carotene may be an important approach to reducing atherosclerosis via its inhibitory effect on the formation of atherogenic oxidized LDL.

Stimulation of Cell-Mediated Low-Density Lipoprotein Oxidative Modification by Oncostatin M

The effect of oncostatin M on low-density lipoprotein oxidative modification by human monocytes or murine endothelial cells was studied by determination of the lipid peroxidation products content and the electrophoretic mobility of the particle. In the range of concentrations from 10 to 30 ng/ml, oncostatin M induced a dose-dependent increase in LDL oxidation. The LDL degradation by J774 macrophage-like cells was also stimulated. Preincubation of cells with oncostatin M also enhanced the subsequent LDL modification This effect was accompanied by a parallel increase in superoxide anion release by cells. Since oncostatin M is produced by immune cells, the described effect suggests a relationship between infection, inflammation and LDL oxidative modification.

Oxidative modification of lipoprotein(a) inhibits binding to lysine-Sepharose and modulates fibrin binding

Oxidative modification of lipoprotein(a) inhibits binding to lysine-Sepharose and modulates fibrin binding

Oxidative modification of lipoproteins and their treatment in primates

Oxidative modification of lipoproteins and their treatment in primates

ApoE-deficient mice are a model of lipoprotein oxidation in atherogenesis. Demonstration of oxidation-specific epitopes in lesions and high titers of autoantibodies to malondialdehyde-lysine in serum.

Apolipoprotein (apo) E-deficient transgenic mice develop marked hyperlipidemia and progressive atherosclerotic lesions. To explore whether oxidative modification of lipoproteins is involved in atherogenesis in this murine model, we performed extensive immunocytochemical studies. Atherosclerotic lesions ranging from early fatty streaks to very advanced plaques were examined from the aortic valve region and the thoracic and abdominal aorta. Using guinea pig antisera against malondialdehyde (MDA)-lysine and 4-hydroxynonenal-lysine, two epitopes generated during the oxidative modification of low-density lipoprotein (LDL), we demonstrated the presence of these "oxidation-specific epitopes" in atherosclerotic lesions. In early lesions, oxidation-specific epitopes were found predominantly in macrophage-rich areas, whereas diffuse extracellular staining predominated in necrotic areas of advanced lesions. We have previously shown that autoantibodies against MDA-lysine are present in the circulation of humans and rabbits and that the immunoglobulin fraction extracted from their lesions contains autoantibodies against several "oxidation-specific" epitopes. Sera from apoE-deficient mice also contained circulating autoantibodies to MDA-lysine, and both early and advanced lesions were rich in murine immunoglobulins. Titers of serum autoantibodies were significantly higher in apoE-deficient mice than in C57BL/6 mice. Autoantibodies in murine plasma recognized MDA-lysine epitopes in atherosclerotic lesions of rabbits, and the immunostaining was competitively inhibited by excess human MDA-LDL. Similar findings were obtained by competitive radioimmunoassay. Finally, a morphometric technique was developed and tested in these mice that allows a quantitative assessment of aortic atherosclerosis. These findings suggest that in apoE-deficient mice, lipoprotein oxidation is involved in atherogenesis and that these transgenic mice constitute an appropriate model with which to study the antiatherogenic effect of antioxidant intervention.

Tumor necrosis factor enhances low density lipoprotein oxidative modification by monocytes and endothelial cells

The effect of tumor necrosis factor on the oxidative modification of LDL by U937 human monocytes or murine endothelial cells was studied by determination of the lipid peroxidation product content and the electrophoretic mobility of the particle. In the range of concentrations from 2.5 to 10 ng/ml, the cytokine induced a dose-dependent increase in cellular-induced oxidation of LDL. This effect was accompanied by a stimulation of LDL degradation by J774 macrophage-like cells. Concurrently, the TNF-treated cells secreted superoxide anion with a higher rate. Since LDL oxidation is believed to be an important feature in the formation of the atherosclerotic plaque, the described effects of TNF might be of importance in long-term exposure to this cytokine during inflammation.

Increased oxidazability of plasma low density lipoprotein from patients with coronary artery disease

Oxidative modification of lipoproteins may play a crucial role in the pathogenesis of atherosclerosis. This study was designed to examine whether increased lipid peroxides and/or oxidative susceptibility of plasma lipoproteins occur in patients with coronary artery disease. The levels of lipid peroxides, estimated as thiobarbituric acid-reactive substances (TBARS), were significantly greater in the plasma and very low density lipoprotein (VLDL) of symptomatic patients with coronary artery disease than in those of healthy persons, but the TBARS levels of low density lipoprotein (LDL) and high density lipoprotein (HDL) showed insignificant difference between patients and normals. To evaluate the oxidative susceptibility of lipoproteins, we employed in vitro Cu 2+ oxidation of lipoproteins monitored by changes in fluorescenece, TBARS level, trinitrobenzene sulfonic acid (TNBS) reactivity, apolipoprotein immunoreactivity and agarose gel electrophoretic mobility. While VLDL and LDL of normal controls were oxidazed at 5–10 μM Cu 2+, pooled VLDL and LDL of patients with coronary artery disease were oxidized at 1–2.5 μM Cu 2+, i.e., at relatively lowver oxidative stress. At 5 μM Cu 2+, VLDL and LDL of patients with coronary artery disease still showed at faster oxidation rate, judged by the rate of fluorescence increase, higher TBARS level, less TNBS reactivity, greater change in apo B immunoreactivity and higher electrophoretic mobility than those of normal controls. However, the difference on the oxidizability of HDL was insignificant for patients vs. normals. In conclusion, we have shown that plasm VLDL and LDL of patients with coronary artery disease are more susceptible to in vitro oxidative modification than those of health persons. The data suggest that enhanced oxidizability of plasma lipoproteins may be important factor influencing the development of coronary artery disease.

Lipoprotein oxidation and measurement of hydroperoxide formation in a single microtitre plate.

The oxidative modification of lipoproteins is of clinical importance because of potential contribution to atherogenesis [1, 2, 3]. An early step in the complex process of oxidation is the peroxidation of polyunsaturated fatty acids. We describe here a method for the Cu(II)-catalyzed oxidation of human low density lipoproteins with the subsequent analysis of hydroperoxides formation in a single microtitre plate. The procedure includes a modification of an iodometric peroxide assay for test tubes using a commercially available reagent. The microtitre plate method correlated well with the test tube procedure (r = 0.99) and showed comparable sensitivity and reproducibility. It was sensitive down to 0.5 nmol hydroperoxides/well and linear up to at least 20 nmol well-1. The method can handle several hundreds of samples a day with considerably less labour than the test tube procedure. It was well suited to monitor the kinetics of lipoprotein oxidation. The method was also used to test the potency of antioxidants, however, some antioxidants may interfere with the iodometric reaction and should be tested in the assay before use.

Dietary supplementation with vitamins C and E inhibits in vitro oxidation of lipoproteins.

The oxidative modification of lipoproteins has been implicated in atherogenesis, suggesting a protective role of circulating antioxidants. Vitamin C (ascorbic acid, 1 g/day) and vitamin E (dl alpha-tocopheryl acetate, 800 IU/day) were administered to healthy female and male volunteers. Lipoproteins with density < 1.063 g/mL were isolated from serum before and after vitamin supplementation and incubated with copper (Cu) or mononuclear cells (MC) plus Cu. Administration of vitamins C and E together to 4 subjects for 10 days resulted in a 57% (range 40-72%) decrease in Cu-catalyzed production of thiobarbituric acid reactive substances (TBARS) under the following conditions of assay: incubation times of 0-8 hours, Cu concentrations of 0-10 microM lipoprotein protein concentrations of 0.1-0.5 mg/mL. Decreases in other parameters of lipoprotein oxidation, i.e,, electrophoretic mobility, production of conjugated dienes and modification of amino groups, were also observed. Vitamin E administration alone produced a 52% inhibition and vitamin C alone a 15% inhibition of TBARS formation. Vitamins C and E supplementation resulted in a 78% decrease in the susceptibility of lipoproteins to MC-mediated oxidation. There was a strong inverse correlation (r = -0.64, p < 0.0007) between vitamin E levels in the lipoproteins and TBARS production in samples from 12 subjects administered vitamins C and E. In 3 individuals vitamin E levels remained low and in 2 of these subjects there was no effect of vitamins C and E administration on TBARS production. These results suggest a protective role of antioxidant vitamins and significant individual variability in response.

Oxidative modification of lipoprotein(a) and the effect of β-carotene

Lipoprotein (a) [Lp(a)] particles isolated and purified from human plasma were found to be oxidatively modified when incubated in vitro with human mononuclear cells or Cu 2+. This modification, which involved lipid peroxidation measured as thiobarbituric acid-reactive substance (TBARS), caused marked changes in the structure and biological properties of Lp(a). Relative to native Lp(a), oxidized particles showed decreases of free amino groups, protein fragmentation, increased negative charge, and high aggregation ability. They were taken up and degraded readily by macrophages in vitro, inducing cholesteryl ester accumulation. When apolipoprotein (a) [apo(a)] was clipped off by exposure to dithiothreitol (DTT), the remaining particle was degraded by macrophages at a significantly lower rate. This observation implies that oxidative modification of apo(a) may have an influence on Lp(a) recognition by scavenger receptors of macrophages. Under the same experimental conditions, low-density lipoprotein (LDL) concentrations equal to those of Lp(a) showed a lower susceptibility to oxidation. This was probably due to higher vitamin E (30% more) and β-carotene (40% more) content compared with Lp(a), when expressed as a function of cholesterol concentration and measured in the same subject. The addition of β-carotene to Lp(a) in vitro partially protected Lp(a) against oxidation and aggregation. As a result, uptake of oxidized Lp(a) by macrophages decreased markedly. We conclude that Lp(a) particles are prone to oxidation and that the increased risk of coronary artery disease associated with elevated Lp(a) levels may be related in part to their oxidative modification and uptake by macrophages, resulting in the formation of macrophage-derived foam cells.

Pathogenesis of the atherosclerotic lesion. Implications for diabetes mellitus.

In this review, we have highlighted pivotal cellular and molecular events in the initiation and progression of atherosclerosis. Key components of lesion initiation are an enhanced focal intimal influx and accumulation of lipoproteins, including LDL in hemodynamically determined lesion-prone areas, focal monocyte-macrophage recruitment, intimal generation of ROS, and oxidative modification of lipoproteins (including LDL [Ox-LDL]). Modified lipoproteins are taken up by the non-downregulating macrophage scavenger receptor, with foam cell formation and the development of the so-called fatty streak. One transitional event in lesion progression is foam cell necrosis, likely attributable to the cytotoxicity of both intimal free radicals and Ox-LDL, with development of an extracellular metabolically inert lipid core. Another is the migration to and proliferation within the intima of medial SMCs, leading to the synthesis of plaque collagens, elastin, and proteoglycans. Mural thrombosis plays a significant role in the late-stage progression of lesions. Regression of lesions is considered a function of the dynamic balance among components of initiation, progression, plaque stabilization, and removal of plaque constituents--the so-called regression quartet. Here, we critically examine how components of diabetes mellitus might impact not only lesion development, but also lesion regression. It is concluded that some components of diabetes mellitus augment key mechanisms in lesion initiation and progression and will likely retard the processes of plaque regression. Specifically, we focus on the various influences of diabetes mellitus on lipoprotein influx and accumulation, free radical generation and Ox-LDL, monocyte-macrophage recruitment, thrombosis and impaired fibrinolysis, and the reverse cholesterol transport system. The importance of nonenzymatic protein glycosylation in modifying a number of these processes is emphasized.

The antioxidant butylated hydroxytoluene protects against atherosclerosis.

Rabbits fed a 1% cholesterol diet with or without the antioxidant butylated hydroxytoluene (BHT) developed typical atherosclerotic lesions. The addition of BHT gave higher levels of total cholesterol (+40%), triglycerides (+250%), low density lipoprotein (LDL), and very low density lipoprotein (VLDL) in plasma. Despite the lower plasma lipid levels, the degree of atherosclerosis of the aortic surface was considerably higher in rabbits fed cholesterol than in the group treated with cholesterol and BHT. The mean atherosclerotic involvement was 18.6 +/- 4.4% in the former group and 5.9 +/- 1.7% in the latter group (p = 0.02). In all animals, there was a high correlation between the area of the arterial lesion and cholesterol content (r = 0.96). Serum levels of cholesterol autooxidation products (7-ketocholesterol and cholesterol 5 alpha,6 alpha-epoxide) were lower in the group of rabbits treated with BHT (p less than 0.005). Serum levels of vitamin E were slightly higher in the BHT group. There was no significant difference in the clearance of beta-VLDL between the two treatment groups after using either beta-VLDL from cholesterol-fed animals or beta-VLDL from BHT-fed animals. The results are in accord with the contention that oxidative modification of lipoproteins is important for the development of atherosclerosis and that antioxidants may have a protective effect. At present, however, other explanations cannot be completely excluded, for example, effects of antioxidants on immunologic factors or monocyte adhesion.

Current Strategies for Atherosclerosis and Lowering Cholesterol

Atherosclerosis is a multifactorial disorder in which nutritional factors are closely related to a number of the manifestations of the disease. Sounder nutritional principles might therefore prevent atherosclerosis itself or its major complications. Optimising nutrients would diminish monocyte adhesion to endothelium, suppress platelet aggregation, prevent oxidative modification of lipoproteins, minimise activation of coagulant factors and reduce the flux of cholesterol and of atherogenic lipoproteins through plasma. Optimising the mix of dietary fatty acids should have the highest priority. Emphasis should be placed on those fatty acids, such as those derived from fish, which modify several cardiovascular risk factors, including plasma lipids, blood pressure and coagulation factors. Reduction of risk factors is also achieved but to a lesser extent by reducing total fat intake, reducing dietary cholesterol, optimising the mix of starches and fibre and increasing plant foods generally. A developing strategy is to identify genetic predisposition to environmentally induced hyperlipidaemia, since most forms of hyperlipidaemia reflect interactions between genetic and dietary factors.