Physiologic levels of ascorbate inhibit the oxidative modification of low density lipoprotein

Oxidatively modified low density lipoprotein (LDL) could contribute to the atherosclerotic process by its cytotoxic effect, uptake by the scavenger receptor and influence on monocyte and macrophage motility. The aim of the present study was to examine the effect of physiologic levels of alpha-tocopherol and ascorbate on Cu2(+)-induced oxidative modification of LDL. Whereas alpha-tocopherol had an inhibitory effect on the oxidative modification of LDL only for 5 h, as evidenced by the electrophoretic mobility and lipid peroxide content, ascorbate inhibited the oxidative modification of LDL for both 5 and 24 h. By inhibiting the oxidative modification of LDL, ascorbate prevented the uptake and degradation of oxidatively modified LDL by the scavenger-receptor mechanism of cultured human monocyte derived macrophages. It thus appears that in this cell-free system (2.5 microM Cu2+), ascorbate is a more potent antioxidant than alpha-tocopherol. These findings indicate that ascorbate in physiologic concentrations should inhibit the oxidate modification of LDL in vivo.     

Aspirin protects low density lipoprotein from oxidative modification.

OBJECTIVE: To examine the effects of aspirin on the potential for oxidative modification of low density lipoprotein (LDL). DESIGN: Before and after trial. SETTING: University department of medicine within a district general hospital campus. PATIENTS: Ten healthy normolipidaemic volunteers drawn from laboratory and medical staff. INTERVENTIONS: Aspirin (enteric coated) 300 mg daily for two weeks. MAIN OUTCOME MEASURES: In vitro oxidation of LDL following ultraviolet C (UVC) irradiation with measurements made of malondialdehyde, conjugated dienes, and electrophoretic mobility. RESULTS: There was a significant decrease in malondialdehyde production from LDL modified by aspirin in vivo following exposure to UVC irradiation for 90 minutes, culminating in a 30% decrease by 240 minutes (mean (SD) 64.2 (9.12) v 89.6 (11.6) nmol/mg LDL protein, P = 0.029). These observations were borne out using LDL modified by aspirin in vitro. The UVC induced increase in relative electrophoretic mobility of LDL was also significantly reduced following aspirin treatment (mean (SD) 2.17 (0.16) v 2.66 (0.24), P = 0.012). CONCLUSIONS: Aspirin, both in vivo and in vitro, protects LDL against subsequent oxidative modification, providing an additional mechanism whereby aspirin may protect against atherosclerosis.     

Semicarbazide-sensitive amine oxidase catalyzes endothelial cell-mediated low density lipoprotein oxidation

OBJECTIVE: Deamination products of semicarbazide-sensitive amine oxidases (SSAO), i.e. aldehydes, superoxide and ammonia have been shown to initiate vascular damage. SSAOs are copper-enzymes, present in endothelial (EC), smooth muscle cells (SMC) and in blood. Transition metals ions (Cu, Fe) mediate the oxidative (atherogenic) modification of LDL by SMC and EC. The physiological source of the active metal ions is still under debate. We hypothesize that SSAOs may catalyze LDL oxidation by endothelial cells via enzyme-complexed Cu++. METHODS: EC isolated from human umbilical veins and cultured in 35 mm wells in RPMI-1640 medium were used as LDL oxidation system. RESULTS: Diamine oxidase (DAO), a SSAO which activity is elevated in tissues and sera of diabetic patients, catalyzes the oxidation of LDL by EC. In the presence of purified DAO (0.07 to 70 U/l) LDL oxidation was increased up to 10-fold as measured by thiobarbituric acid reactive substance (TBARS) formation as well as apoprotein modification of LDL. Chemical blockage of the SSAO substrate binding site did not inhibit the catalytic effect of DAO on LDL oxidation. Denaturation of the enzyme did not destroy the ability of the preparation to facilitate LDL oxidation by EC. The potential of the enzyme to catalyze LDL oxidation was not suppressed in the presence of serum. However, selective removing of enzyme-copper completely abolished the ability of the enzyme to trigger cell-mediated LDL oxidation. CONCLUSION: DAO, beside generating angiopathic deamination products, has the potential to act as a pathophysiological catalyst of LDL atherogenic modification by vascular cells.     

The oxidative modification of low density lipoprotein by human lymphocytes.

Oxidation of low density lipoprotein in atherosclerotic lesions may be involved in converting macrophages into lipid-laden foam cells. Lesions contain endothelial cells, smooth muscle cells, macrophages and lymphocytes. The first three types of cells have been shown previously to modify low density lipoprotein so as to increase its uptake by macrophages. We report here that lymphocytes from human blood are also capable of doing this. The modification process was an oxidative one because there was an increase in the thiobarbituric acid-reactive substances in media containing lymphocyte-modified low density lipoprotein and the modification could be inhibited by the antioxidants butylated hydroxytoluene and probucol. The lymphocyte-modified low density lipoprotein was taken up and degraded by macrophages by their scavenger receptor(s).     

Human plasma trans-sialidase causes atherogenic modification of low density lipoprotein.

In earlier studies we have found that incubation of low density lipoprotein (LDL) with autologous blood plasma-derived serum leads to a loss of sialic acid from lipoprotein particles. In this study we demonstrated that sialic acid removed from LDL was transferred to glycoconjugates of lipoproteins, glycoproteins and sphingolipids of human serum. This showed that human serum contained the trans-sialidase activity. Gel-filtration chromatography of human blood serum demonstrated the presence of trans-sialidase activity in lipoprotein subfractions as well as in lipoprotein-deficient serum. Trans-sialidase (about 65 kDa) was isolated from lipoprotein-deficient serum using affinity chromatography carried out with Neu5Acalpha2-8Neu5Ac-sepharose FF-6. Optimal pH values for the trans-sialidase were 3.0, 5.0 and 7.0. Calcium and magnesium ions stimulated the enzyme activity at millimolar concentrations. Isolated enzyme can remove sialic acid from LDL, IDL, VLDL, and HDL particles (in decreasing rate order). Serum trans-sialidase transferred sialic acid from glycoconjugates of plasma proteins (fetuin, transferrin) and gangliosides (GM3, GD3, GM1, GD1a, GD1b). Sialylated glycoconjugates of human blood erythrocytes also served as substrate for serum trans-sialidase. We have found that sialic acid can also be removed from N- and O-linked glycans, sialylated Le(x) and Le(a), oligosialic acids, and sphingolipid carbohydrate chains. The rate of sialic acid release decreased in the following order: alpha2,6>alpha2,3>alpha2,8. Transferred molecule of sialic acid can form alpha2,6, alpha2,3 and to a lesser degree alpha2,8 linkage with galactose, N-acetyl-galactosamine or sialic acid of acceptors. The glycoconjugates of erythrocytes, lipoprotein particles, plasma proteins, neutral sphingolipids and gangliosides may serve as acceptors of transferred sialic acid. Trans-sialidase-treated native LDL becomes desialylated and then can induce cholesteryl ester accumulation in human aortic intimal smooth muscle cells. Thus, trans-sialidase may be involved in the early stages of atherogenesis characterized by foam cell formation.     

Mechanisms by which cysteine can inhibit or promote the oxidation of low density lipoprotein by copper

Oxidised low density lipoprotein (LDL) may play a role in atherogenesis. We have investigated some of the mechanisms by which the thiol cysteine and the disulphide cystine can influence the oxidation of LDL by copper ions. Cysteine or cystine (100 microM) inhibited the oxidation of native LDL by copper in a simple phosphate buffer. One of the mechanisms by which cysteine (or more likely its oxidation products in the presence of copper) and cystine inhibited LDL oxidation was by decreasing the binding of copper to LDL (97% inhibition). Cysteine, but not cystine, rapidly reduced Cu(2+) to Cu(+). This may help to explain the antioxidant effect of cysteine as it may limit the amount of Cu(2+) that is available to convert alpha-tocopherol in LDL into the prooxidant alpha-tocopherol radical. Cysteine (but not cystine) had a prooxidant effect, however, toward partially oxidised LDL in the presence of a low copper concentration, which may have been due to the rapid breakdown of lipid hydroperoxides in partially oxidised LDL by Cu(+) generated by cysteine. To prove that cysteine can cause the rapid breakdown of lipid hydroperoxides in LDL, we enriched LDL with lipid hydroperoxides using an azo initiator in the absence of copper. Cysteine, but not cystine, increased the rate of lipid hydroperoxide decomposition to thiobarbituric acid-reactive substances (TBARS) in the presence of copper.     

Lipopolysaccharide enhances oxidative modification of low density lipoprotein by copper ions, endothelial and smooth muscle cells

The effect of lipopolysaccharide (LPS, endotoxin) on low density lipoprotein (LDL) oxidative modification by copper ions, endothelial and smooth muscle cells was studied by determination of the level of lipid peroxidation products (thiobarbituric acid reactive substances or TBARS), the diene level and the electrophoretic mobility of the LDL particle. LPS 25-75 microg/ml induced a dose-dependent increase in LDL oxidation by copper ions, endothelial and smooth muscle cells. At 75 microg LPS/ml, the TBARS content was 1.9, 1.6, and 1.8-fold increased, respectively. The LDL degradation by J774 macrophage-like cells was concomitantly stimulated. Preincubation of the LDL particle with LPS induced a marked increase in the subsequent LDL oxidative modification either by copper ions or by endothelial and smooth muscle cells. In addition, pretreatment of endothelial and smooth muscle cells with LPS also induced an enhancement of LDL oxidative modification performed in the absence of LPS. This effect was accompanied by a parallel increase in superoxide anion release by the cells. These results point at one of the mechanisms involved in the described association between bacterial infection and acute myocardial infarction as well as coronary heart disease.     

Cellular oxidative modification of low density lipoprotein does not require lipoxygenases.

The oxidative modification of low density lipoprotein (LDL) may play an important role in the pathogenesis of atherosclerosis. LDL can be oxidatively modified in vitro by endothelial cells, mouse peritoneal macrophages, or copper ions. Studies using lipoxygenase inhibitors have suggested that lipoxygenase(s) is required for the cellular modification of LDL [Rankin, S. M., Parthasarathy, S. & Steinberg, D. (1991) J. Lipid Res. 32, 449-456]. We have reexamined the effect of lipoxygenase inhibitors on cellular modification and found that (i) inhibitors specific for 5-lipoxygenase do not block LDL modification; (ii) inhibitors that block lipoxygenase by donating one electron to the enzyme (reductive inactivation) prevent LDL modification by cells and also modification mediated by copper ions, implying that they act as general antioxidants; (iii) the lipoxygenase inhibitor 5,8,11,14-eicosatetraynoic acid blocks 15-lipoxygenase activity in intact macrophages at concentrations 100 times less than those required to block LDL modification by macrophages; and (iv) 5,8,11,14-eicosatetraynoic acid is cytotoxic at concentrations about twice those required to prevent modification. Furthermore, macrophages and the RECB4 line of endothelial cells modify LDL with similar efficiencies despite dramatic differences in 15-lipoxygenase activity. Thus we conclude that neither 5-lipoxygenase nor 15-lipoxygenase is required for modification of LDL by cultured cells.     

Dietary cholesterol increases the susceptibility of low density lipoprotein to oxidative modification

Evidence suggests that oxidative modification of low density lipoprotein (LDL) occurs in vivo, increasing the atherogenecity of the particle. A total of 13 subjects (age range 46-78 years) with an LDL cholesterol concentration >3.36 mmol/l consumed each of four diets for 32-day periods. The diets contained 30% energy as fat of which 2/3 was either corn oil or beef tallow with and without 115 mg/4.2 MJ of supplemental cholesterol in the form of cooked egg yolk. The susceptibility of LDL to oxidation was assessed during a challenge with hemin and hydrogen peroxide, and results are expressed as lag time to oxidation in minutes. Addition of moderate amounts of cholesterol to either the corn oil or beef tallow enriched diet resulted in increased susceptibility of LDL to oxidation (decreased lag time): 69+/-22 min versus 96+/-24 min in the corn oil diet with versus without supplemental cholesterol, respectively, P = 0.006; 82+/-20 min versus 96+/-26 min in the beef tallow diet with versus without supplemental cholesterol, respectively, P = 0.025. A stepwise equation indicated that as plasma oleic acid concentrations increased and/or linoleic acid concentrations decreased, lag time increased (decreased susceptibility to oxidation), whereas as dietary cholesterol concentrations increased, lag time decreased (increased susceptibility to oxidation). In conclusion, these data suggest that addition of a moderate amount of dietary cholesterol to a reduced fat diet rich in polyunsaturated or saturated fatty acids increased the in vitro susceptibility of LDL to oxidation.     

A role for endothelial cell lipoxygenase in the oxidative modification of low density lipoprotein.

Oxidative modification of low density lipoprotein (LDL) has been implicated as a factor in the generation of macrophage-derived foam cells in vitro and in vivo. However, the exact mechanism of LDL oxidation has not been established. The present studies show that cellular lipoxygenase activity is involved in endothelial cell-induced oxidation of LDL. Inhibitors of lipoxygenase (but not inhibitors of cyclooxygenase) reduced LDL oxidation by as much as 70-85% under the conditions used. In contrast, the addition of pure (recombinant) superoxide dismutase inhibited by only approximately 25% under the same conditions. Oxidation of LDL by smooth muscle cells, on the other hand, was effectively inhibited by superoxide dismutase, as was Cu2+-catalyzed oxidation of LDL. When LDL was added to endothelial cell cultures within a dialysis bag, it did not undergo oxidative modification, suggesting that cell-LDL contact is necessary. We propose that an important element in cell-induced oxidation of LDL depends on (i) lipoxygenase oxidation of cellular lipids, followed by their exchange into LDL in the medium; (ii) direct lipoxygenase-dependent oxidation of LDL lipids during LDL-cell contact; (iii) or both.     

Modification of low density lipoprotein by endothelial cells involves lipid peroxidation and degradation of low density lipoprotein phospholipids.

Low density lipoprotein (LDL) incubated with cultured endothelial cells from rabbit aorta or human umbilical vein is altered in several ways (EC-modified): (i) It is degraded by macrophages much faster than LDL similarly incubated in the absence of cells or incubated with fibroblasts. (ii) Its electrophoretic mobility is increased. (iii) Its density is increased. We report here that antioxidants completely prevent these changes. We also report that these changes do not take place if transition metals in the medium are chelated with EDTA. During EC-modification as much as 40% of the LDL phosphatidylcholine is degraded to lysophosphatidylcholine by a phospholipase A2-like activity. When incubation conditions in the absence of cells were selected to favor oxidation--for example, by extending the time of incubation of LDL at low concentrations, or by increasing the Cu2+ concentration--LDL underwent changes very similar to those occurring in the presence of cells, including degradation of phosphatidylcholine. Hence, some phospholipase activity appears to be associated with the isolated LDL used in these studies. The results suggest a complex process in which endothelial cells modify LDL by mechanisms involving generation of free radicals and action of phospholipase (s).     

Essential role of phospholipase A2 activity in endothelial cell-induced modification of low density lipoprotein.

Previous studies have established that incubation of low density lipoprotein (LDL) with cultured endothelial cells (EC) converts it to a new form (EC-modified LDL) that is now recognized by a specific receptor on macrophages (the acetyl LDL receptor) and is taken up and degraded 3-10 times more rapidly than native LDL (biological modification). The formation of EC-modified LDL depended on generation of free radicals with consequent peroxidation of LDL lipids and was accompanied by extensive hydrolysis of LDL phosphatidylcholine at the 2-position. The present studies show that p-bromophenacyl bromide, a site-specific irreversible inhibitor of phospholipase A2 activity, blocks this hydrolysis and, at the same time, the enhanced macrophage degradation. We show further that during EC modification the apoprotein B of LDL undergoes considerable modification and that this also is prevented by the phospholipase inhibitor. Finally, as reported previously, changes similar to those observed on incubation of LDL with EC can be induced by incubation in the absence of cells but in the presence of a sufficiently high concentration of Cu2+. This also is accompanied by hydrolysis of phosphatidylcholine at the 2-position and breakdown of apoprotein B. These changes are also inhibited by p-bromophenacyl bromide, suggesting the presence of a phospholipase A2 activity associated with LDL as it is isolated. A hypothesis is presented linking lipid peroxidation, phosphatidylcholine hydrolysis, and changes in the LDL apoprotein during EC modification.     

Low density lipoprotein undergoes oxidative modification in vivo.

It has been proposed that low density lipoprotein (LDL) must undergo oxidative modification before it can give rise to foam cells, the key component of the fatty streak lesion of atherosclerosis. Oxidation of LDL probably generates a broad spectrum of conjugates between fragments of oxidized fatty acids and apolipoprotein B. We now present three mutually supportive lines of evidence for oxidation of LDL in vivo: (i) Antibodies against oxidized LDL, malondialdehyde-lysine, or 4-hydroxynonenal-lysine recognize materials in the atherosclerotic lesions of LDL receptor-deficient rabbits; (ii) LDL gently extracted from lesions of these rabbits is recognized by an antiserum against malondialdehyde-conjugated LDL; (iii) autoantibodies against malondialdehyde-LDL (titers from 512 to greater than 4096) can be demonstrated in rabbit and human sera.     

Integrated study of low density lipoprotein metabolism and very low density lipoprotein metabolism in non-insulin-dependent diabetes

The metabolisms of VLDL, IDL, and LDL and their interconversions have been studied in ten obese untreated male Pima Indian diabetics compared to 16 age-, sex-, and weight-matched nondiabetics. VLDL was elevated in the diabetics and had abnormal composition, as indicated by a significantly higher ratio of triglyceride/apo B. Fractional catabolic rates for both VLDL apoB and VLDL triglyceride were lower in diabetics, and diabetics had increased production of VLDL triglyceride but not VLDL apoB compared to obese nondiabetics. A higher proportion of VLDL apoB was removed without conversion to LDL in diabetics. LDL cholesterol and apoB were higher in diabetics, but production of LDL apoB was not different from nondiabetics. Fractional catabolic rate for LDL apoB, however, was significantly lower in the diabetics. The data indicate that the triglyceride-rich VLDL in non-insulin-dependent diabetics are less readily converted to LDL, whereas the elevated LDL in this group of diabetics is due to impaired clearance. Thus, decreased conversion of VLDL to LDL and impaired LDL clearance are two opposing phenomena which may influence the LDL concentration of diabetics in either direction. Thus, despite minimal changes in LDL concentration, there are multiple defects in the metabolism of LDL in non-insulin dependent diabetes which may contribute to the increased atherogenesis in this disorder.     

Rabbit very low density lipoprotein receptor: a low density lipoprotein receptor-like protein with distinct ligand specificity.

A cDNA that expresses a receptor for very low density lipoprotein (VLDL) was isolated from a rabbit heart cDNA library and characterized. The deduced amino acid sequence of the cDNA revealed that the cDNA encodes a protein with striking homology to the low density lipoprotein (LDL) receptor. Like the LDL receptor, the mature protein consists of the following five domains spanning 846 amino acids: 328 N-terminal amino acids including an 8-fold repeat of 40 amino acids homologous to the ligand binding repeat of the LDL receptor; 396 amino acid residues homologous to the epidermal growth factor precursor including three cysteine-rich repeats; a region immediately outside of the plasma membrane rich in serines and threonines; 22 amino acids traversing the plasma membrane; and 54 amino acids including the NPVY sequence that is required for clustering of the LDL receptor in coated pits and that projects into the cytoplasm. LDL-receptor-deficient Chinese hamster ovary cells transfected with the cDNA bound and internalized VLDL, beta-migrating VLDL, and intermediate density lipoprotein but did not bind LDL with high affinity. The 3.6- and 9.5-kilobase mRNAs for the VLDL receptor are highly abundant in heart, muscle, and adipose tissue. Barely detectable amounts of the mRNAs were present in liver. Based on the structural features, ligand specificity, and tissue expression of the mRNAs, we suggest that this VLDL receptor may mediate uptake of apolipoprotein E-containing lipoproteins enriched with triglyceride in nonhepatic tissues that are active in fatty acid metabolism.     

氧化修饰LDL与动脉粥样硬化

动脉粥样硬化（Ａｔｈｅｒｏｓｃｌｅｒｏｓｉｓ,ＡＳ）的发病机理十分复杂,至今没有完全阐明,因而对ＡＳ的防治缺乏有效的措施。近２０年来,大量的研究认为氧化修饰的ＬＤＬ（Ｏｘ－ＬＤＬ）在ＡＳ发生和发展中起着十分重要的作用。随着对Ｏｘ－ＬＤＬ引起ＡＳ作用的深入了解,国外学者已经开始将抗氧化剂用于抗ＡＳ发展的研究。本文将综述Ｏｘ－ＬＤＬ在ＡＳ发展中的作用和抗氧化剂防治ＡＳ的进展。　　一、ＬＤＬ氧化机制最初的ＬＤＬ氧化可能是发生在ＬＤＬ中不饱和脂肪酸的双键位置。在氧自由基作用下,脂肪酸失去Ｈ＋形成含二烯基…     

Effects of cholesterol-lowering treatments on oxidative modification of plasma intermediate density lipoprotein plus low density lipoprotein fraction in Type 2 diabetic patients

To investigate the normalization of enhanced oxidative modification of the lipoprotein such as increased lysophosphatidylcholine (LPC) and lipid hydroperoxide (LPO) contents in diabetic subjects, we studied the effect of cholesterol-lowering treatment on those parameters in 24 hypercholesterolemic Type 2 diabetic patients. Those patients were randomly assigned to two treatment groups, such as 12 patients treated with pravastatin 10 mg daily and 12 patients treated with probucol 500 mg daily for 8 weeks. Characteristics of the patients including age, gender, body mass index (BMI), smoking habit, modality of diabetic treatment and the glycemic control state were comparable between the two groups. LPC content in the lipoprotein fractions obtained from 24 patients with Type 2 diabetes mellitus was significantly higher than that of non-diabetic control subjects. The abnormality was improved to the control level after a significant improvement of serum cholesterol levels following 8 week-treatments with either probucol or pravastatin without any change in glycemic control (P < 0.025). Furthermore, increased LPO content in the lipoprotein fraction in those diabetics was also significantly (P < 0.0025) improved by the probucol treatment and tended to be improved by pravastatin treatment (P = 0.06). LPC contents in the lipoprotein fraction was positively correlated with LPO contents before cholesterol-lowering treatments (r = 0.41, P < 0.05). These results indicate that cholesterol-lowering treatments effectively reduce oxidative modification of the lipoprotein fraction containing intermediate density lipoprotein (IDL) and low density lipoprotein (LDL) in hypercholesterolemic Type 2 diabetic patients.     

Common variants of apolipoprotein A-IV differ in their ability to inhibit low density lipoprotein oxidation

Apolipoprotein A-IV (apoA-IV) inhibits lipid peroxidation, thus demonstrating potential anti-atherogenic properties. The aim of this study was to investigate how the inhibition of low density lipoprotein (LDL) oxidation was influenced by common apoA-IV isoforms. Recombinant wild type apoA-IV (100 microg/ml) significantly inhibited the oxidation of LDL (50 microg protein/ml) by 5 microM CuSO(4) (P<0.005), but not by 100 microM CuSO(4), suggesting that it may act by binding copper ions. ApoA-IV also inhibited the oxidation of LDL by the water-soluble free-radical generator 2,2'-azobis(amidinopropane) dihydrochloride (AAPH; 1 mM), as shown by the two-fold increase in the time for half maximal conjugated diene formation (T(1/2); P<0.05) suggesting it can also scavenge free radicals in the aqueous phase. Compared to wild type apoA-IV, apoA-IV-S347 decreased T(1/2) by 15% (P=0.036) and apoA-IV-H360 increased T(1/2) by 18% (P=0.046). All apoA-IV isoforms increased the relative electrophoretic mobility of native LDL, suggesting apoA-IV can bind to LDL and acts as a site-specific antioxidant. The reduced inhibition of LDL oxidation by apoA-IV-S347 compared to wild type apoA-IV may account for the previous association of the APOA4 S347 variant with increased CHD risk and oxidative stress.