Activated human monocytes oxidize low-density lipoprotein by a lipoxygenase-dependent pathway

Monocyte-mediated oxidation of low-density lipoprotein (LDL) converts the lipoprotein to a potent cytotoxin. The oxidation process requires monocyte activation and requires superoxide anion since it can be blocked by superoxide dismutase. In this study, the requirement for lipoxygenase activity is shown, in that 1) inhibitors of lipoxygenase prevent the alteration of LDL, 2) copper (II) (3,5-diisopropylsalicylic acid), an agent shown to enhance lipoxygenase activity in a cell-free system, similarly enhances monocyte-mediated LDL alteration, and 3) the (3,5-diisopropylsalicylic acid)-enhanced monocyte-mediated modification of LDL can be completely blocked by inhibitors of lipoxygenase or by superoxide dismutase. These data suggest an integral role for monocyte lipoxygenase in the generation by activated monocytes of the extracellular superoxide anion that participates in the oxidation of LDL and the conversion of LDL to a cytotoxin. Monocyte-modified LDL may be a mediator in tissue damage that accompanies atherosclerosis or occurs at sites of inflammation.     

Lipoxygenase-mediated transformation of human low density lipoprotein to an oxidized and cytotoxic complex

We have been studying the mechanisms involved in the oxidative modification of low density lipoprotein (LDL) that lead to its transformation to a cytotoxic complex. Here we examine the direct effect-of soybean lipoxygenase (SLO), a 15-lipoxygenase, on normal human LDL. SLO oxidized LDL and rendered it cytotoxic; agents known to interfere with lipoxygenase activity inhibited this reaction. Enhancement of both the SLO-mediated LDL oxidation and the conversion of LDL to a cytotoxin was observed when either superoxide dismutase or copper (II) (3,5,-diisopropylsalicylic acid)2, both of which dismute superoxide anion, were included during the incubation of SLO with LDL. In contrast, catalase inhibited this reaction in the presence or absence of agents that dismute superoxide anion. Thus, purified lipoxygenase can mediate LDL modification and superoxide anion inhibits this reaction, Furthermore, H2O2 is essential for SLO-mediated LDL oxidation and conversion of LDL to a cytotoxin.     

In vitro knockout of human p47phox blocks superoxide anion production and LDL oxidation by activated human monocytes

We previously reported that superoxide dismutase (SOD) blocked human monocyte oxidation of LDL and therefore concluded that superoxide anion (O(2)(.-)) was required for oxidation. Others, however, have suggested that SOD may inhibit by mechanisms alternative to the dismutation of O(2)(.-). This study definitively addresses the involvement of O(2)(.-) in monocyte oxidation of LDL. Using an antisense ODN designed to target p47phox mRNA, we found that treatment of monocytes with antisense ODN caused a substantial and selective decrease in expression of p47phox protein, whereas sense ODN was without effect. Corresponding functional assays demonstrated that antisense ODN inhibited production of O(2)(.-). As sense ODN caused no inhibition of O(2)(.-) production, these results suggested that inhibition of p47phox expression caused reduction in O(2)(.-) production. Evaluation of the contribution of O(2)(.-) production to monocyte-mediated oxidation of LDL lipids confirmed that O(2)(.-) production is required for LDL lipid oxidation as antisense ODN treatment significantly inhibited LDL oxidation whereas sense ODN treatment caused no inhibition. This is the first report of the reduction of NADPH oxidase activity in intact human monocytes by directly targeting the mRNA of a significant member of this enzyme complex. Our results provide convincing data that O(2)(.-) is indeed required for monocyte-mediated LDL oxidation.     

Interactions of nitric oxide and peroxynitrite with low-density lipoprotein

Nitric oxide (*NO) is a free radical species that diffuses and concentrates in the hydrophobic core of low-density lipoprotein (LDL) to serve as a potent inhibitor of lipid oxidation processes. Peroxynitrite (PN), the product of the diffusion-limited reaction between *NO and superoxide (O2*-) represents a relevant mediator of oxidative modifications in LDL. The focus of this review is the analysis of interactions between *NO and PN and its secondary reactions with oxygen radicals on LDL oxidation, which are relevant in the development of the early steps as well as progression of atherosclerosis. We propose that the balance between rates of PN and *NO production, which greatly depends on oxidative stress processes within the vascular wall, will critically determine the final extent of oxidative LDL modifications leading or not to scavenger receptor-mediated LDL uptake and foam cell formation.     

Oxidative modification of low density lipoprotein (LDL) by activated human monocytes and the cell lines U937 and HL60

Summary Human peripheral blood monocytes, upon activation, have the capacity to oxidize low density lipoprotein (LDL) and render the LDL toxic to cultured cells. Previous studies by our laboratory indicate that this process is mediated by free radicals in that it can be prevented by addition of free radical scavengers and antioxidants during the incubation of monocytes with LDL. Here we report that optimal modification of LDL by monocytes was influenced by media composition. In the absence of added metal ions, oxidation was distinctly dependent on the concentration of monocytes as well as LDL concentration. Exposure of monocytes to lipopolysaccharide or stimulation of phagocytosis by opsonized zymosan resulted in marked enhancement of LDL oxidation compared to other activating agents. After exposure to activated monocytes, lipid oxidation products in the supernatant were found both in a high molecular weight fraction containing LDL (>30 000 Daltons) and in a lipoprotein-free, low molecular weight fraction (<30 000 Daltons), yet only the high molecular weight, LDL-containing fraction was toxic to target cells. In addition, human myelomonocytic cell lines U937 and HL60 were shown to mediate oxidation of LDL. As with monocytes, exposing these cells to opsonized zymosan caused the level of LDL oxidation to be significantly enhanced. These findings offer further insight into the mechanisms of monocyte-mediated oxidation of lipoproteins and will facilitate studies investigating the role of monocyte-modified LDL in tissue injury. This project was funded by grants form the American Heart Association-Northeast Ohio Affiliate and the National Institutes of Health, Bethesda, MD (HL-29582).     

The participation of nitric oxide in cell free- and its restriction of macrophage-mediated oxidation of low-density lipoprotein.

The potential role of nitric oxide radical (NO .) in macrophage-mediated oxidation and conversion of human low density lipoprotein (LDL) to a high-uptake form was examined by exposing LDL to aerobic solutions of either NO . or 3-morpholino-sydnonimine-hydrochloride (SIN-1, a compound that spontaneously forms NO . and superoxide anion radical) or to mouse peritoneal macrophages in the presence and absence of modulators of cellular NO . synthesis. Incubation with NO . alone caused oxidation of LDL's ubiquinol-10 and accumulation of small amounts of lipid hydroperoxides, but failed to form any high-uptake ligand for endocytosis by macrophages and did not alter the LDL particle charge or the integrity of apoB. Exposure of LDL to SIN-1 resulted in complete consumption of all antioxidants and substantial formation of lipid hydroperoxides, but again had little effect on the lipoprotein particle charge or generation of high-uptake form. Preincubation of macrophages with interferon-gamma increased the cells ability to generate reactive nitrogen metabolites. The extent of cell-mediated oxidation of LDL and the generation of high-uptake LDL was substantial in resident cells in which NO . synthesis was barely detectable, depressed in cells active in NO . synthesis and restored when NO . synthesis was suppressed by the arginine analogue, NMMA. These results suggest that, while together with superoxide anion radical, NO . can oxidize LDL, its synthesis is not required for macrophage-mediated oxidation of LDL in vitro; rather it exerts a protective role in preventing oxidative LDL modification by macrophages.     

Cell-mediated LDL oxidation: the impact of transition metals and transferrin

Activated monocytes release oxygen radicals by respiratory burst and oxidative damage can be accelerated by transition metals. We investigated the cell-mediated and metal-catalysed in vitro oxidation of low-density lipoproteins (LDL), as well as the impact of the metal-binding protein transferrin (Tf). LDL oxidation was measured by monitoring the increase in fluorescence (350/440 nm excitation/emission). Maximal respiratory burst by U937 cells was achieved after 96 h differentiation with retinoic acid and dihydroxyvitamin D3 followed by stimulation with opsonised zymosan. Addition of activated cells resulted in the LDL oxidation, even in the absence of transition metals. Moreover, activated cells greatly enhanced metal-catalysed oxidative modifications, especially in the presence of copper. By binding metals, Tf was able to strongly impair this process. In conclusion, by generating oxygen radicals, activated U937 cells were able to oxidise LDL. The oxidising process was most pronounced in the presence of copper and could be blocked by Tf.     

Role of superoxide in endothelial-cell modification of low-density lipoproteins

Cultured endothelial cells and arterial smooth muscle cells have been shown to modify LDL in a way that leads to rapid uptake by macrophages. Previous studies have demonstrated that this modification involves free radical peroxidation of LDL, and that the role of the cells was to accelerate oxidation under conditions where it otherwise would occur slowly. The objective of the present study was to determine whether the modification was mediated by oxygen-derived free radicals, and whether the ability of a given cell type of line to modify LDL was related to its secretion rate of O2- or H2O2. The results showed that modification required the presence of oxygen, and could be specifically inhibited by superoxide dismutase but not by catalase or by mannitol, a hydroxyl radical scavenger. Rabbit aortic endothelial cells, rabbit arterial smooth muscle cells, monkey arterial smooth muscle cells and human skin fibroblasts were all found to modify LDL, and all of these cell types generated more O2- (superoxide dismutase-inhibitable cytochrome c reduction) than a line of bovine aortic endothelial cells that did not modify LDL. The content of superoxide dismutase and catalase was higher in bovine aortic endothelial cells than in the cell lines that modified LDL, but glutathione peroxidase levels were not different. It was concluded that cells that were capable of modifying LDL produced superoxide or a substance that could be converted to superoxide in the medium, and that superoxide was an important, though possibly indirect, mediator of the modification of LDL by cells.     

The participation of nitric oxide in cell free- and its restriction of macrophage-mediated oxidation of low-density lipoprotein

The potential role of nitric oxide radical (NO ·) in macrophage-mediated oxidation and conversion of human low density lipoprotein (LDL) to a high-uptake form was examined by exposing LDL to aerobic solutions to either NO · or 3-morpholinosydnonimine-hydrochloride (SIN-1, a compound that spontaneously forms NO · and superoxide anion radical) or to mouse peritoneal macrophages in the presence and absence of modulators of cellular NO · synthesis. Incubation with NO · alone caused oxidation of LDL's ubiquinol-10 and accumulation of small amounts of lipid hydroperoxidases, but failed to form any high-uptake ligand for endocytosis by macrophages and did not alter the LDL particle charge or the integrity of apoB. Exposure of LDL to SIN-1 resulted in complete consumption of all antioxidants and substantial formation of lipid hydroperoxidases, but again had little effect on the lipoprotein particle charge or generation of high-uptake form. Preincubation of macrophages with interferon-γ increased the cells ability to generate reactive nitrogen metabolites. The extent of cell-mediated oxidation of LDL and the generation of high-uptake LDL was substantial in resident cells in which NO · synthesis was barely detectable, depressed in cells active in NO · synthesis and restored when NO · synthesis was suppressed by the arginine analogue, NMMA. These results suggest that, while longer with superoxide anion radical, NO · can oxidize LDL, its synthesis is not required for macrophage-mediated oxidation of LDL in vitro; rather it exerts a protective role in preventing oxidative LDL modification by macrophages.     

Oxidized low-density lipoprotein induces calcium influx in polymorphonuclear leukocytes

Polymorphonuclear leukocytes (PMN) have been suggested to play a role in atherosclerosis, but intracellular signaling after stimulation with oxidized low-density lipoprotein (LDL) is unknown. We investigated mechanistic aspects of oxidized LDL-induced superoxide production by human PMN, with special emphasis on intracellular Ca(2+) concentration ([Ca(2+)](i)). Oxidized LDL, but not native LDL, evoked an early but sustained increase in [Ca(2+)](i) and a delayed production of superoxide. The increase in [Ca(2+)](i) could be reduced by fucoidan and completely prevented by U73122, suggesting involvement of the scavenger receptor and coupling to the phospholipase C signal transduction pathway. Furthermore, we provide evidence that the increase in [Ca(2+)](i) partly results from protein kinase C-dependent Ca(2+) influx. The relevance of this Ca(2+) entry for oxidized LDL-stimulated effects is illustrated by the finding that superoxide production was markedly reduced in the absence of external Ca(2+). Finally, inhibition of phagocytosis by cytochalasin B abolished oxidized LDL-stimulated superoxide production without affecting, however, the Ca(2+) mobilization. These effects of oxidized LDL on [Ca(2+)](i) and on respiratory burst of PMN may underlie the occurrence of elevated levels of [Ca(2+)](i) of resting PMN in hypercholesterolemia and represent a mechanism by which PMN can amplify processes in the early phase of atherosclerosis.     

Anti- and pro-oxidant effects of urate in copper-induced low-density lipoprotein oxidation.

We reported earlier that urate may behave as a pro-oxidant in Cu2+-induced oxidation of diluted plasma. Thus, its effect on Cu2+-induced oxidation of isolated low-density lipoprotein (LDL) was investigated by monitoring the formation of malondialdehyde and conjugated dienes and the consumption of urate and carotenoids. We show that urate is antioxidant at high concentration but pro-oxidant at low concentration. Depending on Cu2+ concentration, the switch between the pro- and antioxidant behavior of urate occurs at different urate concentrations. At high Cu2+ concentration, in the presence of urate, superoxide dismutase and ferricytochrome c protect LDL from oxidation but no protection is observed at low Cu2+ concentration. The use of Cu2+ or Cu+ chelators demonstrates that both copper redox states are required. We suggest that two mechanisms occur depending on the Cu2+ concentration. Urate may reduce Cu2+ to Cu+, which in turn contributes to formation. The Cu2+ reduction is likely to produce the urate radical (UH.-). It is proposed that at high Cu2+ concentration, the reaction of UH.- radical with generates products or intermediates, which trigger LDL oxidation. At low Cu2+ concentration, we suggest that the Cu+ ions formed reduce lipid hydroperoxides to alkoxyl radicals, thereby facilitating the peroxidizing chain reaction. It is anticipated that these two mechanisms are the consequence of complex LDL-urate-Cu2+ interactions. It is also shown that urate is pro-oxidant towards slightly preoxidized LDL, whatever its concentration. We reiterate the conclusion that the use of antioxidants may be a two-edged sword.     

Myeloperoxidase/nitrite-mediated lipid peroxidation of low-density lipoprotein as modulated by flavonoids

In the presence of a H(2)O(2)-generating system, myeloperoxidase (MPO) caused conjugated diene formation in low-density lipoprotein (LDL), indicating lipid peroxidation which was dependent on nitrite but not on chloride. The oxidation of LDL was inhibited by micromolar concentrations of flavonoids such as (-)-epicatechin, quercetin, rutin, taxifolin and luteolin, presumably via scavenging of the MPO-derived NO(2) radical. The flavonoids served as substrates of MPO leading to products with distinct absorbance spectra. The MPO-catalyzed oxidation of flavonoids was accelerated in the presence of nitrite.     

Urate attenuates oxidation of native low-density lipoprotein by hypochlorite and the subsequent lipoprotein-induced respiratory burst activities of polymorphonuclear leukocytes

Oxidation converts native low-density lipoprotein (LDL) into a signal molecule promoting inflammatory processes during atherogenesis. The exact contribution of different antioxidants in prevention of LDL oxidation is not known. Uric acid efficiently scavenges oxidants including hypochlorite. We investigated the effect of different urate concentrations (25-500 mol/l) on the oxidation of isolated native LDL by sodium hypochlorite (1000 mol/l). While relative electrophoretic mobility declined continuously with increasing urate concentrations in the oxidation medium, lipid peroxidation as measured by TBARS was blunted only at high molar urate/NaOCl ratios. By decreasing oxidative modifications, urate dose-dependently (beginning with a urate/NaOCl ratio of 1:40) diminished stimulatory effects of oxidized LDL on the respiratory burst of resting polymorphonuclear leukocytes (PMNL). Protecting effects of urate against the proinflammatory action of oxidized LDL on activated cells were evident only at a molar urate/NaOCl ratio of 1:2 suggesting different sensitivities of PMNL to LDL oxidation state in dependence on their activity state.     

Plasma thiols inhibit hemin-dependent oxidation of human low-density lipoprotein

Oxidative modification of human low-density lipoprotein (LDL) renders it atherogenic. Previous studies demonstrated that plasma thiols promote oxidation of LDL by free ferric iron (Fe3+). The current study investigated effects of plasma thiols on oxidation of LDL by hemin, a physiological Fe3+-protoporphyrin IX complex thought to be capable of initiating LDL oxidation in vivo. In contrast to free Fe3+ which is incapable of oxidizing LDL in the absence of an exogenous reductant, hemin readily promoted LDL oxidation. During incubation of LDL (0.2 mg of protein/ml) with hemin (10 microM) at 37 degrees C for 6 h, thiobarbituric acid-reactive substances (TBARS), a marker of lipid oxidation, increased from 0.3 (+/-0.1) nmol/mg of LDL protein to a maximal concentration of 45.8 (+/-5.2) nmol/mg of LDL protein. Under the same experimental conditions, lipid-conjugated dienes, another marker of lipid oxidation, increased from non-detectable to near-maximal levels of 78-187 nmol/mg of LDL protein, and lipoprotein polyunsaturated fatty acyl-containing cholesteryl ester content decreased to 15-36% of that present in native (i.e. unoxidized) LDL. Continued incubation of LDL with hemin for up to 24 h resulted in no further significant alterations in lipoprotein levels of TBARS, lipid-conjugated dienes, and cholesteryl esters. In addition to these chemical modifications indicative of lipoprotein oxidation, agarose gel electrophoretic analysis indicated that exposure of LDL to hemin resulted in conversion of the lipoprotein to an atherogenic form as evidenced by its increased anodic electrophoretic mobility. Addition of physiological concentrations of plasma thiols (either cysteine, homocysteine or reduced glutathione; 1-100 microM, each) inhibited hemin-mediated oxidation of LDL. Thus, whereas the maximal TBARS concentration was achieved following 6 h of incubation of LDL with hemin alone, addition of thiol extended the time required to attain maximal TBARS concentration to > or = 12 h. Similar antioxidant effects of thiols on formation of lipid-conjugated dienes, loss of cholesteryl esters, and lipoprotein anodic electrophoretic mobility were also observed. However, all thiols were not equally effective at inhibiting hemin-dependent LDL oxidation. Thus, whereas reduced glutathione was most effective at inhibiting hemin-dependent LDL oxidation, an intermediate effect was observed for homocysteine, and cysteine was least effective. The inhibition of hemin-mediated LDL oxidation by plasma thiols reported here confirms a previous observation that, under certain conditions, thiols can function as antioxidants, but contrasts with the previously documented pro-oxidant effect of the same thiols on oxidation of LDL by free Fe3+. These contrasting effects of plasma thiols on hemin- and free Fe3+-mediated LDL oxidation indicate that, in vivo, the ability of thiols to function as either anti- or pro-oxidants during LDL oxidation may, at least in part, be determined by the type of oxidant stress to which the lipoprotein is exposed.     

Human neutrophils oxidize low-density lipoprotein by a hypochlorous acid-dependent mechanism: the role of vitamin C

Oxidatively modified low-density lipoprotein (LDL) has been strongly implicated in the pathogenesis of atherosclerosis. Peripheral blood leukocytes, such as neutrophils, can oxidize LDL by processes requiring superoxide and redox-active transition metal ions; however, it is uncertain whether such catalytic metal ions are available in the artery wall. Stimulated leukocytes also produce the reactive oxidant hypochlorous acid (HOCl) via the heme enzyme myeloperoxidase. Since myeloperoxidase-derived HOCl may be a physiologically relevant oxidant in atherogenesis, we investigated the mechanisms of neutrophil-mediated LDL modification and its possible prevention by the antioxidant ascorbate (vitamin C). As a sensitive marker of LDL oxidation, we measured LDL thiol groups. Stimulated human neutrophils (5x10(6) cells/ml) incubated with human LDL (0.25 mg protein/ml) time-dependently oxidized LDL thiols (33% and 79% oxidized after 10 and 30 min, respectively). Supernatants from stimulated neutrophils also oxidized LDL thiols (33% oxidized after 30 min), implicating long-lived oxidants such as N-chloramines. Experiments using specific enzyme inhibitors and oxidant scavengers showed that HOCl, but not hydrogen peroxide nor superoxide, plays a critical role in LDL thiol oxidation by neutrophils. Ascorbate (200 microM) protected against neutrophil-mediated LDL thiol oxidation for up to 15 min of incubation, after which LDL thiols became rapidly oxidized. Although stimulated neutrophils accumulated ascorbate during oxidation of LDL, pre-loading of neutrophils with ascorbate did not attenuate oxidant production by the cells. Thus, activated neutrophils oxidize LDL thiols by HOCl- and N-chloramine-dependent mechanisms and physiological concentrations of vitamin C delay this process, most likely due to scavenging of extracellular oxidants, rather than by attenuating neutrophil oxidant production.     

Incubation of endothelial cells in a superoxide-generating system: impaired low-density lipoprotein receptor-mediated endocytosis

Endothelial cells (EC) of blood vessels are submitted to oxidative stress under various circumstances. These conditions may modify EC functions; therefore, in the present work we have studied the receptor-mediated endocytosis of low-density lipoproteins (LDL) and malondialdehyde-modified LDL by the LDL receptor and the "scavenger" receptor, respectively, in cultured human umbilical vein EC after short (0-120 minutes) incubations in a superoxide anion (O2-) generating system. In both receptor-mediated processes, the oxidative stress produces a significant decrease at four different LDL concentrations (5-50 micrograms/ml) after 120 minutes of oxidation. On the other hand, the fluid-phase endocytosis of sucrose by EC seems to be stimulated by these conditions. Furthermore, incorporation of antioxidant enzymes in the O2- -producing system shows that H2O2 is an obligatory intermediate in order to produce the effect on the receptor-mediated processes. Hypotheses concerning the mechanisms involved in the modifications of endocytotic processes and their implications in vivo are discussed.     

Ibuprofen protects low density lipoproteins against oxidative modification

Oxidative modification of LDL by vascular cells has been proposed as the mechanism by which LDL become atherogenic. The effect of ibuprofen on LDL modification by copper ions, monocytes and endothelial cells was studied by measuring lipid peroxidation products. Ibuprofen inhibited LDL oxidation in a dose-dependent manner over a concentration range of 0.1 to 2.0 mM. Ibuprofen (2 mM, 100 microg/ml LDL) reduced the amount of lipid peroxides formed during 2 and 6 h incubation in the presence of copper ions by 52 and 28%, respectively. Weak free radical scavenging activity of ibuprofen was observed in the DPPH test. The protective effect of ibuprofen was more marked when oxidation was induced by monocytes or endothelial cells. Ibuprofen (1 mM, 100 microg/ml LDL) reduced the amount of lipid peroxides generated in LDL during monocyte-mediated oxidation by 40%. HUVEC-mediated oxidation of LDL in the absence and presence of Cu2+ was reduced by 32 and 39%, respectively. More lipid peroxides appeared when endothelial cells were stimulated by IL-1beta or TNFalpha and the inhibitory effect of ibuprofen in this case was more pronounced. Ibuprofen (1 mM, 100 microg/ml LDL) reduced the amount of lipid peroxides formed during incubation of LDL with IL-1beta-stimulated HUVEC by 43%. The figures in the absence and presence of Cu2+ for HUVEC stimulated with TNFalpha were 56 and 59%, respectively. To assess the possibility that ibuprofen acts by lowering the production rate of reactive oxygen species, the intracellular concentration of H2O2 was measured. Ibuprofen (1 mM) reduced intracellular production of hydrogen peroxide in PMA-stimulated mononuclear cells by 69%. When HUVEC were stimulated by IL-1beta or TNFalpha the reduction was 62% and 66%, respectively.     

Inducible nitric oxide synthase knockout mouse macrophages disclose prooxidant effect of interferon-gamma on low-density lipoprotein oxidation.

To test our hypothesis that interferon-gamma (IFN-gamma) has a direct prooxidant effect on macrophage-mediated LDL oxidation behind its antioxidant effect via induction of inducible nitric oxide synthase (iNOS), we incubated LDL with wild-type (iNOS(+/+)) or iNOS knockout mouse (iNOS(-/-)) macrophages preincubated with IFN-gamma or IFN-gamma plus lipopolysaccharide (IFN-gamma/LPS) for 24 h. LDL oxidation was measured in terms of formation of thiobarbituric acid reactive substances (TBARS) and electrophoretic mobility. Thiol production, nitrite production, and superoxide production from macrophages were measured by using Ellman's assay, the Griess reagent, and the SOD-inhibitable cytochrome c reduction method, respectively. IFN-gamma alone or combined with LPS induced iNOS expression and increased nitrite production in iNOS(+/+) macrophages, but not in iNOS(-/-) macrophages. TBARS formation from LDL was suppressed in IFN-gamma- and IFN-gamma/LPS-treated iNOS(+/+) macrophages but was increased in IFN-gamma-treated iNOS(-/-) macrophages. In the presence of N(G)-monomethyl-l-arginine (l-NMMA), a NOS inhibitor, the suppressive effect of IFN-gamma and IFN-gamma/LPS was abolished and TBARS formation was even increased to a level above that of untreated iNOS(+/+) macrophage. NOC 18, an NO donor, dose dependently inhibited macrophage-mediated LDL oxidation. IFN-gamma increased superoxide and thiol productions in both types of macrophages. We conclude that IFN-gamma promotes macrophage-mediated LDL oxidation by stimulating superoxide and thiol production under conditions where iNOS-catalyzed NO release is restricted.     

Chromium and human low-density lipoprotein oxidation

Chromium is a catalytic metal able to foster oxidant damage, albeit its capacity to induce human LDL oxidation is to date unkown. Thus, we have investigated whether trivalent and hexavalent chromium, namely Cr(III) and Cr(VI), can induce human LDL oxidation. Cr(III) as CrCl₃ is incapable of inducing LDL oxidation at pH 7.4 or 4.5. However, Cr(III), specifically at physiological pH of 7.4 and in the presence of phosphates, causes an absorbance increase at 234 resembling a spectrophotometric kinetics of LDL oxidation with a lag- and propagation-like phase. In this regard, it is conceivable that peculiar Cr(III) forms such as Cr(III) hydroxide and, especially, Cr(III) polynuclear hydroxocomplexes formed at pH 7.4 interact with phosphates generating species with an intrinsic absorbance at 234 nm, which increases over time resembling a spectrophotometric kinetics of LDL oxidation. Cr(VI), as K₂Cr₂O₇, can instead induce substantial human LDL oxidation at acidic pH such as 4.5, which is typical of the intracellular lysosomal compartment. LDL oxidation is related to binding of Cr(VI) to LDL particles with quenching of the LDL tryptophan fluorescence, and it is inhibited by the metal chelators EDTA and deferoxamine, as well as by the chain-breaking antioxidants butylated hydroxytoluene and probucol. Moreover, Cr(VI)-induced LDL oxidation is inhibited by mannitol conceivably by binding Cr(V) formed from LDL-dependent Cr(VI) reduction and not by scavenging hydroxyl radicals (OH); indeed, the OH scavengers sodium formate and ethanol are ineffective against Cr(VI)-induced LDL oxidation. Notably, heightened LDL lipid hydroperoxide levels and decreased LDL tryptophan fluorescence occur in Cr plating workers, indicating Cr-induced human LDL oxidation in vivo. The biochemical, pathophysiological and clinical implications of these novel findings on chromium and human LDL oxidation are discussed.     

Mechanism of low-density lipoprotein oxidation by hemoglobin-derived iron

Excellular hemoglobin is an extremely active oxidant of low-density lipoproteins (LDL), a phenomenon explained so far by different mechanisms. In this study, we analyzed the mechanism of met-hemoglobin oxidability by comparing its mode of operation with other hemoproteins, met-myoglobin and horseradish peroxidase (HRP) or with free hemin. The kinetics of met-hemoglobin activity toward LDL lipids and protein differed from that of met-myoglobin and HRP, both quantitatively and qualitatively. Those differences were further clarified by analyzing heme transfer from the above-mentioned hemoproteins to LDL. It appeared that met-hemoglobin transferred most of its hemin to LDL, and the presence of H(2)O(2) accelerated the process. In contrast, met-myoglobin partially released hemin, but only in the presence of H(2)O(2), while HRP could not transfer heme at all. The minor amount of hemin transferred from met-myoglobin to LDL sufficed to trigger ApoB oxidation, forming covalent aggregates via inter-bityrosines. This indicated that heme bound to high affinity site(s) is responsible for oxidation. LDL components providing the sites were analyzed by binding heme-CO monomers to LDL. Soret spectra revealed that the high affinity site of monomeric hemin is located on the LDL protein, ApoB. The complex heme-CO-ApoB underwent instantaneous oxidation to hemin-ApoB, and the bound hemin then slowly disintegrated in conjunction with LDL oxidation. Hemopexin prevented LDL oxidation by trapping hemoprotein transferable heme. We concluded that met-hemoglobin exerts its oxidative activity on LDL via transfer of heme, which serves as a vehicle for iron insertion into the LDL protein, leading to formation of atherogenic LDL aggregates.