Human (THP-1) macrophages oxidize LDL by a thiol-dependent mechanism.

The oxidative modification of low-density lipoprotein by macrophages may be an important mechanism in the pathogenesis of atherosclerosis. The human monocytic leukaemic cell line THP-1, when stimulated with phorbol ester, shares many properties with human monocyte-derived macrophages. Oxidation of LDL by these cells was characterised by depletion of alpha-tocopherol, increases in thiobarbituric acid reactive substances and increases in electrophoretic mobility. The LDL particles were also converted to a form which increased accumulation of cholesteryl esters within macrophages. The oxidative mechanism appeared to be dependent upon the presence of thiols in the cellular medium. Oxidation of LDL by THP-1 macrophages, and production of thiols by these cells, were dependent upon the presence of L-cystine in the medium. Furthermore, cellular oxidation of LDL could be partially mimicked by the addition of cysteine to Hams F10 medium. Macrophage-independent oxidation of LDL, mediated by the addition of copper ions, was inhibited by cystine and cysteine in phosphate buffered saline, but not in Hams F10 medium. The glutathione content of THP-1 macrophages was also dependent upon the presence of cysteine or cystine in the medium, but inhibition of glutathione synthesis by buthionine sulfoximine did not prevent the production of thiols or the oxidation of LDL by THP-1 macrophages.

The resistance of low density lipoprotein to oxidation promoted by copper and its use as an index of antioxidant therapy.

The measurement ex vivo of the resistance of low density lipoprotein (LDL) to oxidation promoted by copper is now being used in surveys of human populations at risk of developing atherosclerosis. However, it is not known whether a relationship between LDL oxidisability measured in this way and the development of atherosclerotic lesions exists. Using Watanabe rabbits as a model of the disease, we have found that dietary supplementation with the antioxidants, probucol and alpha-tocopherol, increased the resistance of LDL isolated from small volumes of plasma to oxidation. The antioxidant effects of probucol incorporated into LDL through dietary supplementation were greater than when incorporated ex vivo. When dietary supplementation was extended to a period of three months, the well established anti-atherosclerotic effects of probucol were confirmed and a highly significant relationship between the probucol content of the LDL particle and the extent of the atherosclerotic lesion in the aorta emerged. These results suggest that the assessment of the resistance of LDL isolated from plasma to oxidation promoted by copper may reflect the response of the arterial atherosclerotic process to antioxidant therapy.

Oxidation of human low-density lipoprotein by soybean 15-lipoxygenase in combination with copper (II) or met-myoglobin.

The enzyme 15-lipoxygenase has been implicated in the oxidation of low-density lipoprotein (LDL) in human atherosclerotic lesions. The biochemical mechanism for this oxidative process is not fully understood, and the interaction of the lipoxygenase-modified lipoprotein with metals or metalloproteins has not been explored. In the present study we have used soybean lipoxygenase to model the interaction of the enzyme with LDL and show that a direct oxygenation of fatty acids occurs, including those esterified to cholesterol, with no lag phase or change in electrophoretic mobility of the LDL particle but with some depletion of alpha-tocopherol. The enzyme-dependent oxidation may involve propagation through the release of peroxyl radicals from its active site but appears to have no requirement for free iron or copper. When lipoxygenase-treated LDL is exposed to either copper (II) or metMb, a rapid oxidation process occurs, resulting in a marked decrease in resistance to oxidation and an increase in the rate of modification to a form with increased electrophoretic mobility. This effect was not seen if lipoxygenase-treated LDL was oxidized by SIN-1, a peroxynitrite donor that oxidizes LDL with no requirement for endogenous lipid hydroperoxides. We propose that a synergistic interaction may occur between the peroxides inserted into LDL as a consequence of the enzymatic action of lipoxygenase with haem proteins or copper, which decreases the potency of the endogenous antioxidants and enhances oxidation.

Human (THP-1) macrophages oxidize LDL by a thiol-dependent mechanism.

The oxidative modification of low-density lipoprotein by macrophages may be an important mechanism in the pathogenesis of atherosclerosis. The human monocytic leukaemia cell line THP-1, when stimulated with phorbol ester, shares many properties with human monocyte-derived macrophages. Oxidation of LDL by these cells was characterised by depletion of alpha-tocopherol, increases in thiobarbituric acid reactive substances and increases in electrophoretic mobility. The LDL particles were also converted to a form which increased accumulation of cholesteryl esters within macrophages. The oxidative mechanism appeared to be dependent upon the presence of thiols in the cellular medium. Oxidation of LDL by THP-1 macrophages, and production of thiols by these cells, were dependent upon the presence of L-cystine in the medium. Furthermore, cellular oxidation of LDL could be partially mimicked by the addition of cysteine to Hams F10 medium. Macrophage-independent oxidation of LDL, mediated by the addition of copper ions, was inhibited by cystine and cysteine in phosphate buffered saline, but not in Hams F10 medium. The glutathione content of THP-1 macrophages was also dependent upon the presence of cysteine or cystine in the medium, but inhibition of glutathione synthesis by buthionine sulfoximine did not prevent the production of thiols or the oxidation of LDL by THP-1 macrophages.

The role of alpha-tocopherol as a peroxyl radical scavenger in human low density lipoprotein.

It is thought that the oxidation of low density lipoprotein (LDL) plays a key role in the pathogenesis of atherosclerosis. It is well known that lipid peroxidation reactions are propagated by peroxyl radicals and it follows, therefore, that the capacity of an individual LDL particle to scavenge these oxidants may be an important indicator of its atherogenic potential. There are several components within LDL which scavenge peroxyl radicals including chain breaking antioxidants and amino acids on the protein. It is not clear at present which of these antioxidants is most important. In attempting to address the question we have used a simple method for the measurement of the total capacity of the LDL particle to scavenge peroxyl radicals. This assay depends upon the ability of antioxidants in LDL to inhibit the peroxyl radical dependent oxidation of luminol. We have found that approximately 80% of the antioxidant capacity of LDL, isolated from a number of donors, could be accounted for by the alpha-tocopherol present in the samples. We have compared these results with those obtained when the identical samples of LDL were oxidized with copper and found, as reported by others, a wide range in the susceptibility of the different LDL preparations to oxidation by this transition metal. We suggest that this variability is unlikely to be due to differences in the ability of an LDL particle to scavenge peroxyl radicals.

Pro-oxidant effects of lipoxygenase-derived peroxides on the copper-initiated oxidation of low-density lipoprotein.

It has been proposed that lipoxygenases, specifically 15-lipoxygenase, may play an important role in promoting the oxidation of low-density lipoprotein (LDL) in the artery wall. It is well known that peroxides are unstable in the presence of transition metals, decomposing to form the alkoxy and peroxy radicals, and so initiating lipid peroxidation. To test whether lipoxygenase-derived peroxides may promote the oxidation of LDL in the presence of copper, the lipoprotein was enriched with lipid peroxides derived from the enzymic action of 5- and 15-lipoxygenases on either linoleic or arachidonic acid. All of these products were found to promote oxidation, whereas the related hydroxy fatty acids had no effect. This suggests that lipoxygenase-derived peroxides associated with the LDL particle may promote peroxidation in the presence of a suitable transition metal catalyst. This result has implications both for the mechanism of the potential pro-oxidant action of lipoxygenases in vivo and for the ex vivo assessment of the oxidizability of LDL samples isolated from different donors.

Mechanisms contributing to ozone-induced bronchial hyperreactivity in guinea-pigs.

The effect of ozone (3 ppm, 15-120 min) on bronchial reactivity in the guinea-pig was studied. Ozone induced marked (6-250-fold) bronchial hyperreactivity (BHR) to a range of inhaled, but not intravenous bronchoconstrictors. The degree of BHR was related to the duration of prior ozone exposure. The glutathione redox status was shifted to a more oxidized state in lung after 120 min ozone treatment, although no changes were found in the energy status of lung tissue, as judged by the concentrations of adenosine phosphates. Ascorbic acid pretreatment prevented BHR induced by 30 min ozone exposure. Neutral endopeptidase inhibitors elicited BHR to both substance P and histamine, but did not further enhance bronchoconstriction to substance P after ozone exposure for 120 min. Neither mepyramine, fentanyl, indomethacin nor a 5-lipoxygenase inhibitor (BW B70C), given prior to ozone exposure prevented the induction of BHR to histamine. Atropine or bilateral vagotomy reduced BHR after a 120-min, but not 30-min exposure to ozone. We conclude that in the guinea-pig, ozone induces non-specific, route-dependent BHR by oxidative injury, reducing airway NEP activity and enhancing the cholinergic and peptidergic component to bronchoconstriction. Neither cyclooxygenase nor 5-lipoxygenase products appear to play a role in ozone-induced BHR in this animal model.

The simultaneous generation of superoxide and nitric oxide can initiate lipid peroxidation in human low density lipoprotein.

Oxidation of low density lipoprotein (LDL) has been shown to occur in the artery wall of atherosclerotic lesions in both animal models and human arteries. The oxidant(s) responsible for initiating this process are under intensive investigation and 15-lipoxygenase has been suggested in this context. Another possibility is that nitric oxide and superoxide, generated by cells present in the artery wall, react together to form peroxynitrite which decomposes to form the highly reactive hydroxyl radical. In the present study we have modelled the simultaneous generation of superoxide and nitric oxide by using the sydnonimine, SIN-1 and have investigated its effects on LDL. SIN-1 liberates both superoxide and nitric oxide during autooxidation resulting in the formation of hydroxyl radicals. We have demonstrated that superoxide generated by SIN-1 is not available to take part in a dismutation reaction since it reacts preferentially with nitric oxide. It follows, therefore, that during the autooxidation of SIN-1 little or no superoxide, or perhydroxyl radical will be available to initiate lipid peroxidation. We have shown that SIN-1 is capable of initiating the peroxidation of LDL and also converts the lipoprotein to a more negatively charged form. The SIN-1-dependent peroxidation of LDL is completely inhibited by superoxide dismutase which scavenges superoxide. Neither sodium nitroprusside or S-nitroso-N-acetyl penicillamine, which only produce nitric oxide, are able to modify LDL. These results are consistent with the hypothesis that a product of superoxide and nitric oxide could oxidize lipoproteins in the artery wall and so contribute to the pathogenesis of atherosclerosis in vivo.

Treatment of macrophages with oxidized low-density lipoprotein increases their intracellular glutathione content.

Macrophages derived from the human monocyte cell line THP-1 or isolated from the peritoneum of C3H/HEJ mice were incubated with oxidized low-density lipoprotein (LDL) and the total glutathione content (oxidized plus reduced) was measured. An initial depletion of glutathione was followed by an increase, such that after a period of 24 h the glutathione content has approximately doubled. This response required the oxidation of the lipid phase of the LDL molecule, since both native LDL and acetylated LDL had little effect on glutathione levels. The response of the cells to oxidized LDL was dependent on the extent of oxidative modification of the protein. It was also found that 4-hydroxynonenal had a similar effect on THP-1 cells, and we suggest that this or other aldehydes present in oxidized LDL causes the induction of glutathione synthesis in response to an initial oxidative stress and consequent glutathione depletion. In addition, we found that both cell types possess transferases and peroxidases capable of detoxifying aldehydes and peroxides. However, treatment of cells with oxidized LDL or 4-hydroxynonenal for a period of 24 h had no effect on the activities of these enzymes.

Oxidation of low-density lipoprotein and macrophage derived foam cells.

Copper-oxidized LDL has many of the characteristics of the modified LDL generated in the artery wall during the initial stages of atherosclerosis. It is not, however, a chemically defined species but shows significant variations in both its chemical composition and behaviour in biological systems depending upon the extent to which the peroxidation reaction has occurred (Fig. 1). Taking care to define the extent of LDL modification we have used this form of oxidized LDL to investigate the effects on the macrophage of this potentially toxic particle. This cell, in contrast to endothelial cells, appears to be particularly well adapted to detoxify lipid peroxidation products since it possesses glutathione peroxidases capable of metabolizing oxidized LDL and responds to oxidized LDL by increasing its GSH content. Acetylated LDL had little or no effect on GSH levels showing that lipid loading per se or recognition by the macrophage scavenger receptor is not sufficient to induce the synthesis of this antioxidant. We have confirmed the observation that oxidized LDL does not activate expression of the gene for TNF and raise the possibility that PGE2 produced by the cells and possibly during the oxidation of LDL may be the mediator suppressing the synthesis of this cytokine. Our results support the hypothesis that the lipid-laden macrophage does not contribute to an inflammatory response in the artery wall and imply a protective role for the macrophage in scavenging oxidized LDL.