In vitro cell injury by oxidized low density lipoprotein involves lipid hydroperoxide-induced formation of alkoxyl, lipid, and peroxyl radicals.

Abstract

Mounting evidence supports current theories linking lipoprotein oxidation to atherosclerosis. We sought the cellular biochemical mechanism by which oxidized LDL inflicts cell injury. Inhibitors of candidate pathways of cell death were used to treat human fibroblast target cells exposed to oxidized LDL. Ebselen, which degrades lipid hydroperoxides, inhibited oxidized LDL toxicity, consistent with our recent report that 7f8-hydroperoxycholesterol (7fi-OOH chol) is the major cytotoxin of oxidized LDL. Intracellular chelation of metal ions inhibited, while preloading cells with iron enhanced, toxicity. Inhibition of oxidized LDL and 7/B-OOH chol toxicity by 2-keto-4-thiolmethyl butyric acid, a putative alkoxyl radical scavenger and by vitamin E, probucol and diphenylphenylenediamine, putative scavengers of peroxyl radicals was consistent with the involvement of these radicals in the lethal sequence. Cell death was thus postulated to occur due to lipid peroxidation via a sequence involving lipid hydroperoxide-induced, iron-mediated formation of alkoxyl, lipid, and peroxyl radicals. Pathways involving other reactive oxygen species, new protein synthesis, or altered cholesterol metabolism were considered less likely, since putative inhibitors failed to lessen toxicity. Understanding the mechanism of cell injury by oxidized LDL and its toxic moiety, 7J3-OOH chol, may indicate specific interventions in the cell injury believed to accompany vascular lesion development. (J. Clin. Invest. 1995Invest. . 96:1866Invest. -1873

Uptake of Oxidized LDL by Macrophages Results in Partial Lysosomal Enzyme Inactivation and Relocation

“…It has even been suggested that cholesterol might be a stabilizer of lysosomal membranes in cells of atherosclerotic lesions, thus preventing the relocation of lysosomal enzymes. 33 However, based on our data and those of others, 34,35 we conclude that the effect of lipoprotein cholesterol on lysosomal stability is largely dependent on the physicochemical status of the former. If the lipids are oxidized, the resulting oxysterols, hydroperoxides, and their toxic carbonylic fragments may affect lysosomal enzymes and membranes, whereas nLDL or AcLDL would not.…”

Oxidized Low Density Lipoprotein and Lysophosphatidylcholine Stimulate Cell Cycle Entry in Vascular Smooth Muscle Cells

“…Cells made quiescent by 48 h of serum deprivation were exposed to oxidized LDL, lysoPC, or serum and [ 3 H]thymidine incorporation was measured as a function of time. Although as expected the response of cells to oxidized LDL or lysoPC was not of the same magnitude as that of cells exposed to serum (9), the time course of thymidine incorporation was the same as after serum stimulation; that is, the peak thymidine incorporation occurred at approximately 28 h from the addition of oxidized LDL, lysoPC, or serum (data not shown). Thus, cells responded temporally to oxidized LDL or lysoPC as they respond to known mitogens.…”

“…Oxidized LDL induces certain genes (1,2), suppresses others (3)(4)(5), alters cellular lipid metabolism (6), and injures cells (7)(8)(9). Because oxidized LDL has been shown to reside in arterial lesions in animals and humans (10), the alterations in cell function observed in vitro have been suspected of involvement in vascular lesion development (11)(12)(13).…”

“…LDL Preparation and Modification-LDL (1.019 g/ml Յ solvent density Յ 1.063 g/ml) was prepared by sequential ultracentrifugation from pooled, citrated human plasma according to a modification (8) of the method of Hatch and Lees (27). Oxidized LDL was prepared by dialyzing native LDL against isotonic saline (pH 7.0 -8.0) containing 5 M CuSO 4 at room temperature until the characteristic color change occurred from golden to yellow to translucent without color (9). The oxidation was stopped, and the lipoprotein bound metal ion was removed (28,29) by dialyzing against isotonic saline containing 0.5 mM EDTA at 4°C with at least three changes of dialysate.…”

Oxidized low-density lipoprotein (Ox-LDL) but not LDL aggravates the manifestations of experimental antiphospholipid syndrome (APS)

“…Several mechanisms have been proposed to explain this effect, including enhanced uptake of Ox-LDL by macrophages, chemotactic potential for circulating monocytes, inhibition of the motility of tissue macrophages, and alteration of the coagulation pathways [16,19]. Ox-LDL has been shown to be cytotoxic to various cells such as vascular cells, monocytes and macrophages [20,21] by mechanisms not yet sufficiently characterized. It does seem conceivable, however, that Ox-LDL may exert some of its deleterious effects, owing to its immunogenicity, with the resultant formation anti-Ox-LDL antibodies and generation of immune complexes.…”