Dehydroascorbic acid prevents apoptosis induced by oxidized low-density lipoprotein in human monocyte-derived macrophages.
Human low-density lipoprotein (LDL) oxidized with Cu2+ or the radical generator 2,2'-azobis(2-methyl-propionamidine) hydrochloride (AAPH) induces apoptosis in mature human monocyte-derived macrophages as assessed by staining with fluorescein-isothiocyanate-labeled annexin V, by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, and by staining of the 7A6 mitochondrial antigen. Oxidized LDL-induced apoptosis was dose and time dependent and clearly distinct from apoptosis induced by serum deprivation. Human autologous serum and lipoprotein-deficient human serum prevented apoptosis induced by oxidized LDL. Supplementation of serum-free culture medium with 25 microM ascorbic or isoascorbic acid only partially protected macrophages from apoptosis, whereas dehydroascorbic acid (DHAA) completely inhibited apoptosis induced by either Cu2+- or AAPH-oxidized LDL. Apoptosis was also inhibited by the structural analogue alloxan. Both cyclic multiketones dose-dependently inhibited oxidized LDL-induced apoptosis with IC50 in the submicromolar range. Prior loading of macrophages with ascorbic acid did not prevent the induction of apoptosis. Apoptosis was reduced by more than 90% after treatment of oxidized LDL with DHAA, whereas after incubation with either ascorbic or isoascorbic acid there was no such reduction. Removal of free DHAA by gel filtration did not reverse the inactivation. Parameters of LDL oxidation such as electrophoretic mobility, alpha-tocopherol content, thiobarbituric-acid-reactive subtances and lipid peroxide levels did not correlate to apoptotic activity. Also, binding and uptake of Texas-red-labeled oxidized LDL was not prevented by DHAA. Dithiothreitol-treatment of oxidized LDL, however, reduced the apoptotic activity by 76%. Our results suggest that oxidized thiols on apoB may be essential for the induction of apoptosis by oxidized LDL in human macrophages.
Institute of Biochemistry, University of Basel, Switzerland. asmis@ubaclu.unibas.ch
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D000496:Alloxan / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D001205:Ascorbic Acid / Q000494:pharmacology; D016895:Culture Media, Serum-Free; D003683:Dehydroascorbic Acid / Q000494:pharmacology*; D004229:Dithiothreitol / Q000494:pharmacology; D006801:Humans; D008658:Inactivation, Metabolic; D008077:Lipoproteins, LDL / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects*; D009000:Monocytes / Q000187:drug effects*; D010084:Oxidation-Reduction
D016895:Culture Media, Serum-Free; D008077:Lipoproteins, LDL; C099252:oxidized low density lipoprotein; D000496:Alloxan; D001205:Ascorbic Acid; D004229:Dithiothreitol; D003683:Dehydroascorbic Acid