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 μM 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, a-tocopherol content, thiobarbituric-acid-reactive substances 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.