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) in duces apoptosis in mature human monocyte-derived macrophages as assess ed by staining with fluorescein-isothiocyanate-labeled annexin V, by t erminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, a nd by staining of the 7A6 mitochondrial antigen. Oxidized LDL-induced apoptosis was dose and time dependent and clearly distinct from apopto sis induced by serum deprivation. Human autologous serum and lipoprote in-deficient human serum prevented apoptosis induced by oxidized LDL. Supplementation of serum-free culture medium with 25 mu M ascorbic or isoascorbic acid only partially protected macrophages from apoptosis, whereas dehydroascorbic acid (DHAA) completely inhibited apoptosis ind uced by either Cu2+- or AAPH-oxidized LDL. Apoptosis was also inhibite d by the structural analogue alloxan. Both cyclic multiketones dose-de pendently inhibited oxidized LDL-induced apoptosis with IC50 in the su bmicromolar 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 inc ubation with either ascorbic or isoascorbic acid there was no such red uction. Removal of free DHAA by gel filtration did not reverse the ina ctivation. Parameters of LDL oxidation such as electrophoretic mobilit y, alpha-tocopherol content, thiobarbituric-acid-reactive subtances an d lipid peroxide levels did not correlate to apoptotic activity. Also, binding and uptake of Texas-red-labeled oxidized LDL was not prevente d by DHAA. Dithiothreitol-treatment of oxidized LDL, however, reduced the apoptotic activity by 76%. Our results suggest that oxidized thiol s on apoB may be essential for the induction of apoptosis by oxidized LDL in human macrophages.