Ceruloplasmin enhances smooth muscle cell and endothelial cell-mediated low density lipoprotein oxidation by a superoxide-dependent mechanism

Cultured vascular smooth muscle cells (SMC) and endothelial cells (EC) stimulate low density lipoprotein (LDL) oxidation by free radical-mediated, transition metal-dependent mechanisms. The physiological source(s) of metal ions is not known; however, purified ceruloplasmin, a plasma protein containing 7 coppers, oxidizes LDL in vitro, We now show that ceruloplasmin also increases LDL oxidation by vascular cells, In metal ion-free medium, human ceruloplasmin increased bovine aortic SMC- and EC-mediated LDL oxidation by up to 30- and 15-fold, respectively, The maximal response was at 100-300 mu g ceruloplasmin/ml, a level at or below the unevoked physiological plasma concentration, Oxidant activity was dependent on protein structure as a specific proteolytic cleavage or removal of one of the seven ceruloplasmin copper atoms inhibited activity, Three lines of evidence indicated a critical role for cellular superoxide (O-2(radical anion)) in ceruloplasmin-stimulated oxidation, First, the rate of production of O-2(radical anion) by cells correlated with their rates of LDL oxidation, Second, superoxide dismutase effectively blocked ceruloplasmin-stimulated oxidation by both cell types, Finally, O-2(radical anion) production by SMC quantitatively accounted for the observed rate of LDL oxidation, To show this, the course of O-2(radical anion) production by SMC was simulated by repeated addition of xanthine and xanthine oxidase to culture medium under cell free conditions, Neither ceruloplasmin nor O-2(radical anion) alone increased LDL oxidation, but together they completely reconstituted the oxidation rate of ceruloplasmin-stimulated SMC, These results are the first to show that ceruloplasmin stimulates EC- and SMC-mediated oxidation of LDL and that cell-derived O-2(radical anion) accounts quantitatively for metal-dependent, free radical-initiated oxidation of LDL by these cells.