ROLE OF REACTIVE OXYGEN SPECIES IN THE SIGNALING CASCADE OF CYCLOSPORINE A-MEDIATED UP-REGULATION OF ENOS IN VASCULAR ENDOTHELIAL-CELLS

1 Cyclosporine A (CsA) increases eNOS mRNA expression in bovine cultur ed aortic endothelial cells (BAEC). As some effects of CsA may be medi ated by reactive oxygen species (ROS), present experiments were devote d to test the hypothesis that the CsA-induced eNOS up-regulation could be dependent on an increased synthesis of ROS. 2 CsA induced a dose-d ependent increase of ROS synthesis, with the two fluorescent probes us ed, DHR123 (CsA 1 mu M: 305+/-7% over control) and H(2)DCFDA (CsA 1 mu M: 178+/-6% over control). 3 Two ROS generating systems, xanthine plu s xanthine oxidase (XXO) and glucose oxidase (GO), increased the expre ssion of eNOS mRNA in BAEC, an effect which was maximal after 8 h of i ncubation (XXO: 168+/-21% of control values. GO: 208+/-18% of control values). The ROS-dependent increased eNOS mRNA expression was followed by an increase in eNOS activity. 4 The effect of CsA on eNOS mRNA exp ression was abrogated by catalase, and superoxide dismutase (SOD). In contrast, the antioxidant PDTC augmented eNOS mRNA expression, both in basal conditions and in the presence of CsA. 5 The potential particip ation of the transcription factor AP-1 was explored. Electrophoretic m obility shift assays were consistent with an increase in AP-1 DNA-bind ing activity in BAEC treated with CsA or glucose oxidase. 6 The presen t results support a role for ROS, particularly superoxide anion and hy drogen peroxide, as mediators of the CsA-induced eNOS mRNA up-regulati on. Furthermore, they situate ROS as potential regulators of gene expr ession in endothelial cells, both in physiological and pathophysiologi cal situations.