0359: Cyclosporine A prevents the induction of replicative senescence in cultured coronary artery endothelial cells: role of eNOS-derived NO and the p53/p21 and p16 pathways

Abstract

Clinical studies have indicated a higher incidence of heart failure and death following myocardial infarction with increasing age, and that cyclosporine A limits myocardial infarction size. Since aging is associated with the induction of vascular and endothelial senescence, an irreversible cell cycle arrest involving an increased activity of p53 and its downstream effector p21, and p16, and a reduced expression of endothelial nitric oxide synthase (eNOS), the possibility that cyclosporine A prevents endothelial senescence was evaluated using cultured porcine coronary artery endothelial cells, and, if so, the underlying mechanism was characterized. Replicative senescence was induced by sequential passaging of primary cultures of endothelial cells up to the fourth passage (P4). Endothelial senescence was assessed by senescence-associated β- galactosidase (SA-β-gal) activity, and protein expression by Western blot analysis. Passaging of cultures of endothelial cells was associated with a gradual increase in SA-β-gal activity, a down-regulation of eNOS protein and an up-regulation of p16, p21 and p53 protein level in cells from P1 to P4. Cyclosporine A prevented the increase in SA-β-gal activity at concentrations as low as 0.3 μg/ml. The eNOS inhibitor, L-N G -nitroarginine methyl ester, increased SA-β-gal activity in cells at P1, and prevented the protective effect of cyclosporine A in cells at P3 and P4. Cyclosporine A prevented the down-regulation of eNOS and the upregulation of p16, p21 and p53 in cells at P3. The present findings indicate that cyclosporine A delays endothelial cell replicative senescence most likely by preventing the down-regulation of eNOS expression and the up-regulation of the p53/p21 and p16 pathways leading to cell cycle arrest.