Abstract 297: Crataegus Special Extract WS 1442 Retards Replicative Senescence in Coronary Artery Endothelial Cells: Role of eNOS-derived NO

Abstract

Endothelial cell senescence promotes endothelial dysfunction, which has been suggested to have a critical role in the initiation and/or progression of atherosclerosis, and also to contribute to the pathogenesis of age-associated vascular disorders. Endothelial senescence is characterized by an irreversible cell cycle arrest, which involves an increased activity of p53 and its downstream effector p21. Endothelial senescence is also associated with a decreased expression of endothelial nitric oxide synthase (eNOS). The present study has evaluated whether the Crataegus special extract WS ® 1442, a rich source of polyphenols and a potent inducer of eNOS activation, prevents replicative senescence of porcine coronary artery endothelial cells, and, if so, to elucidate the underlying mechanism. Replicative senescence was induced by sequential passaging of primary cultures of endothelial cells up to the fourth passage (P4). Changes of endothelial senescence were determined by measuring senescence-associated β-galactosidase (SA-β-gal) activity. Western blot was used to analyze the protein expression of p53, p21 and eNOS. Compared to P1, the SA-β-gal activity was 240% increased in cells at P4 ( P <0.001), and this effect was associated with 93% ( P <0.001) and 56% ( P <0.001) increased expression of p53, p21 and a 87% decreased expression of eNOS ( P <0.001). Treatment of P3 cells with the p53 inhibitor (pifithrin) reduced 43% SA-β-gal activity indicating a role of p53 activity in replicative senescence ( P <0.001). Treatment of endothelial cells with the Crataegus extract reduced by 56% the SA-β-gal activity ( P <0.01), improved by 131 % eNOS expression ( P <0.01) and reduced by 39% the up-regulation of p21 in cells at P4 without affecting the expression level of p53. The inhibitor of eNOS, L-NAME promoted the induction of endothelial senescence at P1 and reduced the inhibitory effect of the Crataegus extract on SA-β-gal activity at P3. In conclusion, the present findings indicate that the Crataegus extract delays endothelial cell replicative senescence most likely by preventing the downregulation of eNOS expression and activity and the upregulation of the p53/p21 pathway.