Abstract 123: Age-Related Endothelial Senescence is Driven by Thrombospondin-1-Activated NADPH Oxidase Nox1

Organismal aging represents an independent risk factor underlying many vascular diseases, including systemic and pulmonary hypertension, and atherosclerosis. While the mechanisms driving aging are largely elusive, a steady persistent increase in tissue oxidative stress has been associated with senescence. Previously we showed TSP1 elicits NADPH oxidase (Nox)-dependent vascular smooth muscle cell oxidative stress. However mechanisms by which TSP1 affects endothelial redox biology are unknown. Here, we tested the hypothesis that TSP1 induces endothelial oxidative stress-linked senescence in aging. Using rapid autopsy disease-free human pulmonary (PA) artery, we identified a significant positive correlation between age, protein levels of TSP1, Nox1 and the cell-cycle repressor p21cip (p<0.05). Age also positively associated with increased Amplex Red-detected PA hydrogen peroxide levels (p<0.05). Moreover, treatment of human PA endothelial cells (HPAEC) with TSP1 (2.2nM; 24h) increased expression (~1.9 fold; p<0.05) and activation of Nox1 (~1.7 fold; p<0.05) compared to control, as assessed by Western blot and SOD-inhibitable cytochrome c reduction. Western blotting and immunofluorescence showed a TSP1-mediated increase in p53 activation, indicative of the DNA damage response. Moreover, TSP1 significantly increased HPAEC senescence in a p53/p21cip/Rb-dependent manner, as assessed by immunofluorescent detection of subcellular localization and senescence-associated β-galactosidase staining. To explore this pathway in vivo, middle-aged (8-10 month) wild-type and TSP1-null mice were utilized. In the TSP1-null, reduced lung senescence, oxidative stress, Nox1 levels and p21cip expression were observed compared to wild-type supporting findings in human samples and cell experiments. Finally, prophylactic treatment with specific Nox1 inhibitor NoxA1ds (10μM) attenuated TSP1-induced HPAEC ROS, p53 activation, p21cip expression and senescence. Taken together, our results provide molecular insight into the functional interplay between TSP1 and Nox1 in the regulation of endothelial senescence, with implications for molecular control of the aging process.