Abstract 336: The Imbalanced Redox Homeostasis in Senescent Endothelial Cells is Due to Dysregulated Thioredoxin-1 and NADPH-Oxidase 4

Abstract

Senescent endothelial cells are not only present in aged vessels, but also in atherosclerotic lesions, where they contribute to endothelial dysfunction. One hallmark of cellular senescence is an imbalanced redox homeostasis. However, the underlying mechanisms how this imbalance occurs are not completely understood. Thioredoxin-1 (Trx-1) is one of the most important redox regulators in primary human endothelial cells. It has previously been shown that Trx-1, in addition to its oxidoreductase activity has anti-apoptotic properties. Cellular generators of reactive oxygen species (ROS) are NADPH oxidases (NOXs), of which NOX4 shows highest expression levels in endothelial cells. Therefore, the aim of the study was to investigate whether and how Trx-1 and NOX4 are regulated during stress-induced premature senescence in endothelial cells, an ex vivo model mirroring the situation in aged or diseased vessels. We treated primary human endothelial cells for two weeks with hydrogen peroxide to generate stress-induced premature senescence in these cells. Senescence induction was confirmed by an increase in senescence-associated beta-Galactosidase and nuclear p21, two established senescence markers. Moreover, cytosolic and mitochondrial reactive oxygen species formation were enhanced. Concomitantly, we observed decreased Trx-1 levels and elevated NOX4 levels, which could explain the imbalance in the cellular redox homeostasis. Moreover, the lysosomal protease Cathepsin D, known to degrade Trx-1 under short-term oxidative stress, was over-activated, providing a mechanistic basis for reduced Trx-1 protein levels. Inhibition of “over-active” Cathepsin D by the specific, cell-permeable inhibitor pepstatin A counteracted the increase in nuclear p21, ROS formation and degradation of Trx-1, thus leading to blockade of stress-induced premature senscence by stabilizing the cellular redox homeostasis. Moreover, aortic Trx-1 levels negatively correlated with NOX4 expression in NOX4 transgenic mice exclusively expressing NOX4 in the endothelium and their wildtype littermates. Our data show that loss of Trx-1 and upregulation of NOX4 may importantly contribute to the imbalanced redox homeostasis in senescent endothelial cells ex vivo and in vivo.