The Anti-Apoptotic Properties of APEX1 in the Endothelium Require the First 20 Amino Acids and Converge on Thioredoxin-1.

The APEX nuclease (multifunctional DNA repair enzyme) 1 (APEX1) has a disordered N-terminus, a redox, and a DNA repair domain. APEX1 has anti-apoptotic properties, which have been linked to both domains depending on cell type and experimental conditions. AIMS: As protection against apoptosis is a hallmark of vessel integrity, we wanted to elucidate whether APEX1 acts anti-apoptotic in primary human endothelial cells and, if so, what the underlying mechanisms are. RESULTS: APEX1 inhibits apoptosis in endothelial cells by reducing Cathepsin D (CatD) cleavage, potentially by binding to the unprocessed form. Diminished CatD activation results in increased Thioredoxin-1 protein levels leading to reduced Caspase 3 activation. Consequently, apoptosis rates are decreased. This depends on the first twenty amino acids in APEX1, because APEX1 (21-318) induces CatD activity, decreases Thioredoxin-1 protein levels, and, thus, increases Caspase 3 activity and apoptosis. Along the same lines, APEX1 (1-20) inhibits Caspase 3 cleavage and apoptosis. Furthermore, re-expression of Thioredoxin-1 via lentiviral transduction rescues endothelial cells from APEX1 (21-318)-induced apoptosis. In an in vivo model of restenosis, which is characterized by oxidative stress, endothelial activation, and smooth muscle cell proliferation, Thioredoxin-1 protein levels are reduced in the endothelium of the carotids. INNOVATION: APEX1 acts anti-apoptotic in endothelial cells. This anti-apoptotic effect depends on the first 20 amino acids of APEX1. CONCLUSION: As proper function of the endothelium during life span is a hallmark for individual health span, a detailed characterization of the functions of the APEX1N-terminus is required to understand all its cellular properties. Antioxid. Redox Signal. 26, 616-629.

The imbalanced redox status in senescent endothelial cells is due to dysregulated Thioredoxin-1 and NADPH oxidase 4.

Environmental stressors as well as genetic modifications are known to enhance oxidative stress and aging processes. Mitochondrial and nuclear dysfunctions contribute to the onset of aging. One of the most important redox regulators in primary human endothelial cells is Thioredoxin-1 (Trx-1), a 12 kD protein with additional anti-apoptotic properties. Cellular generators of reactive oxygen species are NADPH oxidases (NOXs), of which NOX4 shows highest expression levels in endothelial cells. Therefore, the aim of the study was to investigate how Trx-1 and NOX4 are regulated during stress-induced premature senescence in endothelial cells. We treated primary human endothelial cells for two weeks with H2O2 to generate stress-induced premature senescence in these cells. In this model senescence-associated ß-Galactosidase and nuclear p21 as senescence markers are increased. Moreover, total and mitochondrial reactive oxygen species formation is enhanced. An imbalanced redox homeostasis is detected by elevated NOX4 and decreased Trx-1 levels. This can be rescued by lentiviral expression of Trx-1. Moreover, the lysosomal protease Cathepsin D is over-activated, which results in reduced Trx-1 protein levels. Inhibition of "over-active" Cathepsin D by the specific, cell-permeable inhibitor pepstatin A abolishes the increase in nuclear p21 protein, ROS formation and degradation of Trx-1 protein, thus leading to blockade of stress-induced premature senescence by stabilizing the cellular redox homeostasis. Aortic Trx-1 levels are decreased and Cathepsin D activity is increased in NOX4 transgenic mice exclusively expressing NOX4 in the endothelium when compared to their wildtype littermates. Thus, loss of Trx-1 and upregulation of NOX4 importantly contribute to the imbalance in the redox-status of senescent endothelial cells ex vivo and in vivo.