NOX4 upregulation increases superoxide and mitochondrial dysfunction in brain endothelial cells

Abstract

Reactive oxygen species (ROS) are key contributors to cardiovascular pathophysiology. The primary generators of cell ROS are mitochondria and NADPH oxidases (NOX); however possible interactions between these two sources are not clear. We have shown in mouse brain endothelial cells (cultured bEND.3 cells) that mitochondrial superoxide increases with ischemic insults that cause cell death. We hypothesized that angiotensin II (ANGII), another promoter of vascular dysfunction, would increase mitochondrial ROS and do so via its known ability to activate NOX enzymes. We found ANGII (10uM) treatment 24 or 48 h of endothelial (bEND3) cells increased mitochondrial superoxide generation 3‐fold (using Mitosox dye) and decreased mitochondrial membrane potential (using TMRM). These effects were suppressed by diphenylene iodonium chloride (DPI), an inhibitor of all NOX isoforms, and VAS2870 (VAS), selective inhibitor of NOX4. ANGII increased protein levels of NOX4, but not NOX1 and NOX2. We found NOX4 localized to the mitochondria; NOX4 protein levels measured in isolated mitochondria were increased 1.5 fold by ANGII. Together, our data suggest a novel action of ANGII to increase mitochondrial superoxide in endothelial cells through increased expression and mitochondrial localization of NOX4, a constitutively active ROS generator. Chronically, this effect would lead to mitochondrial damage and endothelial dysfunction. Supported by US NIH RO1 HL50775