NADPH OXIDASE 2, 4 AND MITOCHONDRIA-DERIVED REACTIVE OXYGEN SPECIES CONTRIBUTE TO ANGIOPOIETIN-1 INDUCED ANGIOGENESIS

Abstract

The NOX proteins are reactive oxygen species (ROS) generating enzymes and together with Mitochondrial-derived ROS, they play an important role in pathological as well as physiological processes of the vasculature. Recent evidences indicate that ROS generated by NOX and the mitochondria may play important roles in growth factors downstream signaling. Our own study have pointed out that Angiopoietin-1 (Ang-1) induced ROS production is important for Ang-1/Tie-2 receptor signaling in endothelial cells, however, we did not identify the molecular sources of ROS generation in this respect. In this study, we have identified the origins of Ang-1 induced ROS production in endothelial cells and their involvement in Ang-1 induced angiogenesis. The most abundant isoforms of NOX in human umbilical endothelial cells were knocked down using adenoviruses expressing shRNA against NOX2 and NOX4. Mitochondrial-derived ROS were inhibited using SS31 peptide or Mito-Tempol. Cellular ROS levels were measured upon Ang-1 treatment using Amplex Red and Mitochondrial ROS level were measured with MitoSox. To evaluate the involvement of NOX or Mitochondrial-derived ROS in Ang-1 induced angiogenesis we evaluated cell survival by cell count and Caspase-3 cleavage, tubulogenesis by matrigel assay and migration by scratch wound assay. Activation level of the MAPK and PI3K/AKT pathways downstream of Ang-1/Tie-2 were measured by immunoblotting. Ang-1treatment leads to NOX-derived and Mitochondrial ROS production. NOX4 and the mitochondrial ROS are required for Ang-1 induced ROS production. NOX4 knockdown and Mitochondrial ROS blockage lead to impaired Ang-1 induced migration. NOX2 minimally contributes to Ang-1 induced ROS production and appears to be required for Ang-1 induced cell survival. In addition, NOX4 and mitochondrial-derived ROS are required for optimal activation of p38 and inactivation of ERK and AKT. On the other hand NOX2 appears to be required for optimal AKT activation and ERK and p38 inactivation. We conclude that NOX4 and mitochondrial ROS are required for Ang-1 induced migration through p38 activation. We also concluded that NOX2-derived ROS regulates Ang-1 induced cell survival by activation of AKT. This study provides new insight into cellular signaling mechanism downstream of Ang-1/Tie-2 signaling axis.