Inhibition of Notch signaling leads to increased intracellular ROS by up-regulating Nox4 expression in primary HUVECs

• Blockade of Notch signaling increased reactive oxygen species (ROS) in primary HUVECs.
• Abruption of ROS generation abolished Notch blockade-induced HUVEC proliferation, migration and adhesion.
• The enhanced generation of ROS after blocking Notch signal was accompanied by augmented expression of Nox4.
• Increased expression of Nox4 led to enhanced phosphorylation of VEGFR2 and ERK in HUVECs.

The essential roles of Notch pathway in angiogenesis have been reported for years. However, how Notch pathway plays its role in regulating endothelial cells remains largely unknown. In this study we found that blockade of Notch signaling with a γ-secretase inhibitor increased reactive oxygen species (ROS) in primary human umbilical vein endothelial cells (HUVECs) under both normaxic and ischemia/reperfusion (I/R) conditions. Abruption of ROS generation with ROS scavengers or specific inhibitors of ROS production in HUVECs abolished Notch blockade-induced HUVEC proliferation, migration and adhesion, suggesting that the regulation of Notch pathway on endothelial cell behavior is at least partially dependent on its down-regulation of ROS level. We further showed that the enhanced generation of ROS after blocking Notch signal was accompanied by augmented expression of Nox4, which led to increased phosphorylation of VEGFR2 and ERK in HUVECs. In summary, our results have shown that Notch signaling regulates ROS generation by suppressing Nox4, and further modulates endothelial cell proliferation, migration and adhesion.