Activation of NADPH/ROS pathway contributes to angiogenesis through JNK signaling in brain endothelial cells

Abstract

Recent evidences have shown that reactive oxygen species (ROS) are involved in regulating angiogenesis and preventing tissue injury. However, the precise molecular mechanisms behind ROS-induced angiogenesis are still unknown. The aim of the present study was to investigate the effects of ROS-induced angiogenesis in rat brain microvessel endothelial cells (rBMECs) and identify involving the signal pathways. For initial experiments, the rBMECs were incubated with different concentrations of hydrogen peroxide (H2O2). For the second experiments, the rBMECs were respectively treated with ROS scavenger dimethylthiourea (DMTU), NADPH oxidase (Nox) inhibitor apocynin, small interfering RNAs-mediated knock down Nox2 or Nox4, or pretreated with c-Jun N-terminal kinase (JNK) inhibitor SP600125. The cell proliferation, migration, tube formation, and the expressions of several important neuroangiogenic factors including vascular endothelial growth factor (VEGF), brain derived neurotrophic factor (BDNF), matrix metalloproteinase (MMP) -9 and phos-JNK were measured. Low level of H2O2 significantly promoted endothelial cell (EC) proliferation, migration and tube formation and upregulated levels of VEGF, BDNF, MMP-9 and phos-JNK. DMTU and apocynin significantly inhibited endothelial angiogenesis and downregulated these protein levels. As expected, knockdown of Nox2 or Nox4 expression blocked endothelial angiogenesis and downregulated the expressions of pro-neuroangiogenic factors. Furthermore, H2O2-induced endothelial angiogenesis and high expressions of pro-neuroangiogenic factors were decreased by SP600125. In conclusion, Nox-derived ROS were required for endothelial angiogenesis. Low level of ROS may activate JNK signaling pathway and upregulate pro-neuroangiogenic factors, ultimately mediating endothelial angiogenesis.