Recent evidence suggests that ClC-3,a member of the ClC family of Cl-channels or Cl-/H+ antiporters,plays a critical role in NADPH oxidase-derived reactive oxygen species (ROS) generation.However,the underling mechanisms remain unclear.In this study we investigated the effects and mechanisms of CIC-3 on NADPH oxidase activation and ROS generation in endothelial cells.Treatment with angiotensin Ⅱ (Ang Ⅱ,1 μmol/L) significantly elevated ClC-3 expression in cultured human umbilical vein endothelial cells (HUVECs).Furthermore,Ang Ⅱ treatment increased ROS production and NADPH oxidase activity,an effect that could be significantly inhibited by knockdown of CIC-3,and further enhanced by overexpression of ClC-3.SA-β-galactosidase staining showed that ClC-3 silencing abolished Ang Ⅱ-induced HUVEC senescence,whereas ClC-3 overexpression caused the opposite effects.We further showed that Ang Ⅱ treatment increased the translocation of p47phox and p67phox from the cytosol to membrane,accompanied by elevated Nox2 and p22phox expression,which was significantly attenuated by knockdown of ClC-3 and potentiated by overexpression of CIC-3.Moreover,overexpression of ClC-3 increased Ang Ⅱ-induced phosphorylation of p47phox and p38 MAPK in HUVECs.Pretreatment with a p38 inhibitor SB203580 abolished ClC-3 overexpression-induced increase in p47phox phosphorylation,as well as NADPH oxidase activity and ROS generation.Our results demonstrate that ClC-3 acts as a positive regulator of Ang Ⅱ-induced NADPH oxidase activation and ROS production in endothelial cells,possibly via promoting both Nox2/p22phox expression and p38 MAPK-dependent p47phox/p67phox membrane translocation,then increasing Nox2 NADPH oxidase complex formation.