NADPH oxidase is critically involved in CCL2‐induced endothelial cell activation

Abstract

CCL2 plays an important role in vascular diseases via recruitment of leukocytes and by altering integrity of the vascular wall. Molecular mechanisms of direct CCL2‐induced activation of vascular endothelial cells (ECs) are largely unknown. Here we assessed the hypothesis that NADPH oxidase is critically involved in regulating EC responses to CCL2. Studies using EPR spin trap CMH and DCF imaging showed that CCL2 induces a significant and time‐dependent increase in the reactive oxygen species (ROS) which was blocked by pretreating ECs with NADPH oxidase inhibitors or anti‐oxidants. Measurement of directed EC migration showed that CCL2 induced a dose‐dependent increase in EC migration. The increase in migration was blocked by inhibiting NADPH oxidase activity. Immunoblot analysis revealed that CCL2 rapidly activates Akt, ERK, and p38 MAPK. Pretreatment of ECs with NADPH oxidase inhibitors, antioxidants, or the superoxide scavenger, significantly attenuated CCL2‐induced activation of Akt, but did not alter the activation of ERK or p38 MAPK. Inhibiting activity of mitochondrial complex II did not prevent the CCL2‐induced Akt activation. These studies indicate that CCL2‐mediated activation of ECs and induction of EC migration are mediated by NADPH oxidase‐derived ROS and that the process involves the redox dependent activation of Akt.Supported by EY04618, EY11766, and VA (R.B.C.); HL70215 (R.W.C); AHA (W. Z.)