Search

Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis involving its ability to stimulate the phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser1177 which activates eNOS and the production of NO by endothelial cells (ECs). Mitochondria are increasingly recognized to play signalling roles in ECs, frequently involving the production of reactive oxygen species (ROS). In this study, we found that VEGF-induced eNOS activation in ECs required functional mitochondria; inhibition of the mitochondrial respiratory chain at complex I with rotenone or uncoupling the mitochondrial membrane potential with carbonylcyanide-m-chlorophenylhydrazone (CCCP) attenuated VEGF-induced eNOS activation, which also required a proximal signalling pathway involving VEGF receptor-2/Src kinase-dependent activation of phospholipase Cγ1 (PLCγ1)-induced calcium signalling. Mitochondria’s signalling role was independent of ROS as VEGF treatment did not induce a detectable increase in mitochondrial ROS production nor did mitochondria-targeted antioxidants inhibit VEGF-induced signalling. Instead, mitochondria controlled VEGF-induced calcium signalling; rotenone or CCCP inhibited VEGF-induced cytosolic calcium transients. Inhibition of the sodium/calcium exchanger NCLX (a mitochondrial calcium export transporter) with its pharmacological inhibitor CGP37157 or NCLX-targeted siRNA attenuated VEGF-induced cytosolic calcium transients and eNOS activation. VEGF also stimulated a rapid and transient increase in mitochondrial calcium that was significantly prolonged by NCLX inhibition with CGP37157 or NCLX-targeted siRNA. NCLX activity has functional implications as its inhibition with siRNA promoted the migratory capacity of ECs. In contrast to VEGF, NCLX was not important for controlling changes in intracellular calcium in response to oxidative stress induced by H2O2 or myeloperoxidase (MPO)-derived oxidants, despite MPO activating eNOS in a PLC/calcium-dependent manner. Together these findings identify a previously unrecognized role for mitochondrial NCLX in controlling intracellular calcium signalling leading to eNOS activation in response to VEGF but not oxidative stress. NCLX may represent a new molecular target for modulating the angiogenic properties of ECs.