Endothelial dysfunction is associated with oxidative stress and dysregulation of calcium signalling. We previously demonstrated that calcium dependent Nox5 is regulated by Ang II and may be implicated in vascular damage in hypertension. Elevation of intracellular free calcium concentration ([Ca2+]i) influences oxidative stress, which may be related to activation of redox sensitive calcium channel TRPM2. Here, we postulated that in human microvascular endothelial cells (HMEC), Ang II induces ROS generation through TRPM2-dependent processes. HMECs were stimulated with either Ang II (100 nM) or H2O2 (1 mM). ROS production was measured by lucigenin (superoxide) and amplex red (H2O2); redox sensitive MAPKs/eNOS activation by immunoblotting; and AT1R/AT2R mRNA by Q PCR. In some experiments, the following inhibitors were used: aminoethoxydiphenyl borate (APB, TRPM2/SERCA/IP3R inhibitor), N (p amylcinnamoyl)anthranilic acid (ACA, TRPM2/PLA2 inhibitor), 8-bromo-cADP-ribose (8 Br-cADPR, ADPR antagonist), 3,4-Dihydro-5[4-(1-piperindinyl)butoxy]-1(2H)-isoquinoline (DPQ, poly-ADPR polymerase inhibitor) and PD123319 (AT2R antagonist). In hmECs, Ang II increased superoxide levels (1.9±0.2 fold, p<0.05 vs. vehicle), an effect blocked by APB, ACA, 8-Br-cADPR, DPQ and PD123319. In addition, Ang II-induced increase in H2O2 production (1.6±0.21 fold, p<0.05 vs. vehicle) was inhibited by AT2R antagonism. Ang II stimulation increased ERK1/2 activation (185%), but not p38, through AT2R-dependent mechanisms (p<0.05). AT2R, but not AT1R was expressed in HMECs. H2O2, an activator of TRPM2, decreased superoxide production (25%) and increased MAPKs (p38: 82%; ERK1/2: 40%; JNK: 36%), as well as, eNOS (24%) activation, p<0.05. In addition, ROS formation by Ang II was measured in the presence of L-type calcium channel blocker diltiazem, which also blocked Ang II effects on superoxide production. In conclusion, our data demonstrate that Ang II-induced oxidative stress and redox-sensitive MAPK activation in hmEC cells may involve TRPM2 and calcium-dependent signalling, which are mediated via AT2R. Our findings identify a novel signalling pathways whereby Ang II/AT2R regulates endothelial cell ROS generation through TRPM2.
Thrombin stimulates calcium‐dependent production of mitochondrial ROS in pulmonary microvascular endothelial cells