Abstract MP29: Activation Of Transient Receptor Potential Melastatin 2 (trpm2) Cation Channel Contributes To Nox4-induced Protective Effects In Endothelial Cells.

Abstract

NOX4-induced H 2 O 2 production promotes vasodilation and is cardiovascular protective. H 2 O 2 also regulates TRPM2, a redox-sensitive channel that regulates Ca 2 influx. eNOS is a Ca 2+ -dependent enzyme, and hypertension-associated endothelial dysfunction involves eNOS inactivation. As NOX4-derived H 2 O 2 regulates TRPM2 and consequently Ca 2+ influx, we questioned whether downregulation of the H 2 O 2 -TRPM2-Ca 2+ axis in endothelial cells may contributes to impaired vascular relaxation in hypertension. WT and TTRhRen hypertensive mice were crossed with Nox4 KO mice. Vascular function was studied in mesenteric resistance arteries by wire myography. Ca 2+ influx was assessed by fluorescence microscopy in aortic endothelial cells, eNOS activation and TRPM2 expression were assessed by immunoblotting and immunohistochemistry, respectively. Blood pressure in TTRhRen (130.3±7.0 mmHg) and TTRhRen/NOX4 KO mice (141.3±18 mmHg) was significantly increased compared to control mice (98.1±8.0 mmHg). Endothelium-dependent relaxation was impaired in TTRhRen mice (Emax: WT 83.5±4.03 vs TTRhRen 59.1±3.5), effects worsened by NOX4 KO (37.9±5.4), p<0.05. Activation of TRPM2 with ADPR, improved vascular relaxation in TTRhRen/NOX4 KO mice (75.9±7.7); an effect also achieved with H 2 O 2 incubation (74.2±15.4), p<0.05. Ang II stimulated H 2 O 2 generation (% of control: 138.23±9.04) followed by Ca 2+ influx (AUC - Ca 2+ : 19401.25±1940.21), an important regulator of eNOS. These processes were reduced by TRPM2 inhibition (AUC - Ca 2+ : 8-br-cADPR 15232.2±1052.0; Olaparib 14260±843.2 and 2-APB 13095.2±277.4, p<0.05) and by the NOX1/4 inhibitor GKT137831 (AUC - Ca 2+ : Ang II 107357±1940.2 vs GKT 15067.5±255.6, p<0.05). Activation of eNOS (Ser1177) by Ang II in endothelial cells was blocked by PEG-catalase, GKT137831, and the TRPM2 inhibitor 8-br-cADPR. TRPM2 inhibitors also increased MAPK expression in endothelial cells. In conclusion, endothelial dysfunction in TTRhRen/NOX4 KO mice involves impaired TRPM2 activation. Reduced bioavailability of H 2 O 2 due to Nox4 downregulation is a major driver of this process. We identify a new axis in endothelial cells involving Nox4-H 2 O 2 -mediated activation of TRPM2-Ca 2+ -eNOS signalling which is vasoprotective.