Vasoprotective effects of NOX4 are mediated via polymerase and transient receptor potential melastatin 2 cation channels in endothelial cells

Alves-Lopes, Rheure and Lacchini, Silvia and Neves, Karla B. and Harvey, Adam and Montezano, Augusto C. and Touyz, Rhian M. (2023) Vasoprotective effects of NOX4 are mediated via polymerase and transient receptor potential melastatin 2 cation channels in endothelial cells. Journal of Hypertension, 41 (9). pp. 1389-1400. ISSN 0263-6352 (https://doi.org/10.1097/HJH.0000000000003478)

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Abstract

Background: NOX4 activation has been implicated to have vasoprotective and blood pressure (BP)-lowering effects. Molecular mechanisms underlying this are unclear, but NOX4-induced regulation of the redox-sensitive Ca 2+ channel TRPM2 and effects on endothelial nitric oxide synthase (eNOS)-nitric oxide signalling may be important. Method: Wild-type and LinA3, renin-expressing hypertensive mice, were crossed with NOX4 knockout mice. Vascular function was measured by myography. Generation of superoxide (O 2- ) and hydrogen peroxide (H 2 O 2 ) were assessed by lucigenin and amplex red, respectively, and Ca 2+ influx by Cal-520 fluorescence in rat aortic endothelial cells (RAEC). Results: BP was increased in NOX4KO, LinA3 and LinA3/NOX4KO mice. This was associated with endothelial dysfunction and vascular remodelling, with exaggerated effects in NOX4KO groups. The TRPM2 activator, ADPR, improved vascular relaxation in LinA3/NOX4KO mice, an effect recapitulated by H 2 O 2 . Inhibition of PARP and TRPM2 with olaparib and 2-APB, respectively, recapitulated endothelial dysfunction in NOX4KO. In endothelial cells, Ang II increased H 2 O 2 generation and Ca 2+ influx, effects reduced by TRPM2 siRNA, TRPM2 inhibitors (8-br-cADPR, 2-APB), olaparib and GKT137831 (NOX4 inhibitor). Ang II-induced eNOS activation was blocked by NOX4 and TRPM2 siRNA, GKT137831, PEG-catalase and 8-br-cADPR. Conclusion: Our findings indicate that NOX4-induced H 2 O 2 production activates PARP/TRPM2, Ca 2+ influx, eNOS activation and nitric oxide release in endothelial cells. NOX4 deficiency impairs Ca 2+ homeostasis leading to endothelial dysfunction, an effect exacerbated in hypertension. We define a novel pathway linking endothelial NOX4/H 2 O 2 to eNOS/nitric oxide through PARP/TRPM2/Ca 2+ . This vasoprotective pathway is perturbed when NOX4 is downregulated and may have significance in conditions associated with endothelial dysfunction, including hypertension.

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