Central role of c-Src in NOX5- mediated redox signalling in vascular smooth muscle cells in human hypertension

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Camargo, Livia L. and Montezano, Augusto C. and Hussain, Misbah and Wang, Yu and Zou, Zhiguo and Rios, Francisco J. and Neves, Karla B. and Alves-Lopes, Rheure and Awan, Fazli R. and Guzik, Tomasz J. and Jensen, Thomas and Hartley, Richard C. and Touyz, Rhian M. (2022) Central role of c-Src in NOX5- mediated redox signalling in vascular smooth muscle cells in human hypertension. Cardiovascular Research, 118 (5). pp. 1359-1373. ISSN 0008-6363 (https://doi.org/10.1093/cvr/cvab171)

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Abstract

Aims: NOX-derived reactive oxygen species (ROS) are mediators of signalling pathways implicated in vascular smooth muscle cell (VSMC) dysfunction in hypertension. Among the numerous redox-sensitive kinases important in VSMC regulation is c-Src. However, mechanisms linking NOX/ROS to c-Src are unclear, especially in the context of oxidative stress in hypertension. Here, we investigated the role of NOX-induced oxidative stress in VSMCs in human hypertension focusing on NOX5, and explored c-Src, as a putative intermediate connecting NOX5-ROS to downstream effector targets underlying VSMC dysfunction. Methods and results: VSMC from arteries from normotensive (NT) and hypertensive (HT) subjects were studied. NOX1,2,4,5 expression, ROS generation, oxidation/phosphorylation of signalling molecules, and actin polymerization and migration were assessed in the absence and presence of NOX5 (melittin) and Src (PP2) inhibitors. NOX5 and p22phox-dependent NOXs (NOX1-4) were down-regulated using NOX5 siRNA and p22phox-siRNA approaches. As proof of concept in intact vessels, vascular function was assessed by myography in transgenic mice expressing human NOX5 in a VSMC-specific manner. In HT VSMCs, NOX5 was up-regulated, with associated oxidative stress, hyperoxidation (c-Src, peroxiredoxin, DJ-1), and hyperphosphorylation (c-Src, PKC, ERK1/2, MLC20) of signalling molecules. NOX5 siRNA reduced ROS generation in NT and HT subjects. NOX5 siRNA, but not p22phox-siRNA, blunted c-Src phosphorylation in HT VSMCs. NOX5 siRNA reduced phosphorylation of MLC20 and FAK in NT and HT. In p22phox- silenced HT VSMCs, Ang II-induced phosphorylation of MLC20 was increased, effects blocked by melittin and PP2. NOX5 and c-Src inhibition attenuated actin polymerization and migration in HT VSMCs. In NOX5 transgenic mice, vascular hypercontractilty was decreased by melittin and PP2. Conclusion: We define NOX5/ROS/c-Src as a novel feedforward signalling network in human VSMCs. Amplification of this system in hypertension contributes to VSMC dysfunction. Dampening the NOX5/ROS/c-Src pathway may ameliorate hypertension-associated vascular injury.

ORCID iDs

Camargo, Livia L., Montezano, Augusto C., Hussain, Misbah, Wang, Yu, Zou, Zhiguo, Rios, Francisco J., Neves, Karla B. ORCID logoORCID: https://orcid.org/0000-0001-5158-9263, Alves-Lopes, Rheure, Awan, Fazli R., Guzik, Tomasz J., Jensen, Thomas, Hartley, Richard C. and Touyz, Rhian M.;
CORE (COnnecting REpositories)