Institute of Cardiovascular and Medical Sciences, University of Glasgow, 126 University Place, Glasgow G12 8TA, UK.
Diabetes and Cardio-Metabolic Disorders Laboratory, Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Jhang Road, P.O. Box. 577, Faisalabad, Pakistan.
Cardiovasc Res. 2022 Mar 25;118(5):1359-1373. doi: 10.1093/cvr/cvab171.
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.
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.
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.
活性氧（ROS）是血管平滑肌细胞（VSMC）功能障碍中涉及的信号通路的介质，其来源于 NADPH 氧化酶（NOX）。在对 VSMC 调节很重要的众多氧化还原敏感激酶中，c-Src 是其中之一。然而，NOX/ROS 与 c-Src 之间的联系机制尚不清楚，尤其是在高血压的氧化应激背景下。在这里，我们研究了在人类高血压中，NOX 诱导的氧化应激在 VSMC 中的作用，重点研究了 NOX5，并探索了 c-Src，因为它是连接 NOX5-ROS 与 VSMC 功能障碍下游效应靶点的假定中间物。
研究了来自正常血压（NT）和高血压（HT）受试者的动脉中的 VSMC。在不存在和存在 NOX5（蜂毒素）和 Src（PP2）抑制剂的情况下，评估了 NOX1、2、4、5 的表达、ROS 的产生、信号分子的氧化/磷酸化以及肌动蛋白聚合和迁移。使用 NOX5 siRNA 和 p22phox-siRNA 方法下调 NOX5 和 p22phox 依赖性 NOXs（NOX1-4）。作为完整血管中概念验证的方法，通过肌动描记法评估在 VSMC 中特异性表达人源 NOX5 的转基因小鼠的血管功能。在 HT VSMC 中，NOX5 上调，伴有氧化应激、过氧化物（c-Src、过氧化物酶、DJ-1）和过度磷酸化（c-Src、PKC、ERK1/2、MLC20）。NOX5 siRNA 减少了 NT 和 HT 受试者的 ROS 生成。NOX5 siRNA 而非 p22phox-siRNA 减弱了 HT VSMC 中的 c-Src 磷酸化。NOX5 siRNA 减少了 NT 和 HT 中的 MLC20 和 FAK 的磷酸化。在 p22phox 沉默的 HT VSMC 中，Ang II 诱导的 MLC20 磷酸化增加，该作用被蜂毒素和 PP2 阻断。NOX5 和 c-Src 抑制减弱了 HT VSMC 中的肌动蛋白聚合和迁移。在 NOX5 转基因小鼠中，蜂毒素和 PP2 降低了血管的高收缩性。
我们将 NOX5/ROS/c-Src 定义为人类 VSMC 中一种新的前馈信号网络。该系统在高血压中的扩增导致 VSMC 功能障碍。抑制 NOX5/ROS/c-Src 途径可能改善与高血压相关的血管损伤。
