Nox4调控VSMC衰老在动脉粥样硬化中的作用研究

Vascular smooth muscle cells (VSMC) senescence plays a pivotal role in the development process of atherosclerosis; Reactive oxygen species (ROS) have been involved in the development of VSMC senescence. Our previous studies have demonstrated that NADPH oxidase 4 (Nox4) is main source of ROS in atherosclerosis-associated cardiovascular diseases. Therefore, Nox4 may play a key regulatory role in the process of VSMC senescence. Herein, it is the first time for our project to explore the role of modulation of VSMC senescence Nox4 by in development process of atherosclerosis and the underlying mechanism as follows: 1) VSMC senescence was induced by angiotensin II (AngII) and the VSMC senescence, Nox4 expression, ROS production and et al in aging VSMC were measured by different assay. The Nox4 expression positively correlated with VSMC senescence and the key role of Nox4 in AngII-induced VSMC senescence were identified by combining the lentiviral Nox4 overexpression/knockdown or strategies of drug interference; 2) atherosclerosis was induced by AngII in ApoE-/- mice and the plaque size，fibrous cap stability，VSMC senescence, Nox4 expression, ROS production and et al in artery were tested, to elucidate that upregulation of Nox4 induces senescence of VSMC and accelerates the development of atherosclerosis by the lentiviral VSMC-targeted Nox4 overexpression/knockdown; 3) To demonstrate that modulation of sirtuin 1 (SIRT1)by Nox4 resulting in nuclear factor-κB(NF-kB) signaling activation which contributes to VSMC aging and development of athersclerosis in above-mentioned cell and animal models by various molecular biology techniques. These findings will reveal a novel mechanism of development of atherosclerosis and suggest that modulation of VSMC senescence by Nox4 downregulation can offer a therapeutic option for treating atherosclerosis.

VSMC衰老在动脉粥样硬化发展中发挥重要角色；ROS是诱导VSMC衰老关键因素；我们研究表明Nox4是心血管疾病过程中ROS的主要来源。因此，Nox4可能在VSMC衰老过程中发挥关键调控角色；本项目从以下方面首次探讨Nox4调控VSMC衰老在动脉粥样硬化中作用及机制：1)在血管紧张素II诱导的VSMC衰老模型中，结合药物干预和过表达/沉默Nox4，检测细胞衰老水平、Nox4、ROS水平等，确定Nox4与VSMC衰老相关性并阐明Nox4在VSMC衰老中起关键调控作用；2)在ApoE-/-小鼠动脉粥样硬化模型中，结合慢病毒特异性VSMC过表达/沉默Nox4，观察动脉斑块形成、稳定性、VSMC衰老、Nox4等指标，揭示Nox4促VSMC衰老加速动脉粥样硬化作用；3)进一步揭示Nox4通过调控SIRT1对NF-κB信号的抑制作用促衰老机制；本项目将为防治动脉粥样硬化发病机制提供新的理论依据。

烟酰胺腺嘌呤二核苷酸氧化酶4（Nox4）是血管细胞氧自由基（ROS）的主要来源，TGF-β1/Smad3是诱导血管平滑肌细胞（VSMC）产生CTGF表达的重要信号通路；因此，Nox4/ROS可能介导TGF-β1/Smad3信号通路调控CTGF的表达，从而调控血管重构。我们的研究结果发现：1) TGF-β1明显诱导VSMC的CTGF和Nox4表达，Smad3的激活和 ROS产生；2) NADPH氧化酶抑制剂DPI明显减轻TGF-β1诱导CTGF的表达和ROS的产生，此外沉默Smad3和Nox4明显抑制TGF-β1诱导CTGF的表达；3) 沉默Smad3和Nox4明显抑制TGF-β1诱导的Smad3和Nox4的相互作用；4) 在动脉粥样硬化的病人和动物模型上也进一步确证了Nox4的水平与Smad3的激活和CTGF表达的正相关性。上述结果首次表明在血管重构过程中Nox4依赖Smad3的激活增加TGF-β1诱导CTGF的表达，同时也表明Nox4是动脉粥样硬化相关心血管疾病发生、 发展的一个关键调控点。