肺动脉平滑肌细胞Mg2+功能特性及其在肺高压发病过程中作用

Pulmonary hypertension (PH) is a pathophysiological condition associated with a board spectrum of diseases of different pathological features and etiologic mechanisms. It is a severe disease characterized by increasing in pulmonary vasomotor tone and vasoreactivity，alterations in calcium pathways and agonist-contraction coupling, progressive remodeling (proliferation, migration, and apoptosis), as well as increased oxidative stress in pulmonary artery smooth muscle cells (PASMCs). These changes subsequently lead to elevated pulmonary vascular resistance, right heart dysfunction and death. Magnesium, the second most abundant intracellular cation，plays a critical role in vasomotor tone and vascular reactivity in systemic circulation by modulating calcium channel, agonist -contraction coupling, vascular remodeling and oxidative stress of vascular smooth muscle cells. It is related to the pathogenesis of several cardiovascular disorders such as hypertension, atherosclerosis, and coronary artery disease. However, howmagnesium signals are regulated in pulmonary vasculature and translated into the pathological changes of PH is unclear. To test this hypothesis, we will use a combination of biophysical techniques of magnesium and calcium imaging, molecular techniques of gene knockdown and overexpression, and physiological techniques on two different models of PH of distinctively different etiologies to (1) determine the functional characteristics of magnesium homeostasis, and the involvement of specific isoforms of magnesium channel or transport in the different forms of PH, (2) determine and the regulatory effects of magnesium on calcium channels, agonist-contraction coupling and vasoreactivity in PH, (3) establish the roles of magnesium signaling pathway in the modulation of pulmonary vascular remodeling and oxidative stress associated with PH. This proposal will provide novel information on the mechanism of pathogenesis in PH and identify potential targets for the development of new therapeutic strategies.

不同类型肺高压发病过程中存在共同信号通路，包括肺血管紧张性和反应性增强，肺动脉平滑肌细胞（PASMCs）Ca2+通道、激动剂-收缩耦联异常，重构（增殖、迁移和凋亡）与氧化应激增强等改变。Mg2+是胞内第二丰富阳离子，已证明参与调节体循环血管紧张性和反应性，细胞Ca2+通道、激动剂-收缩耦联、重构和氧化应激等，是心血管疾病发病重要机制。但Mg2+对肺血管调节及在肺高压中作用未见报道。本申请拟采用Mg2+荧光成像、基因敲低和过表达，及生理学等技术，在正常、肺高压和高/低Mg2+预处理大鼠上，试图明确：①Mg2+稳态功能特点，哪种Mg2+转运体或通道参与何种肺高压发病。②Mg2+对血管Ca2+通道、激动剂-收缩耦联和反应性影响及肺高压时变化。③Mg2+信号通路在与肺高压相关的血管重构和氧化应激中作用。成果将丰富平滑肌细胞Mg2+信号调控理论，为阐明肺高压发病机制提供理论依据，为其治疗提供新靶点。

在野百合碱（MCT）和慢性低氧（CH）所致的两种肺动脉高压大鼠模型上开展研究，结果表明：1）饮食补充Mg2+可逆转MCT和CH致肺动脉高压大鼠的肺动脉重塑。2）肺动脉高压中，肺动脉平滑肌细胞（PASMCs）胞内游离Mg2+浓度降低。3）肺动脉高压大鼠肺动脉主干（PAs）的Mg2+转运体mRNA表达：HIP14、HIP14L、MagT1、MMgT1、Mrs2、NIPA1、NIPA2、SLC41A、SLC41A2、CNNM2和TRPM7显著增加，而MMgT2、SLC41A3下降；蛋白表达：SLC41A1、SLC41A2、CNNM2和TRPM7增加，而SLC41A3下降。补充Mg2+可以逆转Mg2+转运蛋白mRNA和蛋白表达的改变。4）高Mg2+培养明显抑制PASMC的增殖和迁移，增加细胞凋亡；而低Mg2+则产生相反效果。此外，siRNA靶向性敲减SLC41A1/2的可以减弱细胞的增殖和迁移，促进细胞凋亡；然而，SLC41A3过表达产生相反作用。5）siRNA靶向SLC41A1下调缺氧（3%O2）引起PASMCs中NFATc3的mRNA表达和核转位。6）在MCT预处理，以及无Mg2+或低Mg2+可增强CPA（激活SOCC）介导的PAs和肺动脉分支（sPAs）收缩反应，但仅能增强OAG（激活ROCC）介导的sPAs收缩反应；7）高Mg2+可显著抑制正常和MCT大鼠SOCC和ROCC介导的内皮完整或去内皮PAs和sPAs收缩反应，并提高ACh-诱导的MCT大鼠sPAs的舒张反应。8）去除细胞外Mg2+可抑制PAs和sPAs对ET-1和ACh的血管反应活性。胞外高Mg2+（4.8 mM）可抑制ET-1诱导的正常、慢性低氧小鼠和MCT大鼠的内皮完整或去内皮的PAs和sPAs的血管收缩，并增强正常小鼠PAs中ACh诱导的一氧化氮（NO）产生。9）在正常小鼠/大鼠的PAs中，高Mg2+增强了ACh诱导的血管舒张，而在CH暴露后或MCT预处理后，这种增强被完全消除，但高Mg2+增强sPAs的内皮依赖性舒张的作用依然存在。.本研究为发展补充Mg2+作为一种治疗肺动脉高压方法提供理论基础，以及为开发作用于Mg2+转运体的靶向性治疗肺高压的药物提供新分子靶点。研究成果将丰富平滑肌细胞Mg2+信号调控理论，无论是对肺动脉高压，或是其他各种心血管疾病中发病机制阐明都将提供一个新思路。