胆碱调控TRPC/Ca2+介导的血管平滑肌细胞肥大和增殖的机制研究

Vascular remodeling is the important pathophysiological basis of most vascular disorders such as hypertension and atherosclerosis. The role of Ca2+-dependent signal transduction pathways in vascular remodeling is not yet clear. Our previous studies have shown that improving vagus nerve exerts cardiovascular protective effects in various cardiovascular diseases such as heart failure, ischemic heart disease or ischemia/reperfusion injury. The present project will investigate the molecular mechanisms of choline regulation of transient receptor potential canonical channels (TRPC)/Ca2+-mediated vascular smooth muscle cell (VSMC) hypertrophy and proliferation through establishment of vascular remodeling model induced by pressure overload/AngⅡcombined with improving the vagus nerve drug––choline: our project will 1) examine the effects of choline on VSMC hypertrophy and proliferation, and its intrinsic link to endothelial injury, extracellular matrix deposition by vascular morphology and function assessments; 2) determine the effects of choline on TRPC/Ca2+-activated cell hypertrophy-associated calcineurin and calcium/calmodulin-dependent protein kinase Ⅱ signaling transduction pathways by applying calcium fluorescent probes and western blotting technology, in order to discover the targets of calcium regulation of VSMC hypertrophy; 3) investigate the effects of choline on Ca2+ channel TRPC and regulatory proteins and the phenotypic switch of VSMCs (contractile/synthetic-proliferative phenotype) by applying siRNA and coimmunoprecipitation, in order to disclose the signal pathway of choline regulation of VSMC phenotypic switch-mediated cell proliferation. The present project will help to elucidate the molecular mechanism of choline regulating vascular remodeling, offering a new promising approach for pharmacological interventions of vascular remodeling and targets of intracellular calcium regulation.

血管重构是多种心血管疾病的病理生理基础，钙依赖信号转导途径在血管重构中的作用尚未明确。前期研究发现改善迷走神经可发挥心血管保护作用。本项目拟通过建立压力超负荷/AngⅡ诱导的血管重构模型结合改善迷走神经药物胆碱，研究胆碱调节TRPC/Ca2+介导的血管平滑肌细胞（VSMC）肥大和增殖的分子机制：采用血管形态学、功能学方法明确胆碱对VSMC肥大和增殖的影响，及其与平滑肌细胞病变相关因素内皮损伤、细胞外基质沉淀间内在联系；应用钙荧光探针和免疫印迹技术检测胆碱对TRPC/Ca2+激活的细胞肥大CaN和CaMKⅡ信号转导途径的作用，明确胆碱对VSMC肥大钙调控靶点；运用siRNA和免疫共沉淀技术检测胆碱对钙通道TRPC及调节蛋白的影响及对表型转化的调节作用，明确VSMC表型转化介导细胞增殖的信号通路。本研究将有助于阐明胆碱调控血管重构的分子机制，寻求血管重构的药理学干预措施和钙调控的细胞内靶点。

高血压、肺动脉高压、动脉粥样硬化是导致冠心病、心肌梗死、心衰等心血管疾病的主要诱发因素，作为构成血管壁组织结构及维持血管张力的主要细胞成分——血管平滑肌细胞（vascular smooth muscle cell, VSMC），在该类病变中扮演着重要角色。钙信号在维持血管张力和调控血管平滑肌细胞增殖等过程中发挥了关键作用。本项目通过建立VSMC损伤模型，以VSMC的钙调控为研究核心，探讨VSMC钙调控机制及胆碱在血管紧张素Ⅱ（AngⅡ）诱导的平滑肌细胞表型转化及其介导的VSMC增殖迁移中的作用及其可能机制：（1）建立人肺动脉平滑肌细胞（PASMC）和冠状动脉平滑肌细胞（CASMC）慢性低氧模型，应用钙荧光探针和荧光显微镜、Seahorse细胞能量代谢及分子生物学检测，研究肺动脉和冠状动脉平滑肌细胞钙通道及蛋白调控机制。结果表明CASMC和PASMC的线粒体生物能量学（如基础呼吸和ATP生成）相似, PASMC的糖酵解显著高于CASMC。在常氧情况下，CASMC 中的钙库操纵性钙内流（SOCE）/受体操纵性钙内流（ROCE）高于PASMC，可能与其分子组分STIM1/2，Orai1/2和TRPC6的表达在CASMC中高于PASMC相关。慢性低氧对CASMC的SOCC/ROCC（STIM1/STIM2，Orai1/Orai2和TRPC6）和静息[Ca2+]cyt无明显效应，而显著上调PASMC的SOCC/ROCC并增加静息[Ca2+]cyt。本研究揭示了低氧造成肺血管收缩和肺血管重构，而造成冠状动脉舒张的功能性差异的可能分子机制与低氧可诱导肺血管平滑肌细胞上调钙通道SOCC/ROCC的表达密切相关。（2）通过建立AngⅡ诱导的大鼠VSMC重构模型，给予迷走神经相关药物胆碱和相应的受体阻断剂及应用免疫荧光和激光共聚焦等技术，探讨胆碱对AngⅡ诱导的VSMC表型转化及其介导的迁移和增殖的调节作用机制。胆碱可能通过激活M3AChR，促进Nrf2-ARE信号通路而减轻NOX-1和线粒体ROS的产生，改善AngⅡ诱导的血管平滑肌细胞表型转化(增加平滑肌细胞收缩表型标志物α-SMA的表达，降低合成型标志物OPN表达），抑制血管平滑肌细胞迁移、增殖。本实验探讨了血管重构的发生机制及胆碱的保护效应，有助于揭示血管平滑肌细胞钙调控靶点和探索防治血管重构相关疾病的药理学干预措施。