Caveolae对TRPC1-SOCE的影响在周期性牵张调控血管平滑肌细胞增殖、迁移中的作用及机制研究

Stretch influences the proliferation and migration of vascular smooth muscle cells (VSMC), further leads to cardiovascular diseases. Canonical transient receptor potential 1 (TRPC1) and its mediated store-operated calcium entry (SOCE) play an important role in proliferation and migration of VSMCs and the development of cardiovascular diseases such as neointimal hyperplasia. The localization of TRPC1 with caveolae regulates its characteristics and its mediated SOCE. Furthermore, caveolae is disssembled or assembled responded to stretch. Then raised a question: whether mechanical stretch influences TRPC1 and its mediated SOCE through caveolae, further stretch-regulated VSMC function ? This question has not been answered till today. .This project focuses on the “the role and underlying mechanism of caveolae-impacted TRPC1-SOCE in cyclic stretch-regulated proliferation and migration of VSMCs”, and will investigate: 1) whether and how mechanical stretch influences TRPC1 and its mediated SOCE through caveolae; 2) the role and underlying mechanism of that influence in stretch-regulated alteration of VSMC function, using scanning microscopy, patch clamp, calcium image, as well as co-immunoprecipitation. This study is aimed to provide insight into understanding the mechanism by which cyclic stretch influences the VSMC function and finding novel therapeutic strategy for cardiovascular disease.

牵张调控血管平滑肌细胞（VSMC）的增殖迁移等功能，与血管疾病的发生发展密切相关。离子通道TRPC1及所介导的钙流SOCE与VSMC的增殖迁移和内膜增厚等血管病征呈正相关。TRPC1在质膜上的凹陷—小窝（Caveolae）内的定位调节其特性及所介导的SOCE的活性。小窝结构又受牵张影响。由此提出科学问题：牵张是否通过Caveolae影响TRPC1-SOCE的活性进而实现对VSMC功能的调控？目前相关研究未见报道。鉴于此，本项目拟开展Caveolae对TRPC1-SOCE的影响在周期性牵张调控VSMC增殖迁移中的作用及机制研究，并应用扫描电镜、膜片钳、钙成像、免疫共沉淀等技术重点研究：1）牵张是否以及如何通过Caveolae影响TRPC1-SOCE的活性；2）这种影响在牵张调控VSMC增殖迁移中的作用及机制。以期为深入理解力学致VSMC功能变化的机制、找寻心血管疾病治疗的靶标提供生物力学依据

背景：病理性牵张促使血管平滑肌细胞（VSMC）由收缩表型向合成表型转变、促进其增殖和迁移，进而导致高血压、动脉粥样硬化和血管成型术后再狭窄等血管疾病的发生。离子通道TRPC1与VSMC的增殖迁移及血管疾病的发生发展正相关，其机制与其介导钙池操纵性钙内流（SOCE）有关。在血管内皮细胞上，TRPC1驻留在Caveolae内并与其结构蛋白Caveolin-1（Cav-1）相互作用协同介导SOCE。TRPC1和Caveolae均具有力敏感性。所以提出科学假设：牵张通过调控Caveolae，进而调控TRPC1-SOCE（Caveolae、TRPC1和SOCE三者相互作用）及下游信号通路实现对VSMC功能的调控。 研究内容：对VSMC加载生理性（1Hz、5%张应变）和病理性（1Hz、15%张应变）牵张，未牵拉组做为对照，研究了：1）牵张对Cav-1表达的影响；Cav-1是否参与牵张对VSMC功能的调控及其参与机制；2）牵张对TRPC1表达和活性的影响；TRPC1在牵张调控VSMC钙响应中的作用；TRPC1是否参与牵张对VSMC功能的调控及其参与机制;3）牵张作用下，TRPC1与Cav-1的共定位；4）牵张作用下，TRPC1和SOCE成分之一STIM1的共定位。重要结果：1）病理性牵张促使VSMC向合成表型转变、促进其增殖迁移的同时，反向调控Cav-1和TRPC1：下调Cav-1的表达却上调TRPC1的表达增强其活性；2）Cav-1过表达可逆转病理性牵张所致VSMC向合成表型的转变，即使VSMC维持收缩表型，其机制与调控Myocaedin的表达有关；3）TRPC1介导病理性牵张所致VSMC钙浓度升高，TRPC1阻断或Knockdwon抑制牵张对VSMC增殖的促进，其机制与“Calcium-ERK”信号轴激活有关。4）病理性牵张下，Cav-1和TRPC1不存在共定位；5）生理性和病理性牵张下TRPC1与STIM1均有不同程度的共定位，但共定位程度没有明显区别。结论和意义：Cav-1和TRPC1在牵张调控VSMC功能中所起作用不同，起作用的机制亦不相同。Caveolae对TRPC1的功能无调控作用。TRPC1介导病理性牵张所致VSMC钙浓度升高，但其是否和SOCE有关尚需进一步证明。这些发现加深了对病理性牵张所致血管疾病发生发展机制的理解。此外，受基金资助，发表SCI论文5篇。