FLNA调控蛋白激酶PAK1信号通路参与肺动脉高压血管重构的作用和机制 研究

Pulmonary arterial hypertension (PAH) is a progressive and fatal disease characterized by pulmonary arteriole remodeling. Pulmonary arterial smooth muscle cells (PASMCs) proliferation is one of the most important factors for pulmonary vascular remodeling. Our previously studies have suggested that Filamin A (FLNA), which act as a cytoskeletal protein, was significantly upregulated in PAH model, and mainly located in the PASMCs. FLNA upregulation participated in the mechanisms of hypoxia induced PASMCs proliferation and migration, indicating FLAN may play an important role in the pathogenesis of PAH. However, the exact mechanism is still unclear. FLNA can bind to its ligand small G Protein, and activate downstream PAK1 signaling pathway, thus promote PASMCs proliferation and migration, which ultimately resulting in vascular remodeling. In this study, FLNA-Rho-PAK1 signaling pathway will be regulated by small interfering RNA, specific inhibitors as well as conditional FLNA-knockout in smooth muscle cells at the cell and small animal levels. This study is aimed to explore the new mechanisms of FLNA-Rho-PAK1 signaling pathway in pulmonary vascular remodeling, which will lead to new targets for PAH therapies.

肺动脉高压（PAH）是一种进展迅速、预后极差的疾病，其主要病理特征为肺血管重构。肺动脉平滑肌细胞增殖和迁移是构成肺血管重构的主要病理基础。我们前期研究发现，细丝蛋白A（FLNA）这一细胞骨架蛋白在肺动脉高压大鼠肺动脉平滑肌细胞中表达明显上调，并且下调FLNA表达抑制低氧诱导的肺动脉平滑肌细胞增殖，表明FLNA在肺血管重构中有重要作用，但具体机制尚不清楚。FLNA与小G蛋白Rho结合后，可以磷酸化下游蛋白激酶PAK1，促进平滑肌细胞增殖和迁移，提示FLNA可能通过调节PAK1信号通路参与肺血管重构。本实验拟原代培养大鼠肺动脉平滑肌细胞、构建大鼠PAH模型及FLNA基因条件性敲除小鼠，运用小干扰RNA及特异性抑制剂调控FLNA-Rho-PAK1信号通路，从细胞和整体水平探索FLNA-Rho-PAK1通路对肺动脉平滑肌细胞增殖和迁移的影响及其机制，进而为PAH防治寻找新的靶点。

细丝蛋白A （FLNA）是一种高分子量的细胞骨架蛋白，在细胞运动调节中起着重要作用。FLNA突变与肺动脉高压（Pulmonary arterial hypertension , PAH）之间的关系此前已被报道；然而具体机制尚不清楚。本研究旨在探讨血管平滑肌细胞（Smooth muscle cell, SMC）表达的FLNA在PAH发生过程中的作用。我们构建平滑肌细胞特异性FLNA敲除小鼠，将小鼠暴露于缺氧环境（10% O2）28天，建立小鼠肺动脉高压模型。我们同时原代培养人肺动脉平滑肌细胞（PASMCs），通过转染FLNA小干扰RNA或过表达质粒，研究FLNA敲除或过表达对PASMCs增殖和迁移的影响。我们发现PAH患者肺组织中FLNA表达水平升高，且在肺小动脉的PASMCs中表达更为明显。平滑肌细胞特异性FLNA敲除减弱缺氧诱导的小鼠肺动脉高压和肺血管重塑。离体实验发现，FLNA下调会降低PASMCs的增殖和迁移能力，并且降低p-PAK-1和活性RAC1的表达。而FLNA过表达促进PASMCs增殖和迁移，提高p-PAK-1和活性RAC1表达水平。本研究重点探讨FLNA在肺动脉高压血管重塑中的作用；旨在为肺动脉高压治疗的寻找潜在的靶点。