IQGAP对肺微血管内皮细胞屏障功能的调控及信号转导机制

Hallmark of acute respiratory distress syndrome (ARDS) is pulmonary microvascular endothelial barrier injury and its increase of permeability. Barrier function of pulmonary microvascular endothelial cells(PMVEC) is dynamically regulated by intercellular junctions and cytoskeletal proteins. Subtype of IQRas GTPase-activating protein (IQGAP) are identified in cultured PMVECs from rat. The aim of this study is firstly to examine the expression of IQGAPs on PMVECs and investigate the relation between change of subtype of IQGAP and pulmonary microvascular endothelial barrier disruption in inflammation. The technology of RNA interference through lentiviral vector inhibiting IQGAP expression and plasmid transfection overexpressing IQGAP is used to explore the regulation of those proteins in barrier function disruption of PMVEC induced by inflammation, respectively. The feedback regulation mechanism of FAK，RhoA，Cdc42 and Rac1 on IQGAP are explored. To ameliorate the barrier function injury of PMVEC induced by inflammation, we also intend to intervene in the signal transduction pathway and actin remodeling of PMVEC according to different the sites of action of IQGAPs. These results may further elucidate the sites of action and its signal transduction mechanism of IQGAPs on barrier function of pulmonary microvascular endothelium .

肺微血管内皮屏障功能损伤及通透性增加是急性呼吸窘迫综合征（ARDS）的特征,内皮细胞屏障功能受细胞间连接及骨架蛋白的动态调节。本课题分离和培养大鼠肺微血管内皮细胞（PMVEC），鉴定PMVEC膜异亮氨酸－谷氨酸Ras和GTP酶激活蛋白（IQGAP)亚型、观察炎症诱导PMVEC屏障功能损伤过程中IQGAP亚型的变化及其与肺微血管内皮屏障功能损伤的关系。构建IQGAP基因RNA干扰慢病毒，观察抑制IQGAP基因表达后对肺微血管屏障功能的影响，检测质粒转染高表达IQGAP后上述过程的变化。探讨FAK、RhoA、Cdc42及Rac1对IQGAP的反馈调节机制。针对上述IQGAP亚型的变化及其作用环节，采取干预措施改变细胞内信号转导，通过调控PMVEC肌动蛋白骨架，以期减轻炎症诱导的PMVEC屏障功能损伤。初步明确IQGAP在肺微血管内皮屏障功能的调控作用环节及信号转导机制。

肺微血管内皮细胞（pulmonary microvascular endothelial cells, PMVECs）屏障功能受损及通透性增加被认为是ARDS发病的关键环节，其屏障功能的维持依赖于肌动蛋白骨架与细胞间连接的动态平衡。本研究主要探讨异亮氨酸－谷氨酸Ras和GTP酶激活蛋白（IQGAPs）在炎症诱导PMVECs屏障损伤中的作用及信号转导机制。我们发现：1.大鼠PMVECs上存在IQGAPs三种亚型，以IQGAP1表达量含量最高，且主要位于PMVECs细胞连接处；2.RNA-seq分析显示抑制IQGAP1表达会影响PMVECs屏障的完整性，IQGAP1可能在内皮细胞屏障修复和损伤中起关键作用；3.LPS与TNF-α均呈时间及浓度依赖方式诱导大鼠PMVECs通透性增加，且伴随F-actin骨架重排、粘附连接蛋白VE-cadherin及β-catenin表达减少；4.LPS刺激PMVECs后，IQGAP1在mRNA及蛋白水平均呈时间及浓度依赖方式上调并与F-actin共定位增加；5.抑制IQGAP1表达明显减轻LPS刺激引起的PMVECs通透性增高，肌球蛋白轻链磷酸化，F-actin骨架重排及VE-cadherin的再分布，而过表达IQGAP1加剧LPS诱导的PMVECs通透性增高；6.TNF-α刺激逐渐降低RPMVECs中活性Rac1（Rac1-GTP）含量，激活Rac1可促使细胞外周骨架蛋白增强，cortactin向细胞周边以及细胞膜积聚，从而抵御TNF-α的损伤作用；7.分别抑制FAK及RhoA信号通路均减轻TNF-α诱导的PMVECs通透性增高、F-actin重排及Ezrin磷酸化，抑制FAK还可降低LPS引起的PMVECs高通透性以及IQGAP1的表达上调；8.激活Rap1信号通路减轻LPS诱导的PMVECs通透性增高及F-actin重排；9.LPS激活NF-КB信号通路上调IQGAP1表达，IQGAP1可反馈调节NF-КB信号通路；10. IQGAP1在LPS诱导的大鼠肺损伤动物模型中表达升高；11.抑制IQGAP1蛋白表达通过增加Rap1的活性，从而降低LPS引起PMVECs高通透性及F-actin重排；特异性抑制Rap1的活性，可削弱IQGAP1敲除带来的内皮屏障保护效应。