ADAR1调控Caveolin-1对肺微血管内皮细胞通透性的影响及机制研究

Acute lung injury is a common severe disease with high fatality of 40%. Its core pathological changes are the pulmonary microvascular endothelial cells (PMECs) injury, and the following increase of permeability of PMECs induces the serious leakage, inflammation and edema. The clinical treatment for ALI is still arduous. To investigate the upstream regulatory mechanisms could provide a new strategy for basic research and clinical treatment of lung permeability lesions. Through adenosine deaminase acting on RNA 1（ADAR1）endothelial tissue-specific knockout mice model in the preliminary experiments, we found that the deletions of ADAR1 may significantly increase the pulmonary permeability, lower the Caveolin-1（CAV-1）protein which is correlated with the connections of vascular endothelial cells, and increase plasma NO levels. Synthesizing the preliminary research and existing literatures, we propose that "ADAR1 deletions may lead to increasing permeability of PMECs, and ADAR1 mediating the NO release by regulating CAV-1 could be the key mechanism". We intend to verify the important role of ADAR1 in PMECs permeability regulation and clarify its effects and relevant mechanisms of regulating CAV-1 expression and the following downstream pathway impacting the pulmonary permeability through the model of ADAR1 endothelium tissue-specific knockout mice and primary pulmonary vascular endothelial cells. This project will propose and confirm a new regulatory molecule with an important role in pulmonary microvascular endothelial permeability. The results may provide a new direction for the vascular endothelial cells regulation study, the basic research and the future clinical treatments of lung injury.

急性肺损伤为常见危重疾病，病死率高达40％。其核心病理变化之一是肺微血管内皮细胞（PMECs）通透性增加导致的大量渗出、炎症和水肿，临床治疗仍很棘手，研究其上游调控机制可为其基础研究和临床治疗提供新策略。前期通过RNA腺苷脱氨酶1（ADAR1）血管内皮特异敲除小鼠模型发现ADAR1缺失可致肺通透性和血浆NO浓度显著增高，体外血管内皮细胞中下调ADAR1可致窖蛋白（Caveolin-1）明显下调。结合前期研究，我们初步提出“ADAR1缺失可致PMECs通透性增高，其调控Caveolin-1影响NO释放是其关键作用机制”。下一步我们拟通过ADAR1内皮特异敲除小鼠和原代PMECs模型，证实ADAR1在PMECs通透性调控中的重要作用并阐明其调控机制。本项目将提出并证实一个在PMECs通透性调控中具有重要作用的新分子，可为血管内皮细胞调控研究、肺损伤相关的基础研究和将来的临床治疗提供新的方向。

急性肺损伤为常见危重疾病，病死率高达40％。其核心病理变化之一是肺微血管内皮细胞（PMECs）通透性增加导致的大量渗出、炎症和水肿，临床治疗仍很棘手，研究其上游调控机制可为其基础研究和临床治疗提供新策略。本项目通过系列分子、细胞和动物实验，建立了ADAR1ECKO小鼠稳定繁殖体系，发现1周时ADAR1ECKO小鼠存活率为61.8%，4周后小鼠存活率为52.3%，显著低于ADAR1flox/flox小鼠。ADAR1ECKO小鼠肺泡可见塌陷，融合，肺泡间隔增厚，肺间质水肿，肺泡内出血，而ADAR1flox/flox小鼠肺组织病理切片未见异常。证实了ADAR1ECKO小鼠ADAR1蛋白和CAV-1蛋白的表达显著低于ADAR1flox/flox小鼠。发现了ADAR1ECKO小鼠较ADAR1flox/flox小鼠肺组织通透性明显增加，ADAR1ECKO组小鼠肺功能储备下降，肺功能受损。使用CD31磁珠分选成功施行ADAR1ECKO和ADAR1flox/flox小鼠肺血管内皮原代细胞分离和培养，进一步证实了ADAR1ECKO肺血管原代内皮细胞中CAV-1蛋白表达下降，而eNOS蛋白表达无差别。证实了ADAR1过表达可以降低IL-1β诱导的内皮细胞炎症激活，而ADAR1敲减可以增强此效应，miRNA-143直接靶向结合ADAR1调控此过程：miRNA-143过表达可以抑制ADAR1表达，而miRNA-143敲减可以促进ADAR1表达，从而调控下游内皮细胞炎症激活。通过项目实施，研究团队申请的国家实用新型专利1项，发表SCI论文2篇，国内核心期刊论文1篇。资助项目参与人国际学习与交流2次。培养硕士研究生2名。本项目发现ADAR1是肺内皮细胞结构和功能的重要全新调控因子，且其可以被miRNA-143调控，为肺血管内皮损伤的机制研究提供了全新的通路，为后续临床治疗和药物研究提供了全新的靶点。