Cai Junting, Wei Jianxin, Li Shuang, Suber Tomeka, Zhao Jing
Acute Lung Injury Center of Excellence, Division of Pulmonary, Asthma, and Critical Care Medicine, Department of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA; Third Affiliated Hospital of Xiangya Medical School, Changsha, Hunan 410013, China.
Acute Lung Injury Center of Excellence, Division of Pulmonary, Asthma, and Critical Care Medicine, Department of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA.
Mediators Inflamm. 2017;2017:6893560. doi: 10.1155/2017/6893560. Epub 2017 Feb 27.
Maintenance of pulmonary endothelial barrier integrity is important for reducing severity of lung injury. Lysophosphatidic acid (LPA) regulates cell motility, cytoskeletal rearrangement, and cell growth. Knockdown of LPA receptor 1 (LPA1) has been shown to mitigate lung injury and pulmonary fibrosis. AM966, an LPA1 antagonist exhibiting an antifibrotic property, has been considered to be a future antifibrotic medicine. Here, we report an unexpected effect of AM966, which increases lung endothelial barrier permeability. An electric cell-substrate sensing (ECIS) system was used to measure permeability in human lung microvascular endothelial cells (HLMVECs). AM966 decreased the transendothelial electrical resistance (TEER) value immediately in a dose-dependent manner. VE-cadherin and f-actin double immunostaining reveals that AM966 increases stress fibers and gap formation between endothelial cells. AM966 induced phosphorylation of myosin light chain (MLC) through activation of RhoA/Rho kinase pathway. Unlike LPA treatment, AM966 had no effect on phosphorylation of extracellular signal-regulated kinases (Erk). Further, in LPA1 silencing cells, we observed that AM966-increased lung endothelial permeability as well as phosphorylation of VE-cadherin and focal adhesion kinase (FAK) were attenuated. This study reveals that AM966 induces lung endothelial barrier dysfunction, which is regulated by LPA1-mediated activation of RhoA/MLC and phosphorylation of VE-cadherin.
维持肺内皮屏障完整性对于减轻肺损伤的严重程度至关重要。溶血磷脂酸（LPA）可调节细胞运动、细胞骨架重排和细胞生长。已证明敲低LPA受体1（LPA1）可减轻肺损伤和肺纤维化。AM966是一种具有抗纤维化特性的LPA1拮抗剂，被认为是一种未来的抗纤维化药物。在此，我们报告了AM966的一种意外作用，即增加肺内皮屏障通透性。使用电场细胞基质传感（ECIS）系统测量人肺微血管内皮细胞（HLMVECs）的通透性。AM966立即以剂量依赖性方式降低跨内皮电阻（TEER）值。VE-钙黏蛋白和f-肌动蛋白双重免疫染色显示，AM966增加了内皮细胞之间的应力纤维和间隙形成。AM966通过激活RhoA/Rho激酶途径诱导肌球蛋白轻链（MLC）磷酸化。与LPA处理不同，AM966对细胞外信号调节激酶（Erk）的磷酸化没有影响。此外，在LPA1沉默细胞中，我们观察到AM966增加的肺内皮通透性以及VE-钙黏蛋白和黏着斑激酶（FAK）的磷酸化均减弱。这项研究表明，AM966诱导肺内皮屏障功能障碍，这是由LPA1介导的RhoA/MLC激活和VE-钙黏蛋白磷酸化所调节的。
