Zhou Qun, Jiang Jianjun, Chen Guanjun, Qian Cheng, Sun Gengyun
Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of Anhui Medical University, Hefei, China.
Department of Geriatric Respiratory Medicine, The First Affiliated Hospital of Anhui University of Chinese Medicine, Hefei, China.
Front Pharmacol. 2021 May 12;12:676817. doi: 10.3389/fphar.2021.676817. eCollection 2021.
One of the important pathogenesis of acute respiratory distress syndrome (ARDS) is the dysfunction of pulmonary microvascular endothelial barrier induced by a hyperinflammatory immune response. However, the potential mechanisms of such an imbalance in pulmonary microvascular endothelial cells (PMVECs) are not yet understood. Explore the molecular mechanism of endothelial barrier dysfunction induced by inflammatory immune cytokines in ARDS, and find a therapeutic target for this syndrome. Rat PMVECs were cultured to form a monolayer. Immunofluorescence, flow cytometry, and Western blotting were selected to detect the distribution and the expression level of phosphorylated Ezrin protein and Ezrin protein. Transendothelial electrical resistance (TER) and transendothelial fluxes of fluorescein isothiocyanate (FITC)-labeled bovine serum albumin (BSA) were utilized to measure the permeability of the cell monolayer. Ezrin short hairpin RNA (shRNA) and Ezrin 567-site threonine mutant (Ezrin) were used to examine the role of Ezrin protein and phosphorylated Ezrin protein in endothelial response induced by tumor necrosis factor-alpha (TNF-α), respectively. The function of focal adhesion kinase (FAK) and Ras homolog gene family, member A (RhoA) signaling pathways were estimated by inhibitors and RhoA/FAK shRNA in TNF-α-stimulated rat PMVECs. The activation of FAK and RhoA was assessed by Western blotting or pull-down assay plus Western blotting. The TER was decreased after TNF-α treatment, while the Ezrin protein phosphorylation was increased in a time- and dose-dependent manner. The phosphorylated Ezrin protein was localized primarily at the cell periphery, resulting in filamentous actin (F-actin) rearrangement, followed by a significant decrease in TER and increase in fluxes of FITC-BSA. Moreover, FAK and RhoA signaling pathways were required in the phosphorylation of Ezrin protein, and the former positively regulated the latter. The phosphorylated Ezrin protein was induced by TNF-α the FAK/RhoA signaling pathway leading to endothelial hyperpermeability in PMVECs.
急性呼吸窘迫综合征（ARDS）的重要发病机制之一是由过度炎症免疫反应诱导的肺微血管内皮屏障功能障碍。然而，肺微血管内皮细胞（PMVECs）中这种失衡的潜在机制尚不清楚。探索ARDS中炎症免疫细胞因子诱导内皮屏障功能障碍的分子机制，并找到该综合征的治疗靶点。培养大鼠PMVECs以形成单层。选择免疫荧光、流式细胞术和蛋白质免疫印迹法来检测磷酸化埃兹蛋白（Ezrin）和埃兹蛋白的分布及表达水平。利用跨内皮电阻（TER）和异硫氰酸荧光素（FITC）标记的牛血清白蛋白（BSA）的跨内皮通量来测量细胞单层的通透性。分别使用埃兹蛋白短发夹RNA（shRNA）和埃兹蛋白567位点苏氨酸突变体（Ezrin）来研究埃兹蛋白和磷酸化埃兹蛋白在肿瘤坏死因子-α（TNF-α）诱导的内皮反应中的作用。通过抑制剂和RhoA/黏着斑激酶（FAK）shRNA评估TNF-α刺激的大鼠PMVECs中黏着斑激酶（FAK）和Ras同源基因家族成员A（RhoA）信号通路的功能。通过蛋白质免疫印迹法或下拉测定法加蛋白质免疫印迹法评估FAK和RhoA的激活情况。TNF-α处理后TER降低，而埃兹蛋白磷酸化呈时间和剂量依赖性增加。磷酸化埃兹蛋白主要定位于细胞周边，导致丝状肌动蛋白（F-肌动蛋白）重排，随后TER显著降低，FITC-BSA通量增加。此外，FAK和RhoA信号通路参与埃兹蛋白的磷酸化，前者对后者起正向调节作用。TNF-α通过FAK/RhoA信号通路诱导磷酸化埃兹蛋白，导致PMVECs内皮通透性增加。
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