Dysfunction of vascular barriers is a critical step in inflammatory diseases. Endothelial tight junctions (TJs) control barrier function, and the cytoplasmic adaptor protein cingulin connects TJs to signalling pathways. However, local events at TJs during inflammation are largely unknown. In this study, we investigate the local response of TJ adaptor protein cingulin and its interaction with Rho guanine nucleotide exchange factor H1 (GEF-H1, also known as ARHGEF2) upon vascular barrier disruption to find a new approach to counteract vascular leak. Based on transendothelial-electrical-resistance (TEER) measurements, cingulin strengthened barrier integrity upon stimulation with histamine, thrombin and VEGF. Cingulin also attenuated myosin light chain 2 (MLC2; also known as MYL2) phosphorylation by localising GEF-H1 to cell junctions. By using cingulin phosphomutants, we verified that the phosphorylation of the cingulin head domain is required for its protective effect. Increased colocalisation of GEF-H1 and cingulin was observed in the vessels of vasculitis patients compared to those in healthy skin. Our findings demonstrate that cingulin can counteract vascular leak at TJs, suggesting the existence of a novel mechanism in blood endothelial cells that protects barrier function during disease.

中文翻译：

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磷酸化的 cingulin 将 GEF-H1 定位在紧密连接处，以保护血液内皮细胞中的血管屏障。

血管屏障功能障碍是炎症性疾病的关键步骤。内皮紧密连接 (TJ) 控制屏障功能，细胞质衔接蛋白 cingulin 将 TJ 连接到信号通路。然而，炎症期间 TJ 的局部事件在很大程度上是未知的。在这项研究中，我们研究了 TJ 衔接蛋白 cingulin 的局部反应及其与 Rho 鸟嘌呤核苷酸交换因子 H1（GEF-H1，也称为 ARHGEF2）在血管屏障破坏时的相互作用，以寻找一种新的方法来对抗血管渗漏。基于经内皮电阻 (TEER) 测量，cingulin 在组胺、凝血酶和 VEGF 刺激下增强了屏障完整性。Cingulin 还通过将 GEF-H1 定位到细胞连接处来减弱肌球蛋白轻链 2 (MLC2；也称为 MYL2) 磷酸化。通过使用 cingulin 磷酸突变体，我们验证了 cingulin 头部结构域的磷酸化是其保护作用所必需的。与健康皮肤相比，在血管炎患者的血管中观察到 GEF-H1 和 cingulin 的共定位增加。我们的研究结果表明，cingulin 可以抵消 TJ 处的血管渗漏，这表明血液内皮细胞中存在一种在疾病期间保护屏障功能的新机制。