Cardeza Center for Hemostasis, Thrombosis, and Vascular Biology, Cardeza, Foundation for Hematologic Research, Department of Medicine, Division of Hematology, Sydney Kimmel Medical College, Thomas Jefferson University, Philadelphia, USA.
Cardeza Center for Hemostasis, Thrombosis, and Vascular Biology, Cardeza, Foundation for Hematologic Research, Department of Medicine, Division of Hematology, Sydney Kimmel Medical College, Thomas Jefferson University, Philadelphia, USA.
Vascul Pharmacol. 2022 Aug;145:107088. doi: 10.1016/j.vph.2022.107088. Epub 2022 Jul 4.
Thrombin-induced endothelial permeability is associated with various pathological conditions. Apoptosis signal-regulating kinase-1 (ASK1), one of the upstream MAP3K, has been reported to be an important regulator of endothelial stress and apoptosis. Despite this, its role in endothelial permeability is unknown. The aim of this study was to determine the role of ASK1 in thrombin-induced endothelial permeability. To do so, a live cell monitoring system and transwell assay were used to evaluate in vitro endothelial permeability, while a Miles assay was used for in vivo permeability. Immunofluorescence and western blotting were used to visualize integrity of the junctions and phosphorylation of various proteins, respectively. We observed that in vivo thrombin-induced vascular permeability was attenuated in Ask1 mice. Pretreatment of human primary endothelial cells (ECs) with GS-4997 (ASK1 inhibitor) and deficiency of ASK1 in primary mouse lung ECs significantly attenuated the thrombin-induced endothelial permeability. Furthermore, in the presence of GS-4997, the following were also significantly reduced: thrombin-induced para-cellular gap formation, VE-cadherin proteolysis, and dislocation of VE-cadherin, JAM-A, and ZO1 from the junctions. Inhibition of ASK1 restored peripheral location of F-actin, similar to that induced by sphingosine-1-phosphate. These results suggest a unique role for ASK1 in regulating thrombin-induced endothelial permeability.
凝血酶诱导的内皮通透性与各种病理状况有关。凋亡信号调节激酶 1（ASK1）作为上游 MAP3K 之一，已被报道是内皮应激和凋亡的重要调节因子。尽管如此，其在内皮通透性中的作用尚不清楚。本研究旨在确定 ASK1 在凝血酶诱导的内皮通透性中的作用。为此，使用活细胞监测系统和 Transwell 测定法评估体外内皮通透性，而 Miles 测定法用于评估体内通透性。免疫荧光和 Western blot 分别用于可视化连接的完整性和各种蛋白质的磷酸化。我们观察到，在 Ask1 小鼠中，体内凝血酶诱导的血管通透性减弱。人原代内皮细胞（ECs）用 GS-4997（ASK1 抑制剂）预处理和原代小鼠肺 ECs 中 ASK1 的缺乏显著减弱了凝血酶诱导的内皮通透性。此外，在存在 GS-4997 的情况下，凝血酶诱导的细胞旁间隙形成、VE-钙粘蛋白的蛋白水解和 VE-钙粘蛋白、JAM-A 和 ZO1 从连接处的位置改变也显著减少。ASK1 的抑制作用恢复了外周 F-肌动蛋白的位置，类似于鞘氨醇 1-磷酸诱导的位置。这些结果表明 ASK1 在调节凝血酶诱导的内皮通透性方面具有独特的作用。
