Institute for Personalized Respiratory Medicine, Section of Pulmonary, Critical Care, Sleep, and Allergy, University of Illinois at Chicago, College of Medicine, Chicago, IL 60612, USA.
Eur Respir J. 2011 Jul;38(1):78-88. doi: 10.1183/09031936.00047810. Epub 2010 Nov 11.
Strategies to improve pulmonary endothelial barrier function are needed to reverse the devastating effects of vascular leak in acute respiratory distress syndrome. FTY720 is a pharmaceutical analogue of the potent barrier-enhancing phospholipid sphingosine 1-phosphate (S1P). FTY720 decreases vascular permeability by an incompletely characterised mechanism that differs from S1P. Here, we describe its barrier-promoting effects on intracellular signalling and junctional assembly formation in human pulmonary endothelium. Permeability of cultured human pulmonary endothelial cells was assessed using transendothelial electrical resistance and dextran transwell assays. Junctional complex formation was assessed using membrane fractionation and immunofluorescence. Pharmacological inhibitors and small interfering (si)RNA were utilised to determine the effects of individual components on permeability. Unlike S1P, FTY720 failed to induce membrane translocation of adherens junction or tight junction proteins. β-catenin, occludin, claudin-5 or zona occludens protein (ZO)-1/ZO-2 siRNAs did not alter FTY720-induced barrier enhancement. FTY720 induced focal adhesion kinase (FAK) phosphorylation and focal adhesion formation, with FAK siRNA partially attenuating the prolonged phase of barrier enhancement. Inhibition of Src, protein kinase (PK)A, PKG, PKC or protein phosphatase 2A failed to alter FTY720-induced barrier enhancement. FTY720 increased c-Abl tyrosine kinase activity and c-Abl siRNA attenuated peak barrier enhancement after FTY720. FTY720 enhances endothelial barrier function by a novel pathway involving c-Abl signalling.
需要采取策略来改善肺血管内皮屏障功能，以逆转急性呼吸窘迫综合征中血管渗漏的破坏性影响。FTY720 是一种强效的屏障增强磷脂鞘氨醇 1-磷酸（S1P）的药物类似物。FTY720 通过一种与 S1P 不同的不完全特征的机制降低血管通透性。在这里，我们描述了它对人肺内皮细胞内信号转导和连接组装形成的促进屏障作用。使用跨内皮电阻和葡聚糖 Transwell 测定法评估培养的人肺内皮细胞的通透性。使用膜分馏和免疫荧光法评估连接复合体的形成。利用药理学抑制剂和小干扰 (si)RNA 来确定各个成分对通透性的影响。与 S1P 不同，FTY720 未能诱导细胞黏附连接或紧密连接蛋白的膜易位。β-连环蛋白、occludin、claudin-5 或 zona occludens 蛋白 (ZO)-1/ZO-2 siRNA 不会改变 FTY720 诱导的屏障增强。FTY720 诱导粘着斑激酶 (FAK) 磷酸化和粘着斑形成，FAK siRNA 部分减弱了屏障增强的延长阶段。Src、蛋白激酶 (PK)A、PKG、PKC 或蛋白磷酸酶 2A 的抑制作用未能改变 FTY720 诱导的屏障增强。FTY720 增加 c-Abl 酪氨酸激酶活性，c-Abl siRNA 减弱 FTY720 后峰值屏障增强。FTY720 通过涉及 c-Abl 信号的新型途径增强内皮屏障功能。
