Mehta Dolly, Tiruppathi Chinnaswamy, Sandoval Raudal, Minshall Richard D, Holinstat Michael, Malik Asrar B
Department of Pharmacology, College of Medicine, The University of Illinois, 835 S Wolcott Avenue, Chicago, IL 60612, USA.
J Physiol. 2002 Mar 15;539(Pt 3):779-89. doi: 10.1113/jphysiol.2001.013289.
The adhesive force generated by the interaction of integrin receptors with extracellular matrix (ECM) at the focal adhesion complex may regulate endothelial cell shape, and thereby the endothelial barrier function. We studied the role of focal adhesion kinase (FAK) activated by integrin signalling in regulating cell shape using cultured human pulmonary artery endothelial cells. We used FAK antisense oligonucleotide (targeted to the 3'-untranslated region of FAK mRNA (5'-CTCTGGTTGATGGGATTG-3') to determine the role of FAK in the mechanism of thrombin-induced increase in endothelial permeability. Reduction in FAK expression by the antisense augmented the thrombin-induced decrease in transendothelial electrical resistance (decrease in mock transfected cells of -43 +/- 1 % and in sense-transfected cells of -40 +/- 4 %, compared to the decrease in antisense-transfected cells of -60 +/- 3 %). Reduction in FAK expression also prolonged the drop in electrical resistance and prevented the recovery seen in control endothelial cells. Thus, the thrombin-induced increase in permeability is both greater and attenuated in the absence of FAK expression. Inhibition of actin polymerization with latrunculin-A prevented the translocation of FAK to focal adhesion sites and tyrosine phosphorylation of FAK and paxillin, and concomitantly reduced the thrombin-induced decrease in electrical resistance by approximately 50 %. Thus, the modulatory role of FAK on endothelial barrier function is dependent on actin polymerization. FAK translocation to focal adhesion complex in endothelial cells guided by actin cables and the consequent activation of FAK-associated proteins serve to reverse the decrease in endothelial barrier function caused by inflammatory mediators such as thrombin.
整联蛋白受体与细胞外基质（ECM）在黏着斑复合体处相互作用产生的黏附力，可能调节内皮细胞形状，进而调节内皮屏障功能。我们使用培养的人肺动脉内皮细胞，研究了由整联蛋白信号激活的黏着斑激酶（FAK）在调节细胞形状中的作用。我们使用FAK反义寡核苷酸（靶向FAK mRNA的3'非翻译区（5'-CTCTGGTTGATGGGATTG-3'））来确定FAK在凝血酶诱导的内皮通透性增加机制中的作用。反义寡核苷酸使FAK表达降低，增强了凝血酶诱导的跨内皮电阻降低（与反义转染细胞-60±3%的降低相比，mock转染细胞降低-43±1%，正义转染细胞降低-40±4%）。FAK表达降低还延长了电阻下降时间，并阻止了对照内皮细胞中出现的恢复。因此，在没有FAK表达的情况下，凝血酶诱导的通透性增加更大且减弱。用Latrunculin-A抑制肌动蛋白聚合可阻止FAK转位至黏着斑位点以及FAK和桩蛋白的酪氨酸磷酸化，并同时使凝血酶诱导的电阻降低约50%。因此，FAK对内皮屏障功能的调节作用依赖于肌动蛋白聚合。由肌动蛋白丝引导的FAK转位至内皮细胞的黏着斑复合体以及随之而来的FAK相关蛋白的激活，有助于逆转由凝血酶等炎症介质引起的内皮屏障功能降低。
