Focal adhesion kinase in neutrophil-induced microvascular hyperpermeability
Focal adhesion kinase in neutrophil-induced microvascular hyperpermeability
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DOI：
10.1080/10739680590905251
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发表时间：
2005-03-01
期刊：
影响因子：
2.4
通讯作者：
Yuan, SY
中科院分区：
文献类型：
--
作者：
Guo, MZ;Wu, MH;Yuan, SY
目的：最近的实验证据表明，粘着斑激酶(FAK)在物理应激和化学刺激下介导内皮细胞的黏附、收缩和迁移中起重要作用。然而，FAK在炎症过程中对微血管屏障特性的功能影响尚未被揭示。本研究的目的是探讨FAK在中性粒细胞依赖的微血管高通透性中的潜在作用。方法：在C5a激活的中性粒细胞刺激下，测定完整的猪冠状动脉微静脉白蛋白的表观通透性系数。同时，在暴露于C5a激活的中性粒细胞的培养的小静脉内皮细胞单层中，对白蛋白的跨内皮流量进行了定量。Western blotting和免疫细胞化学分别检测FAK酪氨酸在内皮细胞中的磷酸化和分布。为明确FAK在中性粒细胞诱导的内皮高通透性中的信号转导作用，表达、纯化FAK相关的非激酶(FrnK)，并将其直接导入小静脉内皮细胞，检测FAK抑制中性粒细胞对中性粒细胞的通透性反应。结果：C5a激活的中性粒细胞可诱导小静脉通透性增加，且呈时间和浓度依赖性。微静脉转染FRANK不改变基础屏障功能，但显著减轻中性粒细胞诱导的高通透性，并呈剂量依赖关系。小静脉内皮细胞单层对中性粒细胞也有类似的通透性反应，但在FRNK转染后这种反应减弱。此外，Western印迹分析显示，激活的中性粒细胞引起FAK酪氨酸磷酸化水平呈浓度依赖性增加，其时间进程与静脉高通透性相关。FRANK可阻断中性粒细胞诱导的FAK酪氨酸磷酸化。此外，免疫荧光显微镜显示FAK的形态发生了显著的变化，从正常情况下点状的点状图案转变为在中性粒细胞刺激下与细胞纵轴平行的拉长的短划线状。结论：粘着斑激酶在中性粒细胞激活的内皮高通透性反应中起重要作用。FAK的磷酸化可能在炎症过程中微血管屏障功能的调节中发挥重要的信号作用。
Objective: Recent experimental evidence indicates an essential role of focal adhesion kinase (FAK) in mediating endothelial adhesion, contraction, and migration under physical stress and chemical stimulation. However, the functional impact of FAK on microvascular barrier property during inflammation has not been revealed. The aim of this study was to explore the potential contribution of FAK to neutrophil-dependent microvascular hyperpermeability.Methods: The apparent permeability coefficient of albumin was measured in intact, isolated porcine coronary venules during stimulation by C5a-activated neutrophils. In parallel, the transendothelial flux of albumin was quantified in cultured venular endothelial cell monolayers exposed to C5a-activated neutrophils. Western blotting and immunocytochemistry were performed to asses FAK tyrosine phosphorylation and distribution in endothelial cells, respectively. To specify the signaling effect of FAK on neutrophil-elicited endothelial hyperpermeability, FAK-related nonkinase (FRNK) was expressed, purified, and directly transfected into the endothelium of venules, and the permeability response to neutrophils was measured during inhibition of FAK.Results: C5a-activated neutrophils induced a time- and concentration-dependent increase in venular permeability. Transfection of venules with FRNK did not alter the basal barrier function but greatly attenuated neutrophil-induced hyperpermeability in a dose-related manner. A similar permeability response to neutrophils was observed in venular endothelial cell monolayers, which was diminished after FRNK transfection. In addition, Western blot analysis showed that activated neutrophils caused a concentration-dependent increase in FAK tyrosine phosphorylation with a time course correlating with that of venular hyperpermeability. Transfection of FRNK blocked neutrophil-evoked FAK tyrosine phosphorylation. Furthermore, immunofluorescence microscopy revealed a significant morphological change of FAK from a punctuated, dot-like pattern under normal conditions to an elongated, dash-like straining that aligned with the longitudinal axis of cells upon neutrophil stimulation.Conclusion: The results suggest that focal adhesion kinase significantly contributes to the endothelial hyperpermeability response to neutrophil activation. Phosphorylation of FAK may play an important signaling role in the regulation of microvascular barrier function during inflammation.