Guo Mingzhang, Wu Mack H, Granger Harris J, Yuan Sarah Y
Department of Surgery, Texas A & M University Health Science Center, Temple, Texas, USA.
Microcirculation. 2005 Mar;12(2):223-32. doi: 10.1080/10739680590905251.
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.
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 assess 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.
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 staining that aligned with the longitudinal axis of cells upon neutrophil stimulation.
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.
最近的实验证据表明，黏着斑激酶（FAK）在介导物理应激和化学刺激下的内皮细胞黏附、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明确。本研究旨在探讨FAK对中性粒细胞依赖性微血管高通透性的潜在作用。
在C5a激活的中性粒细胞刺激下，测量完整分离的猪冠状动脉小静脉中白蛋白的表观渗透系数。同时，在暴露于C5a激活的中性粒细胞的培养小静脉内皮细胞单层中，对白蛋白的跨内皮通量进行定量。分别进行蛋白质免疫印迹法和免疫细胞化学法，以评估内皮细胞中FAK酪氨酸磷酸化和分布情况。为明确FAK对中性粒细胞诱导的内皮细胞高通透性的信号传导作用，表达、纯化FAK相关非激酶（FRNK）并将其直接转染至小静脉内皮细胞，在抑制FAK的过程中测量对中性粒细胞的通透性反应。
C5a激活的中性粒细胞诱导小静脉通透性呈时间和浓度依赖性增加。用FRNK转染小静脉未改变基础屏障功能，但以剂量相关方式显著减弱了中性粒细胞诱导的高通透性。在小静脉内皮细胞单层中观察到对中性粒细胞的类似通透性反应，FRNK转染后该反应减弱。此外，蛋白质免疫印迹分析表明，激活的中性粒细胞导致FAK酪氨酸磷酸化呈浓度依赖性增加，其时间进程与小静脉高通透性相关。FRNK转染可阻断中性粒细胞诱发的FAK酪氨酸磷酸化。此外，免疫荧光显微镜显示，FAK在形态上有显著变化，从正常条件下的点状、斑点状模式变为在中性粒细胞刺激后与细胞纵轴对齐的细长、短线状染色。
结果表明，黏着斑激酶显著促成了内皮细胞对中性粒细胞激活的高通透性反应。FAK的磷酸化可能在炎症过程中微血管屏障功能的调节中起重要的信号传导作用。
