Seebach Jochen, Mädler Hans-Jürgen, Wojciak-Stothard Beata, Schnittler Hans-Joachim
Institut of Physiology, TU-Dresden, Dresden, Germany.
Thromb Haemost. 2005 Sep;94(3):620-9. doi: 10.1160/TH05-01-0015.
Endothelial barrier function depends on the integrity of intercellular adherens junctions controlled by the association of VE-cadherin/catenin complex with cortical actin filaments. Both tyrosine phosphorylation/dephosphorylation of junctional proteins and actin reorganization mediated by rho-GTPases regulate barrier function but the relationship between these regulatory mechanisms is unclear. Here we studied the effects of factors increasing protein tyrosine phosphorylation, pervanadate (PV) and VEGF, on distribution of VE-cadherin, F-actin polymerization and transendothelial electrical resistance (TER) in human umbilical vein endothelial cells (HUVECs). Changes in protein tyrosine phosphorylation of cytoplasmic and junctional proteins, as well as the activity of rho-GTPase racl, were also measured. We report for the first time that PV and VEGF induced a rapid transient increase in endothelial barrier function accompanied by rac1 activation, a differentiated tyrosine phosphorylation of the VE-cadherin/catenin complex, recruitment of actin filament to cell junctions and ruffle formation. A sustained decrease in endothelial barrier function was observed at later times of PV and VEGF treatment. Expression of dominant negative rac1, N17rac1 abolished the barrier-enhancing effects of PV and VEGF, while the sustained decrease in barrier function was unaffected. These observations bring into focus early short-term effects of protein tyrosine phosphorylation in cells, often overshadowed by more pronounced and long-lasting later effects and may play an important role in the regulation of endothelial barrier function.
内皮细胞屏障功能取决于由血管内皮钙黏蛋白/连环蛋白复合体与皮质肌动蛋白丝结合所控制的细胞间黏附连接的完整性。连接蛋白的酪氨酸磷酸化/去磷酸化以及由rho-GTP酶介导的肌动蛋白重组均调节屏障功能，但这些调节机制之间的关系尚不清楚。在此，我们研究了增加蛋白酪氨酸磷酸化的因子（过氧钒酸盐（PV）和血管内皮生长因子（VEGF））对人脐静脉内皮细胞（HUVECs）中血管内皮钙黏蛋白的分布、F-肌动蛋白聚合和跨内皮电阻（TER）的影响。我们还检测了细胞质和连接蛋白的蛋白酪氨酸磷酸化变化以及rho-GTP酶rac1的活性。我们首次报道，PV和VEGF诱导内皮细胞屏障功能迅速短暂增强，同时伴有rac1激活、血管内皮钙黏蛋白/连环蛋白复合体的特异性酪氨酸磷酸化、肌动蛋白丝募集到细胞连接处以及微绒毛形成。在PV和VEGF处理的后期，观察到内皮细胞屏障功能持续下降。显性负性rac1（N17rac1）的表达消除了PV和VEGF对屏障的增强作用，而屏障功能的持续下降则不受影响。这些观察结果使人们关注到细胞中蛋白酪氨酸磷酸化的早期短期效应，这些效应常常被更明显和持久的后期效应所掩盖，并且可能在内皮细胞屏障功能的调节中发挥重要作用。
