Department for Physiology, VU University Medical Center, Institute for Cardiovascular Research, van der Boechorststraat 7, Amsterdam 1108 BH, The Netherlands.
Cardiovasc Res. 2013 Aug 1;99(3):471-82. doi: 10.1093/cvr/cvt075. Epub 2013 Mar 27.
Endothelial cells (ECs) control vascular permeability by forming a monolayer that is sealed by extracellular junctions. Various mediators modulate the endothelial barrier by acting on junctional protein complexes and the therewith connected F-actin cytoskeleton. Different Rho GTPases participate in this modulation, but their mechanisms are still partly resolved. Here, we aimed to elucidate whether the opening and closure of the endothelial barrier are associated with distinct localized RhoA activities at the subcellular level.
Live fluorescence resonance energy transfer (FRET) microscopy revealed spatially distinct RhoA activities associated with different aspects of the regulation of endothelial monolayer integrity. Unstimulated ECs were characterized by hotspots of RhoA activity at their periphery. Thrombin receptor activation in the femoral vein of male wistar rats and in cultured ECs enhanced RhoA activity at membrane protrusions, followed by a more sustained RhoA activity associated with cytoplasmic F-actin filaments, where prolonged RhoA activity coincided with cellular contractility. Unexpectedly, thrombin-induced peripheral RhoA hotspots were not spatially correlated to the formation of large inter-endothelial gaps. Rather, spontaneous RhoA activity at membrane protrusions coincided with the closure of inter-endothelial gaps. Electrical impedance measurements showed that RhoA signalling is essential for this protrusive activity and maintenance of barrier restoration.
Spontaneous RhoA activity at membrane protrusions is spatially associated with closure, but not formation of inter-endothelial gaps, whereas RhoA activity at distant contractile filaments contributes to thrombin-induced disruption of junctional integrity. Thus, these data indicate that distinct RhoA activities are associated with disruption and re-annealing of endothelial junctions.
内皮细胞 (ECs) 通过形成由细胞外连接封闭的单层来控制血管通透性。各种介质通过作用于连接蛋白复合物和与之相连的 F-肌动蛋白细胞骨架来调节内皮屏障。不同的 Rho GTPases 参与这种调节，但它们的机制仍部分解决。在这里，我们旨在阐明内皮屏障的开放和关闭是否与亚细胞水平上特定的局部 RhoA 活性有关。
活荧光共振能量转移 (FRET) 显微镜显示，与内皮单层完整性调节的不同方面相关的 RhoA 活性具有空间上的差异。未受刺激的 ECs 的特征是其外围存在 RhoA 活性热点。凝血酶受体在雄性 Wistar 大鼠股静脉和培养的 EC 中的激活增强了膜突起处的 RhoA 活性，随后是与细胞质 F-肌动蛋白丝相关的更持续的 RhoA 活性，其中持续的 RhoA 活性与细胞收缩性一致。出乎意料的是，凝血酶诱导的外周 RhoA 热点与大的内皮细胞间隙的形成没有空间相关性。相反，膜突起处自发的 RhoA 活性与内皮细胞间隙的闭合同时发生。电阻抗测量表明 RhoA 信号对于这种突起活性和屏障恢复的维持是必不可少的。
膜突起处自发的 RhoA 活性与内皮细胞间隙的闭合有关，但与间隙的形成无关，而远处的收缩丝处的 RhoA 活性有助于凝血酶诱导的连接完整性的破坏。因此，这些数据表明，不同的 RhoA 活性与内皮连接的破坏和再连接有关。
