Aslam Muhammad, Tanislav Christian, Troidl Christian, Schulz Rainer, Hamm Christian, Gündüz Dursun
Department of Cardiology and Angiology, Justus Liebig University, Giessen, Germany.
Department of Neurology, Justus Liebig University, Giessen, Germany.
Physiol Rep. 2014 Oct 24;2(10). doi: 10.14814/phy2.12175. Print 2014 Oct 1.
Inflammatory mediators like thrombin disrupt endothelial adherens junctions (AJs) and barrier integrity leading to oedema formation followed by resealing of AJs and a slow recovery of the barrier function. The molecular mechanisms of this process have not yet been fully delineated. The aim of the present study was to analyse the molecular mechanism of endothelial barrier recovery and thrombin was used as model inflammatory mediator. Thrombin caused a strong increase in endothelial permeability within 10 min accompanied by loss of Rac1 but not cdc42 activity, drop in cellular cAMP contents, and a strong activation of the endothelial contractile machinery mainly via RhoA/Rock signalling. Activation of RhoA/Rock signalling precedes and is dependent upon a rise in the cytosolic Ca(2+) concentration. Inhibition of cytosolic Ca(2+) rise but not MLCK or Rock enhances the recovery of endothelial barrier function. The cellular cAMP contents increased gradually during the barrier recovery phase (30-60 min after thrombin challenge) accompanied by an increase in Rac1 activity. Inhibition of Rac1 activity using a specific pharmacological inhibitor (NSC23766) abrogated the endothelial barrier recovery process, suggesting a Rac1-dependent phenomenon. Likewise, inhibition of either adenylyl cyclase or the cAMP-effectors PKA and Epac (with PKI and ESI-09, respectively) caused an abrogation of Rac1 activation, resealing of endothelial AJs and recovery of endothelial barrier function. The data demonstrate that endothelial barrier recovery after thrombin challenge is regulated by Rac1 GTPase activation. This Rac1 activation is due to increased levels of cellular cAMP and activation of downstream signalling during the barrier recovery phase.
凝血酶等炎症介质会破坏内皮细胞黏附连接（AJs）和屏障完整性，导致水肿形成，随后AJs重新封闭，屏障功能缓慢恢复。这一过程的分子机制尚未完全阐明。本研究的目的是分析内皮屏障恢复的分子机制，并将凝血酶用作炎症介质模型。凝血酶在10分钟内使内皮通透性显著增加，同时Rac1活性丧失，但cdc42活性未丧失，细胞内cAMP含量下降，并且主要通过RhoA/Rock信号通路强烈激活内皮收缩机制。RhoA/Rock信号通路的激活先于并依赖于胞质Ca(2+)浓度的升高。抑制胞质Ca(2+)升高而非肌球蛋白轻链激酶（MLCK）或Rho相关卷曲螺旋蛋白激酶（Rock）可增强内皮屏障功能的恢复。在屏障恢复阶段（凝血酶刺激后30 - 60分钟），细胞内cAMP含量逐渐增加，同时Rac1活性增强。使用特异性药理抑制剂（NSC23766）抑制Rac1活性可消除内皮屏障恢复过程，提示这是一种依赖Rac1的现象。同样，抑制腺苷酸环化酶或cAMP效应物蛋白激酶A（PKA）和交换蛋白直接激活cAMP（Epac）（分别使用PKI和ESI - 09）会导致Rac1激活被消除、内皮AJs重新封闭以及内皮屏障功能恢复。数据表明，凝血酶刺激后内皮屏障的恢复受Rac1 GTP酶激活的调节。这种Rac1激活是由于屏障恢复阶段细胞内cAMP水平升高和下游信号通路的激活。
