cAMP controls the restoration of endothelial barrier function after thrombin-induced hyperpermeability via Rac1 activation.
cAMP controls the restoration of endothelial barrier function after thrombin-induced hyperpermeability via Rac1 activation.
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DOI：
10.14814/phy2.12175
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发表时间：
2014-10-01
影响因子：
2.5
通讯作者：
Gündüz D
中科院分区：
文献类型：
--
作者：
Aslam M;Tanislav C;Troidl C;Schulz R;Hamm C;Gündüz D
炎症介质，如凝血酶，破坏内皮细胞黏附连接(AJs)和屏障的完整性，导致水肿性形成，随后AJs重新封闭，屏障功能恢复缓慢。这一过程的分子机制尚未完全阐明。本研究以凝血酶为模型炎症介质，分析内皮屏障修复的分子机制。凝血酶在10分钟内引起内皮通透性的显著增加，伴随着rac1而不是cdc42活性的丧失，细胞内cAMP含量下降，并主要通过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细胞内钙离子浓度的升高，并依赖于细胞内钙离子浓度的升高。抑制胞浆Ca~(2+)升高而不是抑制MLCK或Rock可促进内皮屏障功能的恢复。在屏障恢复期(凝血酶攻击后30~60min)，细胞内cAMP含量逐渐升高，并伴随着rac1活性的升高。使用一种特定的药理抑制剂(NSC23766)抑制rac1的活性可消除内皮屏障的恢复过程，这提示了一种rac1依赖的现象。同样，抑制腺苷环化酶或cAMP效应物PKA和EPAC(分别用PKI和ESI-09)都会导致rac1的激活被取消，内皮AJ重新封闭，内皮屏障功能恢复。这些数据表明，凝血酶攻击后内皮屏障的恢复是由rac1 GTP酶激活调节的。这种Rac1的激活是由于屏障恢复阶段细胞内cAMP水平的增加和下游信号的激活。E12175在本研究中，我们分析了Rho家族GTP酶成员动态活性的变化以及内源性cAMP信号在凝血酶诱导的EC高通透性恢复中的作用。为了模拟体内的条件，凝血酶在整个实验过程中都存在。研究表明，用凝血酶攻击人脐静脉内皮细胞(HUVEC)单层会导致RhoA/Rock信号的迅速激活和rac1活性的抑制，同时细胞内cAMP含量减少。在EC屏障功能的恢复期(30-60min)，细胞内cAMP水平升高，而rac1而不是cdc42被激活。抑制腺酰环化酶(AC)或下游cAMP信号通路可在恢复期取消rac1的激活，并阻碍EC屏障功能的恢复。
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 Ca2+ concentration. Inhibition of cytosolic Ca2+ 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. e12175 In the present study, we analysed the changes in the dynamic activities of members of the Rho family of GTPases and the role of endogenous cAMP signalling in the restoration of thrombin‐induced EC hyperpermeability. To imitate the in vivo conditions, the thrombin was present during whole experiments. The study demonstrates that challenging the human umbilical vein endothelial cell (HUVEC) monolayers with thrombin results in a prompt activation (within first 10 min) of RhoA/Rock signalling and inhibition of Rac1 activity accompanied by a reduction in cellular cAMP contents. During the recovery phase of EC barrier function (30–60 min), an activation of Rac1 but not cdc42 occurs which is accompanied by an increase in intracellular levels of cAMP. Inhibition of adenylyl cyclase (AC) or downstream cAMP signalling abrogates Rac1 activation during the recovery phase and impedes the restoration of EC barrier function.