Rho and ROCK signaling in VEGF-induced microvascular endothelial hyperpermeability
Rho and ROCK signaling in VEGF-induced microvascular endothelial hyperpermeability
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DOI：
10.1080/10739680600556944
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发表时间：
2006-04-01
期刊：
影响因子：
2.4
通讯作者：
Wu, MH
中科院分区：
文献类型：
--
作者：
Sun, HR;Breslin, JW;Wu, MH
目的：血管内皮生长因子（VEGF）在多种生理和病理条件下对微血管通透性的调节起重要作用。作者检验了小GTdR Rho及其下游效应物ROCK（Rho相关卷曲螺旋蛋白激酶）介导VEGF诱导的微静脉通透性增加的假设。他们还研究了肌球蛋白轻链（MLC）的磷酸化和肌动蛋白聚合，两个良好的特征的Rho-ROCK通路的目标，涉及在内皮屏障functions.Methods的调节：白蛋白（P-α）的表观渗透系数测定在完整的分离的猪冠状静脉和培养的冠状静脉内皮细胞（CVEC）单层。使用Rhotekin-琼脂糖下拉测定法测定RhoA活化。MLC磷酸化进行了评估，通过免疫印迹与磷酸化特异性抗体，和内皮细胞F-肌动蛋白被认为是使用荧光microscopic.Results：VEGF增加P-A在两个孤立的冠状静脉和CVEC单层。高通透性反应发生在一个类似的时间过程中的Rho激活，MLC磷酸化，肌动蛋白应力纤维的形成。用Y27632选择性阻断ROCK可剂量依赖性地抑制VEGF诱导的微静脉通透性增高。此外，抑制Rho与外切酶C3或ROCK与Y-27632衰减VEGF诱导的增加，在CVEC monolayers.Conclusions的渗透性，MLC磷酸化，和肌动蛋白应力纤维的形成，这些研究结果表明，Rho-ROCK信号通路有助于VEGF诱导的高渗透性。肌球蛋白轻链磷酸化和肌动蛋白应力纤维形成伴随着VEGF刺激后通透性的增加而发生。
Objectives: Vascular endothelial growth factor (VEGF) plays an important role in the regulation of microvascular permeability under various physiological and pathological conditions. The authors tested the hypothesis that the small GTPase Rho and its downstream effector ROCK (Rho-associated coiled-coil-containing protein kinase) mediate VEGF-induced increases in venular permeability. They also investigated myosin light chain (MLC) phosphorylation and actin polymerization, two well-characterized targets of the Rho-ROCK pathway that are implicated in the regulation of endothelial barrier function.Methods: The apparent permeability coefficient of albumin (P-a) was measured in intact isolated porcine coronary venules and in cultured coronary venular endothelial cell (CVEC) monolayers. RhoA activation was determined using a Rhotekin-agarose pull down assay. MLC phosphorylation was evaluated by immunoblotting with phospho-specific antibodies, and endothelial cellular F-actin was viewed using fluorescence microscopy.Results: VEGF increased P-a in both isolated coronary venules and CVEC monolayers. The hyperpermeability response occurred in a similar time course to that of Rho activation, MLC phosphorylation, and actin stress fiber formation. Selective blockage of ROCK with Y27632 dose-dependently inhibited VEGF-induced venular hyperpermeability. Moreover, inhibition of either Rho with exoenzyme C3 or ROCK with Y-27632 attenuated VEGF-induced increases in permeability, MLC phosphorylation, and actin-stress fiber formation in CVEC monolayers.Conclusions: Collectively, these findings suggest that the Rho-ROCK signal pathway contributes to VEGF-induced hyperpermeability. Myosin light-chain phosphorylation and actin stress fiber formation occur concomitantly with the increase in permeability upon VEGF stimulation.