Rho and rho kinase modulation of barrier properties: cultured endothelial cells and intact microvessels of rats and mice
Rho and rho kinase modulation of barrier properties: cultured endothelial cells and intact microvessels of rats and mice
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DOI：
10.1113/jphysiol.2001.013117
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发表时间：
2002-02-15
影响因子：
5.5
通讯作者：
Drenckhahn, D
中科院分区：
文献类型：
--
作者：
Adamson, RH;Curry, FE;Drenckhahn, D
先前使用培养的内皮细胞单层的实验表明，Rho家族小GTP酶通过调节细胞肌动蛋白丝支架的组装、基于肌球蛋白的收缩性的活性和Ca 2+依赖性内皮细胞粘附分子VE-钙粘蛋白的连接分布参与内皮细胞单层通透性的调节。我们使用培养的内皮细胞（来自猪肺动脉和小鼠心脏）和血管内皮细胞研究这些机制。原位（小鼠主动脉，以及小鼠和大鼠肠系膜的单独灌注的微静脉微血管）。暴露于艰难梭菌毒素B（100 ng ml（-1））在60-90 min内灭活了50- 90%的所有内皮Rho蛋白。这伴随着培养的内皮单层和小鼠主动脉内皮原位中肌动蛋白丝应力纤维和连接F-肌动蛋白的显著减少。此外，VE-钙粘蛋白沿沿着内皮连接变得不连续。用Y-27632（30 μ M）抑制Rho激酶90-120分钟诱导体外和原位F-肌动蛋白减少，但不引起VE-钙粘蛋白染色的再分布或减少。灌注毒素B的微血管增加的基础液压渗透性（L-p），但并没有减弱的瞬时增加L-p暴露于缓激肽的微血管。微血管灌注Y-27632（30 μ M）长达100分钟，降低基础L-p，但不减弱血小板活化因子（PAF）或缓激肽诱导的渗透性增加。这些结果表明，毒素B介导的内皮屏障性能的降低是由于RhoA以外的小GTP酶的失活。Rho蛋白以及RhoA介导的收缩机制不参与缓激肽或PAF诱导的完整微血管通透性过高。
Previous experiments using cultured endothelial monolayers indicate that Rho-family small GTPases are involved in modulation of endothelial monolayer permeability by regulating assembly of the cellular actin filament scaffold, activity of myosin-based contractility and junctional distribution of the Ca2+-dependent endothelial cell adhesion molecule, VE-cadherin. We investigated these mechanisms using both cultured endothelial cells (from porcine pulmonary artery and mouse heart) and vascular endothelium. in situ (mouse aorta, and individually perfused venular microvessels of mouse and rat mesentery). Exposure to Clostridium difficile toxin B (100 ng ml(-1)) inactivated 50-90 % of all endothelial Rho proteins within 60-90 min. This was accompanied by considerable reduction of actin filament stress fibres and junctional F-actin in cultured endothelial monolayers and in mouse aortic endothelium in situ. Also, VE-cadherin became discontinuous along endothelial junctions. Inhibition of Rho kinase with Y-27632 (30 muM) for 90-120 min induced F-actin reduction both in vitro and in situ but did not cause redistribution or reduction of VE-cadherin staining. Perfusion of microvessels with toxin B increased basal hydraulic permeability (L-p) but did not attenuate the transient increase in L-p of microvessels exposed to bradykinin. Perfusion of microvessels with Y-27632 (30 muM) for up to 100 min reduced basal L-p but did not attenuate the permeability increase induced by platelet activating factor (PAF) or bradykinin. These results show that toxin B-mediated reduction of endothelial barrier properties is due to inactivation of small GTPases other than RhoA. Rho proteins as well as RhoA-mediated contractile mechanisms are not involved in bradykinin- or PAF-induced hyperpermeability of intact microvessels.