van Nieuw Amerongen G P, Vermeer M A, van Hinsbergh V W
Gaubius Laboratory TNO-PG, Leiden, The Netherlands.
Arterioscler Thromb Vasc Biol. 2000 Dec;20(12):E127-33. doi: 10.1161/01.atv.20.12.e127.
In the present study, the roles of the small GTPase RhoA and its target Rho kinase in endothelial permeability were investigated in vitro. We have shown previously that, in addition to a rise in the intracellular Ca(2+) concentration (Ca(2+)), RhoA is involved in the prolonged thrombin-induced barrier dysfunction. To study the role of RhoA and Rho kinase more specifically, endothelial cells were stimulated with lysophosphatidic acid (LPA), a commonly used RhoA activator. LPA induced a 2- to 3-fold increase in the passage of horseradish peroxidase (HRP) across endothelial monolayers that lasted for several hours, whereas thrombin induced a 5- to 10-fold increase. Comparable to the thrombin-induced barrier dysfunction, the LPA-induced barrier dysfunction was accompanied by a reorganization of the F-actin cytoskeleton and the formation of focal attachment sites. LPA induced only a transient increase in myosin light-chain (MLC) phosphorylation, which returned to basal level within 10 minutes. In endothelial cells, Ca(2+) was not elevated by LPA. Chelation of Ca(2+)(i) ions by 1, 2-bis(2-aminophenoxy)ethane-N:,N:,N:',N:'-tetraacetic acid did not prevent the LPA-induced passage of HRP. Apparently, a low degree of MLC kinase activation occurred, because the MLC kinase inhibitor KT5926 reduced the levels of both basal and LPA-stimulated HRP passage. Inhibition of RhoA by the C3 transferase from Clostridium botulinum inhibited the LPA-induced cytoskeletal changes and prevented the LPA-induced HRP passage. Inhibition of Rho kinase by Y-27632 completely prevented the LPA-induced increase in HRP passage without affecting basal permeability. These data indicate that LPA-induced endothelial hyperpermeability occurs without a change in Ca(2+) and requires activation of RhoA and Rho kinase.
在本研究中，我们在体外研究了小GTP酶RhoA及其靶标Rho激酶在内皮细胞通透性中的作用。我们之前已经表明，除了细胞内Ca(2+)浓度（Ca(2+)）升高外，RhoA还参与凝血酶诱导的长时间屏障功能障碍。为了更具体地研究RhoA和Rho激酶的作用，用溶血磷脂酸（LPA）刺激内皮细胞，LPA是一种常用的RhoA激活剂。LPA诱导辣根过氧化物酶（HRP）穿过内皮单层的通过率增加2至3倍，持续数小时，而凝血酶诱导的通过率增加5至10倍。与凝血酶诱导的屏障功能障碍类似，LPA诱导的屏障功能障碍伴随着F-肌动蛋白细胞骨架的重组和粘着斑的形成。LPA仅诱导肌球蛋白轻链（MLC）磷酸化的短暂增加，10分钟内恢复到基础水平。在内皮细胞中，LPA不会使Ca(2+)升高。用1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸螯合Ca(2+)(i)离子并不能阻止LPA诱导的HRP通过。显然，发生了低度的MLC激酶激活，因为MLC激酶抑制剂KT5926降低了基础和LPA刺激的HRP通过水平。肉毒杆菌的C3转移酶对RhoA的抑制作用抑制了LPA诱导的细胞骨架变化，并阻止了LPA诱导的HRP通过。Y-27632对Rho激酶的抑制作用完全阻止了LPA诱导的HRP通过率增加，而不影响基础通透性。这些数据表明，LPA诱导的内皮细胞高通透性在Ca(2+)无变化的情况下发生，并且需要RhoA和Rho激酶的激活。
