Division of Nephrology, Department of Medicine, The Third Affiliated Hospital of Sun Yat-sen University, 600 Tianhe Av, 510630, Guangzhou, GD, People's Republic of China.
Acta Diabetol. 2010 Dec;47 Suppl 1:57-63. doi: 10.1007/s00592-009-0121-8. Epub 2009 Apr 29.
Despite growing evidence for a pathogenic role of vascular endothelial growth factor (VEGF) in microvascular complications of diabetes, the underlying mechanism responsible for its detrimental effect remains unknown. In the current study, we hypothesized that some of the detrimental effects of VEGF on microvascular endothelial cells in the diabetic milieu stem from its aberrant signaling, which leads to perturbed tight junction assembly and increased endothelial permeability. Using an integrated in vitro approach, we investigated whether the effect of VEGF on endothelial cell permeability involves Rac1 GTPase activation and tight junction disassembly. Rac1 activity was detected by Western blotting in cell membrane protein as well as pull-down assay. The permeability of glomerular endothelial cells monolayer was detected as transendothelial electronic resistance. Then tyrosine phosphorylated occludin protein was detected by Western blotting after immunoprecipitation. N17Rac1 cells are obtained by transfection of glomerular endothelial cells with a dominant negative mutant of Rac1. The data obtained in this study indicate that activation of Rac1 GTPase contributes to VEGF-induced endothelial cell hyperpermeability. We also observed that Rac1 activation leads to increased endothelial permeability through tyrosine phosphorylation of occludin. Indeed, N17Rac1 cells dramatically attenuated the effect of VEGF on phospho-occludin and endothelial cell permeability. These results, when taken together, provide a framework for understanding the role of VEGF-induced Rac1/phospho-occludin pathway in the integrity of endothelial barrier function in the glomerulus.
尽管越来越多的证据表明血管内皮生长因子 (VEGF) 在糖尿病微血管并发症中具有致病作用，但导致其产生有害影响的潜在机制尚不清楚。在本研究中，我们假设 VEGF 在糖尿病环境中对微血管内皮细胞的一些有害影响源自其异常信号转导，这导致紧密连接组装受到干扰和内皮通透性增加。我们采用综合的体外方法，研究了 VEGF 对内皮细胞通透性的影响是否涉及 Rac1 GTP 酶的激活和紧密连接的解体。通过 Western blot 在细胞膜蛋白和下拉实验中检测 Rac1 活性。肾小球内皮细胞单层的通透性通过跨内皮电阻检测。然后通过免疫沉淀后 Western blot 检测酪氨酸磷酸化 occludin 蛋白。通过用 Rac1 的显性失活突变体转染肾小球内皮细胞获得 N17Rac1 细胞。本研究获得的数据表明 Rac1 GTP 酶的激活有助于 VEGF 诱导的内皮细胞高通透性。我们还观察到 Rac1 激活通过 occludin 的酪氨酸磷酸化导致内皮通透性增加。事实上，N17Rac1 细胞显著减弱了 VEGF 对磷酸化 occludin 和内皮细胞通透性的作用。这些结果表明，VEGF 诱导的 Rac1/磷酸化 occludin 途径在肾小球内皮屏障功能完整性中具有重要作用。
