E1A激活基因阻遏子促进人血管内皮细胞VEGF分泌和单层通透性增加

目的: 探讨E1A激活基因阻遏子(CREG)诱导的人血管内皮细胞(ECs)单层通透性改变中的作用及机制。方法: 用CREG过表达及CREG表达下调的ECs为模型,Transwell chamber弥散模型观察ECs单层通透性的改变; 荧光倒置显微镜观察细胞骨架肌动蛋白F-actin及黏附连接蛋白VE-cadherin在ECs中的分布和形态学改变;酶联免疫吸附实验(ELISA)检测ECs血管内皮生长因子(VEGF)分泌。结果: CREG过表达的ECs (EO组) 较EN组单层通透性明显增高 (P<0.05);CREG表达下调的ECs(ES组)较EN组单层通透性有所下降(P<0.05)。与EN组相比较,EO组细胞中F-actin排列紊乱,形成大量应力纤维; ES组F-actin则主要呈细丝状分布于细胞周边,中央分布较少。同时,EO组VE-cadherin在细胞周边的正常拉链状结构减少或缺失,细胞间隙增宽;而ES组VE-cadherin在细胞周边呈正常拉链状分布,细胞之间连接紧密。ELISA检测显示EO组细胞上清中VEGF分泌较EN组明显增加(P<0.05);ES组VEGF分泌较EN组减少(P<0.05)。应用VEGF中和抗体阻断后,CREG过表达引起的EO通透性增加的现象明显受到抑制。结论: CREG过表达可能通过VEGF介导的信号途径引起F-actin重构及VE-cadherin减少,使血管内皮细胞单层通透性增加。

Effects of cellular repressor of E1A-stimulated genes on VEGF release and monolayer permeability of human vascular endothelial cells

AIM: To study the effects and mechanism of cellular repressor of E1A-stimulated genes (CREG) on VEGF release and monolayer permeability of human vascular endothelial cells (ECs). METHODS: The monolayer permeability of ECs was measured by transwell chamber model. The expression and localization of F-actin and VE-cadherin were examined by immunofluroscence using Olympus IX-70 fluorescent microscope. Enzyme-linked immunosorbent assay (ELISA) were performed to determine the concentration of vascular endothelial growth factors(VEGF) in the culture medium. VEGF neutrilization antibody was used to block the expression of VEGF in the cells. RESULTS: The monolayer permeability of CREG over-expressing ECs (EO group) was significantly higher than that of the normal control ECs (EN group, P<0.05). The monolayer permeability of CREG suppressing ECs (ES group) was lower than that in EN group (P<0.05). F-actin cytoskeleton in EO group showed disorganized, polymerized and bundled obviously to form large quantity of stress fibers in the central portion of the cells, whereas F-actin in EO group was mainly observed in the peripheral portion of the cells and only small amounts in the central portion of the cells. A widespread gap formation and a loss of VE-cadherin staining at the periphery were found in the cells of EO group. Inversely, the cells in ES group showed the localization of VE-cadherin at the cell-cell contacts tightly and the formation of zipper-like structures. Compared with EN group, the secretion of VEGF in the cell culture supernatants increased in EO group, but decreased in ES group (P<0.05). Furthermore, the changes of ECs permeability, cytoskeleton reorganization and loss of VE-cadherin induced by CREG were abolished by the addition of anti-VEGF neutralizing antibody. CONCLUSION: CREG over-expression increases the monolayer permeability of ECs, induces the cytoskeleton reorganization and reduces VE-cadherin expression by enhancing the secretion of VEGF in vitro.