血管扩张刺激磷蛋白对抵抗素促进冠状动脉内皮细胞增殖和迁移的影响

Role of vasodilator stimulated phosphoprotein in resistin-induced proliferation and migration of human coronary endothelial cells

目的抵抗素可通过导致内皮细胞功能的失调,参与动脉粥样硬化的发生和发展,然而其作用机制研究较少。文中旨在研究抵抗素对冠状动脉内皮细胞功能的影响,探讨影响抵抗素作用的可能机制。方法构建针对人血管扩张刺激磷蛋白(VASP)的慢病毒表达载体的VASP mRNA的shRNA干扰序列(LV-si VASP组)和阴性对照载体(LV-sicntr组),并将其感染高表达VASP的人冠状动脉内皮细胞,RT-PCR检测VASP mRNA的表达,Western blot测定蛋白的表达。用不同浓度的抵抗素(0、10、50、100、200 ng/m L)体外作用于人冠状动脉内皮细胞系(HCAECs),MTT法检测抵抗素对细胞增殖的作用,Transwell小室实验检测细胞迁移能力的改变。在相同抵抗素浓度100 ng/m L下,检测2组对内皮细胞增殖、迁移影响。免疫荧光法分析2组细胞表面受体血管内皮生长因子受体2(VEGFR2)表达情况。采用pull-down法分析2组细胞Rho A活性。结果LV-si VASP组RNA表达抑制率[(68.1±0.8)%]、VASP蛋白表达抑制率[(59.3±1.7)%]较LV-sicntr组(100%)明显降低(P<0.05)。与0 ng/m L比较,50~200 ng/m L抵抗素作用48 h后,可以显著促进细胞的增殖(P<0.05)。50~200 ng/m L抵抗素处理24 h后,HCAEC呈剂量依赖性促进细胞迁移(P<0.05)。沉默VASP后,抑制了抵抗素对细胞增殖和迁移的作用(P<0.05)。荧光显微镜观察结果显示,与LV-sicntr组比较,LV-si VASP组VEGFR2表达未见明显变化,而LV-si VASP组Rho A活性较LV-sicntr组减弱[(41.3±3.1)%vs 100%],差异有统计学意义(P<0.05)。结论抵抗素呈浓度依赖性的促进冠状动脉内皮细胞增殖、迁移,敲除VASP对抵抗素功能的影响与减弱的Rho A活性有关。

Objective Resistin,also known as an adipose tissue-specific secretory factor,is involved in the development and progression of artherosclerosis by inducing dysfunction of endothelial cells,but its action mechanism has been rarely studied. The purpose of this study was to investigate the effect of resistin on the function of human coronary artery endothelial cells( HCAECs) and the underlying mechanisms. Methods We constructed the lentiviral vasodilator-stimulated phosphoprotein( VASP) shRNA interfering sequence( the LV-si VASP group) and a negative control vector( the LV-sicntr group),transfected HCAECs with VASP siRNA or control siRNA,and determined the VASP mRNA and protein expressions by quantitative RT-PCR and Western blot,respectively. We treated the HCAECs with resistin at the concentrations of 0,10,50,100,and200 ng/m L followed by detection of its effects on the proliferation and migration of the cells by MTT and Transwell chamber assay,respectively. We made comparisons between the LV-si VASP and LV-sicntr groups in the proliferation and migration of the HCAECs treated with 100 ng/m L resistin,the expression of vascular endothelial growth factor receptor-2( VEGFR2) by immunofluorescent staining,and the activity of Rho A by pull-down assays. Results Compared with the LV-sicntr group,the LV-si VASP group showed significantly inhibited expressions of RNA( 100% vs [68.1±0.8〗%,P<0.05) and the VASP protein( 100% vs [59.3±1.7]%,P< 0.05) Treatment with resistin at 50-200 ng/m L markedly increased the proliferation of the HCAECs at 48 hours( P<0.05) and induced a dose-dependent promotion of their migration at 24 hours( P<0.05).No significant difference was observed in the expression VEGFR2 between the two groups,while the activity if Rho A was remarkably reduced in the LV-si VASP group( [41.3±3.1]%) as compared with the LV-sicntr group( P<0.05). Conclusion Resistin promotes the proliferation and migration of HCAECs,and the influence of VASP ablation on the action of resistin is associated with the decreased activity of Rho A.