生长因子受体结合蛋白2接头蛋白2缺失促进下肢动脉血管内皮细胞自噬的机制

Loss of growth factor receptor-binding protein 2 adaptor protein 2 associated binding protein 2 promotes autophagy of vascular endothelial cells

目的观察缺氧通过抑制血管内皮细胞生长因子受体结合蛋白2接头蛋白2(GAB2)的表达,导致下肢动脉内皮损伤的分子机制。方法血管标本取材于上海交通大学医学院附属仁济医院血管外科,粥样硬化股动脉标本取自于下肢动脉粥样硬化闭塞(ASO)截肢患者,正常动脉血管取自于意外车祸截肢患者。通过组织免疫荧光(IF)、实时定量反转录聚合酶链反应(RT-qPCR)和蛋白质印迹法(Western blot)检测正常动脉血管与ASO病变动脉血管内膜组织中GAB2表达差异。采用RT-qPCR和Western blot检测缺氧条件下内皮细胞中微小RNA(miR)-30a及GAB2表达改变。内皮细胞中沉默或过表达GAB2后Western blot检测自噬标志物LC3Ⅱ/Ⅰ的表达。组间比较采用t检验。结果组织免疫荧光(IF)和Western blot检测显示内皮细胞自噬水平ASO组织高于正常组织(5.252±0.449,t=15.950,P<0.01;1.805±0.210,t=5.471,P<0.01),同时GAB2表达ASO组织低于正常组织(0.349±0.028,t=32.120,P<0.01;0.399±0.059,t=5.180,P<0.01);体外实验显示内皮细胞中GAB2表达缺氧组低于对照组(0.668±0.088,t=3.774,P<0.01);内皮细胞自噬标志物LC3Ⅱ/Ⅰ水平沉默GAB2组高于对照组(1.197±0.036,t=9.376,P<0.01)。进一步机制研究结果显示,内皮细胞中miR-30a表达量缺氧组高于对照组(2.349±0.118,t=9.257,P<0.01),GAB2 mRNA水平过表达miR-30a组低于对照组(0.207±0.040,t=6.031,P<0.01)。结论缺氧促进血管内皮细胞表达miR-30a,通过降解GAB2 mRNA,引起血管内皮细胞自噬水平升高,导致动脉内皮损伤。

Objective To explore the molecular mechanism of endothelial injury induced by hypoxia in lower limb arteries by inhibiting the expression of vascular endothelial growth factor receptor-binding protein 2 adaptor protein 2(GAB2).Methods Vascular samples were obtained from the Department of Vascular Surgery,Renji Hospital,Shanghai Jiaotong University School of Medicine,atherosclerotic femoral artery samples were obtained from arteriosclerosis obliterans(ASO)amputees,and normal arterial vessels were obtained from accidental car accident amputees.Tissue immunofluorescence assay,real-time quantitative reverse transcription-polymerase chain reaction(RT-qPCR),and Western blotting were used to detect differential intimal GAB2 expression between normal and ASO vessels.RT-qPCR and Western blotting were used to detect hypoxia-induced miR-30a upregulation and GAB2 downregulation.To investigate the effect of GAB2 protein on autophagy of human umbilical vein endothelial cells(HUVECs),after silencing and overexpression of GAB2 in HUVECs,Western blotting was used to detect the autophagy marker LC3Ⅱ/Ⅰ.T test was applied for statistical analysis using GraphPad 8.0.Results Tissue immunofluo-rescence(IF)and Western blotting showed that endothelial cell autophagy levels were higher in ASO tissues than in normal tissues(5.252±0.449,t=15.950,P<0.01;1.805±0.210,t=5.471,P<0.01),while GAB2 expression in ASO tissues was lower than in normal tissues(0.349±0.028,t=32.120,P<0.01;0.399±0.059,t=5.180,P<0.01):in vitro experiments revealed that GAB2 expression in endothelial cells was lower in the hypoxia group than in the control group(0.668±0.088,t=3.774,P<0.01);endothelial cell autophagy marker LC3Ⅱ/Ⅰ levels were higher in the silenced GAB2 group than in the control group(1.197±0.036,t=9.376,P<0.01).Further mechanistic studies revealed that miR-30a expression in endothelial cells was higher in the hypoxia group than in the control group(2.349±0.118,t=9.257,P<0.01),and GAB2 mRNA level overexpression was lower in the miR-30a group than in the control group(0.207±0.040,t=6.031,P<0.01).Conclusion Hypoxia upregulated miR-30a expression in vascular endothelial cells.miR-30a promotes GAB2 mRNA degradation and regulates autophagy in vascular endothelial cells,leading to arterial endothelial injury.