YAP通过增加HIF-1α转录活性进而促进小鼠下肢缺血后动脉生成  

YAP promotes arteriogenesis after hind-limb ischemia in mice by upregulating transcriptional activity of HIF-1α

作　　者：李智昱 李梦琦[2] 张成虎 李博川 何金龙[1] 梁德刚[2] 艾玎 LI Zhi-yu;LI Meng-qi;ZHANG Cheng-hu;LI Bo-chuan;HE Jin-long;LIANG Degang;AI Ding(Collaborative Innovation Center of Tianjin for Medical Epigenetics and Department of Physiology and Pathophysiology,Tianjin Medical University,Tianjin 300070,China;Department of Cardiothoracic Surgery,Tianjin Medical University General Hospital,Tianjin 300052,China)

机构地区：[1]天津医科大学生理学与病理生理学系,天津医学表观遗传学协同创新中心,天津300070 [2]天津医科大学总医院心血管外科,天津300052

出　　处：《中国病理生理杂志》2020年第9期1537-1542,共6页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.91849105;No.81870313;No.81670388)。

摘　　要：目的:探讨内皮细胞Yes相关蛋白(YAP)在小鼠下肢缺血后动脉生成过程中的作用与机制。方法:缺氧处理人脐静脉内皮细胞(HUVECs),并使用Western blot检测YAP第127位丝氨酸的磷酸化水平,采用免疫荧光染色检测YAP的细胞亚定位情况;免疫共沉淀实验检测HUVECs中YAP与缺氧诱导因子1α(HIF-1α)结合的情况;萤光素酶报告基因实验检测YAP野生型(YAP-WT)质粒以及YAP丝氨酸位点突变(YAP-5SA)质粒对HIF-1α转录活性的调节;构建内皮细胞特异性YAP过表达(EC-YAP^tg)小鼠,于结扎股动脉手术后1、8、15和22 d使用激光多普勒血流仪检测小鼠下肢血流恢复情况;免疫荧光染色检测小鼠腓肠肌CD31、α-平滑肌肌动蛋白(α-SMA)及巨噬细胞分化抗原3(Mac3)的表达情况。结果:缺氧可以时间依赖性地下调YAP第127位丝氨酸的磷酸化,同时促进YAP在细胞核定位;免疫共沉淀实验证实,YAP可以与HIF-1α在缺氧条件下相互结合;YAP-WT或者YAP-5SA均可以显著增加HIF-1α的转录活性;与对照(YAP^flox)小鼠相比,EC-YAP^tg小鼠下肢缺血后血流恢复情况更好,腓肠肌组织染色显示新生血管的数量显著增加。结论:YAP可以与HIF-1α相互结合并增加其转录活性;内皮细胞过表达YAP可以促进缺血后的动脉生成。AIM:To investigate the function and the underlying mechanism of endothelial Yes-associated protein(YAP)in the process of arteriogenesis.METHODS:Human umbilical vein endothelial cells(HUVECs)were treated with hypoxia,the phosphorylation of YAP was determined by Western blot,and subcellular localization of YAP was analyzed by immunofluorescence staining.The binding of YAP to hypoxia-inducible factor-1α(HIF-1α)in HUVECs was detected by co-immunoprecipitation.The transcriptional activity of HIF-1α was detected by luciferase reporter assay.Endothelial cell-specific YAP-overexpression(EC-YAP^tg)mice were constructed,and the perfusion of hind limb was measured by laser Doppler flowmetry at 1,8,15 and 22 d after femoral artery ligation.The expression of CD31,α-smooth muscle actin(α-SMA)and macrophage differentiation antigen-3(Mac3)in gastrocnemius was observed by immunofluorescence staining.RESULTS:Hypoxia decreased the phosphorylation level of YAP at Ser127 in a time-dependent manner,and increased nuclear localization of YAP.Co-immunoprecipitation assay confirmed that YAP bound to HIF-1αunder hypoxia.YAP transfection significantly increased the transcriptional activity of HIF-1α.Compared with YAP^flox mice,EC-YAP^tg mice showed better blood flow recovery after hind-limb ischemia and significantly promoted arteriogenesis in gastrocnemius.CONCLUSION:YAP binds to HIF-1αand increases its transcriptional activity.YAP promotes the arteriogenesis after ischemia.

关 键 词：Yes相关蛋白 缺氧诱导因子1Α 动脉生成 内皮细胞 

分 类 号：R543.5[医药卫生—心血管疾病] R363.2[医药卫生—内科学]