内皮细胞特异性骨形态发生蛋白2对血管新生的影响:生物信息学分析和实验验证

Endothelial cell-specific bone morphogenetic protein 2 affects angiogenesis:bioinformatics analysis and experimental validation

背景:血管新生是心血管疾病的主要干预靶点,骨形态发生蛋白2具有调控血管新生作用,但内皮细胞特异性骨形态发生蛋白2对血管新生的调控作用不清楚。目的:探讨内皮细胞特异性骨形态发生蛋白2对血管新生的影响。方法:(1)生物信息学分析:通过Panglao DB公共基因表达数据库单细胞转录组荟萃分析观察骨形态发生蛋白2细胞群表达丰度和定位。血管新生小鼠和内皮(心内膜)过表达骨形态发生蛋白2小鼠转录组测序数据集探索内皮细胞骨形态发生蛋白2对血管新生信号通路的调控作用。(2)体内实验验证:建立小鼠后肢缺血模型,对比模型小鼠患侧与健侧缺血后肢7,14和21 d血流灌注情况,免疫荧光和免疫组织化学染色评估小鼠骨形态发生蛋白2和CD31的表达定位情况。(3)体外实验验证:体外培养人脐静脉内皮细胞,分为对照组、缺氧组和骨形态发生蛋白2抑制剂(Noggin蛋白)干预组,培养24 h,观察各组内皮细胞血管新生情况。结果与结论:(1)内皮细胞是表达骨形态发生蛋白2的重要细胞亚群,在血管新生内皮细胞和骨形态发生蛋白2过表达内皮细胞转录组再分析均发现骨形态发生蛋白2表达明显升高,血管新生通路明显激活。(2)缺血7 d小鼠新生血管周围骨形态发生蛋白2阳性血管明显增加(P<0.05),缺血2周骨形态发生蛋白2阳性血管明显减少(P<0.001)。(3)体外培养人脐静脉内皮细胞,缺氧干预后,内皮细胞迁移能力和血管出芽明显增加,血管新生因子血管内皮生长因子和血小板衍生生长因子的表达明显升高,Noggin明显减少了缺氧诱导的内皮细胞血管新生(P<0.001),并下调血管内皮生长因子和血小板衍生生长因子的表达(P<0.01)。(4)结果证实,内皮细胞特异性骨形态发生蛋白2具有调控血管新生作用,靶向性内皮细胞骨形态发生蛋白2可望改善血管新生。

BACKGROUND:Angiogenesis is the main treatment target of cardiovascular diseases.Bone morphogenetic protein 2 can modulate angiogenesis,but the regulatory effect of endothelial cell-specific bone morphogenetic protein 2 on angiogenesis is unclear.OBJECTIVE:To investigate the effect of endothelial-specific bone morphogenetic protein 2 on angiogenesis.METHODS:(1)Bioinformatics analysis:Cellular expression specificity and abundance of bone morphogenetic protein 2 were meta-analyzed by the Panglao DB single-cell transcriptome database.The endothelial cell transcriptome sequencing dataset of the mouse hindlimb model and endocardial transcriptome dataset of mice overexpressing bone morphogenetic protein 2 were reanalyzed to evaluate the effect of endothelial cell bone morphogenetic protein 2 on the angiogenesis pathway.(2)Validation in vivo:After establishing the mouse hindlimb model,we compared the blood perfusion between the affected and sham limb at 7,14,and 21 days.The expression of the colocation of bone morphogenetic protein 2 and CD31 was explored by immunofluorescence and immunohistochemical staining.(3)Validation in vitro:The cultured human umbilical vein endothelial cells in vitro were divided into a control group,a hypoxia group,and a bone morphogenetic protein 2 inhibitor Noggin intervention group.After being cultured for 24 hours,the angiogenesis of endothelial cells in each group was observed.RESULTS AND CONCLUSION:(1)Endothelial cells are an important cell subgroup expressing bone morphogenetic protein 2.Both in the mouse hindlimb ischemia model and endocardial cells overexpressing bone morphogenetic protein 2,bone morphogenetic protein 2 was significantly up-regulated,and the angiogenesis pathway was significantly activated.(2)In the mouse hindlimb model,bone morphogenetic protein 2-positive blood vessels around neoangiogenesis increased significantly at 7 days of ischemia(P<0.05),and decreased significantly after 2 weeks of ischemia(P<0.001).(3)In umbilical vein endothelial cells cultured in vitro,after hypoxic intervention,the migration and sprouting of endothelial cells increased significantly,and the expression of angiogenesis factors vascular endothelial growth factor and platelet-derived growth factor was significantly increased.Noggin significantly reduced hypoxiainduced endothelial cell angiogenesis(P<0.001)and down-regulated the expression of vascular endothelial growth factor and platelet-derived growth factor(P<0.01).(4)These findings verify that endothelial cell-specific bone morphogenetic protein 2 can regulate angiogenesis,and targeting endothelial cell bone morphogenetic protein 2 is a promising way to improve angiogenesis.