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

doi:10.12307/2024.737

Abstract

Abstract:

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 PanglaoDB 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 hypoxia-induced 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.

Key words: endothelial cell, bone morphogenetic protein 2, angiogenesis, single-cell RNA sequencing, bulk RNA sequencing, signaling pathway, hindlimb ischemia model, tube formation experiment

CLC Number:

Yan Ru, Wang Kairu, Zhang Feiyan, Jia Shaobin, Cong Guangzhi. Endothelial cell-specific bone morphogenetic protein 2 affects angiogenesis: bioinformatics analysis and experimental validation[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(1): 103-110.

Figures/Tables 3

2.1 生物信息学分析结果 2.1.1 单细胞测序分析显示内皮细胞是骨形态发生蛋白 2 表达的主要细胞亚群通过PanglaoDB数据库单细胞测序数据集进行荟萃分析显示，共纳入42个表达骨形态发生蛋白 2 的小鼠单细胞测序样本，分析标记出18个细胞簇，显示内皮细胞是骨形态发生蛋白 2 表达的主要细胞亚群。其中10个内皮细胞簇表达骨形态发生蛋白 2 。以样本SRS3348009为例，共测序出2 450个细胞，t-SNE降维分析标记出14个细胞簇，其中702个为内皮细胞，内皮细胞簇高表达骨形态发生蛋白 2 ，见图1。 2.1.2 后肢缺血模型内皮细胞骨形态发生蛋白 2 表达明显升高，激活血管新生通路通过对后肢缺血小鼠模型分离出的内皮细胞Bulk RNA测序数据集GSE155012再分析，PCA显示模型组与对照组内皮细胞表现出完全不同的转录组特征，见图2A。火山图分析显示，与对照组比较，上调基因为441个，下调基因为280个，见图2B。模型组内皮细胞骨形态发生蛋白 2 表达明显升高，见图2C，调控血管新生的基因血管内皮生长因子A，血小板衍生生长因子A和血小板衍生生长因子B转录表达差异不明显(FDR-P > 0.05)，见图2D-F。将差异基因进行28个经典血管新生信号通路GO富集分析可见，共10个血管新生通路明显富集，见图2G-H，依次为血管新生(GO：0001525)，调控血管新生(GO：0045765)，负性调控血管新生(GO：0045765)，正性调控血管新生(GO：0045766)，血管新生出芽(GO：0002040)，血管新生出芽细胞迁移(GO：0090049)，负性血管新生出芽细胞迁移(GO：0090051)，负性调控血管新生出芽(GO：1903671)，创伤愈合血管新生(GO：0060055)和调控血管出芽(GO：1930670)。以上结果显示：在后肢缺血小鼠模型内皮细胞高表达骨形态发生蛋白 2 ，同时血管新生通路明显激活，骨形态发生蛋白 2 高表达可能与血管新生通路激活有关。"

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