Department of Orthopedic Surgery, The 2nd Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang Province, 150001, P. R. China.
College of Bioinformatics Science and Technology, Harbin Medical University, Harbin, Heilongjiang Province, 150081, P. R. China.
Cell Mol Life Sci. 2024 Aug 12;81(1):344. doi: 10.1007/s00018-024-05371-4.
Osteogenesis is tightly coupled with angiogenesis spatiotemporally. Previous studies have demonstrated that type H blood vessel formed by endothelial cells with high expression of CD31 and Emcn (CD31 Emcn ECs) play a crucial role in bone regeneration. The mechanism of the molecular communication around CD31 Emcn ECs and bone mesenchymal stem cells (BMSCs) in the osteogenic microenvironment is unclear. This study indicates that exosomes from bone mesenchymal stem cells with 7 days osteogenic differentiation (7D-BMSCs-exo) may promote CD31 Emcn ECs angiogenesis, which was verified by tube formation assay, qRT-PCR, Western blot, immunofluorescence staining and µCT assays etc. in vitro and in vivo. Furthermore, by exosomal miRNA microarray and WGCNA assays, we identified downregulated miR-150-5p as the most relative hub gene coupling osteogenic differentiation and type H blood vessel angiogenesis. With bioinformatics assays, dual luciferase reporter experiments, qRT-PCR and Western blot assays, SOX2(SRY-Box Transcription Factor 2) was confirmed as a novel downstream target gene of miR-150-5p in exosomes, which might be a pivotal mechanism regulating CD31 Emcn ECs formation. Additionally, JC-1 immunofluorescence staining, Western blot and seahorse assay results showed that the overexpression of SOX2 could shift metabolic reprogramming from oxidative phosphorylation (OXPHOS) to glycolysis to enhance the CD31 Emcn ECs formation. The PI3k/Akt signaling pathway might play a key role in this process. In summary, BMSCs in osteogenic differentiation might secrete exosomes with low miR-150-5p expression to induce type H blood vessel formation by mediating SOX2 overexpression in ECs. These findings might reveal a molecular mechanism of osteogenesis coupled with type H blood vessel angiogenesis in the osteogenic microenvironment and provide a new therapeutic target or cell-free remedy for osteogenesis impaired diseases.
成骨作用与血管生成具有时空紧密的偶联性。先前的研究表明，由高表达 CD31 和 Emcn（CD31EmcnECs）的内皮细胞形成的 H 型血管在骨再生中起着至关重要的作用。在成骨微环境中，围绕 CD31EmcnECs 和骨髓间充质干细胞（BMSCs）的分子通讯机制尚不清楚。本研究表明，具有 7 天成骨分化的骨髓间充质干细胞来源的外泌体（7D-BMSCs-exo）可能通过体外和体内的管形成试验、qRT-PCR、Western blot、免疫荧光染色和 µCT 等方法促进 CD31EmcnECs 的血管生成。此外，通过外泌体 miRNA 微阵列和 WGCNA 分析，我们确定下调的 miR-150-5p 作为最相关的枢纽基因，将成骨分化与 H 型血管生成偶联起来。通过生物信息学分析、双荧光素酶报告实验、qRT-PCR 和 Western blot 分析，证实 SOX2（SRY-Box Transcription Factor 2）是外泌体中 miR-150-5p 的一个新的下游靶基因，可能是调节 CD31EmcnECs 形成的关键机制。此外，JC-1 免疫荧光染色、Western blot 和 Seahorse 分析结果表明，SOX2 的过表达可以将代谢重编程从氧化磷酸化（OXPHOS）转向糖酵解，从而增强 CD31EmcnECs 的形成。PI3k/Akt 信号通路可能在这个过程中发挥关键作用。总之，成骨分化中的 BMSCs 可能会分泌表达低 miR-150-5p 的外泌体，通过介导 ECs 中 SOX2 的过表达来诱导 H 型血管形成。这些发现可能揭示了成骨微环境中成骨作用与 H 型血管生成偶联的分子机制，并为成骨受损疾病提供了新的治疗靶点或无细胞治疗方法。
