METTL3-mediated m6A methylation of PDGF regulates the angiogenesis dependent bone formation

Background Recent research has identified CD31 ^hi EMCN ^hi vessels, known as type H vessels, which play a crucial role in the specialized bone metabolic microenvironment. These vessels are reported to integrate angiogenesis with osteogenesis, thereby influencing angiogenesis-dependent bone formation. N6-methyladenosine (m6A) modification, mediated by METTL3, is essential in eukaryotic mRNA; however, its role in angiogenesis-dependent bone formation remains unclear. Methods Herein, H-BMECs were screened and identified via magnetic bead cell sorting and immunofluorescence analysis, and the methylation level of m6A was assessed using m6A dot blot assay. METTL3 was knocked down to investigate its impact on angiogenesis in vivo and vitro . Besides, candidate targets and mechanisms of METTL3 were assessed through m6A sequencing and RNA sequencing. The underlying mechanisms were further investigated using qPCR, Western blotting, and MeRIP-qPCR assays. RNA stability, migration, and tube formation assays, as well as qPCR and Western blotting, were performed to illuminate the potential biological functions of METTL3 and PDGF in BMECs in vitro . The consequences of METTL3-mediated m6A alterations in angiogenesis-dependent bone formation were further investigated using an in vivo Cdh5-Cre;Mettl3 ^fl/fl mouse model. Results The findings demonstrated that METTL3 promoted angiogenesis-dependent bone formation by enhancing the m6A methylation of PDGF. Mechanistically, it was observed that both the m6A methylation level and METTL3 expression were reduced in osteoporosis. Furthermore, METTL3 knockout markedly impaired the function of bone microvascular endothelial cells (BMECs) and suppressed angiogenesis-dependent bone formation in mice, whereas upregulation of PDGF expression effectively restored angiogenesis and bone formation. Conclusions It can be hereby concluded that METTL3-mediated m6A methylation regulates the angiogenesis function of BMECs and promotes angiogenesis-dependent bone formation. Meanwhile, METTL3-PDGF/PI3K/Akt is identified as a critical axis and potential mechanism in osteoporosis.