Abstract
Distraction osteogenesis (DO) is used to treat large bone defects in the field of oral and maxillofacial surgery. Successful DO-mediated bone regeneration is dependent upon angiogenesis, and endothelial progenitor cells (EPCs) are key mediators of angiogenic processes. The N6-methyladenosine (m6A) methyltransferase has been identified as an important regulator of diverse biological processes, but its role in EPC-mediated angiogenesis during DO remains to be clarified. In the present study, we found that the level of m6A modification was significantly elevated during the process of DO and that it was also increased in the context of EPC angiogenesis under hypoxic conditions, which was characterized by increased METTL3 levels. After knocking down METTL3 in EPCs, m6A RNA methylation, proliferation, tube formation, migration, and chicken embryo chorioallantoic membrane (CAM) angiogenic activity were inhibited, whereas the opposite was observed upon the overexpression of METTL3. Mechanistically, METTL3 silencing reduced the levels of VEGF and PI3Kp110 as well as the phosphorylation of AKT, whereas METTL3 overexpression reduced these levels. SC79-mediated AKT phosphorylation was also able to restore the angiogenic capabilities of METTL3-deficient EPCs in vitro and ex vivo. In vivo, METTL3-overexpressing EPCs were additionally transplanted into the DO callus, significantly enhancing bone regeneration as evidenced by improved radiological and histological manifestations in a canine mandibular DO model after consolidation over a 4-week period. Overall, these results indicate that METTL3 accelerates bone regeneration during DO by enhancing EPC angiogenesis via the PI3K/AKT pathway.
Highlights
And efficiently treating mandibular defects remains a major clinical challenge
To illustrate the dynamic changes that occur in the context of Distraction osteogenesis (DO), we detected m6Arelated gene expression patterns associated with different phases in DO, revealing that Methyltransferase-like 3 (METTL3), Wilms tumor associated protein (WTAP), YTHDC1, and YTHDC2 were expressed at significantly higher levels after 2 weeks of consolidation (DO28) relative to DO10 samples, as were levels of the angiogenesis-related cytokine vascular endothelial growth factor (VEGF) and hypoxia-related HIF-1α (Figure 1C)
To simulate the endothelial progenitor cells (EPCs) angiogenesis conditions during DO in vitro, EPCs were cultured in a hypoxic (1% O2) incubator, and we found that METTL3, YTHDF1, YTHDF3, and YTHDC2 were significantly upregulated in EPCs in response to hypoxia at the mRNA level as compared with cells cultured under normoxic conditions (Figure 1D)
Summary
And efficiently treating mandibular defects remains a major clinical challenge. Distraction osteogenesis (DO) is a surgical procedure wherein large bone defects arising as a consequence of injury, congenital malformation, or tumor excision can be repaired (Earley and Butts, 2014; Hatefi et al, 2019). The mechanisms governing DO-related regeneration remain to be clarified, and further research aimed at understanding these underlying molecular mechanisms may aid in shortening the DO-related treatment period, potentially improving the clinical utility of this procedure. Insufficient vascularization is thought to be a primary cause of delayed union or non-union during bone repair (Deshpande et al, 2015; Stegen et al, 2015), and endothelial progenitor cells (EPCs) play a vital role in angiogenesis and vasculogenesis, homing to sites of tissue regeneration and remodeling and therein supporting these important processes (George et al, 2011). EPCs play an essential role by coupling angiogenesis and osteogenesis, and it is critical that the mechanistic role of EPCs in the context of DO-related angiogenesis be clarified
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