VEGFA is essential for the potentiation of ATRA on BMP9-induced osteogenesis of preadipocytes and osteoporotic fracture healing.

The induction of osteogenic differentiation in preadipocytes may serve as a potential therapeutic approach for treating osteoporosis and osteoporotic fractures. All-trans retinoic acid (ATRA) promotes the bone morphogenetic protein 9 (BMP9)-induced osteogenic differentiation of preadipocytes. The present study further investigated whether vascular endothelial growth factor A (VEGFA) may play a role in this process and the effect of ATRA and BMP9 on osteoporotic fracture healing in rats. The results indicated that ATRA and BMP9 synergistically upregulated VEGFA expression in preadipocytes. Furthermore, knockdown of VEGFA expression abolished the stimulatory effect of ATRA on the BMP9-induced early and late osteogenic differentiation of preadipocytes in vitro, as evidenced by a decrease in alkaline phosphatase (ALP) activity, osteopontin and osteocalcin expression as well as mineralization. The in vivo cell implantation assay showed that ATRA failed to augment BMP9-induced ectopic bone formation in the absence of VEGFA. Subsequently, an osteoporotic femoral fracture rat model was established and micro-CT scans, alongside quantitative analysis, revealed that ATRA effectively promoted BMP9-stimulated callus formation during osteoporotic fracture healing. Moreover, ATRA and BMP9 acted together to significantly elevate VEGFA expression in bone calluses. Mechanistically, ATRA and BMP9 synergistically stimulated the osteogenic transcription factor, runt-related transcription factor 2 (Runx2). Transcriptomic and bioinformatic analyses further revealed that the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway may be crucial for mediating the synergistic effects of ATRA and BMP9, as validated by detecting the phosphorylation of PI3K and Akt. However, the increases in Runx2 expression and Akt phosphorylation induced by the combination of ATRA and BMP9 were inhibited by VEGFA silencing. ATRA also failed to increase the BMP9-induced ALP activity in preadipocytes treated with an Akt inhibitor. These findings suggest that VEGFA may influence the potentiation effect of ATRA on the BMP9-mediated osteogenesis of preadipocytes and in osteoporotic fracture healing through Runx2 and the PI3K/Akt signaling pathway.