mpECs with high Piezo2 expression promote fracture healing by driving angiogenesis through the Notch signaling pathway.

Abstract

Fractures will impair or disrupt angiogenesis, resulting in delayed union or non-union. Exploring angiogenic signaling molecules and related pathways can promote fracture healing. In this study, the roles of different endothelial cell (EC) subsets in fracture healing were observed using single-cell RNA sequencing (scRNA-seq). It was found that mpECs did affect the repair and regeneration of fracture sites, and could up-regulate genes related to the Notch signaling, angiogenesis, and cell cycle. In addition, in this study, Piezo2 expression was successfully knocked down by transfection of shRNA in human umbilical vein endothelial cells (HUVECs) for in vitro assays. The results suggested that the reduced expression of Piezo2 in HUVECs can suppress cell proliferation and cell cycle and further impair the activation of the Notch signaling pathway, inhibiting angiogenesis. Subsequently, HUVECs were intervened with the Notch pathway inhibitor DAPT and agonist Jagged1. It was found that inhibition of the Notch signaling pathway by Piezo2 knockdown was more significant in the presence of DAPT, whereas Jagged1 reversed the Piezo2 knockdown-caused changes in the downstream protein expression of the Notch pathway. With Jagged1, Piezo2 knockdown-induced decrease in HUVEC tube formation disappeared. Moreover, the tube formation was significantly enhanced, with a marked increase in tube length. Cell counting kit-8 (CCK-8) assay and flow cytometry demonstrated that Jagged1 can promote cell proliferation and trigger cell cycle entry. In conclusion, Piezo2 affects the phenotype of ECs by modulating the Notch signaling pathway and further promotes angiogenesis, thus accelerating fracture healing.