Abstract
BACKGROUND: Macrophage exosomes and vascular endothelial cells (VECs) are critical to bone healing. However, few studies explore the molecular regulation of them in the bone fracture microenvironment. METHODS: In this study, we explored the effects of adenosine receptor A2A (ADA2AR) in macrophage exosomes and VECs during bone healing. CGS21680 (an ADA2AR agonist) and ZM241385 (an ADA2AR antagonist) were used. First, the effects of the ADA2AR on VECs during bone healing were studied in vivo in a rat tibial fracture model. Second, the effects of ADA2AR on VECs and in the regulation of VECs by macrophages were examined in the bone fracture microenvironment. Third, the effects of ADA2AR on the regulation of macrophage exosomes on VECs were analyzed. Finally, the genes and long non-coding RNAs (lncRNAs) associated with the regulation of VECs by the ADA2AR were examined by high-throughput sequencing and bioinformatics analysis. RESULTS: CGS21680 accelerated VEC proliferation in the early stage of bone healing and that ZM241385 suppressed VEC proliferation in vivo. ZM241385 inhibited cell viability and tube formation in vitro. However, CGS21680 did not promote tube formation, cell proliferation, or cell migration in vitro. The inhibition of macrophage exosomes could suppress tube formation and VEC migration. CGS21680 had no effects on tube formation in a macrophage-VEC co-culture. The macrophage exosomes were purified and CGS21680 promoted the macrophage secretion of exosomes. In contrast, ZM241385 inhibited the macrophage secretion exosomes. Finally, the lncRNA and mRNA involved in the activation of the ADA2AR in VECs were analyzed. CGS21680 upregulated 3274 mRNAs and downregulated 2236 mRNAs, and upregulated 1696 lncRNAs and downregulated 1882 lncRNAs. The hub genes involved in angiogenesis were Flt1, Fgf2, Mapk14, Fn1, and Jun. CONCLUSION: The activation of ADA2AR was essential for angiogenesis and the secretion of exosomes by macrophages during bone healing; moreover, the inactivation of the ADA2AR led to poor angiogenesis and bone nonunion.