supplemental material:The activated adenosine A2A receptor (ADA2AR) is essential for angiogenesis during bone healing
Poor angiogenesis causes bone nonunion and vascular endothelial cells (VECs) are essential for angiogenesis. The activated adenosine A2A receptor (ADA2AR) promotes bone healing but the role of ADA2AR in angiogenesis during bone healing has not been discussed, in addition, the effects of ADA2AR on VECs are still controversial. This study explored the effects of ADA2AR on VECs in the bone fracture microenvironment. CGS21680 was used to activate ADA2AR and ZM241385 inhibited the ADA2AR. Firstly, we found that CGS21680 accelerated angiogenesis in the early stage of bone healing and ZM241385 suppressed the angiogenesis. Secondly, we constructed the bone fracture microenvironment and found that ZM241385 inhibited VECs proliferation and vascular tube formation in vitro. Macrophages are the important cells to activate VECs and promote angiogenesis in the early stage of bone healing, so we analyzed the effects of ADA2AR in macrophage regulation on VECs. We found that activated ADA2AR enhanced the regulation of macrophage on VECs, in addition, promoted VECs migration and vascular tube formation in bone fracture microenvironment, while, ZM241385 still suppressed vascular tube formation. Meanwhile, we inhibited the exosomes secreted from macrophages, which could reverse the effects of activated ADA2AR on VECs. Besides, we found that CGS21680 promoted macrophages to secrete exosomes to activate VECs and ZM241385 inhibited the exosomes secreted. Thirdly, we explored the role of exosomes secreted by macrophages and found that it was the key factor in activated ADA2AR on VECs. Finally, the involved lncRNA and mRNA in activated ADA2AR on VECs were analyzed. CGS21680 upregulated 3274 mRNAs and downregulated 2236 mRNAs, in addition, upregulated 1696 lncRNAs and downregulated 1882 lncRNAs. The hub genes involved in angiogenesis were Flt1, Fgf2, Mapk14, Fn1 and Jun. Overall, the activated ADA2AR was essential for angiogenesis during bone healing, in addition, ADA2AR inactivated lead to poor angiogenesis and bone nonunion.