HIF-1 Activation in Macrophages Promotes Angiogenesis in BCP-Instructed Bone Formation

Objectives: Osteo-inductive materials draws attention for inducing osteogenesis among micropores in non-skeletal sites. By prefabricated the biphasic calcium phosphate(BCP) subcutaneously for 9 months, we succeeded in repairing left mandible defect in a man without osteogenous cells or growth factors. However, mechanism for the special osteogenesis is still controversial. In terms of previous researches and our histological results, hypoxia and hypoxia-inducible factor 1(HIF-1) signaling pathway may involves in initiation of material-inducing osteogenesis.
Methods: Porous BCP were provided by Kuros Biosciences. In vivo, HIF-1 signaling pathway or macrophage is supressed separately to investigate their effect in osteogenesis and superior expressing cells in early stage after implantation subcutaneously on FVB mice’s back. In vitro, influence Raw264.7 cell cultured under hypoxia perform on angiogenesis is further explored by tube formation assay, transwell assay, RNA-sequencing, etc.
Results: Suppression of HIF-1 seriously impacts vessel regeneration and subsequent osteogenesis. Then inducing macrophage apoptosis causes similar impact in angiogenesis, and in immunofluorescence, F4/80 and HIF-1 almost expressed in same cells. So it's deducted that HIF-1 expressing macrophages is a dominant cell line in early stage and affect angiogenesis and coupling osteogenesis process. Additionally, liquid supernatant from Raw264.7 cells cultured on material under hypoxia significantly promotes angiogenesis, proliferation, and migration of Human Umbilical Vein Endothelial Cell(HUVEC), compared to supernatant from macrophage cultured under normoxia. Finally, in RNA-seq, vital molecules in angiogenesis such as VEGF and Ang upregulated, and several KEGG pathways and GO biological processes are activated.
Conclusions: In bone formation instructed by calcium phosphate ceramics, HIF-1 upregulated in recruited macrophages due to locally hypoxia. Its down-stream genes highly express, and angiogenesis in the system is promoted and may prompt subsequent osteogenesis.