A dual role of HIF1α in regulating osteogenesis-angiogenesis coupling

Objectives: The hypoxia-inducible factor 1-alpha (HIF1 alpha), a key molecule in mediating bone-vessel crosstalk, has been considered a promising target for treating osteoporosis caused by gonadal hormones. However, senile osteoporosis, with accumulated senescent cells in aged bone, has a distinct pathogenesis. The study aimed at revealing the unknown role of HIF1 alpha in aged bone, thus broadening its practical application in senile osteoporosis. Materials and methods: Femurs and tibias were collected from untreated mice of various ages (2 months old, 10 months old, 18 months old) and treated mice (2 months old, 18 months old) underwent 4-w gavage of 2-methoxyestradiol (a kind of HIF1 alpha inhibitor). Bone-vessel phenotypes were observed by microfil infusion, micro-CT and HE staining. Markers of senescence, osteogenesis, angiogenesis, oxidative stress and expression of HIF1 alpha were detected by senescence beta-galactosidase staining, qRT-PCR, western blot and immunostaining, respectively. Furthermore, bone mesenchymal stem cells from young mice (YBMSCs) and aged mice (ABMSCs) were transfected by knockout siRNA and overexpression plasmid of HIF1 alpha. Senescence beta-galactosidase staining, Cell Counting Kit-8, transwell assay, alkaline phosphatase staining, alizarin red-S staining and angiogenesis tests were utilized to assess the biological properties of two cell types. Then, Pifithrin-alpha and Nutlin-3 alpha were adopted to intervene p53 of the two cells. Finally, H2O2 on YBMSCs and NAC on ABMSCs were exploited to change their status of oxidative stress to do a deeper detection. Results: Senescent phenotypes, impaired osteogenesis-angiogenesis coupling and increased HIF1 alpha were observed in aged bone and ABMSCs. However, 2-methoxyestradiol improved bone-vessel metabolism of aged mice while damaged that of young mice. Mechanically, HIF1 alpha showed opposed effects in regulating the cell migration and osteogenesis-angiogenesis coupling of YBMSCs and ABMSCs, but no remarked effect on the proliferation of either cell type. Pifithrin-alpha upregulated the osteogenic and angiogenic markers of HIF1 alpha-siRNA-transfected YBMSCs, and Nutlin-3 alpha alleviated those of HIF1 alpha-siRNA-transfected ABMSCs. The HIF1 alpha-p53 relationship was negative in YBMSCs and NAC-treated ABMSCs, but positive in ABMSCs and H2O2-treated YBMSCs. Conclusion: The dual role of HIF1 alpha in osteogenesis-angiogenesis coupling may depend on the ROS-mediated HIF1 alpha-p53 relationship. New awareness about HIF1 alpha will be conducive to its future application in senile osteoporosis.