State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, Department of Prosthodontics, West China Hospital of Stomatology, Sichuan University, No 14, Sec. 3, Renminnan Road, Chengdu, 610041, People's Republic of China.
Department of Oral Prosthodontics, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan, China.
Stem Cell Res Ther. 2022 Feb 5;13(1):59. doi: 10.1186/s13287-022-02742-1.
The hypoxia-inducible factor 1-α (HIF1α), 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 HIF1α in aged bone, thus broadening its practical application in senile osteoporosis.
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 HIF1α inhibitor). Bone-vessel phenotypes were observed by microfil infusion, micro-CT and HE staining. Markers of senescence, osteogenesis, angiogenesis, oxidative stress and expression of HIF1α were detected by senescence β-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 HIF1α. Senescence β-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-α and Nutlin-3a were adopted to intervene p53 of the two cells. Finally, HO on YBMSCs and NAC on ABMSCs were exploited to change their status of oxidative stress to do a deeper detection.
Senescent phenotypes, impaired osteogenesis-angiogenesis coupling and increased HIF1α were observed in aged bone and ABMSCs. However, 2-methoxyestradiol improved bone-vessel metabolism of aged mice while damaged that of young mice. Mechanically, HIF1α 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-α upregulated the osteogenic and angiogenic markers of HIF1α-siRNA-transfected YBMSCs, and Nutlin-3a alleviated those of HIF1α-siRNA-transfected ABMSCs. The HIF1α-p53 relationship was negative in YBMSCs and NAC-treated ABMSCs, but positive in ABMSCs and HO-treated YBMSCs.
The dual role of HIF1α in osteogenesis-angiogenesis coupling may depend on the ROS-mediated HIF1α-p53 relationship. New awareness about HIF1α will be conducive to its future application in senile osteoporosis.
缺氧诱导因子 1-α（HIF1α）是介导骨血管相互作用的关键分子，已被认为是治疗性腺激素引起的骨质疏松症的有前途的靶点。然而，老年性骨质疏松症在老年骨中积累衰老细胞，具有明显的发病机制。本研究旨在揭示 HIF1α在老年骨中的未知作用，从而拓宽其在老年性骨质疏松症中的实际应用。
从小鼠（2 个月、10 个月、18 个月）和处理过的小鼠（2 个月、18 个月）中收集股骨和胫骨，未经处理的小鼠接受了 2 个月的 2-甲氧基雌二醇（一种 HIF1α 抑制剂）灌胃。通过微管输注、微 CT 和 HE 染色观察血管表型。通过衰老β-半乳糖苷酶染色、qRT-PCR、western blot 和免疫染色分别检测衰老、成骨、血管生成、氧化应激和 HIF1α表达的标志物。此外，从小鼠（YBMSCs）和老年鼠（ABMSCs）的骨髓间充质干细胞（BMMSCs）中转染 HIF1α 的敲除 siRNA 和过表达质粒。通过衰老β-半乳糖苷酶染色、细胞计数试剂盒-8、Transwell 测定、碱性磷酸酶染色、茜素红 S 染色和血管生成试验评估两种细胞类型的生物学特性。然后，采用 Pifithrin-α 和 Nutlin-3a 干预两种细胞的 p53。最后，利用 HO 处理 YBMSCs 和 NAC 处理 ABMSCs 以改变其氧化应激状态，进行更深入的检测。
在老年骨和 ABMSCs 中观察到衰老表型、成骨-血管生成偶联受损和 HIF1α 增加。然而，2-甲氧基雌二醇改善了老年小鼠的骨血管代谢，而损害了年轻小鼠的骨血管代谢。在调节 YBMSCs 和 ABMSCs 的细胞迁移和成骨-血管生成偶联方面，HIF1α 表现出相反的作用，但对两种细胞类型的增殖均无明显影响。Pifithrin-α 上调了 HIF1α-siRNA 转染的 YBMSCs 的成骨和血管生成标志物，而 Nutlin-3a 减轻了 HIF1α-siRNA 转染的 ABMSCs 的这些标志物。在 YBMSCs 中，HIF1α-p53 关系为负相关，而在 NAC 处理的 ABMSCs 中为正相关，在 ABMSCs 中，HIF1α-p53 关系为正相关，而在 HO 处理的 YBMSCs 中为负相关。
HIF1α 在成骨-血管生成偶联中的双重作用可能取决于 ROS 介导的 HIF1α-p53 关系。对 HIF1α 的新认识将有助于其在老年性骨质疏松症中的未来应用。
