Activation of BMP4/SMAD pathway by HIF-1α in hypoxic environment promotes osteogenic differentiation of BMSCs and leads to ectopic bone formation.

OBJECTIVE: Heterotopic ossification (HO), also known as ossifying myositis, is a condition that produces abnormal bone and cartilage tissue in the soft tissues. Hypoxia inducible factor lα (HIF-lα) regulates the expression of various genes, which is closely related to the promotion of bone formation, and Drosophila mothers against decapentaplegic protein (SMAD) mediates the signal transduction in the Bone morphogenetic protein (BMP) signaling pathway, which affects the function of osteoblasts and osteoclasts, and thus plays a key role in the regulation of bone remodeling. We aimed to investigate the mechanism by which HIF-1α induces osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in a hypoxic environment. METHODS: A cellular hypoxia model was constructed to verify the expression of HIF-1α, while alizarin red staining was performed to observe the osteogenic differentiation ability of bone marrow mesenchymal stem cells (BMSCs). Alizarin red staining was used to analyze the late mineralization ability of the cells. Western blot analysis was performed to analyze the expression levels of osteogenesis-related factors OCN, OPN proteins as well as the pathway proteins BMP4, p-Smad1/5/8, and Smad1. We also constructed a rat model of ectopic bone formation, observed ectopic ossification by X-ray, and verified the success of the rat model by ELISA of HIF-1α. HE staining was used to observe the matrix and trabecular structure of bone, and Masson staining was used to observe the collagen and trabecular structure of bone. Immunohistochemistry analyzed the expression of OCN and OPN in ectopic bone tissues, and WB analyzed the expression of pathway proteins BMP4, p-Smad1/5/8 and Smad1 in ectopic bone tissues to verify the signaling pathway of ectopic bone formation. RESULTS: Our results indicate that hypoxic environment upregulates HIF-1a expression and activates BMP4/SMAD signaling pathway. This led to an increase in ALP content and enhanced expression of the osteogenesis-related factors OCN and OPN, resulting in enhanced osteogenic differentiation of BMSCs. The results of our in vivo experiments showed that rats inoculated with BMSCs overexpressing HIF-1α showed bony structures in tendon tissues, enhanced expression of the bone signaling pathways BMP4 and p-Smad1/5/8, and enhanced expression levels of the osteogenic-related factors OCN and OPN, resulting in the formation of ectopic bone. CONCLUSIONS: These data further suggest a novel mechanistic view that hypoxic bone marrow BMSCs activate the BMP4/SMAD pathway by up-regulating the expression level of HIF-1α, thereby promoting the secretion of osteogenic factors leading to ectopic bone formation.

Exosome MiR-21-5p Upregulated by HIF-1α Induces Adipose Stem Cell Differentiation to Promote Ectopic Bone Formation.

Heterotopic bone occurs after burns, trauma and major orthopedic surgery, which cannot be completely cured by current treatments. The development of new treatments requires more in-depth research into the mechanism of HO. Available evidence suggests that miR-21-5p plays an important role in bone formation. However, its mechanism in traumatic HO is still unclear. First, we identified exosomes extracted from L6 cells using TEM observation of the structure and western blotting detection of the surface marker CD63. Regulation effect of HIF-1α to miR-21-5p was confirmed by q-PCR assay. Then we co-cultured L6 cells with ASCs and performed alizarin red staining and ALP detection. Overexpression of miR-21-5p upregulated BMP4, p-smad1/5/8, OCN and OPN, which suggests the BMP4-smad signaling pathway may be involved in miR-21-5p regulation of osteogenic differentiation of ASCs. Finally in vivo experiments showed that miR-21-5p exosomes promoted ectopic formation in traumatized mice. This study confirms that HIF-1α could modulate miR-21-5p exosomes to promote post-traumatic ectopic bone formation by inducing ASCs cell differentiation. Our study reveals the mechanisms of miR-21-5p in ectopic ossification formation after trauma.

High cholesterol and low triglycerides are associated with total lumbar bone mineral density among adults aged 50 years and over: The NHANES 2017-2020.

Background: The association between cholesterol and triglycerides with the lumbar bone mineral density (BMD) was widely investigated, but the results remained conflicting. This study aimed to investigate the relationship between total cholesterol, triglycerides, and total lumbar BMD in adults. Materials and methods: This cross-sectional study included 1,985 individuals aged 50 years and over. The data on total cholesterol, triglycerides, total lumbar BMD, and other covariates were obtained from the National Health and Nutritional (NHANES) between 2017 and March 2020 pre-pandemic. Multivariate logistic regression models were utilized to investigate the association between cholesterol, triglycerides, and total lumbar BMD. Smooth curve fittings and generalized additive models were also used to analyze the potential non-linearity. Results: A total of 901 men and 1,084 women with a mean age of 63.02 ± 8.72 years (age 50-80 years) were included in this study. In multivariate regression analysis, the association between cholesterol and total lumbar BMD was negative (ß = -0.026, 95% CI: -0.033, -0.020). This relationship still existed after adjusted for gender and race (ß = -0.018, 95% CI: -0.025, -0.012) and fully adjusted for all covariates (ß = -0.022, 95% CI: -0.029, -0.015). The association between triglycerides and total lumbar BMD was positive (ß = 0.024, 95% CI: 0.017, 0.031). This relationship still existed after adjusted for gender and race (ß = 0.021, 95% CI: 0.015, 0.028) and fully adjusted for all covariates (ß = 0.021, 95% CI: 0.014, 0.028). In threshold effect analysis, the relationship between triglycerides and total lumbar BMD was an inverted U-shaped curve with the inflection point at 2.597 mmol/L. Conclusion: High levels of total cholesterol and relatively low levels of triglycerides are significantly associated with the total lumbar BMD in adults aged 50 years and over.