Artikel
Tissue non-specific alkaline phosphatase activity regulates adipogenic program and bioenergetics of bone marrow adipose tissue cells obtained from osteoarthritis patients
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Veröffentlicht: | 25. Oktober 2022 |
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Gliederung
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Objectives: Accumulation of bone marrow adipose tissue (MAT) within bone during life interferes with bone health. Up to now, MAT content in bone marrow (BM) of osteoarthritis (OA) patients was investigated by magnetic resonance imaging, while there is no data on cell populations contributing to MAT expansion in OA patients. We established the culture of stromal cells associated to MAT and explored whether inhibition of tissue non-specific alkaline phosphatase (TNAP) can regulate adipogenesis in MAT-derived cell.
Methods: BM tissues were obtained from OA patients undergoing hip arthroplasty (Ethical approval 187/18) and used for BM-mesenchymal stromal cells (MSC) isolation. BM floating fatty fraction was used for isolation of MAT-cells, while subcutaneous adipose tissue from matched donors was used for extraction of peripheral adipose tissue (PAT) progenitors. Adipogenic differentiation was assessed in absence or presence of the reversible inhibitor of TNAP activity (levamisole hydrochlodride). TNAP expression was estimated by TNAP enzymatic activity assay, and immunofluorescence microscopy combined with mitochondrial staining. Energy metabolism was determined using extracellular flux analyzer. Flow cytometry was applied to investigate functional surface markers and mitochondrial content, while western blot was applied for total cellular protein analyses. Adipogenesis-related markers and Alpl gene expression was analyzed by qPCR. Results are expressed as means ± standard deviations.
Results and conclusion: MAT and PAT cells displayed stronger adipogenic potential associated with their stronger upregulation of adipogenic gene (CEBP α, PPAR γ, AdipoQ, Fabp4) expression than BM-MSC. In addition, enzymatic activity, protein and gene expression suggested higher basal expression of TNAP in MAT and PAT cells than in BM-MSC. An increase of TNAP activity and Alpl mRNA level during adipogenesis was observed at a higher extent in MAT and PAT cells. Inhibition of TNAP activity led to decreased lipid droplet formation as well as reduction of CD36 (from 12.2±3.5 to 7.3±3.4% in BMMSC, from 20.4±4.2% to 14.2±2.4% in MAT, from 21.2±5.3 to 11.6±5.3% in PAT) and increment of CD142 (from 10.3±3.4 to18.6±5.8% in BMMSC, from 5.4±0.8 to 9.6±1.2% in MAT, from 7.8±2.3 to 9.4±3.7 in PAT) expression. TNAP inhibition reduced adipogenesis-associated mitochondrial mass increase and oxidative phosphorylation level in all three cell types and particularly in BM-MSC. Along with identified impact on cell bioenergetics, results suggest that TNAP activity plays an important role in adipogenic differentiation of isolated cells. The elucidation of the main cellular and molecular players in marrow adipogenesis fosters the understanding of the role of MAT in OA pathology and may help to conceive novel biomarkers and therapeutics for OA.