Di-san junyi daxue xuebao (Feb 2020)
Effects of ITD-1, a small-molecule inhibitor of TGF-β type Ⅱ receptor, on osteogenic differentiation and angiogenesis of bone marrow stromal cells
Abstract
Objective To investigate the effects of ITD-1, a small-molecule inhibitor of transforming growth factor-β (TGF-β) type Ⅱ receptor (TGFBR2), on osteogenic differentiation and angiogenesis of bone marrow stromal cells (BMSCs). Methods BMSCs from C57/BL6 mice were treated with 10 μmol/L ITD-1 or 0.1% DMSO, and the changes in the cell proliferation and apoptosis were examined using cell counting kit-8 (CCK-8) and flow cytometry, respectively. Real-time quantitative PCR (qPCR) was performed to detect the mRNA expression of the osteogenic markers including alkaline phosphatase (ALP), ostrix (OSX), collagen type Ⅰ (COL1) and osteocalcin (OCN) and the angiogenesis-related markers including vascular endothelial growth factor (VEGF) and angiopoietin 1 (ANGPT1). ALP staining and alizarin red staining were used to assess the activity of ALP and matrix mineralization in the cells, respectively; immunofluorescence assay was used to detect the expression of the osteogenic marker OPN and the angiogenic marker VEGF. Mouse tissue culture experiment was conducted to test the bone mineral density of mouse humerus cultured in the presence of ITD-1. Results The intensity of ALP staining was obviously lowered in ITD-1-treated mouse BMSCs as compared with DMSO-treated cells. Alizarin red staining revealed significantly reduced matrix mineralization in ITD-1-treated BMSCs. Treatment with 10 μmol/L ITD-1 for 12 h significantly suppressed the proliferation of the BMSCs but did not cause obvious cell apoptosis. Treatment with ITD-1 significantly decreased the mRNA expression of the osteogenic markers ALP, OSX, COL1 and OCN (P < 0.05) but increased the mRNA expression of the angiogenesis-related markers VEGF and ANGPT1 (P < 0.05). Immunofluorescence assay also confirmed the decrease of OPN and increase of VEGF expression. The tissue culture experiment showed that culture in the presence of ITD-1 significantly reduced bone mineral density of mouse humerus. Conclusion ITD-1 inhibits the proliferation and osteogenic differentiation and enhances angiogenesis of mouse BMSCs without affecting cell apoptosis.
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