Angiopoietin-2 Enhances Osteogenic Differentiation of Bone Marrow Stem Cells

Our previous studies revealed that co-transplantation of bone marrow stem cells (BMSCs) and adipose-derived stem cells (ADSCs) can enhance bone regeneration and angiogenesis. However, it is unclear which genes are involved in the regulation of osteogenesis and/or angiogenesis during the co-culturing of BMSCs and ADSCs. The expression patterns of genes associated with osteogenesis and/or angiogenesis were analyzed in osteogenesis-induced BMSCs and ADSCs using an oligonucleotide microarray. Significant difference in the expression patterns of several genes were identified from hierarchical clustering and analyzed on co-cultured BMSCs and ADSCs. Angiopoietin-2 (ANGPT2) and activin receptor-like kinase-1 were significantly down-regulated in co-culture than culture of either BMSCs or ADSCs, while fibroblast growth factor-9 was significantly up-regulated in co-culture. The effect of ANGPT2 in osteogenesis-induced BMSCs was validated using recombinant protein and siRNA of ANGPT2. Treatment of the ANGPT2 protein significantly increased the expressions of osteogenic makers and the intensity of Alizarin red-S staining in BMSCs. Down-regulation of ANGPT2 significantly decreased the expression of osteogenic makers. The treatment of ANGPT2 protein to BMSCs induced significantly increased tube formation in Transwell-co-cultured human umbilical vein endothelial cells (HUVECs) compared with untreated control. ANGPT2 siRNA transfection showed the opposite effects. These results suggest that the treatment of ANGPT2 in BMSCs increase osteogenesis and angiogenesis in vitro, and that the enhancement of osteogenesis and angiogenesis in the co-cultured BMSCs and ADSCs seems to be mediated by a mechanism that makes the activation of ANGPT2 unnecessary. These observations provide the first evidence for positive regulation of osteogenesis by ANGPT2 in vitro. J. Cell. Biochem. 118: 2896-2908, 2017.