Figure 1.
A to F Von Kossa staining of VSMCs. Cells were culture in different medium for 14 days. (A) Von Kossa staining of VSMCs cultured in control DMEM medium. (B) Von Kossa staining of VSMCs cultured in osteogenic medium. (C) Von Kossa staining of VSMCs treated with DAPT. (D) -(F) Von Kossa staining of VSMCs treated with different concentrations of OPG: 0.1ng/ml(d), 1ng/ml(e) and 10ng/ml(f) (arrows showed the positive stained calcium particles, black color). G. Cellular calcium content of VSMCs. Calcium content in VSMCs in osteogenic medium was increased by 5 folds in the VSMCs cultured with compared to the control; OPG reduced the calcium deposition in VSMCs induced by osteogenic stimulation by 20 to 60% in a dose dependent manner (p<0.05, compared to VSMCs cultured in osteogenic medium). These observations together indicate the OPG plays an important role in the regulation of the osteoblastic differentiation of VSMC. *, P<0.05 vs control, #, P<0.05 vs osteogenic medium, and &, P<0.05 vs OPG 0.1ng/ml. n=3.
Figure 2.
Alkaline Phosphatase activity of VSMCs.
The ALP activity was measured and normalized by protein amount in VSMC from different media. ALP activity was significantly increased by 3.5 folds in VSMCs treated with osteogenic media compared to the control, confirming a osteogenic conversion in the VSMCs. The enhanced ALP activity induced by osteogenic medium was repressed significantly by 30% with the addition of DAPT and also significantly reduced by OPG dose dependently. *, P<0.05 vs control, #, P<0.05 vs osteogenic medium, and &, P<0.05 vs OPG 0.1ng/ml. n=3.
Figure 3.
Expression of Notch1 and RBP-Jκ in VSMCs.
(A) mRNA level of Notch1 and RBP-Jκ were measured by real-time RT-PCR in VSMCs cultured in different media. (B) Protein level of Notch1 and RBP-Jκ were measured by Western blotting in VSMCs cultured in different media. Notch1 and RBP-Jκ significantly increased by 3 to 4 folds in VSMC cultured in osteogenic medium compared to control at both mRNA and protein levels, suggesting that the Notch1-RBP-Jκ signaling pathway is activated in osteogenic conversion of VSMCs. DAPT reduced the expression of Notch1 by 50% and RBP-Jκ by 30% in VSMCs compared to osteogenic cells at bothe mRNA and protein levels. Similarly, OPG reduced the expression of Notch1 and RBP-Jκ compared to VSMCs in the osteogenic medium in a dose dependent manner. *, P<0.05 vs control, #, P<0.05 vs osteogenic medium, and &, P<0.05 vs OPG 0.1ng/ml. n=3.
Figure 4.
(A) mRNA level of msx2 was measured by real-time RT-PCR in VSMCs cultured in different media in VSMCs cultured in different media. (B) Protein level of Msx2 was measured by Western blotting in VSMCs cultured in different media. The expression of Msx2 was significantly increased with the treatment of osteogenic medium by 3 to 4 fold compared with control at both mRNA and protein levels. The activation of Msx2 by osteogenic media was deteriorated by the addition of DAPT; the addition of OPG significantly reduced the expression of Msx2 in a dose dependent manner, indicating that OPG represses expression of Msx2 through the reduction of the Notch1-RBP-Jκ-signaling pathway. *, P<0.05 vs control, #, P<0.05 vs osteogenic medium, and &, P<0.05 vs OPG 0.1ng/ml. n=3.