Induction of osteogenic differentiation and calcification model in VSMCs by β-GP
To establish osteogenic differentiation and calcification model, we stimulated VSMCs with 10mM β-GP for 72h, and the calcium deposition(purple-red spots)were induced in VSMCs (Figure 3A). The calcium content in the β-GP group was also higher than that in control group (Figure 3B, p < 0.01). The expression levels of osteogenic marker proteins Runx2 and OPN were elevated in comparison to the control group (Figure 3C.3D, p < 0.05). Meanwhile, the expression level of RANKL in the β-GP group was elevated in comparison to the control group (Figure 3C.3D, p < 0.05), but the expression levels of OPG in the β-GP group was not significantly higher than that in the control group (Figure 3C.3D, p > 0.05).
Effect of N-BP on VSMCs calcification
ZOL (a type of N-BP) reduced the purple-red calcium deposition induced by β-GP in VSMCs at 72h. Compared with 1µM ZOL, 5µM ZOL had a more obvious effect for calcified VSMCs in reducing the purple-red calcium deposition (Figure 4A). Also, ZOL reduced calcium content in calcified VSMCs at 72h (Figure 4B, p < 0.05).Compared with 1µM ZOL, 5µM ZOL had a more obvious effect for calcified VSMCs in reducing calcium content (Figure 4B, p < 0.01).
Effect of N-BP on RANKL, OPG and osteogenic marker proteins( Runx2 and OPN) expression in VSMCs of osteogenic differentiation and calcification
ZOL reduced the protein expression level of RANKL in VSMCs of osteogenic differentiation and calcification at 72h (Figure 5A.5B, p < 0.05). Compared with 1µM ZOL, 5µM ZOL had a more obvious effect in reducing the protein expression level of RANKL (Figure 5A.5B, p < 0.01). The protein expression levels of OPG in the β-GP group was slightly higher than that in the control group at 72h, but the difference was not statistically significant (Figure 5A.5C, p > 0.05). Compared with the control group, ZOL plus β-GP group had the higher protein expression level of OPG at 72h (Figure 5A.3C, p < 0.05). Compared with the β-GP group, 1µM ZOL plus β-GP group increased the protein expression level of OPG at 72h, but the difference was not statistically significant (Figure 5A.5C, p > 0.05). Compared with the β-GP group, 5µM ZOL plus β-GP group had the higher protein expression level of OPG at 72h (Figure 5A.5C, p < 0.05). In addition, ZOL reduced the osteogenic marker proteins Runx2 and OPN in VSMCs of osteogenic differentiation and calcification at 72h (Figure 5A.5D.5E, p < 0.05). Compared with 1µM ZOL, 5µM ZOL had a more obvious effect in reducing the protein expression level of Runx2 and OPN (Figure 5A.5D.5E, p < 0.05).
Effect of N-BP on VSMCs calcification due to inhibition of FPPS
In order to reverse the inhibition effect of N-BP on VSMCs calcification due to inhibition of FPPS , we added the downstream products of FPPS in mevalonate pathway such as FOH or GGOH. ZOL reduced the purple-red calcium deposition induced by β-GP in VSMCs at 72h, which was reversed by FOH or GGOH (Figure 6A). Meanwhile, ZOL reduced calcium content in calcified VSMCs at 72h (Figure 6B, p < 0.0001), which was reversed by FOH or GGOH (Figure 4B, p < 0.01).
Effect of N-BP on RANKL, OPG and osteogenic marker proteins( Runx2 and OPN) expression in VSMCs of osteogenic differentiation and calcification due to inhibition of FPPS
In order to reverse the regulatory effect on RANKL, OPG and osteogenic marker proteins( Runx2 and OPN) expression in VSMCs of osteogenic differentiation and calcification due to inhibition of FPPS , we added the downstream products of FPPS in mevalonate pathway such as FOH or GGOH. 5µM ZOL induced enhancement of OPG protein expression and inhibition of RANKL protein expression in VSMCs of osteogenic differentiation and calcification at 72h (Figure 7A.7B.7C, p < 0.001), which were reversed by FOH or GGOH (Figure 7A.7B.7C, p < 0.05). In addition, 5µM ZOL reduced the osteogenic marker proteins Runx2 and OPN in VSMCs of osteogenic differentiation and calcification at 72h (Figure 7A.7D.7E, p < 0.0001),which were reversed by FOH or GGOH (Figure 7A.7D.7E, p < 0.01).