Nitrogen-containing bisphosphonate induces enhancement of OPG expression and inhibition of RANKL expression via inhibition of farnesyl pyrophosphate synthase to inhibit the osteogenic differentiation and calcification in vascular smooth muscle cells

Background The osteogenic differentiation of vascular smooth muscle cells (VSMCs) plays a key role in the progression of Vascular calcification（VC）. The previous research showed that nitrogen-containing bisphosphonate（N-BP）had inhibitory effect on the osteogenic differentiation and calcification induced by high phosphorus in VSMCs. However, it is not clear how N-BP inhibits the osteogenic differentiation and calcification in VSMCs via specific regulatory mechanisms. Methods β-glycerophosphate (β-GP) was used to induce the osteogenic differentiation and calcification in VSMCs. In some experiments, VSMCs were cultured with β-GP , or β-GP plus different concentrations of N-BP. In some other experiments, the VSMCs were cultured with β-GP plus N-BP, β-GP plus N-BP and farnesol (FOH), or β-GP plus N-BP and geranylgeraniol (GGOH). Alizarin red S staining and determination of calcium content were used to detect calcium deposition.Western Blotting were used to detect expressions of proteins(OPG and RANKL ) and osteogenic marker proteins (Runx2 and OPN). Results β-GP induced the osteogenic differentiation and calcification in VSMCs , increased RANKL protein expression and had no significant effect on OPG protein expression. N-BP increased OPG protein expression and decreased RANKL protein expression in VSMCs of osteogenic differentiation and calcification induced by β-GP. In addition, N-BP reduced the osteogenic marker proteins (Runx2 and OPN) expression and calcium deposition in VSMCs of osteogenic differentiation and calcification induced by β-GP. These effects of N-BP on the osteogenic differentiation and calcification in VSMCs were concentration-dependent, which could be reversed by the downstream products of farnesyl pyrophosphate synthase(FPPS) in mevalonate pathway such as FOH or GGOH. Conclusion N-BP increases OPG expression and decreases RANKL expression via inhibition of FPPS to inhibit the osteogenic differentiation and calcification of VSMCs