Calcium Phosphate Crystals Promote Osteogenic Differentiation Of Vascular Smooth Muscle Cells Through BMP-2/Smad And NF-?B Signaling Pathways

Objectives: The present study is to investigate the effect of calcium phosphate(CaP)crystals induced by high-phosphate(high-Pi)on the osteogenic differentiation of human aortic smooth muscle cells(HASMCs),and whether BMP-2/Smad and NF-?B signaling pathways involved in this process.Methods: 1.High-Pi medium(3 mmol/L)was incubated at 37? for 3 days,then ultracentrifugated to isolate CaP crystals and supernatant.The physical and chemical characteristics of crystals were analyzed using SEM and EDX.Cultured HASMCs were divided into 4 groups: control,high-Pi,crystals and supernatant group.Calcified nodules were observed by Alizarin red staining and calcium deposition was measured using o-cresolphthalein complexone method.The levels of osteogenic protein BMP-2,RUNX2 and OPN were determined by western blotting.The effect of CaP crystals inhibition by PPi 10 ?mol/L was examined.2.The protein levels of p-Smad1 in HASMCs stimulated by CaP crystals were tested using western blotting.HASMCs were pre-treated with 1 ?g/ml BMP-2 inhibitor noggin,BMP-2 shRNA and Smad1 shRNA respectively,then the osteogenic protein levels were measured.3.Confocal laser scanning microscopy monitored the fluorescence intensity of mitochondrial ROS in HASMCs treated with CaP crystals.After incubation with mitochondrial respiratory chain inhibitor rotenone 10 ?mol/L,calcified nodules,calcium deposition and osteogenic proteins were measured.Western blotting determined the protein levels of p-IKK??I?B? and nuclear p65 in HASMCs treated with CaP crystals.P65 shRNA was transfected into HASMCs,and calcification was observed.After adding rotenone 10 ?mol/L,the protein levels of p-IKK??I?B? and nuclear p65 were tested in HASMCs.Results: 1.High-Pi medium induced the formation of CaP crystals,which appeared as sperical particles with diameter of 30-500 nm and Ca/P ratios reaching 1.45±0.03.Compared with the cells in control group,CaP crystals significantly induced the formation of calcified nodules,increased calcium deposition and upregulated the protein levels of BMP-2,RUNX2 and OPN(p all<0.05).PPi significantly inhibited the protein levels of BMP-2,RUNX2 and OPN induced by CaP crystals(p all<0.05).2.After incubation with CaP crystals,the protein levels of p-Smad1 in HASMCs increased and peaked at 30 min.Noggin and BMP-2 shRNA blocked the protein levels of p-Smad1,and partly downregulated the protein levels of RUNX2 and OPN,in HASMCs stimulated by CaP crystals(p all<0.05).Transfection with Smad1 shRNA partly blocked the protein levels of RUNX2 and OPN,in HASMCs stimulated by CaP crystals(p all<0.05).3.CaP crystals promoted mitochondrial ROS production in a time-dependent manner.Rotenone blocked mitochondrial ROS production,reduced calcified nodules and calcium deposition,and partly inhibited the protein levels of BMP-2,RUNX2 and OPN,in HASMCs stimulated by CaP crystals(p all<0.05).CaP crystals activated p-IKK?,degraded I?B? and promoted nuclear translocation of p65 in HASMCs in a time-dependent manner.The transfection with p65 shRNA partly reduced calcified nodules formation and calcium deposition in HASMCs stimulated by CaP crystals(p<0.05).Rotenone inhibited the upregulation of p-IKK?,the degradation of I?B?,and the nuclear translocation of p65(p all<0.05).Conclusion: 1.High-Pi promoted the osteogenic differentiation of HASMCs through the induction of CaP crystals.CaP crystals could accelerate the osteogenic differentiation of HASMCs through signaling pathways of BMP-2/Smad and NF-?B mediated by mitochondrial ROS.