AGEs Promote The Calcification Of Human Aortic Smooth Muscle Cell By Up-regulation Of PTEN

Objective:Discuss the potential relationships between PTEN and The calcification Of HASMC to detect whether PTEN can mediate AGEs to promote vascular calcification.Methods : Apply AGEs with different concentrations(25mg/L ? 50mg/L ?100mg/L)to three groups of HASMC for intervention cultivation. Given the HASMC cultivated under normal conditions(91% DMEM high glucose culture medium+6% fetal calf serum+2% growth factor +1% double resistant) as a blank control group, extract protein from part of cells after 2 days' cultivation.Use western blotting to detect osteogenesis related protein OPG, Runx2, BMP2 expression levels of each HASMC group.The remaining cells should be dyed alizarin red after 10 days' cultivation.In order to make clear whether PTEN took part in the process in which AGEs facilitated HASMC's calcification, we check the protein expression level of PTEN in HASMCs by means of Western blot after giving AGEs treated cells of different concentration(0mg/L?25mg/L?50mg/L?100mg/L)and length of time(0h?12h?24h?48h). Then carrying bpv(Hopic),one specific inhibitor of PTEN, we use Western blot to select the best inhibition concentration further to cultivate HASMC with intervention,extract protein from part of cells after 2 days' cultivation. Use western blotting to detect osteogenesis related protein OPG, Runx2, BMP2 expression levels of each HASMC group.The remaining cells should be dyed alizarin red after 10days' cultivation.In the end,In order to make clear whether PTEN took part in the process in which ?-glycerol phosphate facilitated HASMC's calcification,Apply 10mmol/L ?-glycerol phosphate,10mmol/L ?-glycerol phosphate+100mg/L AGEs,10mmol/L ?-glycerol phosphate+100mg/L AGEs+200?mol/L bp V(Hopic) to three groups of HASMC for intervention cultivation. Set a blank control group, extract protein from part of cells after 2days' cultivation. Use western blotting to detect osteogenesis related protein OPG, Runx2, BMP2 expression levels of each HASMC group. The remaining cells should be dyed alizarin red after 10 days' cultivation.(6)Apply 10mmol/L?-glycerol phosphate, 10mmol/L ?-glycerol phosphate +200?mol/L bp V( Hopic)to two groups of HASMC for intervention cultivation. Set a blank control group, extract protein from part of cells after 2 days' cultivation. Use western blotting to detect osteogenesis related protein OPG,Runx2, BMP2 expression levels of each HASMC group. The remaining cells should be dyed alizarin red after 10 days' cultivation.Results:(1)The calcification degrees and osteogenesis related protein OPG,Runx2, BMP2 expression levels in cells of experimental groups implemented with intervention cultivation with different concentrations AGEs are higher than blank control groups and will enhance following the increase of AGEs concentrations for intervention.(2)The cultivated HASMC with intervention using AGEs of different concentration, its protein expression level of PTEN is higher than that of blank control group. Also, the protein expression level goes up as the rising concentration of used AGEs. It shows that when the intervened time on AGEs comes to 48 h, PTEN's protein expression gets the highest level.(3)The calcification degrees and osteogenesis related protein OPG, Runx2,BMP2 expression levels in cells of AGEs+bp V(Hopic)intervention groups are lower than cells in simple AGEs intervention group, but are higher than blank control groups.(4)The calcification degrees and osteogenesis related protein OPG, Runx2, BMP2 expression levels in cells of ?-glycerol phosphate group,?-glycerol phosphate+AGEs group, ?-glycerol phosphate+AGEs+bp V(Hopic)group are higher than blank control group. Among them, the calcification degrees and osteogenesis related protein OPG, Runx2, BMP2 expression level in cells of ?-glycerol phosphate+AGEs group is the highest.(5)The calcification degrees and osteogenesis related protein OPG, Runx2, BMP2 expression levels in cells of ?-glycerol phosphate group and ?-glycerol phosphate+bp V(Hopic)group are higher than blank control group. But there is no obvious differences between the two group.Conclusion:1. AGEs can increase the PTEN protein expression in HASMC cells.2.AGEs can promote the osteogenesis related protein OPG, RUNX2,BMP2 expression levels in HASMC. After PTEN protein activity is inhibited,AGEs can promote the decrease of functions of the osteogenesis related protein expression in HASMC.3. PTEN has no obvious influences on ?-glycerol phosphate in promoting cell calcification functions in HASMC.