The Dynamic Change Of The MRNA Expression Of Cbfa1and Sox9of Rat Vascular Smooth Muscle Cells Induced By β-glycerophosphate

Objective: Elevated phosphate is an very important non-traditional riskfactor for cardiovascular events in chronic kidney disease (CKD). Thepresence and extent of vascular calcification are independent predictors ofcardiovascular events. Medial calcification is a prominent feature of vascularcalcification in patients with CKD. As the major cell type in vessel mediallayer, the transdifferenciation of vascular smooth muscle cells (VSMCs) toosteo/chondrocyte-like cells is the key point in vascular calcification with theupregulation of osteogenic genes, especially core binding factor a1(Cbfa1)and sex determine region Y-box9(Sox9) seen as the marker oftransdifferenciation. But whether VSMCs transdifferent to osteoblast-like orchondrocyte-like cells is still unknown. We observe the dynamic change of theexpression of gene associated with transdiffereciation in osteo/chondrocyte toinvestigate the effect of β-glycerophosphate on the transdifferentiation of ratVSMCs to provide the theoretical basis for the pathology and prevention ofvascular calcification in patients with CKD.Methods:1The culture and identification of VSMCsPrimary VSMCs were obtained from rat vessel wall by tissue explantsadherent method. After naturally purified, we confirmed the cell type withmorphological and immunocytochemistry methods.2Groups and the treatmentVSMCs were randomly divided into the control group, the β–glycerolphosphate group (HP group) and the phosphonoformic acid group(PFA group). The control group were cultured with low-glucose DMEMcontaining10%fetal bovine serum (FBS), while HP group and PFA group were cultured in10%FBS+10mmol/L β–glycerolphosphate and10%FBS+10mmol/L β–glycerolphosphate+1mmol/L PFA. We changed themedium everyday. After6h,12h,1d-5d stimulation, we examined the dynamicchange in the expression of associated genes.3The mRNA expression of related genes induced by β–glycerolphosphateUsing RT-PCR to detect mRNA expression of core binding factor a1(Cbfa1), sex-determining region Y-box9(Sox9), type II collagen a1(Col IIa1)and type X collagen a1(Col Xa1) at different time points in control and HPgroup. GAPDH and target gene were amplified in the same EP.4The expression of Cbfa1in HP group and PFA groupUsing RT-PCR and Western Blot to detect the mRNA and proteinexpression of Cbfa1in HP and PFA group.5Statistical methodsAll data were analysed with SPSS13.0software. The measurement datawere described in (x s). Differences between the two groups werecompared with t test, differences among multi-groups were compared withone-way analysis of variance (ANOVA) and Student-Newman-Keuls (SNK)was used as pairwise comparison. Statistical significance was defined asP<0.05.Results:1The primary culture of VMSCsCells could be seen to climb out of tissue on the3rd day in primary culture.On the6th to7th day, when cells became schistic that could be subcultured.Using inverted phase contrast microscope, we observed that cells showed atypical fascicules growth and "peak-valley" appearance with spindle (inmajor), star or irregular shape. By HE staining, cells were with abundantcytoplasm and round or ellipse nucleus and one or more nucleoli. To identifythese cells, we performed S-P immunohistochemical stainings for α-smoothmuscle actin (α-SMA actin), which were strong positively expressed incytoplasm with brown yellow immune reaction product arranged in long axisand more than98%cells could be seen with strong positive expression. 2The mRNA expression of related genes in HP groupAfter changed to the HP medium, the expression of Cbfa1mRNAexperienced a trend as that first increased, then fell down and after thatincreased again (P<0.05), like “peak-valley-peak”. However Sox9showed a“single peak”(P<0.05), and the time it reached peak was between the valleyand the second peak of Cbfa1. As the downstream of Sox9, the change of typeII collagon a1(Col IIa1) was similar to Sox9shown as “single peak”. Whiletype X collagon a1(Col Xa1) expressed at low level at first and significantlyincreased at4d and5d (P <0.05).3The mRNA and protein expression of Cbfa1in HP and PFA groupAfter changed to the HP medium, the mRNA and protein expression ofCbfa1both showed a “double peak”(P <0.05) which reached peak at6h and3d respectively. While in PFA group the second peak moved forward whichreached peak at6h and1d respectively(P <0.05).Conclusion:1After stimulation of β-glycerophosphate, the dynamic change of mRNAexpression of Cbfa1, Sox9and their downstream gene in rat VSMCs werecoincidence with their expression characteristic in the differeciation ofchondrocyte. So we presumed that rat VSMCs may experience adifferentiation procedure like chondrocytes induced by β-glycerophosphate.3After inhibition of phosphate intake of rat VSMCs by PFA, the secondpeak of Cbfa1moved forward, which may suppress the furthertransdiffereciation of VSMCs.