1.Alteration Of Runx2Methylation Status During Vascular Smooth Muscle Cells Differentiation Into Osteoblast-like Cells2.Mechanism Of The Effects Of Vaspin On The Apoptosis Of Human Osteoblasts

Objective To establish a cellular model of mouse VMSCs differentiation into osteoblast-like cells induced by β-GP.Methods Mouse VSMCs were induced to differentiate into osteoblast-like cells by medium containing lOmM β-GP and the alterations in smooth muscle cells lineage were observed in SMa-actin and osteoblast lineage:Runx2, OC, OPN, ALP activity and formation of mineralized nodes after exposure to β-GP. VSMCs were cultured with medium containing10mM β-GP. The mRNA expression of Runx2, OC, OPN after culturing for0or12days were validated by real-time PCR. The ALP activity was measured and the mineralized node staining was performed. Protein was extracted after culturing for0,3,8and12days, the expression of SMa-actin and Runx2were measured by Western blot.Results After culturing for12days under calcification medium, expression of Runx2, OC, OPN mRNA were enhanced to a level of8.125±0.105,8.482±0.172and9.472±0.064respectively, ALP activity was also increased and the formation of typical mineralized nodes was observed. The protein expression of Runx2increased, while that of SMa-actin decreased by β-GP.Conclusion We have successfully constructed a cellular model for inducing mouse VSMCs to differentiate into osteoblast-like cells. β-GP enhance the expression of osteogenic markers and inhibited the expression of smooth muscle cellular marker. Objective To investigate the methylation status of CpG sites in Runx2promoter region before and after osteogenic differentiation and after exposure to5-Aza-dC, and to observe the effect of5-Aza-dC on expression of Runx2during the process of osteogenic differentiation. Elucidate the effect of Runx2methylation on VSMCs differentiation into osteoblast-like cells.Methods Extract the DNA of VMSCs in the control. The DNA methylation status in Runx2promoter was detected by bisulfite sequencing. To evaluate appropriate dosage for subsequent experiments, proliferation of VMSCs was assessed by MTT assay. Mouse VSMCs were exposed to calcification medium containing various concentrations of5-Aza-dC for12days, total RNA and protein were extracted respectively. The expression of Runx2in both mRNA and protein levels were evaluated.VSMCs were treated with15μM of5-Aza-dC for0,3,8and12days respectively, then total RNA were extracted and the expression of Runx2mRNA was validated by real-time PCR. The expression of Runx2protein was observed by western blot.Extract the DNA of VSMCs induced by P-GP or by5-aza-dC together with β-GP for12days. After bisulfite modification, the PCR products were cloned into the pGEM-T easy vector. The methylation status of Runx2promoter was detected by bisulfite sequencing.Results Pretreatment with20μM5-aza-dC for24hours, the cell density decreased significantly(P<0.05). No significant decrease in cellular proliferation was seen in VSMCs treated for24hours with5-aza-dC at concentrations below20μM(5,10,15μM). Therefore, concentrations lower than20μM were considered to be moderate and were chosen for subsequent experiments.5-Aza-dC upregulated expression of Runx2in VSMCs in a dose-and time-dependent manner.15μM5-Aza-dC is the optimal dose. Bisulfite sequencing analysis revealed that the methylation levels of Runx2promoter in the control were higher than those induced by p-GP alone or by5-aza-dC together with β-GP. The methylation levels of Runx2promoter in VSMCs induced by β-GP were higher than those induced by5-aza-dC together with β-GP.Conclusion After VSMCs differentiation into osteoblast-like cells, the methylation levels of Runx2promoter decreased.5-Aza-dC upregulated Runx2expression in a dose-and time-dependent manner by demethylating Runx2promoter. Demethylation of Runx2promoter may facilitate VSMCs differentiation into osteoblast-like cells. Objective Vaspin is a newly discovered adipokine secreted from viseral adipose tissue and has an insulin-sensitizing effect. Recently, it has been shown that vaspin suppresses the apoptosis of vascular endothelial cells through PI3/K-Akt signaling pathway. However, the main roles of vaspin and exact mechanism are still unknown. The aim of this study was to investigate the effects of vaspin on human osteoblast(hOB) apoptosis. Methods Cell apoptosis was measured by ELISA and TUNEL. Western blot analysis the effect of vaspin on the expression of Bcl-2and Bax in hOB. Western blot detected the expression of p-ERK1/2, ERK1/2, p-JNK, JNK, p-p38, p38, p-Akt and Akt in cultured primary hOB after exposure to vaspin. Specific inhibitor of ERK(PD98059) was used to evaluate the role of vaspin in signaling pathway.Results (1)Vaspin inhibited hOB apoptosis.(2)Vaspin facilitated the expression of Bcl-2protein, while attenuated the expression of Bax protein in a dose-dependent manner.(3)Vaspin activated the phosphorylation of ERK, pretreatment of hOB with ERK inhibitor PD98059abolished vaspin-induced activation of ERK. Vaspin had no activation on the phosphorylation of p38, JNK and Akt.(4)PD98059deminished the depression of vaspin on hOB apoptosis.Conclusion Vaspin inhibits hOB apoptosis through MAPK/ERK signaling pathway.