Ghrelin Attenuates The Differentiation Of Vascular Smooth Muscle Cells Into Osteoblast-like Cells Through The MAPK-ERK Pathway

PART ONE Ghrelin attenuates the differentiation of vascular smooth muscle cells into osteoblast-like cellsObjective:To determine the effect of ghrelin on the differentiation of vascular smooth muscle cells (VMSCs) into osteoblast-like cells.Methods:Mice VMSCs were isolated from normal mice.The cell protein and conditioned medium were extracted after treatment with10-6mol/L ghrelin for0,12,24,48and72h. The ALP activity, calcium deposition and Runx2expression were determined at different time points.Results:Compared with the control group, ghrelin significantly inhibited ALP activities in a time-dependent manner. The inhibitory effects of ghrelin on ALP activities were observed within24h and reached to the peak levels at48h. The effects of ghrelin on calcium deposition in VSMCs were determined after an incubation period of12days and the results showed that ghrelin remarkably decreased the calcium deposition in VSMCs, and similarly the Runx2expression was significantly inhibited in VSMCs cultured with ghrelin for48h.Conclusion:Ghrelin significantly decreased calcium deposition, the ALP activity and Runx2expression, resulting in an inhibition of mineralization in a time-dependent manner, which suggest that ghrelin attenuated the differentiation of VSMCs into osteoblast-like cells. PART TWO Ghrelin induces the MAPK-ERK signaling pathwayObjective:To determine whether ghrelin stimulates the MAPK signaling pathway in VSMCs.Methods:VSMCs were treated with ghrelin at0,5,10,15,30, and60min. MAPK signaling molecules including ERK, c-jun N-terminal kinases(JNK) and p38MAP kinases (p38) and their phosphorylated isoforms were determined with western blotting.Results:The protein levels of phosphorylated ERK were increased5min after incubation with ghrelin and a peak effect was reached at30min. No changes in the levels of phosphorylated JNK and P38were observed.Conclusion:Ghrelin stimulates the MAPK-ERK pathway in VSMCs. PART THREE Signaling pathway involved in the Ghrelin-attenuated differentiation of VSMCs into osteoblast-like cellsObjective:To further determine whether Ghrelin induces the differentiation of VSMCs into osteoblast-like cells via MAPK pathway.Methods:Expression of growth hormone secretagogue receptor(GHSR) inVSMCs was reduced by siRNA and the activity of ERK was inhibited by the specific inhibitor PD98059. Cells were then treated with10-6M Ghrelin. In the control groups, cells were treated with control siRNA or the vehicle for PD98059.After72h incubation, cells were harvested and levels of phosphorylated and total ERK protein were determined by western blotting, and then the ALP activities were determined.Results:Expression of GHSR protein was significantly reduced by siRNA. Silence of GHSR inhibited the phosphorylation of ERK and abolished the decreased ALP activity by ghrelin. Inhibition of ERK by PD98059blocked effects of ghrelin on ALP activities.Conclusion:Ghrelin inhibits the differentiation of VSMCs into osteoblast-like cells via the MAPK-ERK pathway. PART FOUR GHSR-Ia, Runx2and Ghrelin expression is associated with the arterial media calcification.Objective:To determine whether GHSR-Ia, Runx2and Ghrelin expression is associated with arterial media calcification.Methods:Arterial media calcification was confirmed using Van Kossa stain.The expression of GHSR-Ia, Runx2and Ghrelin in the calcified artery or normal artery was detected using immunohistochemistry.Results:The expression of GHSR-Ia in the calcified artery significantly decreased than that in normal artery. and tThe expression of Runx2in calcified artery significantly increased. The expression ratio between two groups were negatively correlated. It has no significant difference in Ghrelin expression between calcified artery and normal artery was observed.Conclusion:Downregulaiton of GHSR-Ia and upregulation of Runx2are associated with arterial media calcification.