Mechanism Of Endothelin-1in The Regulation Of Aortic Valve Interstitial Cells To Osteoblastic Differentiation

Objective:To isolate and cultivate aortic valve interstitial cells and To establish the osteoblastic differentiation model of aortic valve interstitial cells by pro-calcific medium.Methods:collagenase digestion was obtained to dissolve extracellular matrix of the aortic valves from patients with acute dissection(type A). Cell phenotypic identification was achieved by immunochemistry to measure the following:SM a-actin, vimentin, CD31, SM-myosin and desmin. Pro-calcific medium (containing β-glycerophosphate disodium, dexamethasone and ascorbic acid) was used to promote the osteoblastic differentiation of cultured cells. This cell model was evaluated by alkaline phosphatase activity/staining, calcified nodules(von Kossa staining) and the expression of alkaline phosphatase and Cbfal by western blotting.Results:Isolated cells proliferated well, expressed SM a-actin and vimentin, but no CD31, SM-myosin or desmin. On the21st day of intervention with pro-calcific medium cultured cells, the cell model was achieved by the positive staining of alkaline phosphatase and von Kossa and more expression of alkaline phosphatase and Cbfal compared with blank control.Conclusion:Isolated aortic valve interstitial cells grew well in vitro, expressed specific proteins and were not contaminated by endothelial cells or muscle cells. Pro-calcific medium could promote the osteoblastic differentiation of cultured cells by the expression of alkaline phosphatase and Cbfal and forming calcified nodules. Objective:To prove that ET-1can promote the osteoblastic differentiation of aortic valve interstitial cells, and explore the role of ETAR and ERK pathway.Methods:Different concentrations of ET-1or ET-1plus the ERK pathway blocker PD98059or ET-1plus the ETAR blocker BQ-123were added into the pro-calcific medium. The activity/expression of alkaline phosphatase was detected on the21th day of intervention. The activation level of ERK pathway was measured on different time point (5min,10min,30min,60min,120min) after the addition of ET-1into the medium. The blockade effect of BQ-123on the activation of ERK pathway by ET-1was detected after20min. Results:ET-1promote the expression of alkaline phosphatase on a dose dependent manner which could be attenuated by PD98059and BQ-123. ET-1could activate the ERK pathway whose effect could be attenuated by BQ-123and PD98059.Conclusion:ET-1promote the osteoblastic differentiation of aortic valve interstitial cells via ETAR and ERK pathway.