The Mechanism Of Wnt/?-catenin Signaling On The Osteogenic Differentiation Of Aortic Valve Myofibroblast By TNF-?

Objectives: Calcific Aortic Valve Disease(CAVD)is a common disease in the cardiovascular system and is one of the common causes of heart failure,arrhythmia,syncope,sudden death and other diseases as well.The incidence of CAVD is high in the elderly,and will be higher with the prolongation of life expectancy.However,there is no effective treatment to relieve the progession of valve calcification so far.Once progressed,the surgical valve replacement will be the only available way to lengthen the survival time and to improve the quality of life.The pathogenesis of CAVD is still unclear and needs more to research.In this study,Tumor Necrosis Factor-?(TNF-?)was used to establish a model of osteogenic differentiation of aortic valve myofibroblast,and DKK-1(a inhibitor of Wnt signaling pathway)was used to reveal the effect and the mechanisms of Wnt/?-catenin signaling on the osteogenic differentiation of aortic valve myofibroblast.Methods: 1.Valve myofibroblast were obtained from the heart valves of New Zealand rabbits,and were identified by cellular immunofluorescence technique;2.The CCK-8 kit was used to detect the survival rate of myofibroblast with different concentrations of TNF-?.3.The cells of 3-7 generation were used for experiment and randomly divided into 4 groups:(1)Control group:cells were treated with a certain volume of phosphate buffer;(2)TNF-? group:cells were treated with 10 ng/ml TNF-?;(3)TNF-?+DKK-1 group: cells were treated with 10 ng/ml TNF-? and 10 ng/ml DKK-1;(4)DKK-1 group:cells were treated with 10 ng/ml DKK-1;groups were cultured for 7 days under the same conditions;ALP activity was detected by biochemical assay kit,and the protein expression of ?-catenin,RUNX2,and ?-SMA were detected by Western blotting.The mRNA expression of ?-catenin and RUNX2 were detected by Real Time PCR.Results: 1.The primary rabbbit aortic valve myofibroblast were successfully isolated and the staining of ?-smooth muscle actin(?-SMA)and Vimentin were positive,while the staining of von Willebrand factor(vWF)were negative;2.CCK-8 assay showed that TNF-? had no obvious cytotoxicity to myofibroblast;3.Compared with Control group,the protein expression of RUNX2,?-catenin and ?-SMA,the activity of ALP were significantly increased in TNF-? group(P<0.05),as well as the mRNA expression of RUNX2 and ?-catenin(P<0.05).Besides,the above indexes were significantly lower in TNF-?+DKK-1 group than TNF-?(P<0.05).Conclusion:,Wnt/?-catenin signaling pathway is involved in the progress of osteogenic differentiation induced by TNF-?,and the calcification progression of the valves can be slowed down by inhibiting the Wnt/?-catenin signaling pathway.