Matrix Gla Protein Expression Alters In Aortic Valve Interstitial Cells In The Process Of Calcification

BackgroundCalcific aortic valve disease(CAVD)has been more and more frequently diagnosed,in today's aging society.While,the pathogenesis of this common disease is still not well understood.Recently,evidence is accumulating that CAVD is an active disease process,one in which mechanisms of osteogenesis play an important role.Besides,many cellular processes have been implicated as well.Matrix gla protein(MGP),a potent inhibitor of vascular calcification,is also expressed in heart valves.Studies show that,endogenous MGP expression,as well as its carboxylation,is damaged in the process of vascular calcification.In return,it will absolutely weaken its ability to inhibit calcification.However,the changes of MGP in the process of CAVD are still rarely studied.ObjectiveTo detect the changes of MGP in aortic valve interstitial cells(AVICs)in the process of calcification.Methods1.Isolation and culture of the aortic valve interstitial cells: male healthy adult pigs were killed.Hearts were removed from the chest cavity and carefully rinsed in ice-cold saline solution,then went back to the lab.Aortic valve leaflets were sterilely removed from the hearts.The leaflets were slightly scraped to remove the remaining endothelial cells and cut into pieces,then cultured in Dulbecco's modified Eagle medium(DMEM)containing 20% fetal bovine serum(FBS).The culture medium was changed every two or three days.Cells were grown to passage 2-5 before further researches conducted.Then,the cells were divided into two groups: the control group(cells were cultured in DMEM with 10% FBS)and calcification group(cells were cultured in DMEM with 10% FBS and osteogenic additives,including 50 ?g/ml ascorbic acid,2 m M?-glycerophosphate and 100 n M dexamethasone).Cells from the two groups in different time segments(1.7.14 d)were collected for further experimentation.2.Immunofluorescence was used to identity the phenotype of the isolated cells: the isolated cells were firstly made into suspensions at a density of 1×105 /m L,and plated onto 6-chamber slides.After adhering to the chamber slides,the expression of smooth muscle ?-actin,von Willebrand factor and vimentin was detected by immunofluorescence.3.After one day,seven days and fourteen days,Alizarin Red S staining was used to stain the calcium deposit of both groups,and calcified nodules were directly counted under microscope.4.The expression of MGP was detected by semi-quantitative rt-PCR on mRNA level and by Western Blot on activated protein level after the two groups being treated one day,seven days and fourteen days.Results1.Cells were successfully isolated and cultured using tissue explant method.The cultured cells were spindle-shaped and overlapped layers of parallel.The cells were positive stained for ?-SMA and vimentin,while negative stained for v WF.This indicates that the isolated cells are fibroblast phenotype.2.After 7 days and 14 days,red stained calcified deposit was formed in both groups,while,quantification of calcified nodules and calcification deposit were obviously higher in calcification group than that in control group [(37.55±6.70)/hole vs(2.38±0.97)/hole,(78.29±9.14)/hole vs(5.47±1.59)/hole,(P<0.05)].3.Semi-quantitative rt-PCR showed that,the expression of MGP mRNA in calcification group was significantly down regulated,compared with that of the control group,and the difference was statistically significant(P<0.05).4.Western Blot results showed that,compared with the control group,the amount of activated MGP in calcification group was significantly decreased,and the difference was statistically significant(P<0.05).ConclusionsThe anti-calcification effects of MGP is deficient in the process of calcification.