The Role Of Notch1 Protein In The Relationship Between Apoptosis And Calcification Of Human Heart Valve Interstitial Cells

Background Calcific valvular heart disease(CVHD)is the most common valvular heart disease in the elderly,and has become one of the main cause of heart failure,arrhythmia,syncope,sudden death in the elderly.Aortic valve disease is the third most common disease after hypertension and coronary artery disease in all cardiovascular disease,the aortic valve calcification stenosis is more common among aortic valve diseases,The only effective treatment is valve replacement at present,there is no effective drugs.In the past,Aortic valve calcification was considerd a degenerative disease,but with the recent researchs,it is an active progressive pathological process,but its pathogenesis is still not clear.Studies have shown that calcification of aortic valve are very similar to bone formation,Apoptosis plays an important role in the calcification process of bone formation.Human aortic valve interstitial cells(AVICs)as the major cell type in three layer structure of heart valve,played the most important role in valvular lesions,which involved in the whole process of valvular calcification.Researchs suggest that Notch1 signaling pathway can promote calcification of vascular smooth muscle cells,lipopolysaccharide(LPS)can increase calcification of AVICs by activate the expression of Notch1 signaling pathway.Studies have shown that Notch1 can induce cell apoptosis by inhibit cell cycle protein expression in tumor cells.Therefore,we can hypothesis that Notch1 protein promote valvular calcification may via induce AVICs apoptosis,but there is no related research at present.In this study,to establish valve calcification model,AVICs were cultured by calcification liquid(beta glycerol phosphoric acid,ascorbic acid,dexamethasone,recombinant type human bone morphogenetic protein 2).Analysing the correlation among the expression of Notch1 protein,apoptosis and h VICs calcification.Activating the expression of Notch1 by lps,and inhibiting the expression of Notch1 via gamma secretase inhibitor(DAPT),to reveal the role of Notch1 protein in the relationship between h VICs apoptosis and calcification.To provide a new target for the treatment of calcified valvular disease.Aim1.To establish valve calcification model,AVICs were cultured by calcification liquid(beta glycerol phosphoric acid,ascorbic acid,dexamethasone,recombinant type human bone morphogenetic protein 2).To provide a model for study the mechanism of calcification in vitro.Observing the expression of Notch1 protein,apoptosis in the process of h VICs calcification,To investigatie the correlation among AVICs calcification and the expression of Notch1 protein and apoptosis.2.Based on the cell calcification model in vitro,activating the expression of Notch1 by LPS,and inhibiting the expression of Notch1 via DAPT,then apoptosis and calcification of AVICs were detected,to investigate the role of Notch1 protein in the relationship between h VICs apoptosis and calcification.Methods1.AVICs calcification in vitro and the expression of Notch1 protein/apoptosis in the process of calcificationAVICs were planted in 12 well plates at the density of 1×10/ml,and randomly divided into two groups,calcification group and blank control group.Calcification group cells were divided into 4 subgroups: 1day,3days,5days,7days according to different induction time.There were 3 wells in each group.Calcification group was treated with 200?l calcification induced liquid and 800?l I-GRO PLUS medium(containing 10% fetal bovine serum);The blank control group was cultured by 1ml I-GRO PLUS medium(containing 10% fetal bovine serum);All cells were cultured for7 days,cells were collected after 7 days.The expression of cell calcification was detected by Von Kossa staining,Western Blot and ELISA.Western Blot and PT-PCR were used to detect the expression of Notch1 protein and gene at different time points in the process of cell calcification.Cell apoptosis was detected by fluorescent staining and flow cytometry at different time points in the process of cell calcification.2.The role of Notch1 protein in the relationship between apoptosis and calcification of h VICsCalcified cells in the first part of the experiment were randomly divided into 3groups: The control group(the calcification liquid induced cells in the first part);LPS group: adding 0.2mg/L LPS at 4?l/well on the basis of the control cells,to upregulate the expression of Notch1 protein;DAPT group: adding 50?mol/L DAPT at 4?l/well on the basis of the control cells,to Inhibition the expression of Notch1 protein;Cells in 3groups were cultured for 3 days.The expression of Notch1,BMP-4 and Casepase3 protein was detected by Western Blot in order to reveal the changes of AVICs calcification and apoptosis after activation / inhibition of Notch1 signaling pathway.Results1.AVICs calcification in vitro and the expression of Notch1 protein/apoptosis in the process of calcificationThe blank control group cells structure were clear after 7 days under optical microscope,radially arranged,no particle deposition;Calcification group cells staggered,reticular arranged,black granular sediments around cells.The number of calcified nodules in calcification group(57.3±7.2/well)were higher than blank control(10.2±3.3/well)afer Von Kossa staining(P<0.05),and the number of calcified nodules increased along with the induced days.Western Blot and ELISA show that the expression of BMP4,a molecular markers of calcification,in calcification group were higher than blank control group and increased along with the induced days.In the process of cell calcification,RT-PCR assay showed that the expression of Notch1 gene in calcification group was higher than that in control group(P<0.05).The results of Notch1 protein expression detected by Western Blot were consistent with the RT-PCR,the expression of Notch1 gene(after calcification induction for 1 days)was earlier than Notch1 protein and apoptosis(after calcification induction for 3 days).The result of fluorescent staining and flow cytometry showed that the apoptosis rate was the highest at the 3 day group(20.78%),which was significantly higher than the control group(P<0.05),and then there was a gradual downward trend.All data difference were statistically significant.2.The role of Notch1 protein in the relationship between apoptosis and calcification of h VICsWestern Blot confirmed the expression of Notch1 protein in DAPT group was significantly decreased compared with control group(P<0.05).Notch1 protein expression in LPS group was significantly increased than control group(P < 0.05)and DAPT group(P < 0.001),the difference was statistically significant;After Notch1 signaling pathway was activated by LPS,the expression of Caspase3 and BMP4 protein was significantly higher than control group(P< 0.05)and DAPT group(P< 0.001);The expression of Notch1 was inhibited by DAPT,the expression of Caspase3 and BMP4 protein was significantly decreased compared with control group(P< 0.05),the difference was statistically significant.Conclusion1.AVICs calcification model were established successfully in vitro,Notch1 and apoptosis were upregulated in the dynamic process of AVICs calcification,and the expression of Notch1 gene significantly earlier than apoptosis and cell calcification,AVICs calcification was confirmed to be associated with Notch1 and apoptosis.2.AVICs apoptosis and calcification were significantly increased after Notch1 was activated;AVICs apoptosis and calcification were reduced when Notch1 was inhibited? The result showed that Notch1 has a regulatory effect on AVICs apoptosis and plays an important role in the relationship between apoptosis and calcification of AVICs.