| Background and AimCalcific aortic valve disease?CAVD?which is characterized by pathological calcification is the most common valvular heart disease in elderly patients.Valvular interstitial cells?VICs?are the principal cell type in aortic valve.Previous studies have confirmed that the pathogenesis of calcification can be explained by passive calcium deposition induced by apoptosis of VICs and active phenotype transformation of VICs into osteoblast-like cell.The signals of endoplasmic reticulum stress?ERS?have been reported to be activated in the animal model of CAVD such as ApoE-/-mice,but the role of these proteins related to ERS in valvular calcification is not clear.There are few reports about the effects of ERS on apoptosis,phenotype transformation and proliferation of VICs.Based on the above research background,this study explored the expression change of ERS related proteins in calcified valves firstly and analyzed its effects on apoptosis and phenotypic transformation of VICs.MethodThis study is divided into the following three parts:1.Expression level of ERS related proteins in an osteogenic differentiation model in vitro:Porcine aortic VICs were isolated by type II collagenase and cultured with DMEM supplied with 10%FBS and osteogenic induced medium containing 10-7 mmol/L insulin,50?g/ml ascorbic acid,2 mmol/L sodium dihydrogen phosphate.Calcium deposition in such cell model was measured by alizarin red staining.At the same time,the expression of ERS related proteins was detected by Western blot.2.Expression level of activating transcription factor 6?ATF6?in tissue specimens of patients diagonosed with CAVD:Collecting calcified aortic valve tissue from patients diagonosed with CAVD who was treated with aortic valve replacement.The normal aortic valve tissue was harvested from heart recipient as control.The expression level of ATF6 was detected by immunohistochemistry,apoptosis was detected by TUNEL staining and calcium deposition in CAVD tissues was measured by alizarin red staining.3.The effects of ATF6 on cell growth and phenotypic transformation:The expression of ATF6 in VICs was inhibited by RNAi.Real-time quantitative PCR and Western blot was used to detect expression levels of ATF6 mRNA and protein in VICs after transfection with Ad-shRNA-ATF6.Calcium deposition was measured by alizarin red staining after downregulation of ATF6.Meanwhile,the proliferation and apoptosis of VICs were detected by CCK-8 and flow cytometry,respectively;At last,the proteins related to apoptotic signaling pathway and the markers of osteoblast phenotype transformation were detected by Western blot.Result1.Expression level of ERS related proteins in an osteogenic differentiation model in vitro:A large amount of calcium deposition was observed in osteogenic differentiation model after 7 days,which is confirmed by alizarin red staining.Western blot analysis showed that the expression level of ATF6 increased from the first day and reached its peak on the third day during the osteogenic induction process compared to untreated group with statistical significance?P<0.05?.The expression level of ATF6 decreased gradually after the third day and reached an identical level on the 7th day compared to the untreated group?P>0.05?.Similarly,the expression profile of activating transcription factor 4?ATF4?,x-box binding protein 1?Xbp1?also increased from the first day,reached its peak on the third day during the compared to untreated group without statistical significance?P>0.05?.The expression level of eukaryotic initiation factor 2??eif-2??,growth arrest and DNA damage-inducible gene 34?GADD34?increased from the first day and then decreased,the expression level of phosphorylated eukaryotic translation initiation factor 2??p-eif-2??increased from the second day,but the expression of cells in each group has no statistically significance compared with untreated cells?P>0.05?.2.Expression level of ATF6 in valve samples of CAVD:The results of immunohistochemistry showed that the positive staining rate of ATF6 in CAVD group and control group was 44.30±10.70%and 9.03±7.14%with statistical significance,respectively?P<0.05?.The result of TUNEL staining showed that the apoptotic rate in CAVD was 84.76±9.30%.The expression level of ATF6 was identified to be positively correlated with apoptosis level by correlation analysis?r=0.62,P<0.05?.The degree of calcification of valve samples of CAVD was mostly moderate to severe,the expression level of ATF6 was identified by correlation analysis which has no statistically significance?P>0.05?.3.The effects of ATF6 on cell growth and phenotypic transformation:The mRNA expressionlevelofATF6wasdown-regulatedaftertransfectionwith Ad-shRNA-ATF6?P<0.05?.Then,the amount of calcium deposition was significantly reduced after downregulation of ATF6 in VICs confirmed by alizarin red staining?P<0.05?.The apoptotic rates of VICs were 2.34±0.75%in sh-ATF6 group and 8.56±1.08%in control group with statistical significance?P<0.05?which is confirmed by flow cytometry.In contrast,the proliferation rate was significantly upregulated in sh-ATF6 group than that of control group after transfection with Ad-shRNA-ATF6?P<0.05?.Result of Western blot indicated that the expression level of Caspase-3/8 and Bax which can promote apoptosis were significantly reduced after transfection with Ad-shRNA-ATF6,accompanied with the upregulation of the expression of Parp which can inhibit apoptosis.On the other hand,the expression of p-Fak which can inhibit apoptosis was downregulated.In addition,targeted inhibition of expression of ATF6 had no significant effect on the expression of osteoblast phenotype markers,including Runx2?Runt related transcription factor 2??OPN?Osteopontin??OSX?Osterix?.Conclusion1.ATF6,which is a representative of ERS related protein,was highly expressed in both osteogenic differentiation model in vitro and human valve tissue of patients diagnosed with CAVD.The expression level of ATF6 was positively correlated with apoptosis rate of VICs.2.The calcium deposition and apoptotic rate were significantly reduced after downregulation of the expression of ATF6.And inhibit the expression of proapoptosis related protein Caspase-3/8 and Bax,promotes the expression of Parp which can inhibit apoptosis.3.Targeted inhibition of ATF6 expression had no significant effect on the expression of osteogenic phenotype markers.These results suggest that ATF6 may promote the progression of CAVD by inducing apoptosis of VICs. |
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