Notch 2 Promotes Cardiac Valvular Interstitial Cells BMP_S Secretion To Accelerate Valvular Calcification Via Modulation Of JAK-STAT Activation

BackgroundWith the aging of society, and the examination of the means of continuous enrichment,the incidence of calcified heart valve disease increased significantly. That attracts a lot of attention. In fact, there is no obvious clinical manifestation in the early stage of the calcified valvular disease, but with the occurrence and development of the disease, it will be more serious complications, and even threat to life. So far, there is no effective drug and means to interfere the calcified valvular disease, the main reason is that the pathogenesis of valve calcification is not very clear. The essence of cardiac valve calcification is the connective tissue valve occurred fibrosis and degenerative changes,the valve was changed to harden, thickening, deformation and phosphate deposition,leading to valve stenosis and(or) insufficiency, which attenuates valve function severely, even threat to life. Existing research shows that valvular calcification may be related to chronic infection and autoimmunity, but there are still a lot of controversy,It’s difficult to build the ideal valvular calcification model in vitro. To a certain extent,it also limits the study of the mechanism of valve calcification.Heart valve contains valve endothelial cells(VECs), valve interstitial cells and extracellular matrix and including collagen, elastin and glycosaminoglycans. The aortic valve is divided into three layers: fiber layer, spongiosa layer and muscular layer. The fiber layer is close to the outflow tract, which is composed of collagen, and provides the strength for the valve. The muscular layer of is close to the ventricular muscle,which is full of elasticity. The spongy layer is the central layer, which is mainly composed of the loose connective tissue. The valve endothelial cells are arranged on the surface of the valve in contact with the blood. The valve interstitial cells are the backbone of the valve structure, and participate in the whole process of valve calcification. The calcified valve tissue of surgical removal found that the valve has the formation of osteoblast like cells, which BMP-2, BMP-4 expression increased significantly. Therefore, the bone related protein accumulation is one of the main pathogenesis of valvular calcification leads to dysfunction. The main physiological function of the heart valve is the valve interstitial cells. The phenotypic change of the valve interstitial cells is also an important factor to promote the valve calcification.Inhibition of change of heart valve interstitial cell phenotype maintains valvular interstitial cells in a quiescent phenotype,which can significantly reduce calcification.Notch is a transmembrane protein receptor, human has 4 kinds of Notch receptors(Notch1- 4) and 5 kinds of Notch ligands(Delta-like 1, 3, 4, Jagged1 and Jagged2).Related studies have found that the Notch signal is necessary for the development of T lymphocyte, and participate in human immune response. Lipopolysaccharide(LPS)induced by Notch protein and ligand binding after γ-secretase active form of NICD.NICD enters the cell nucleus, acting NF- kappa B, ERK, JAK-STAT and other cellular inflammatory signaling pathway. It promotes the secretion of IL-1, IL-6, IL-8,TNF- and other inflammatory factors, involved in the inflammatory response of the heart valve. NICD binds the transcription factor CSL by cdc/ankyrin, and raises MAML family to form the NICD-CSL-MAML transcription activation complex. To activate the target genes of HES, HERP, HEY and other transcription factor families.To raise histone acetylation, histone acetylation to inhibit gene transcription. On the other hand, NICD can activate the expression of Bcl-2 protein in valve interstitial cells, and eventually activate the Caspase enzyme, which can lead to the apoptosis of valve interstitial cells.JAK-STAT is a non receptor tyrosine protein kinase, JAK is janus kinase,and STAT is signal transducers and activators of transcription. 4 kinds of JAK(JAK1-3, Tyk2) and6 kinds of STAT(Stat1-6) have been cloned successfully. The ligand induced the formation of the dimer to activate the tyrosine phosphorylation of the receptor,activated STAT then enters the nucleus, binds to specific proteins on DNA, and induces the expression of target genes that encode a variety of proteins JAK-STAT participates in inflammation and cell proliferation in vivo. Therefore, we concluded that JAK-STAT participates in the process of valve chronic inflammation and plays a role in the calcification of valvular interstitial cells.In this study, we used β-glycerol phosphate, ascorbic acid, dexamethasone, human BMP-2 and other factors, to establish the model of human heart valve interstitial cells in vitro. The interstitial cell surface Notch2 expression was higher than that of normal valve interstitial cells. Notch 2 promotes cardiac valvular interstitial cells BMPs secretion to accelerate valvular calcification via modulation of JAK-STAT activation.Therefore, this study provides a theoretical basis for the pathogenesis and treatment of calcified aortic valve disease. In this study, we establish the model of human heart valve interstitial cells in the first, then enhancing the expression of Notch2 and inhibiting its expression to investigate the regulation of downstream channel. This study is divided into three parts.Part 1 Establishment of calcification model of cardiac valve interstitial cells in vitroObjectiveHuman interstitial cells were cultured in vitro, we used β-glycerol phosphate, ascorbic acid, dexamethasone, human BMP-2 and other factors to establish the model of human heart valve interstitial cells in vitro. We investigate the correlation between BMP-2 and interstitial cells of calcified valve. The aim is to find a new target for the intervention of calcification, which provides a new direction for the development of valve calcification.MethodThe density of 1×105/ml was planted in 6 hole plate in the 4 generation of h VIC, and divided into two groups, 6 holes in each group. In the control group, 10% fetal bovine serum and 60ng/ml recombinant human BMP-2 protein(2μl) were added in the cell culture medium. In the experimental group, 10% fetal bovine serum 60ng/ml human BMP-2 protein(2μl), 100nmol/L dexamethasone(80μl), 80μg/ml L-ascorbic acid(120μl), 6mmol/L β-glycerophosphate(300μl). Under these conditions, cells are cultured for 14 days. Von Kossa staining reflects the situation of cell calcification.Western-Blot was used to detect the expression of BMP-2, BMP-4. BMP-2 content in cell culture supernatant was quantified by ELISA.ResultVon Kossa staining was performed in the experimental group for 14 days. The visible cells can form nodules with different sizes, and the cytoplasm of the cells is stained with black and calcium deposition. Von Kossa staining was performed in the control group, the cell morphology and structure were clear, and there were only a few non-specific black particles in the cytoplasm and around the cell. In the experimental group, there were calcification nodes(57±7). In the control group, there were calcification nodes(10±3). The two groups were statistically significant(P<0.03).The expression of inflammatory factors(ICAM-1, IL-8) and calcification related factors(BMP-2/4) in the experimental group were higher than those in the control group. In the experimental group, the expression level of BMP-2 in supernatant of h VIC medium was(92.49±4.92pg/ml),in the control group, the expression level of BMP-2 in supernatant of h VIC medium was(22.22±1.88pg/ml). The two groups were statistically significant(P<0.02).Part 2 Activation of Notch and inflammatory response of heart valve interstitial cellsObjectiveLipopolysaccharide(LPS) induced by Notch protein and ligand binding after γ-secretase active form of NICD. NICD can induce the inflammatory response of valve interstitial cells, so that it can make the phenotypic change and accelerate the valve calcification. By comparing the expression of Notch protein in calcified valve interstitial cells and normal valve, the relationship between valvular inflammation and valve calcification was clearly defined.MethodThe density of 1×105/ml was planted in 6 hole plate in the part1 of h VIC, and divided into two groups, 6 holes in each group. Cell culture medium was added to the control group. In the experimental group, the same amount of cell culture solution was added into the experimental group, and the LPS 0.2μg/ml(4μl) was added at the same time to stimulate the activation of Notch protein for 12 hours. Western-Blot was used to detect the expression of Notch. Immunofluorescence method to detect the expression of Notch. Von Kossa staining reflects the situation of cell calcification. The density of 1×105/ml was planted in 6 hole plate of normal h VIC, The density of1×105/ml was planted in 6 hole plate in the part1 of h VIC, and divided into two groups, 6 holes in each group. LPS 0.2μg/ml(4μl) was added at the same time into two groups to stimulate the activation of Notch protein for 12 hours. Western-Blot was used to detect the expression of BMP-2. BMP-2 content in cell culture supernatant was quantified by ELISA.ResultThe expression of Notch2、DLL4、NICD in the experimental group were higher than those in the control group. Immunofluorescence results showed that the expression of Notch2 protein in the experimental group was significantly higher than that in the control group h VIC. In the experimental group, there were calcification nodes(45±3).In the control group, there were calcification nodes(10±2). The two groups were statistically significant(P<0.04) after Von Kossa staining. In the experimental group,the expression level of BMP-2 in supernatant of h VIC medium was(69.74± 2.32pg/ml), in the control group, the expression level of BMP-2 in supernatant of h VIC medium was(31.02±1.68pg/ml). The two groups were statistically significant(P<0.03).Part 3 Notch 2 promotes cardiac valvular interstitial cells BMPs secretion to accelerate valvular calcification via modulation of JAK-STAT activationObjectiveThe interaction between Notch signaling pathway and JAK-STAT signaling pathway is involved in the process of the differentiation of mesenchymal cells into osteoblasts.Inhibition of Notch activation or inhibition of Notch signaling downstream pathway activation can delay the process of valve calcification. It provides a new target and basis for clinical treatment of valvular calcification disease.MethodThe density of 1×105/ml was planted in 6 hole plate in the part1 of h VIC, and divided into two groups, 6 holes in each group. LPS 0.2μg/ml(4μl) was added to the two group of h VIC to stimulate the activation of Notch protein for 12 hours. The Notch protein specific inhibitor DAPT 50μmol/L(4μl) was added to the experimental group. The control group injected nothing. Western-Blot was used to detect the expression of BMP-2/4 and p-JAK-STAT. BMP-2 content in cell culture supernatant was quantified by ELISA. Von Kossa staining reflects the situation of cell calcification. LPS 0.2μg/ml(4μl) was added to the 6 hole plate after stimulating for1h、2h、4h、8h、12h. Western-Blot was used to detect the expression of p-JAK-STAT.JAK-STAT phosphorylation specific inhibitor GLPG0634(10μg/ml) was added to the experimental group. The amount of cell culture medium was added in the control group. Western-Blot was used to detect the expression of BMP-2/4.ResultThe expression of BMP-2/4 in the experimental group were lower than those in the control group. The expression level of BMP-2 in supernatant of h VIC medium was (28.94±2.28pg/ml) in the control group, the expression level of BMP-2 in supernatant of h VIC medium was(79.44±2.93pg/ml). The two groups were statistically significant(P < 0.03). In the experimental group, there were calcification nodes(12±3). In the control group, there were calcification nodes(30±2). The two groups were statistically significant(P<0.05) after Von Kossa staining. After stimulating for1h、2h、4h、8h、12h, the 5 different experiments confirmed that Notch2 can make JAK-STAT phosphorylation delay. The two groups(4h and 8h) were statistically significant(P<0.05 and P<0.02).Conclusion1.The expression of ICAM-1, IL-8 and BMP-2/4 was increased in calcification of inflammatory cytokines.2.TLR4 activation promotes the activation of Notch protein in the inflammatory response of valve.3.Notch 2 promotes cardiac valvular interstitial cells BMP-2/4 secretion to accelerate valvular calcification via modulation of JAK-STAT activation.