Study On The Influence And Mechanism Of CCAAT/Enhancer-binding Protein Homologous Protein(CHOP) In Aortic Valve Calcification

Part1 Influence of CHOP on aortic valve calcificationObjective: To define the change of CHOP in aortic valve(Ao V)calcification and explore the mechanism of CHOP on Ao V calcification.Methods: Collect valves from patients diagnosed with Ao V calcification,and collect normal Ao V from patients with Bentall surgery because of acute aortic dissection and heart transplantation because of dilated cardiomyopathy.Calcium nodule deposition of human Ao V was detected by von kossa staining,immunohistochemistry was used to detect CHOP,and western bolt and q RT-PCR were used to test the expression of CHOP and Runx2.To study the influence of CHOP on Ao V calcification,we used Apoe-/-mice(n=10)and established Apoe-/-CHOP-/-mice(n=10)model.To evaluate their cardiac function by echocardiography after they were fed western diet for 24 weeks.Calcium nodules of Ao V by von Kossa staining,and blood glucose,cholesterol,LDLs,and triglyceride levels by ELISA were determined.Meanwhile,the level of apoptosis of mouse Ao V was detected by TUNEL staining,the expressions of caspase-3 and CD68 were detected by immunofluorescence staining,and the expressions of Runx2,osteocalcin and ALP were detected by q RT-PCR.The statistical method was independent sample t test,and P < 0.05 was considered statistically significant.Results: There were lots of calcium nodules in calcific Ao V,and the expression of CHOP was much higher than it was in normal Ao V.Also,the calcification marker protein of Runx2 was increased in calcific valves.In animal model,deficiency of CHOP prevented diet-induced Ao V calcification,decreased the transvalvular peak jet velocity and Ao V leaflet thickness in Apoe-/-mice.Compared with Apoe-/-mice,Apoe-/-CHOP-/-mice exhibited significantly reduced apoptosis in Ao V leaflets and expression level of caspase-3,Runx2,osteocalcin and ALP.No significant different of left ventricular function,blood glucose,cholesterol,LDLs,triglyceride levels was observed between groups.Conclusion: CHOP was activated in calcific Ao V,and genetic ablation of CHOP attenuated Ao V calcification,pro-calcification signaling activation,and apoptosis in the leaflets of Apoe-/-mice without altering lipid and glucose metabolism.Part2 study on the influence and mechanism of CHOP in calcification of aortic valve intestinal cellsObjectives: To investigate the influence and mechanism of CHOP in calcification of aortic valve intestinal cells(AVIC).Methods: Collect normal Ao V from patients with Bentall surgery because of acute aortic dissection and heart transplantation because of dilated cardiomyopathy,and got AVIC by collagenase digestion.Treated AVIC with four different strategies,ox LDL+ scramble si RNA or sh RNA(Scr si RNA or sh RNA),ox LDL + CHOP si RNA or sh RNA,Scr si RNA or sh RNA and CHOP si RNA,and detected CHOP,Runx2 and ALP by Western blot,determined ALP activity by flowcytometry,and tested calcium deposit by Alizarin red staining.The expression of Caspase-3 was detected by Western blot,and Flowcytometry analysis of Annexin/PI double labeled VIC was used to evaluate apoptosis of AVIC.To explore whether CHOP participated in paracrine pathway in ox LDL-induced AVIC osteoblastic differentiation,conditioned media(CM)was prepared by collecting media in cells cultured by above-mentioned four strategies.Different CM were used to culture AVIC,and then the expression of CHOP,Runx2,ALP,ALP activity and calcium deposit were tested by the methods used above.In order to further understanding the mechanism of apoptosis bodies(ABs)in ox LDL-induced AVIC osteoblastic,CM from ox LDL+ Scr si RNA or sh RNA,ox LDL+ CHOP si RNA or sh RNA,ox LDL+ Scr si RNA or sh RNA but ABs free,ox LDL+ CHOP si RNA or sh RNA but ABs free were used to culture AVIC,and then detected the expression of CHOP,Runx2,ALP,ALP activity and calcium deposit by the methods described above.All datas were normally distributed.Independent sample t test was used between two groups,and one-way ANOVA or chi-square test was used among multiple groups.P < 0.05 was considered statistically significant.Results: Ox LDL induced CHOP as well as pro-calcification signaling Runx2 and ALP expression,ALP activity and calcium deposits in AVIC.However,all of those effects were significantly attenuated by silencing CHOP.As expected,ox LDL significantly triggered cleaved caspase-3 expression,while CHOP silencing markedly suppressed the effect.The Annexin V/PI double staining further confirmed that CHOP silencing suppressed ox LDL-induced AVIC apoptosis.Compared with CM from Scr si RNA treated cells,CM from ox LDL+Scr si RNA incubated AVIC significantly promoted ALP and Runx2 expression as well as ALP activity in AVIC.However,the effects were significantly blocked when AVIC was challenged with CM from ox LDL+ CHOP si RNA treated cells without altering CHOP expression.CM from ox LDL+Scr si RNA treated AVIC significantly promoted calcium deposits in AVIC compared to CM from Scr si RNA incubated cells.As expected,AVIC treated with CM from ox LDL+ CHOP sh RNA incubated cells had less calcium deposits compared to ox LDL+ Scr sh RNA incubated cells.Compared to ox LDL+ Scr si RNA treated AVIC'CM,AVIC treated with AB-free CM from ox LDL+ Scr si RNA or sh RNA incubated cells suppressed osteogenic mediators Runx2 and ALP expression,as well as ALP activity and calcium deposits without altering CHOP expression.Although AVIC incubated with CM from ox LDL+ CHOP si RNA or sh RNA incubated cells had reduced Runx2 and ALP expression,ALP activity,and calcium deposits,free of AB did not further promote the protective effects.What's more,there was no significant different of Runx2 and ALP expression,ALP activity,and calcium deposits between AVIC treated with AB-free ox LDL+ Scr si RNA or sh RNA incubated cells' CM and AB-free ox LDL+ CHOP si RNA or sh RNA incubated cells' CM.Conclusion: AVIC-derived ABs mediate ox LDL/CHOP signaling-induced osteoblastic differentiation of AVIC.