Study On The Role Of Autophagy In CML Regulating Calcification Outcome Mediated By RAGE/Gal3

Objective: To investigate the role of autophagy in CML regulating calcification outcome mediated by RAGE/ Galectin-3.Methods:(1)In clinical study,we selected diabetics who were suspected diagnosis or clinical diagnosis as coronary artery disease in the Department of Cardiology of the Affiliated Hospital of Jiangsu University from July 2016 to December 2016.According to CCTA,the patients were categorized based on calcification score into control group,mild calcification group,moderate calcification group and severe calcification group.Data regarding gender,age,fasting blood glucose,total cholesterol levels,triglyceride levels,low-density lipoprotein cholesterol levels,high-density lipoprotein cholesterol levels,serum CML levels,and calcification scores were then collected for statistical analysis.(2)In in vitro study,firstly,we observed the effects of CML on A7r5 cells calcification,autophagy protein expression and calcification morphology;secondly,we observed the effects of RAGE and Galectin-3(Gal3)silencing on A7r5 cells calcification,autophagy protein expression and calcification morphology;finally,we observed the effects of RAGE and Gal3 silencing with or without autophagy inhibitor bafilomycin A1(Bfa1)on A7r5 cells calcification,autophagy protein expression,calcification morphology,and autophagic flow.Alizarin red staining,immunofluorescence staining,western blot assay were proformed.(3)In in vivo study,6-week-old male Apo E-/-mice were rendered diabetic by five-consecutive-day intraperitoneal injection of STZ(40 mg/kg/day)and mice who blood glucose levels were more than 16.7 mmol/L after injection of STZ for two weeks were considered diabetic.Firstly,we observed the effects of CML on the calcification and autophagy of atherosclerotic plaques.The mice were randomly devided into three groups: control group(normal feed,n=5),model group(high lipid feed,n=5),and CML group(high lipid feed + tail vein injection of CML,n=5);secondly,on the basis of CML group,left ventricular injection of RAGE or Gal3 silencing expression vectors with or without intragastric injection of autophagy inducer rapamycin were investigated to observe RAGE,Gal3,autophagy inducer on the calcification and autophagy of diabetic atherosclerotic plaque.The mice were randomly devided into six groups: control group(high lipid feed + CML,n = 5),autophagic agonism group(high lipid feed + CML + rapamycin,n=5),RAGE blocking group(high lipid feed + CML + RAGE silencing expression vector,n=5),RAGE blocking + autophagic agonism group(high lipid feed + CML + RAGE silencing expression vector + rapamycin,n = 5),Gal3 blocking group(high lipid feed + CML + Gal3 silencing vector,n = 5),Gal3 blocking + autophagic agonism group(high lipid feed + CML + Gal3 silencing expression vector + rapamycin,n=5).Mice were euthanized after six months feeding and serological analysis(serum glucose,serum lipid,serum CML level),morphological analysis(HE staining,Von kossa staining,immunohistochemical staining,TRAP staining)and molecular biology analysis were then performed.Results:(1)Compared with the control group,the serum levels of FBG,TC,TG,LDL-C,and CML were significantly increased in calcification groups,while the serum levels of HDL-C were significantly decreased(P<0.05).With calcification score increasing,the serum levels of FBG,TC,TG,LDL-C,and CML in the mild,moderate and severe calcification groups were gradually increased while the serum levels of HDL-C were gradually decreased(P<0.05).(2)There were few atherosclerotic plaques and calcified plaques in coronary arteries in the control group,while there were varing degrees of plaque calcification in the coronary arteries.With calcification score increasing,the number of calcifying nodules were increasing and coronary artery calcification is not limited to one lesion and often occur in two of three lesions.(3)Serum CML levels were positively correlated with calcification score of coronary atherosclerotic plaques(r=0.9917,P< 0.0001).(4)CML promotes A7r5 cells calcification and upregulates the expression of Runx2 and LC3 II in calcified cells.(5)RAGE silencing induced a typical macrocalcification and a reduced number of calcifying nodules while Gal3 silencing showed a typical microcalcification and an increased number of calcifying nodules.(6)CML enhanced the expression of autophagic markers,but blocked autophagy flux in a7r5 cells,RAGE silencing and/or Gal3 silencing mitigated this phenomenon to a certain extent.The expression of TRAP,TNAP protein and the content of calcification were increased after interfered with bafilomycin A1,but there were no significant difference in the expression of RAGE,Gal3 protein protein compared with groups without interfered with bafilomycin A1.(7)Atherosclerotic plaque formation in the mouse aorta and sharp diamond cholesterol crystals in the lipid pool of the fibrous cap were observed in all groups.Aortic atherosclerotic plaques calcification and the number of cholesterol crystals increased in the CML group.A large number of osteogenic-cells were observed in the plaques.(8)RAGE silencing decreased calcification and showed typical macrocalcifition while Gal3 silencing increaced calcification and showed typical microcalcification.The calcification and number of cholesterol crystals in autophagic agonism groups were decreased than which in groups without interfered with rapamycin.(9)Immunohistochemical staining and TRAP staining suggested that the expression of Gal3 and TNAP increased in groups with RAGE silencing,while the expression of RAGE and the activity of TRAP decreased;the expression of RAGE and the activity of TRAP increased in groups with Gal3 silencing,while the expression of Gal3 and TNAP decreased;the expression of TNAP and the activity of TRAP in the autophagic agonism groups were decreased than which in groups without interfered with rapamycin,while there were no significant difference in the expression of RAGE,Gal3 protein protein compared with groups without interfered with rapamycin.Conclusions:(1)There is a certain correlation between serum CML levels and calcification in coronary atherosclerotic plaques.(2)CML mediates calcification outcome of A7r5 SMCs through RAGE/ Galectin-3.CML promotes calcification of VSMCs by inhibiting fusion of the autophagosome and lysosome.(3)CML mediates calcification outcome within plaque through RAGE/ Galectin-3.Promoting autophagy may reduce plaque calcification.Autophagy may not be a key link in regulating calcification outcome through RAGE/ Galectin-3.