N?-carboxymethyllysine Regulating Smooth Muscle Cells Matrix Vesicles Promote Diabetic Plaque Calcification Via Sortilin

ObjectiveTo explore the specific mechanism by which CML regulates the release of matrix vesicles to promote diabetic plaque calcification.Methods?1?In the first part,71 patients who were admitted to the Department of Cardiology,Affiliated Hospital of Jiangsu University from 2017 to 2020 were selected.According to the inclusion and exclusion criteria,patients were divided into diabetes group?group A?,diabetic atherosclerosis?group B?,and diabetic plaque calcification group?group C?.Serological samples of all patients were collected,MVs were separated by differential centrifugation,ELISA detection and statistical analysis were performed.?2?In the second part,6-week-old male ApoE^-/-mice were acclimated to the environment for 1 week,and 40mg/kg/day streptozotocin was intraperitoneally injected for 5 days.After 2 weeks,ApoE^-/-mice with blood glucose levels exceeding 300 mg/dL were enrolled.Diabetic ApoE^-/-mice were randomly divided into three groups:NC group?common diet,n=8?,HFD group?high-fat diet,n=8?,CML group?high-fat diet+tail vein injection of CML,n=8?.After 32 weeks of continuous feeding,the mice were euthanized.Aortic tissues were selected for morphological staining?HE staining,oil red O staining,immunofluorescence staining,Von Kossa staining?and molecular biological detection.?3?In the third part,mouse aortic smooth muscle cells?ASMCs?were divided into three groups:NC group,ox-LDL group,and CML group.All cells were cultured under calcification conditions for 4 days,and MVs were separated for NTA detection.A diabetic ApoE^-/-mouse model was constructed,and MVs secreted by ASMCs in CML group were injected into the diabetic ApoE^-/-mice through the tail vein.All mice were randomly divided into 5 groups:NC group,CML group,low concentration MVs group,medium concentration MVs group and high concentration MVs group.After 16 weeks of continuous feeding,Von Kossa staining and calcium content detection were performed.The same treatment was performed at the cell level for alizarin red staining.?4?In the fourth part,MOVAS were divided into seven groups:NC MVs group,ox-LDL MVs group,ox-LDL MVs+IgG group,ox-LDL MVs+Anti-Sortilin group,CML MVs group,CML MVs+IgG group,CML MVs+Anti-Sortilin group.After 4 days of calcification induction,Western blot and Von Kossa staining were performed.Results?1?ELISA results showed that compared with group A and group B,the concentrations of CML in the serum,CD9 and Sortilin in MVs were significantly increased in group C.Pearson correlation analysis showed a positive correlation between CD9 and CML concentrations,and Sortilin is positively correlated with the concentration of CD9.?2?Gross perfusion,oil red O staining,and Von Kossa staining showed that CML can obviously promote the formation of atherosclerosis and plaque calcification in diabetic ApoE^-/-mice compared with NC group and HFD group.?3?Immunofluorescence staining and Western blot showed that compared with NC group and HFD group,the expression of Sortilin and CD9 in CML group was increased.?4?NTA showed that compared with NC group and ox-LDL group,the number of MVs secreted by ASMCs in CML group increased significantly.The degree of calcification in ASMCs and mouse plaques aggravated with increasing MVs concentration from CML group.?5?ELISA showed that CML could increase the concentration of Sortilin in MVs.?6?Western blot and Von Kossa staining results showed that compared with ox-LDL MVs group,CML MVs group promoted cell calcification more obviously,while Anti-Sortilin could reduce the expression of calcification-related proteins and weaken the degree of cell calcification.Conclusions?1?MVs secreted by CML-induced SMCs accelerating the progression of atherosclerosis and plaque calcification.?2?The promotion effect of MVs on calcification under CML regulation may be achieved by Sortilin.