RAGE/galectin-3 Yields In Traplaque Calcification Transformation Via Sortilin

Objective To investigate the role of sortilin in the regulation of intraplaque calcification outcomes by RAGE/galectin-3.Methods(1)Clinical study: From March 2016 to December 2017,a total of 30 patients underwent amputation surgery at the Department of Orthopaedics,Jiangsu University Hospital for "diabetic foot" were enrolled.According to the calcium content of the anterior tibial artery,they were divided into the less calcification group(<5 ?mol/mg)and the more calcification group.Clinical data were analyzed for both groups,including age,gender,BMI,duration of diabetes,past medical history,fasting blood glucose,cholesterol,triglycerides,and serum CML levels.Von Kossa staining showed the morphology of calcification in the anterior tibial artery and analyzed the diameter of the calcium nodules.Immunohistochemical staining revealed the expression of RAGE and galectin-3 in the anterior tibial artery.(2)In vitro experiments: Primary mouse aortic smooth muscle cells were cultured,and an in vitro model of diabetic vascular calcification was constructed with osteogenic medium,CML and oxLDL.After the virus transfection,calcification morphology was observed with Von Kossa staining,and the mean diameter of calcium nodules was analyzed to determine the type of calcification.The effect of RAGE/galectin-3 silencing on sortilin expression was confirmed by western blot.Based on the silence of RAGE/galectin-3,siRNA or recombinant protein was used to block or increase the expression of sortilin.Von Kossa staining and calcium quantitative analysis were carried out to further explore the effect of sortilin on calcium nodule size and calcium content in arterial smooth muscle cells.(3)In vivo experiments: A diabetic atherosclerosis model was constructed using 4 week old apoE-/-mice with C57 BL / 6J background.Mice were injected with streptozotocin(STZ;40 mg / kg / day)for 5 days.And 2 weeks later mice with blood glucose of over 300 mg/dL were included in the experiment.Firstly,the effects of RAGE/galectin-3 on calcification outcome and sortilin expression in plaque were investigated.Groups were as follows: NC(general food,n=5),HFD group(high-fat diet,n=5),CML group(high fat diet + CML,n=5),AAV-shscramble group(high fat diet + CML and AAV-shscramble),AAV-shRAGE group(high fat diet + CML and AAV-shRAGE),AAV-shgalectin-3(high-fat diet + CML and AAV- shgalectin-3)and double intervention group(high-fat diet + CML,AAV-shRAGE and AAV-shgalectin-3).The calcification morphology was observed by Von Kossa staining,and the calcification type was determined.The location and expression of sortilin were revealed by immunofluorescence.Furthermore,Sortilin recombinant protein or sortlin neutralizing antibody was used to intervene RAGE/galectin-3 blocked mice.Von Kossa staining and calcium nodule size analysis were used to determine the effect of sortilin on calcification in vascular plaque of diabetic mice.Results(1)There were no significant differences in age,BMI,fasting blood glucose,total cholesterol,and triglycerides between the more calcification group and the lesscalcification group except for diabetes duration and serum CML level.(2)There were macrocalcification and microcalcification in the anterior tibial artery of the less calcification group and the more calcification group,and there was no significant difference in serum CML levels between the macrocalcification and microcalcification of each group.(3)The expression of galectin-3 in the anterior tibial artery with large calcification was significantly higher than that in RAGE,while the expression of RAGE in the microcalcified anterior tibial artery was significantly higher than galectin-3 in macrocalcified vascular.(4)Primary mouse aortic smooth muscle cells were cultured for 7 days under osteogenic medium,oxLDL and 10 ?mol/L CML.And the calcification was at the highest degree.Meanwhile,the expression of RUNX2 was at the highest level.This condition was used to construct an in vitro model of diabetic vascular calcification.(5)After silencing RAGE,the expression of galectin-3 in mouse aortic smooth muscle cells increased significantly.Meanwhile,the level of sortilin increased and the size of calcification nodules enlarged to form macrocalcification.After the silence of galectin-3,the expression of cellular RAGE was up-regulated.But sortilin expression was reduced,and calcification nodules was significantly smaller,which exhibits microcalcification.(6)After the treatment with sortilin recombinant protein,the calcium content and mean diameter of calcium nodules increased significantly.And after siRNA intervention, the calcium content and long diameter of calcium nodules were significantly reduced.(7)Mice fed with high-fat diet and CML tail vein injection reached the maximum body weight at around the 9th week,then steadily decreased.The blood glucose level of mice fed with high-fat diet and combined with CML tail vein injection increased gradually.(8)After silencing RAGE,the galectin-3 content in the mice vascular increased.And the expression of sortilin also increased significantly.The calcium nodules in the plaques tend to form large calcification.After silencing galectin-3,the RAGE expression of mice vascular was measured to show increased greatly.However,the expression of sortilin was downregulated.The number and size of calcium nodules in the plaque were both increased significantly,which formed microcalcification.(9)CML interacts with RAGE or galectin-3,while RAGE does not interact with galectin-3.(10)After the intervention of sortilin recombinant protein,the mean diameter and calcium content of calcium nodules in the vascular plaques of mice increased significantly.After the intervention of sortilin neutralizing antibody,the size of calcium nodules become smaller.Though the calcium content also decreased after the intervention of antibody,it has no statistically significant.Conclusion Sortilin,acts as a molecular switch for calcification transformation,mediates the formation of microcalcification/macrocalcification downstream of RAGE/galectin-3.RAGE inhibits the expression of sortilin to form intraplaque microcalcification,and galectin-3 promotes the expression of sortilin to regulate the formation of large calcification.