The Research Of HSMCs Calcification Regulated By AGEs/RAGE

Arteriosclerosis, diabetes, vascular complications is the leading cause ofheart and cerebrovascular disease.70%of patients with type2diabetics dieof cardiovascular disease (CVD), the quantity is2-4times higher thannon-diabetic population【1】. Artery atheromatous plaque formation is the mostcommon pathological changes leading to cardiovascular disease, but recentstudies in diabetic vascular complications have found that except for thetraditional risk factors-artery atheromatous plaque formation, vascularcalcification is the important factor leading to cardiovascular disease inpatients with diabetes. Recent studies have found that vascular calcificationis similar to formation of bone, it is an actively adjust process mediated bycells, at the same time there are smooth muscle cell phenotypic changes[4].Vascular smooth muscle cells (VSMCs) are the main cells of arterycalcification, whose induced in vitro calcification has become an importantmodel to the pathogenesis of cardiovascular diseases. Recent studies have found that AGEs may play an important role in theoccurrence of diabetes vascular calcification[5]. It has been proved that thereare receptors (RAGE) exist in VSMCs. The metabolism of AGEs enhancedin DM patients’ body leads to AGEs increased【6】. Long-term highconcentrations of AGEs combining with its receptor RAGE cause allergicreaction and oxidative stress, which is the reason of diabetes vascular lesions【7】. The AGEs expression of diabetics is higher than people without diabetes.The combination of AGEs and its receptor RAGE changes the signalexpression in cells, makes the function of endothelial cells disorder, multiplefactor expression changes, so as to stimulate the proliferation of smoothmuscle cell migration, speed up the further evolution of diabeticatherosclerosis【9】. Smooth muscle cells transform to osteoblast induced byAGEs, and promote the occurrence of vascular calcification.This study found that AGEs promotes HSMCs calcification. AfterAGEs acting on smooth muscle cells, their RAGE expression is obviouslyincreased, and shows concentration gradient dependency, and the expressionof OPG and β-catenin accumulate in the nucleus. The studies have foundthat RAGE, OPG and β-catenin expression level can promote the smoothmuscle cells calcification, further confirm the role of RAGE in diabeticarterial calcification. Part1The Ultrastructure of HSMCsObjective:To establish HSMCs in vitro model.Methods:By tissue adherent method, develop HSMCs successfully, make cellmorphology observation under inverted microscope and the do SM a-actinimmunofluorescence identification for cultured cells.Results:The cells grew well, and the growth of smooth muscle cells is typical"peak valley shape". By immunofluorescence detection found that highexpression of SMα-actin cells, all of which declares that smooth muscle cellshave been successfully developed.Conclusion:Successfully cultivated HSMCs by human femoral artery tissueadherent method, after immunofluorescence detection, SMα-actin cells highexpression, which declare smooth muscle cells have been successfullydeveloped. This provided a basis for follow-up studys. Part2Experimental research for AGEs regulatingsmooth muscle cells calcificationObjective:To study the mechanism of AGEs promote the smooth muscle cellscalcification.Methods:1. Test the extracellular and intracellular calcium ion concentrationof each experimental group;2. By VON KOSSA staining method explains that AGEs promotingHSMCs calification;3.Immunofluorescence test the expression of β-catenin;4.Test the expression of RAGE, β-catenin,OPG，and E-cadherinprotein by western blot;5. Expression of western blot detection of RAGE, β-catenin, OPGand E-cadherin protein after RAGEsiRNA intervention. Compare it withwhat was before.Results:1.By observation of each cell culture form, calcified plaque ofcalcification+AGES group are obviously more than calcification group;2. VON KOSSA cells staining results show that with the increase ofthe AGE-BSA concentration, its amount of calcification expression gradually increases, while cells number of calicification added AGEs（20mol/L）group was obviously more than calcification group, the numberof cytoplasm, nucleus shows pink also increased;3Intracellular Ca2+concentration determination results showedcalcium ion concentration of calcification+AGEs20group was obviouslyhigher, in the second place AGEs40group also increases, the two groupsP<0.01, there was statistically significant difference, while the intracellularcalcium ion concentration of calcification group and AGEs20alsoincreased significantly, P <0.05;4.β-catenin expression increased with the increase of AGEsconcentration, that may be due to RAGE, the receptor of AGEs activatedwnt/β-catenin signal pathway, while the expression of its downstream OPG;5.After intervene of siRNA, the expression of RAGE，β-catenin，OPGdecreased.Conclusion:1.AGEs promotes HSMCs calcification, and the concentration showsgradient;2.The expression of RAGE increased with the increase of AGEsintervention concentration, indicate that HSMCs proliferation induced byAGEs related to RAGE expression;3. OPG,β-catenin expression was enhanced with the increase of theRAGE receptor of AGEs. That is probably because AGEs receptor RAGE activated the Wnt/β-catenin signal pathway and at the same timedownstream protein OPG, E-cadherin protein expression also enhanced,promote smooth muscle cells calcification.