Annexin A2is Phosphorylated By Advanced Glycation End Products And Modulates Endothelial Cytoskeleton Rearrangements And Migration

ObjectiveIn the cellular model of advanced glycation end products（AGEs）， we study the roleof Annexin A2（ANXA2）in the actin cytoskeleton remodelling and cell migration.MethodsPart1The siRNA technology is applied in the culture of the human umbilical veinendothelial cells（HUVECs）to make sure the down-regulation of the annexin A2（ANXA2）expression, and then the knock-down effect is testified with Western Blot.In the part, HUVECs are cultured respectively according to the random subgroupingbelow：untreated group(cultured in DMEM including25mmol L^-1glucose)，controlsiRNA group (cultured in DMEM including25mmol L^-1glucose after the transfectionof control siRNA) and ANXA2siRNA group(cultured in DMEM including25mmol L^-1glucose after the transfection of ANXA2siRNA).Part2FITC-Phalloidine is applied to indicate the rearrangements of the cell cytoskeleton under the fluorescence microscope and the cell motility is manifested by the restitutionassay. In the part, HUVECs are cultured respectively according to the randomsubgrouping below：untreated group(cultured in DMEM including25mmol L^-1glucose)， BSA group(cultured in DMEM including25mmol L^-1glucose with theaddition of0.2g L^-1BSA)， AGE-BSA group(cultured in DMEM including25mmol L^-1glucose with the addition of0.2g L^-1AGE-BSA)， control siRNA group(cultured in DMEM including25mmol L^-1glucose with the addition of0.2g L^-1AGE-BSA after the transfection of control siRNA) and ANXA2siRNA group(culturedin DMEM including25mmol L^-1glucose with the addition of0.2g L^-1AGE-BSA afterthe transfection of ANXA2siRNA).Part3The phosphorylation of ANXA2is testified by the Immunoprecipitation andWestern Blot. In the part, HUVECs are cultured respectively according to the randomsubgrouping below：untreated group(cultured in DMEM including25mmol L^-1glucose)， BSA group(cultured in DMEM including25mmol L^-1glucose with theaddition of0.2g L^-1BSA)and AGE-BSA group(cultured in DMEM including25mmol L^-1glucose with the addition of0.2g L^-1AGE-BSA).The data all above is to beanalysed by the one-way ANOVA（LSD）.ResultsPart1The blot density of ANXA2of the untreated group, control siRNA group, andANXA2siRNA group respectively is1.170±0.027、1.182±0.037and0.676±0.053.There is no statistical difference between the untreated group and the control siRNAgroup, but compared with the first two groups, the blot density of ANXA2of theANXA2siRNA group decreases statistically.Part224hours after random subgrouping, there is no difference between the untreated group and BSA group in the cell cytoskeleton arrangements, with the same even andthin stress fibers; compared with the first two groups, the distribution of the stress fibersof the AGE-BSA group is dense and likely concentrated to some direction. There issimilar cell cytoskeleton arrangements between the control siRNA group and theAGE-BSA group. However, the ANXA2siRNA group represents the same even andthin cell cytoskeleton arrangements with the untreated group.24hours after random subgrouping, the number of the cells migrating into thescratch of the untreated group, BSA group, AGE-BSA group, control siRNA group, andANXA2siRNA group is respectively153±12、143±16、192±8、176±6and142±14.Compared with the untreated group, BSA group or the ANXA2siRNA grouprespectively, the number of the cells migrating into the scratch of either AGE-BSAgroup or control siRNA group increases statistically. The differences among theuntreated group, BSA group and ANXA2siRNA group, also between the AGE-BSAgroup and control siRNA group, are not statistically significant.Part324hours after random subgrouping, the blot density of ANXA2of the untreatedgroup, BSA group and AGE-BSA group is respectively1.011±0.014、0.939±0.089and1.064±0.119, and there is no statistical difference among them. The blot density of thetyrosine-phosphrylated ANXA2of the untreated group, BSA group and AGE-BSAgroup is respectively0.942±0.057、0.945±0.032and1.118±0.095. There is no statisticaldifference between the untreated group and BSA group, but compared with the first twogroups, the blot density of the tyrosine-phosphrylated ANXA2of the AGE-BSA groupstatistically increases.Conclusion1、AGE-BSA could enhance HUVEC cytoskeleton remodeling，and then cell motility.2、ANXA2is involved in the cell cytoskeleton remodeling and migration induced bythe AGE-BSA. 3、ANXA2probably contributes to the cell activity above by itstyrosine-phosphorylation.