Advanced Glycation End Products Induce The Expression Of Vascular Cell Adhesion Molecular-1 In Rat Retinal Microvascular Endothelial Cells And Signal Transduction

IntroductionDiabetic retinopathy is one of diabetic microvascular disease.Accelerated formation and accumulation of advanced glycation end products(AGEs),as well as increased flux of glucose through polyol pathway,have been implicated in the pathogenesis of diabetic retinopathy.AGEs interaction with RAGE affect signal transduction and induce oxidative stress,then make endothelial cells damage and inflammation develops.Statins also possess broad immunomodulatory and anti-inflammatory properties.Probucol is a potent low-density lipoprotein (LDL)-lowering agent with powerful antioxidant property.Our aim is to investigate the effect of AGEs on RMECs and the expression of related factors and its prevention of pravastatin,probucol and combination of pravastatin and probucol.MethodsCultured rat retinal microvascular endothelial cells(RMECs)in vitro and prepared advanced glycation end products - AGE-BSA.Cells were incubated with AGE-BSA in different concentration and the same concentration but different time,then the expression of Receptor for AGEs(RAGE)mRNA,Nicotinamide-adenine dinucleotide phosphate(NADPH)oxidase mRNA and Vascular cell adhesion molecular-1(VCAM-1) protein were analyzed by RT-PCR and Western blot method.Reactive oxygen species (ROS)were examined by flow cytometry and nuclear factor-kappa B(NF-κB) activation was detected by fluorescence microscope.We also studied AGE-BSA induced the expression RAGE in microvascular endothelial cells in the presence or absence of pravastatin,probucol,combination of pravastatin and probucol.ResultsAGE-BSA induced RAGE mRNA,NADPH oxidase mRNA,VCAM-1 protein expression and ROS production in the time/dose dependent pattern.After treatment with AGE-BSA and apocynin,intracellular ROS production decreased.NF-κB was mainly in cytoplasm without AGE-BSA.After RMECs were stimulated by AGE-BSA, NF-κB translocated into nucleus,and reached to the peak in 30 min.Both pravastatin and probucol caused decrease of AGE-induced the expression of RAGE,respectively. But minimal inhibitory effects were obtained after treatment with combined use of pravastatin and probucol.DiscussionThe diabetic retinopathy is one of the chronic complications of diabetes mellitus, the pathogenesis of which remains unclear.Among several pathogenic mechanisms that may contribute to diabetic retinopathy are the formation and accumulation of AGEs. AGEs may exert their biologic effects by receptor-independent or receptor-dependent pathways.By receptor-independent means,AGEs may directly impact on the structure integrity of the vessel wall.Receptor-dependent mechanisms are likely to work in the AGE-induced tissue dysfunction and the best-characterized AGE receptor is RAGE.Our results show that incubation of cultured RMECs with AGE-BSA significantly increased the expression of RAGE mRNA in a concentration-dependent pattern. Interaction of AGEs with RAGE produces downstream signal transduction.In general,NF-κB combination of NF-κB with IκBαresides in cytoplasm.When cells were stimulated outside,IκBαwas in phosphorylation ang degradated.NF-κB (P50 and P65)entered nucleus,which combined with corresponding gene promoter, and then began geng transcription and expression.We studied NF-κB activation by fluorescence microscope.NF-κB was mainly in cytoplasm without AGE-BSA.After RMECs were stimulated by AGE-BSA,NF-κB translocated into nucleus,and reached to the peak in 30 min.NF-κB activation is a crucial.VCAM-1 is an important adhesion molecular on the surface of vascular endothelial cells.The expression of VCAM-1 is low normal condition.In our studies, the increase expression of VCAM-1 indicate inflammation.Pravastatin and probucol caused decrease of AGE-induced the expression of RAGE,respectively.But minimal inhibitory effects were obtained after treatment with combined use of pravastatin and probucol.Combined use of pravastatin and probucol might be a promising therapeutic strategy for treatment of patients with diabetic microvascular complications.Conclusions1.AGEs can induce the expression of RAGE in a certain ranges showing a dose-dependent and time-dependent manner.2.AGEs incubated with RMECs,then NF-κB translocated into nucleus and ROS production decreased.3.AGEs can induce the expression of VCAM-1 in a certain ranges showing a dose-dependent and time-dependent manner.4.Minimal inhibitory effects of AGEs induced the expression of RAGE were obtained after treatment with combined use of pravastatin and probucol.