Effect Of AGEs-Cyr61 Signal Pathway On Laser-induced Choroidal Neovascularization Of Diabetic Mice And Its Mechanism Underlying

[Background]Choroidalneovascularization(CNV)isnowknowntooccurasafinal common pathway in nearly 40 ophthalmic diseases,suh as age-relatedmaculardegeneration(AMD),ocular histoplasmosis,myopic macular degeneration,idiopathic CNV and ocular trauma.AMD is now known the leading cause of vision loss inolderadults of developed countries.Because CNVformation isanusual pathologic characteristic of multiple blinding diseases,exploring occurrence mechanism and the clinical control strategy of CNV has become one of hotspots and difficulties in the research field of ophthalmology in recent years.At present,it has been confirmed that CNV risk factors include age,smoking,genetic and cardiovascular diseases[1].And as a causative factor for cardiovascular disease,correlation research between diabetes and CNV progress has attracted more and more attention [2,3].Thoughthere is a lot of disease-associated study for clinical epidemiological investigation at present,the molecular mechanism that diabetes affect CNV occurrence remains unclearbecause of a lack of basic experimental evidence supporting.Our previous study found that hyperglycemia play an important role on the development and progression of CNV.The main evidence is as follows:(1)Diabetes could increase CNV severity in diabetic mice induced by STZ.(2)Antioxidant could reduce CNV formation through lesseningoxidative damage degree of diabetic mice and downregulating cytokine expression and signal pathway related to angiogenesis.(3)Hyperglycemia increases the level of oxidative stress in RPE cellsand increases the expression of VEGF.(4)Hyperglycemia promoted bone marrow-derived mesenchymal stem cell gathered in the area of CNV and took part in angiogenesis through upregulating VEGFproduction secreted by RPE cell,and then increased CNV sevity.As a kind of very important cell matrix adjustment factors,Cyr61 play an important role in regulating cell adhesion,migration,proliferation,angiogenesis,inflammation and tissue reconstruction.By using Agilent Mouse gene expression profile,Cyr61 was identifed as one ofdifferential genes in choroid tissue of diabetic mice.Whether Cyr61 and signal pathway it mediated involved in CNV formation? What is specific molecular mechanism underlying? Answering the above questions will offer us new strategy for prevention of CNV.[Objective]We aims at exploringthe effect of AGEs-Cyr61 signaling pathway on CNV formation in diabetic mice,revealingthe effect of AGEs-Cyr61 signaling pathway on CEC cell function regulated by RPE cell and illuminatingthe effect of AGEs-Cyr61 signaling pathway on VEGF expression.And with above research we can lay the foundation for finding the new targets for CNV treatment.[Methods]1.C57BL/6J mice were randomly divided into three groups: normal control group,diabetes group and diabetes mellitus plus aminoguanidine treatment group,20 rats in each group.Mice received intraperitoneally injection of STZ for 5 days.After 2 weeks,the CNV was induced by 532 nm frequency laser.The CNV severityofeachgroupweretested by Choroidalflatmoutand HEstaining onday14 afterphotocoagulation.Immunofluorescence staining and ELISA were used to examinethe effect of aminoguanidine on Cyr61 and VEGF expression.2.C57BL/6J mice were randomly divided into three groups: normal control group,diabetes group and diabetes mellitus plus aminoguanidine treatment group,20 rats in each group.Mice received intraperitoneally injection of STZ for 5 days.After 2 weeks,the CNV was induced by 532 nm frequency laser.Choroidalflatmoutand HEstainingwere applied to analysisthe effect of Cyr61 antibodyon CNV formation.3.Establishing co-culture system between RPE cells and CEC cell,and then MTT,Transwell and tube formation assay were carried out to verify the effect of RPE cell with Cyr61 gene silencing on behavioral function of CEC cell.4.Real-time PCR,Western-blot and Dual-luciferase reporter gene assay were performed to observe the effect of AGEs on Cyr61 and its mechanism.5.Real-time PCR,Western-blot,ELISA and immunocytochemistry were used to test the impact of Cyr61 on VEGF and its mechanism.[Results]1.Compared with control group,diabetic mice appeared RPE cell and Bruch’s membranerupture,obvious proliferation and migration and new blood vessels increased.We found that diabetic mice treated with aminoguanidine could ameliorate CNV severity.Cyr61 and VEGF were high expression in CNV area.Aminoguanidine could not only suppress AGEs formation,but also could reduce the production of Cyr61 and VEGF.2.We demonstrated that intravitreal injection with Cyr61 antibody could significantly reduce the CNV severity of the diabetes mice.Specific blocking Cyr61 in CNV area could decrease VEGF expression and secretionin diabetic mice.3.The results showed that inhibiting the formation of AGEs or gene silencing of Cyr61 could markedly repress CEC cell proliferation,migration and tube formation.4.AGEs could combine with RAGE receptor on RPE cell surface,activate JNK signaling pathways,induce the activation of transcription factor Stat3,and with the combination of Cyr61 gene transcription start site-1351~-1333 bp upstream promoter regions,thus promote the expression of Cyr61.5.Cyr61 could combine with Intergrin receptorαVβ3 on CEC cell surface,activate FAK-PI3K/AKT signaling pathways,induce the activation of NF-κB,and thus promotepromote the expression of VEGF and MMP2/13.[Conclusion]In this study,we found for the first time that AGEs could be combined with RAGE receptor,induce Stat 3 activation and regulate the expression of Cyr61.Cyr61 could not only activate the intergrin-PI3K/AKT signal pathway,accelerate NF-κBnuclear translocation and promote VEGF expression in CEC cell,but alsoregulate MMPs expression.Inhibiting AGEs formation or specific blocking Cyr61 could significantly restrain CEC cell proliferation,migration and tube cavity formation,thus alleviate CNV severity caused by diabetes.Taken together,AGEs-Cyr61-VEGF signal pathwayinvolved in the regulation of CNV formation process,which has not been reported previously.Specificblocking the above pathway can significantly inhibit CNV formation,which will provide new strategy for clinical prevention and treatment of CNV diseases.