The Role Of Mitochondrial Ros And Complement Activation Fragmnts C3a And C5a In The Amyloid β Induced Angiogenic Factors By ARPE-19Cells

BackgroundAge-related macular degeneration（AMD）is the aging lesions of macular,the pathogenesis of AMD is complex and still not completely clear, it is acommon retinal disease which can lead to irreversible blindness in theelderly population of developed countries. The main change is the declineof RPE consuming ability, which makes the rod outer segment stayextracellular or deposit beneath the RPE and then form drusen. At the earlystage of AMD development, the prime target is the retinal pigmentepithelium (RPE). The RPE are essential to maintain the function andsurvival of photoreceptors and constitute the blood-retina barrier (BRB).RPE dysfunction leads to photoreceptor apoptosis, BRB disturbance, andunder certain conditions to the production of angiogenic cytokines that takepart in the process of choroidal neovascularization(CNV). These newvessels originate from the choroidal vascular endothelial cells and invadethe retina, finally resulting in vision loss. Drusen are the earliest clinicalsign of AMD and represent deposits lying beneath the RPE. The presence of drusen is considered to be one of the contributing factors for theformation of CNV by stimulating RPE cells to secrete angiogeniccytokines.Amyloid β (Aβ) is one of the main components of drusen, butthe role of it in the formation of CNV and related mechanism are still notclear. Earlier studies also showed that Aβ can increase reactive oxygenspecies (ROS) production by RPE cells. In view of the fact that oxidativestress is considered as an important mediator in the process of angiogenesis,the effect of mitochondrial ROS on the Aβ induced production ofangiogenesis factors is not very clear. The complement components C3aand C5a are also a constituent of drusen and have been implicated in thegeneration of angiogenic and pro-inflammatory cytokines, but whether Aβmight affect the expression of C3a and C5a by ARPE-19cells and whetherthese complement components might play a role in the Aβ mediatedproduction of proangiogenic cytokines is still a question.Collectively，this project will provide a new insight into the role ofamyloid-β in the pathogenesis of AMD and possible mechanism involvedin this process and be hopefully expected to offer a new strategy in thestudy of the prevention and treatment of AMD.PurposeInvestigate the influence of Aβ on the production of angiogenesis factors,and the role of mitochondrial ROS and complement fragments C3a and C5a in the amyloid β induced production of angiogenic factors byARPE-19cells.MethodsCulture the ARPE-19cells line that purchased from ATCC (AmericanType Culture Collection), then stimulate the cells with or without differentconcentrations of Aβ(1-42) or it’s inactive reverse control peptide Aβ(42-1)or the DMSO carrier for24hours. The protein levels of the angiogeniccytokines VEGF, IL-8and MCP-1in the culture supernatants were assessedby ELISA; the production of mitochondria-associated ROS (reactiveoxygen species) following stimulation was detected by Flow cytometry;The protein levels of C3a and C5a were subsequently measured in theculture supernatants by ELISA. ROS blocking experiments were performedby using ROS inhibitor diphenyleneiodonium chloride (DPI) prior to theaddition of Aβ. C3a and C5a blocking experiments were performed byadding anti-C3a antibody or anti-C5a antibody respectively just before Aβwas added into the ARPE-19serum free culture system. CCK-8assay wasused to investigate the effects of Aβ(1-42) and DPI on viability ofARPE-19cells.ResultsA significant dose dependent increase in the level of all these three cytokines was observed when ARPE-19cells were stimulated withAβ(1-42) as compared with the reverse control peptide Aβ(42-1) or theDMSO carrier. At a concentration of20μM Aβ(1-42), a significant twofoldincrease was observed in the level of mitochondria-associated ROS ascompared with Aβ(42-1) or DMSO, no significant effect was observed forthe lower concentrations of Aβ(1-42). Addition of Aβ(1-42) to the serumfree ARPE-19cultures dramatically increased the level of C3a and C5a in adose dependent fashion. The level of C3a and C5a in Aβ(42-1) andDMSO-treated groups were all below the detection limit of the assay. Inrelated experiment, DPI significantly decreased the expression of C3a, C5a,VEGF, IL-8and MCP-1by Aβ(1-42)-treated ARPE-19cells.The obtaineddata also showed that the production of these complement fragments andangiogenic cytokines that were induced by Aβ(1-42) could be mediated bymitochondrial ROS. Anti-C3a and anti-C5a antibodies were added to theculture system, but no significant effects were observed on the level ofVEGF, IL-8and MCP-1.ConclusionsOur results support the hypothesis that Aβ is involved in thepathogenesis of CNV formation by promoting the production of theangiogenic cytokines VEGF, IL-8and MCP-1by RPE cells. Aβ alsoinduced the production of Mitochondrial ROS and formation of C3a and C5a by RPE cells. Mitochondrial ROS played a role in the regulation of Aβinduced expression of angiogenic factors. Although Aβ also induced theformation of C3a and C5a by RPE cells, these factors did not play a role inthe Aβ mediated release of angiogenic cytokines.