Antiangiogenesis Of A Neovasculature Specific Binding Peptide GX1on Retinal Neovascularization Of Diabetic Retinopathy

【Background】Diabetic retinopathy (DR) is one of the most common and serious diabeticmicrovascular complications, and is one of four major reasons for new cases ofblindness among adults. DR is characterized as retinal neovascularizationaccompanied with a serial of pathological changes, such as exudation,hemorrhage and proliferation, which further lead to vascular proliferation andsevere visual impairment. Thus, antiangiogenesis is considered to be an importantstrategy in the therapy of DR. Traditional therapy, such as vitrectomy, panretinalphotocoagulation, are risky and costly, and have some visual impairment.Furthermore DR is easy to recur after traditional treatment. At present, moleculartarget therapy in therapeutic field of tumor provides a new strategy onretinal neovascularization of DR, which is highly effective and low poisonous.Searching for those molecules specifically expressed on retinal neovasculature iskey link of that strategy. Recently study, some special molecules expressed on tumor vascular, such asaminopetidases and integrin receptor, are also expressed onretinal neovascularization of DR, which can mediate phage targeting andinternalizing in endothelial cells. Oligepoptide RGD-4C targeting αⅤβ3/αⅤβ5cansuppressing retinal neovascularization induced by hyperoxia by activatingselectively apoptosis retinal endothelial cells, which illustrates that somemolecules are common expressed in retinal neovasculature and tumor vascularand those molecules promise to be new target forsuppressing retinal neovascularization of DR.By using in vivo phage display technology, a cyclic9-mer peptideCGNSNPKSC homing specifically to vasculature of human gastricadenocarcinoma was obtained and named as GX1which has got the state patent.In vitro studies showed that GX1could suppress the proliferation ability and tubeformation ability of human umbilical vein endothelial cell(HUVEC) andco-cultured human umbilical vein endothelial cell(Co-HUVEC) whichco-cultured with human gastric adenocarcinoma cells SGC7901. In vivo studiesshowed that GX1could suppress neovascularization of chicken embryo allantoismembrane and recombinant fusion protein GX1-rmhTNFα could suppress thetumor growth of gastric cancer bearing nude mice. All these results indicated thatGX1was a novel vascular target of human gastric cancer and could suppresstumor angiogenesis. In another study, GX1could specifically bind with rat retinalneovasculature induced by hyperoxia, which showed that the receptor of GX1might play an important role in retinal neovascularization and might be acharacteristic molecule of retinal neovasculature. So, it’s necessary to investigatethe function of GX1and it’s receptor in retinal neovascularization. 【Objectives】1. To verificate the binding specificity of GX1to retinal neovasculatureendothelial cells of DR.2. To study in vitro effects of GX1on proliferation, migration, tube formation,cell cycle, cell apoptosis and other characteristics of retinal neovasculatureendothelial cells.3. To study in vivo effects of GX1on rat retinal neovascularization induced byhyperoxia.【Methods】1. GX1peptide, control peptide (CNKSPSGNC, Pep2) together withbio-binding GX1(bio-GX1) and bio-binding Pep2(bio-Pep2) was chemicallysynthesized and purified. Immunocytochemical staining andimmunocytofluorescence were used to detect the binding specificity of GX1torat retinal microvascular endothelial cells (rRMECs).2. GX1effects on rRMECs proliferation, migration, tube formation and othercharacteristics were analyzed by in vitro MTT assay, migration assay, tubeformation assay, etc.3. Cell cycle distribution and cell apoptosis induced by GX1treatment weredetected by flow cytometry to look into the possible mechanisms of GX1effectson rRMECs.4. GX1effects on rat retinal neovascularization induced by hyperoxia wereanalyzed by counting the endothelial nuclei of new vessels extending from theretina to the vitreous in histological sections with H&E staining. Retinal flatmount was used to observe the changes of retinal vessels.【Results】1. GX1was shown by immunocytochemical staining andimmunocytofluorescence to bind specifically to rRMECs, while no positive staining was observed in Pep2or PBS groups, indicating that GX1couldselectively target retinal neovasculature of DR..2. MTT assay showed that compared with control peptide, GX1significantlyinhibited the cell proliferation of rRMECs in a dose-dependent manner. In tubeformation assay and migration assay, GX1significantly inhibited the micro-tubeformation and the migration of rRMECs, while no such effect was seen in Pep2treated cells. On the other hand, GX1showed the effects to inhibit theenhancement of the cell proliferation, the micro-tube formation and the migrationinduced by VEGF165.3. Flow cytometry analysis showed that compared with Pep2, GX1increasedthe proportion of apoptotic cells in rRMECs(5.33%vs.7.33%, P<0.05), while nosignificant changes in cell cycle distribution was detected in GX1treatedrRMECs.4. The model of retinal neovascularization in mice induced by hyperoxia wassuccessfully established, The results show that the number of the endothelialnuclei of new vessels extending from the retina to the vitreous was significantlyless in the model mice with GX1injection than that in the model mice with Pep2injection, PBS injection and without GX1injection. In the retinal flat mounts，thedensity and abnormality of retinal new vessels was noted much less in the modelmice with GX1injection compared to that of the model mice with Pep2injection,PBS injection and without GX1. No obvious changes were found in normalcontrol group.【Conclusions】1. Besides the binding specificity of GX1to retinal neovasculature of DR,GX1is also able to inhibit the proliferation, the micro-tube formation and themigration of the retinal neovasculature endothelial cells of DR in vitro, as well asinduce cell apoptosis of the retinal neovasculature endothelial cells of DR. So, we can conclude that GX1could inhibit retinal neovascularization of DR in vitro.2. GX1could inhibit rat retinal neovascularization induced by hyperoxia,which supplies a new method for clinical treatment of retinal neovascularizationof DR.