The Expression And Relationship Between ET-1 And VEGF In The Coculture System Of Bovine Trabecular Meshwork Cells And Retinal Pigment Epithelial Cells Under Hypoxia

BackgroundNeovascular glaucoma(neovascular glaucoma,NVG)is a set of diseases characterized by new blood vessels on iris and angle.This is a kind of refractory glaucoma.The etiologies are mainly about retinal ischemia,hypoxia,inflammation or surgery,trauma,etc.The whole causes can be up to more than 40 different kinds of diseases.And NVG secondary to central retinal vein occlusion and diabetic retinopathy accounted for nearly 70%of the causes,is a kind of destructive,serious blinding eye disease.The formation of NVG is a dynamic development process,divided into glaucoma early stage,primary open-angle glaucoma and primary closed-angle glaucoma.Iris neovascularization(iris neovascularization,NVI)formation is the precondition of NVG,namely that NVI is in a timely manner to be prevented and controlled so that it may not be the trigger and secondary NVG elevated intraocular pressure.A large number of studies have confirmed that the levels of ET-1 and VEGF in the plasma and aqueous of neonatal vascular glaucoma are both elevated,but the specific relationship is not clear.ObjectiveTo explore the change and relationship between ET-1 and VEGF expression in the coculture system of bovine TMCs(BTMCs)and RPE cells under hypoxia.MethodsBTMCs were cultured in vitro by explant culture method and pancreatic enzyme digestion method to extract fresh bovine retinal pigment epithelium cells,and identified by morphological evaluation.The third-generation of BTMCs vaccinated into the under layer of the 6 hole Transwell room culture system,and the third-generation of bovine RPE cells inoculated into the upper layer of the 6 hole Transwell room culture system.All the holes are divided into normal control group,hypoxia group,the hypoxia + ET-1 antagonist group,the hypoxia + VEGF antagonist group,respectively in 2 ml medium to join 40?l PBS,40?l CoCl2 working liquid(final concentration to 200?mol/L),40 ? lCoCl2 working liquid(final concentration to 200 ?mol/L)+ 2 ?l endothelin receptor antagonist working liquid(final concentration of 10-5 ?mol/L),40?lCoCl2 working liquid(final concentration to 200 ?mol/L)+ 20?lVEGF receptor antagonist working liquid(final concentration for 10-8 ?mol/L),continue to cultivate 24h.Fluorescence quantitative PCR(rt-pcr)was used to detect the relative expression of ET-1mRNA and VEGF mRNA in each group.Immunocytochemistry was used to detect the change of antibody expression in each group.ResultsRt-PCR test results show that the relative expression of ET-1 mRNA in the Control group is 0.90±0.15,Hypoxia group is 1.77 ± 0.08,Hypoxia + Anti-ET-1 receptor antagonist group is 0.54 ± 0.04 and Hypoxia +Anti-VEGF receptor antagonist group is 1.26±0.09,differences between each two groups are statistically significant.The relative expression of VEGF mRNA in Control group is 0.99±0.01,Hypoxia cells is 1.68 ±0.06,Hypoxia + Anti-ET-1 receptor antagonist group is 1.26±0.07 and Hypoxia + Anti-VEGF receptor antagonist group is 0.59±0.04.The differences are statistically significant.The results of immunocytochemistry show(table 3-3)that the positive target integral optical density of ET-1 in the Control group is 44167±727.9,Hypoxia group is 80288±960,Hypoxia + Anti-ET-1 receptor antagonist group is 35672± 1090,Hypoxia +Anti-VEGF receptor antagonist group is 54294±1304,the differences were statistically significant.Table 3-4 results show that the positive target integral optical density of VEGF in the Control group is 228264±10021,Hypoxia group is 418205±23377,Hypoxia +Anti-ET-1 receptor antagonist group is 377198±37025 and Hypoxia +Anti-VEGF receptor antagonist group is 538731±9662,differences are statistically significant(P<0.05).ConclusionsUnder the condition of hypoxia,bovine trabecular meshwork cells and retinal pigment epithelium cells co-culture system raised the level of ET-1 and VEGF expression.After adding ET-1 and VEGF receptor antagonists respectively,the expressions of both were down-regulated in different degree.So we speculate that there are some synergistic effects between ET-1 and VEGF during the process of neovascular glaucoma pathogenesis.