Research Of A Novel Double Blocker VF28 Combined With VEGF And FGF And MicroRNA In A Oxygen-induced Retinopathy Mouse Model

Background Retinal neovascularization(RNV)is a major cause of blind-causing eye disease worldwide.On one hand,the important effects of growth factors on retinal neovascularization have been widely recognized,retinal neovascularization can be inhabitted with target drugs by intravitreal injection.Vascular endothelial growth factor(VEGF)and fibroblast growth factor(FGF)are the two key factors to promote retinal neovascularization.The fact that anti VEGF and anti FGF drugs can inhibit retinal neovascularization has been confirmed by several studies,but there is no report about simultaneously using these two factors in eye treatment both at home and abroad.On the other hand,although the anti-VEGF intravitreal injection is the effective method of treating ocular neovascular disease,but there are complications,side effects,and many other problems,so it is particularly important to look for other VEGF-independent pathways.MicroRNA is vital in ocular tissue development,differentiation,apoptosis,metabolism and regeneration after injury,so its mechanism in gene expression of retinal neovascularization and regulating function is getting more and more attention of people.Objective Observe and study the inhibitory effect and possible molecular mechanism of double-targets(VEGF,FGF)drug VF28 for oxygen induced retinal neovascularization.Test microRNA expression of oxygen induced retinopathy mice model,discussing its relationship with the formation of retinal neovascularization.Methods Healthy C57BL/6J female mice and pups were randomly divided into normal and OIR group at postnatal day 7(P7).The normal group was raised in a conventional cage and exposed to room air for 10 d.The OIR group was raised in a sealed chamber and exposed to(75±2)%oxygen.The moms were alternated between the two groups every day to promote their survival under hyperoxia.The OIR group was returned to the room air at P12.Each eye was injected with low(0.5μg),high(1μg)dose of VF28,VEGF trap(0.5μg)and FGF trap(0.5μg)immediately by intravitreal injection,and mices were divide into different groups according to the injected drugs.At P17,mice from either group were retro-orbitally injected with high molecular weight fluorescein isothiocyanate-dextran(FITC-dextran),the eye balls were fixed in 4%paraformaldehyde,and the retinal whole mounts were prepared.The retinal vessels labeled with FITC-dextran were observed under a fluorescence microscope;the eye balls were also processed for paraffin sections and Hematoxylin and Eosin(H&E)staining.The cell nucleis in the newly-formed vessels beyond the inner limiting membrane were quantified.Western blot was used to detect protein changes of PEDF、VEGF and FGF in retina,so as to prove the possible molecular mechanisms of VF28 in restraining retinal neovascularization.In the microRNA expression analysis experiment,P17,the miR was extracted from the eyes,reverse transcribed,and subjected to a customized miR array analysis.The real-time PCR was preformed to verify the results of the miR array.Results Retinal whole mounts labeled with FITC-dextran showed that the peripheral retinal microvessels in the OIR group were tortuous,disorganized with neovascular buds,and the avascular area was prominent in central retina.The avascular area of each drug injection groups is significantly lower than OIR group,VF28 low and high dose group and VEGF trap group are significantly better than that of FGF trap in improvement of retinal avascular area;Low dose of VF28 was significantly better than that of high dose;And compared the same dose of VF28(low dose group)with VEGF the trap group,avascular area has a downward trend,but there was no statistical significance,for vein circuity and capillary tidy distribution degree,VF28 low-dose group look better than that of VEGF trap.HE results show that the vascular endothelial nuclei number of high oxygen treated group were significantly more than the control group.VF28,VEGF trap and FGF trap group can significantly reduce the number of retinal neovascularization of OIR the mice,but the effect of VF28 low dose group is most obvious,significantly better than VF28 high dose group,VEGF trap and FGF trap group,and closed to the normal control group.Western blot result shows PEDF and VEGF had opposite changes in OIR group,characterized by significantly increased VEGF levels and PEDF decreased levels.VF28(0.5μg per eye)and VEGF trap(0.5 μg per eye)could significantly lower VEGF levels and increase PEDF levels significantly(P<0.001 vs model group),and VEGF trap has no effect on FGF upregulation.FGF trap(0.5μg per eye)can significantly reduce the FGF levels(P<0.05 vs OIR group),and has no impact on VEGF.Result for part two:The percentage of avascular area in total retina area in OIR group(25.81±2.12)%was 4-fold that in normal group(6.57±3.6)%(p<0.01,normal vs OIR).H&E staining showed that the number of the cell nuclei beyond inner limiting membrane was(28.41 ±4.01)in OIR retina,which was substantially higher than that(0.16±0.31)in normal retina(p<0.01,normal vs OIR).More interestingly,the results of miR array showed that 21 out of the 80 miRs examined exhibited more than 1.5-fold changes at expression level.Among these 21 miRs,9 were up-regulated,12 were down-regulated;4 miRs showed more than 3-fold expression changes,3 were down-regulated and 1 was up-regulated.The expression of the 4 miRs was verified by real-time PCR.The expression trends of miR-3078,miR-140,miR-29b and miR-29c were consistent with those revealed by the miR array.MiR-3078 was significantly up-regulated(t=-2.380,P<0.05 normal vs OIR),and the other 3 miRs were significantly down-regulated(t=2.638,t=2.323,t=2.415,p<0.05 normal vs OIR).Conclusions Optimal dose of VF28 can effectively decrease retinal avascular area,significantly reduce the number of retinal vascular endothelial cell,whose effect was better than that of VEGF trap and FGF trap of the same dose.The protection function of VF28 in OIR mice may be due to the reduced expression of VEGF and FGF,and increased expression of PEDF.Differential expression of the microRNAs,including miR-3078,140,29b and 29c,was detected in normal and OIR mouse retinas.These miR expression changes may be associated with retinal neovascularization.These results would provide the new leads for further studying pathogenic mechanisms and therapeutic targets for neovascular retinopathy.