| Purpose: To evaluate the anti-angiogenic effect,molecular mechanism and safety profile of two treatments on corneal neovascularization(NV),a miRNA therapeutics and a novel peptide ZY-1.Methods:(1)Nanostring and bioinformatics analyses were used for angiogenesis-related miRNAs profiling and their target genes detection in alkali-burn induced corneal NV mouse model.The miRNA expression was confirmed by RT-qPCR analysis.The miRNA therapeutics was constructed and packaged by recombinant adeno-associated virus(rAAV).The miRNA therapeutics,control virus and PBS were injected intra-stromally before alkali-burn or subconjunctivally after alkali-burn.The corneal NV was then calculated through in vivo observation and immunofluorescence analysis.RT-qPCR and Western Blot were used to analyze the expression of selected miRNAs and their target genes as well as the downstream molecular pathways.The miRNA therapeutics and PBS were injected intra-stromally or subconjunctivally into normal mouse corneas,and the safety profile was investigated by in vivo observation and histological analysis.(2)The novel peptide ZY-1 was derived from placenta growth factor-1(PlGF-1).CCK-8 and Matrigel were used to detect the effects of peptide ZY-1 on VEGF/PlGF induced cell proliferation and tube formation of vascular endothelial cells.The inhibitory effects of topical ZY-1 were evaluated on three animal models,including alkali-burn and intra-stromal suture induced corneal NV rat models and mouse micropocket model.CCK-8 was used to detect the effect of peptide ZY-1 on human corneal epithelial cells(HCECs)at high concentration.The tear film break-up time(BUT)and histopathological analyses were used to estimate the safety profile of ZY-1 for long-term topical application.Results:(1)Among 618 mouse miRNAs profiled,we found 35 up-regulated and 3 down-regulated miRNAs in the neovascularized corneas.We selected miR-184 and miR-204,two miRNAs that were down-regulated > 10-folds in neovascularized corneas,as our candidate targets to test the concept of in vivo gene delivery for therapeutic miRNA to treat corneal NV.Fourteen serotypes of rAAV.EGFP were evaluated for gene transfer efficiency in mouse corneas,and we chose rAAVrh.10 for overexpressing pri-miR-184/204 in the corneas with alkali-burn induced corneal NV.Compared to the control groups,rAAVrh.10-mediated overexpression of miR-184/204 by intra-stromal and subconjunctival injections effectively inhibited alkali-burn induced corneal NV.The anti-angiogenic effect of rAAV.pri-miR-184 was achieved by targeting Fzd4 gene,thus suppressing the canonical Wnt/?-catenin signaling;while rAAV.pri-miR-204 exerted its anti-angiogenic effect by targeting angiopoietin-1 gene(Angpt1),thus suppressing the Angiopoietin-1/Tie2/PI3K/Akt pathway.The in vivo observation and histological analyses indicated that no obvious abnormality was caused by rAAVrh.10 pri-miR184/204 in the surface and fundus of normal mouses.(2)Peptide ZY-1 significantly inhibited VEGF/PlGF induced endothelial cell proliferation and tube formation in vitro.Topical application of peptide ZY-1 effectively suppressed corneal NV induced by alkali-burn,intra-stromal suture and VEGF in vivo.High concentration of peptide ZY-1 had no effect on HCEC cell viability,rat BUT and the structures of rat cornea and conjunctiva.Conclusion:(1)Overexpression of miR-184/204 could effectively prevent or treat corneal NV,and the inhibitory effects were achieved by targeting Fzd4/Wnt/?-catenin and Angpt1/Tie2/PI3K/Akt pathways,respectively.(2)Topical application of peptide ZY-1 could effectively inhibit corneal NV. |
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