Mining For Genes And Peptide Inhibitors Related To Ocular Neovascularition By Bioinformatics Methods

Purpose: Mining new CNV related genes and newly antiangiogenic peptides by bioinformatics methods,investigating inhibitory effects in vitro and vivo,and exploring mechanisms.Method:(1)We designed a computational method to identify novel CNV-related genes using the protein–protein interaction information and shortest paths method.(2)Two novel biologic peptides derived from kringle structure of Neurotrypsin were selected by bioinformatics method,named NT/K-CRS and NT/K-CFY,which investigated antiangiogenic effect in vitro,vivo and mechanisms.Results:(1)33 of putative CNV-related candidate genes were accessed by our method.These genes are deemed to have strong relationships with CNV.(2)Two novel biologic peptides were named NT/K-CRS and NT/K-CFY,with their amino acid sequence RNPDGDAKPW and CFYGDARGKVDWGY.In vitro,MTS,wound healing,chemotaxis assay with transwell chamber,tube formation assay on Matrigel,cell viability assay and TUNEL assay were carried out to confirm the safety and antiangiogenic effect of NT/K-CRS/CFY on VEGF induced HUVECs proliferation,migration,chemotaxis,and tube formation.The antiangiogenic effect of NT/K-CRS/CFY was further confirmed in chorioallantoic membrane angiogenesis assay and retinal neovascularization in oxygen-induced retinopathy(OIR)in vivo.In OIR model,real time PCR and western blot assays were used to explore the influence of NT/K-CRS/CFY on the mRNA and protein expression of the angiogenesis stimulator-VEGF and the angiogenesis-inhibitor PEDF in mouse retina.Conclusions:(1)In this study,we designed a computational method to identify novel CNV-related genes using the protein–protein interaction information and shortest paths method.A list of putative CNV-related candidate genes was accessed and deemed to have strong relationships with CNV.(2)NT/K-CRS and NT/K-CFY effectively inhibit angiogenesis in vitro and vivo,with good security.NT/K-CRS and NT/K-CFY inhibit pathological retinal neovascularization by down-regulation of VEGF mRNA and protein expression and up-regulation of PEDF mRNA and protein,restoring the balance between angiogenic stimulator and inhibitor.