Study On Retinal Proteomics Of Oxygen-induced Retinopathy Model

Objective:Ischemic retinopathies,such as retinopathy of prematurity(ROP)and proliferative diabetic retinopathy(PDR),are the main causes of visual impairment in different age groups.Retinal neovascularization(RNV)is the common pathological feature of ischemic retinopathies.Fully understanding the formation mechanism of RNV is very crucial for the prevention and treatment of ROP and other ischemic fundus diseases.Oxygen-induced retinopathy(OIR)model is a good candidate to simulate two stages of RNV formation of ROP patients.A quantitative proteomic approach,SWATH-MS,was employed to establish the mouse retina proteome,analyze the expression of retinal protein in OIR model mice and explore the potential mechanism of RNV formation.Besides,hypoxia-induced human retinal microvascular endothelial cells(hRMECs)was applied to verify the expression of the target genes identified by proteomics from OIR model.The present study may help to clarify the roles of these genes in hREMCs and provide the insights into the mechanism of the ischemia-induced neovascularization of retina.Methods:1.OIR model was developed in C57BL/6 mice by a five-day exposure to 75%oxygen from postnatal(PN)7 to PN12.Retina flatmounts and retina frozen sections of 2 stages(PN12 and PN17)were stained with Isolectin B4 to assess the retina vasculature.2.Retinas from control and OIR model group of 2 stages(PN12 and PN17)were extracted respectively.SWATH-MS was applied to detect the differential protein between the two groups.The SWATH-MS method was used to detect the differential protein between the two groups.Screening of the credible proteins and differential proteins,followed by Gene Ontology(GO)enrichment analysis and KEGG pathway enrichment of differential protein analysis.The target protein was further screened according to the function and expression site,and the protein-protein interaction relationship was analyzed through the STRING database.qRT-PCR was used to verify the expression level of the genes corresponding to the target proteins.3.hRMECs were verified by immunofluorescence staining and RT-PCR,and induced by hyperoxia and then room air.The effects of hyperoxia and relative hypoxia on the proliferation of hRMECs were verified by CCK-8 experiment and qPCR.RT-PCR was used to screen the expression of target protein corresponding genes in hRMECs.Results:1.OIR mouse model was successfully induced,avascular area could be seen from PN12 OIR mouse retina while neovascularization was observed from PN17 OIR mouse retina.It was also confirmed by frozen section staining.2.Total protein concentration of the retina was determined to be 1.0-1.8μg/mL.5190 proteins were detected by SWATH-MS,283 and 188 differential proteins were detected in PN12 and PN17,respectively.FN1,the highest FC in the OIR model group at PN17,is involved in wound response and wound healing by GO enrichment analysis.The protein that is the most down-regulated in the OIR model group at PN17 is SPRED3,which regulates the activation of growth factor-induced MAP kinase.KEGG pathway analysis showed that the differential proteins of PN12 were mainly enriched in homologous recombination,endocytosis,oxidative phosphorylation and insulin signaling pathway;the differential proteins of PN17 were mainly enriched in RNA translocation,Huntington’s disease and PPAR signaling pathway.STRING database analysis found 19 target proteins related to endothelial cell regulation,including Anxa2,Anxa3,Anxa5,Apoal and Vashl.Of the 19 target proteins,78.9%were expressed in mouse endothelial cells and 57.9%were expressed in human endothelial cells,and Apoal,Fgb,Fgg,and Sparc all vibrantly interact with other target proteins.qRT-PCR verified that the target protein mRNA expression levels in OIR model were consistent with the proteomics results.3.Cell proliferation assay proved that hyperoxia inhibited the proliferation of hRMECs,while relative hypoxia promoted the upregulation of VEGF mRNA.The expression of Anaxa2,Anaxa5 and VASP mRNA was up-regulated,and the expression of Vash1 mRNA was down regulated in relative hypoxia hRMECs.Conclusion:1.OIR model with C57BL/6 mice was successfully constructed,and the feature of RNV accorded with the pathological characteristics of OIR model could be seen in the present study,which provided the experimental animal basis for the follow-up study.2.Proteomic analysis of OIR mice retina provides various differential proteins,including Anxa 2,Anxa 3,Anxa 5 and Vasp,involved in the regulation of endothelial cells(ECs)function.These proteins may be the key proteins that affect the formation of RNV in mouse OIR model.3.Relative hypoxia induced proliferation of hRMECs and led to the upregulation of Anxa2,Anxa5 and Vasp,and the downregulation of Vash1,which was consistent with the proteomic results of PN17.This not only further explained that these target proteins played an important role in RNV formation,but also may be helpful for the future exploration of RNV mechanism.