Proteomic analysis of Oxygen Induced Retinopathy Model using SWATH -MS proteomics reveals novel potential therapeutic target molecules

Abstract

Purpose : Mouse oxygen-induced retinopathy (OIR) model is an animal model sharing many hallmarks with human neovascular eye diseases. We performed characterization of proteins involved in OIR pathogenesis by a recently developed technique, sequential window acquisition of all theoretical mass spectra (SWATH-MS) proteomics.

Methods : Retinas from control and OIR retinas were harvested at P13 (n=5/5 retinas), P17 (n=9/6 retinas) and P42 (n=6/7 retinas) and subjected to SWATH-MS and bioinformatics data analysis, including Ingenuity pathway analysis (IPA). To validate the results obtained from SWATH-MS, mouse retinas were subjected to immunohistochemistry for chosen proteins. In addition, neovascular membranes from human proliferative diabetic retinopathy (PDR) and retinal vein occlusion (RVO) were used to study the clinical relevance of one of the proteins quantified with proteomics.

Results : We were able to relatively quantify almost 3 000 unique proteins and their expression differences between normal and OIR retinas. When comparing OIR retinas to controls, there were 17 differently expressed proteins at P13 and 22 at P17 (adjusted p<0.05). Most upregulated proteins at P13 OIR (by fold change (FC)) were crystallins (log₂ FC 2.67-4.32, non-adjusted p<0.05) IPA analysis connected the differing proteins at P13 OIR to phototransduction pathway (p=4.95e-10), protein kinase A signaling (p=2.29e-04) and glutamine biosynthesis I (p=8.05e-04). IPA analysis revealed increased angiogenesis (P=2.53e-04, activation z-score ≥ 2) at P17 OIR, due to upregulation of 7 proteins, such as Filamin-A (log₂ FC = 0.84, p=0.03) and Myosin-9 (log₂ FC = 0.92, p=0.05). Majority of the changes in protein expression return to normal at P42, but there is evidence to suggest that proteins involved in neurotransmission remain at reduced level.

Conclusions : We have carried out the most extensive proteomic profiling of OIR to date and the results reveal new potential targets to address pathological angiogenesis in retinal diseases. Our analysis reveals novel molecular networks that could contribute to the pathogenesis of neovascular retinal diseases.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.