Chronic Oxidative Stress in Primary Retinal Pigmented Epithelium Monolayers Increases Basolateral Exosome Release and Changes Both Exosome and Extracellular Matrix Proteomes

Abstract

Purpose : Oxidative stress is implicated in the pathobiology of age-related macular degeneration (AMD). Exosomes are small extracellular nanovesicles (EVs) of endocytic origin that mediate different signals between cells and can be modified by oxidative stress. We hypothesize that oxidative stress affects retinal pigmented epithelium (RPE) exosome release and content and contributes to basal deposit extracellular matrix (ECM) changes. Here, we characterize ECM changes and RPE exosome release and content in response to oxidative stress.

Methods : We have previously shown that exosomes can be isolated apically and basally from fully differentiated and polarized primary porcine RPE cells grown on Transwell cell culture inserts. To induce chronic oxidative stress, these cultures were treated daily with 0.2mM H₂O₂ for 4 weeks. Basolateral exosome yield was assessed by Nanoparticle Tracking Analysis (NTA) and immunoblotting for exosomal markers along with total protein quantitation. Purity and protein content of exosome preparations were analyzed by immunoblotting for exosomal markers, and markers for known contaminants. In-depth proteomic mass spectrometry analyses were done on both exosomes and ECM preparations.

Results : Unbiased proteomic analyses of the content of highly purified basolateral exosomes isolated from RPE cultures under chronic oxidative stress revealed changes to a number of ECM proteins. One such protein, Keratin 10 (KRT10), was increased 3-fold in basolateral exosomes and this was mirrored by a 6.5-fold increase of KRT10 in proteomic analyses of the basal extracellular matrix (ECM) and correlated with a 4-fold increase in basolateral exosome secretion as measured by NTA. Treatment of RPE cultures with an inhibitor of exosome release effectively reduced basolateral EV secretion. Proteomic analyses of exosomes and ECM from these cultures will also be presented.

Conclusions : We show for the first time that chronic oxidative stress in primary RPE monolayers induced proteomic changes in exosomes and increased basolateral EV secretion which could be prevented by inhibition of exosome release. We also show examples of protein changes in exosomes that correlate with protein changes in the underlying ECM. These findings have the potential to open up a completely novel avenue for therapeutic intervention in AMD.

This is a 2021 ARVO Annual Meeting abstract.