Directional Exosome Proteomes Reflect Polarity-Specific Functions in Retinal Pigmented Epithelium Monolayers

Abstract

The retinal pigmented epithelium (RPE) forms the outer blood-retinal barrier in the eye and its polarity is responsible for directional secretion and uptake of proteins, lipoprotein particles and extracellular vesicles (EVs). Such a secretional division dictates directed interactions between the systemic circulation (basolateral) and the retina (apical). Our goal is to define the polarized proteomes and physical characteristics of EVs released from the RPE. Primary cultures of porcine RPE cells were differentiated into polarized RPE monolayers on permeable supports. EVs were isolated from media bathing either apical or basolateral RPE surfaces, and two subpopulations of small EVs including exosomes, and dense EVs, were purified and processed for proteomic profiling. In parallel, EV size distribution and concentration were determined. Using protein correlation profiling mass spectrometry, a total of 631 proteins were identified in exosome preparations, 299 of which were uniquely released apically, and 94 uniquely released basolaterally. Selected proteins were validated by Western blot. The proteomes of these exosome and dense EVs preparations suggest that epithelial polarity impacts directional release. These data serve as a foundation for comparative studies aimed at elucidating the role of exosomes in the molecular pathophysiology of retinal diseases and help identify potential therapeutic targets and biomarkers.

Roles of exosomes in the normal and diseased eye

“…PCP provides a powerful approach to both identify bona fide resident proteins and to exclude contaminating proteins from a proteome dataset. We found that the vast majority of RPE-derived exosomal proteins that were highly co-enriched with the exosome-specific marker Syntenin-1 differed between the apical and basolateral side (Klingeborn et al, 2017), (Fig. 5), which was not unexpected if exosomes contribute to different apical versus basolateral signaling and pathways in these polarized cells.…”

“…Supporting this notion, we recently identified A Disintegrin and Metalloproteinase Domain-Containing Protein 10 , also known as ADAM10, as a major component in highly purified exosomes released basolaterally from polarized RPE cultures (Klingeborn et al, 2017). Members of the ADAM family are transmembrane proteinases with a unique structure possessing both adhesion and catalytic domains.…”

“…Our own studies using polarized primary RPE cultures show similar results under FBS-supplemented culture conditions. However, under culture conditions using a serum supplement instead of FBS, exosome release is shifted to roughly equal apical and basolateral release (Klingeborn et al, 2017), suggesting that serum components also mediate polarized release of exosomes.…”

“…Thus, currently it is still unclear if exosomes and/or small EVs in aqueous humor from patients with neovascular AMD can be used as biomarkers of the disease. Interestingly, ongoing studies in our own laboratory recently identified Pigment Epithelium-Derived Factor (PEDF) in highly purified apically released exosomes from polarized RPE cell monolayers, but to a much lesser extent in basolaterally released exosomes (Klingeborn et al, 2017). PEDF is secreted primarily from RPE cells on their apical side to maintain an anti-angiogenic milieu in the outer retina (Ablonczy et al, 2011; Tombran-Tink et al, 1995).…”

“…To aid in answering this question and potentially identifying novel AH exosomal biomarkers, foundational characterization of the composition of exosomes released from these cell types in vitro is needed. Ongoing studies in our laboratory aimed at careful characterization of exosomes and other EVs released from RPE (Klingeborn et al, 2017), can be used as a resource to identify and validate potential exosomal biomarkers in AH.…”