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

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.