Early Remodeling of Polarized Small Extracellular Vesicle Secretion in Retinal Pigment Epithelium Cells under Subtoxic AMD-Relevant Stress

Abstract Retinal pigment epithelium (RPE) dysfunction is central to age-related macular degeneration (AMD), yet the early cellular alterations that precede overt degeneration remain incompletely understood. In this study, we investigated the impact of subtoxic hypoxia-mimetic stress (STH) on the polarized secretion of small extracellular vesicles (sEVs) by human iPSC-RPE monolayers, using this system to model early AMD-relevant RPE stress. The STH regimen induced hypoxia-mimetic stress without causing overt cytotoxicity, while preserving epithelial barrier integrity. However, it markedly remodeled the sEV secretome and disrupted apicobasal cargo allocation, as exemplified by basolaterally biased SERPINA5 enrichment and apically biased CTSC reduction. Proteomic analysis further revealed increased AMD-relevant cargo, such as APOE, VEGFA, and ANGPTL4, while reducing lysosomal hydrolases. Concurrently, STH impaired RPE phagocytic and degradative function. Notably, these sEV changes were largely uncoupled from bulk intracellular proteomic alterations, suggesting that polarized sEV profiling may serve as a sensitive indicator of RPE dysfunction under AMD-relevant stress. At the structural level, STH disrupted apical microvilli and reduced microtubule-associated cytoskeletal stability. Importantly, CoCl₂ washout partially reversed these secretory, structural, and functional abnormalities. Taken together, our findings suggest that early and reversible remodeling of polarized sEV secretion reflects a subtoxic AMD-related stress state in RPE cells, which provides a potential opportunity to detect and modulate RPE dysfunction before overt degeneration.