Modeling senescence in ARPE19 cells to investigate the role of small extracellular vesicles in age‐related macular degeneration

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Aims/Purpose: Age‐related macular degeneration (AMD) is a degenerative disease causing significant vision loss in the elderly. The molecular mechanisms of AMD onset and progression are not fully understood. Senescent cells are known to secrete increased amounts of small extracellular vesicles (sEV). However, the impact of sEVs on RPE dysfunction and AMD through senescence spreading remains unclear. Our study aimed to develop an in vitro model of RPE cell senescence.Methods: We used the retinal pigment epithelial cell line, ARPE‐19. Senescence induction in RPE cells was achieved through two different approaches: repeated passaging (replicative senescence) and ionizing radiation (DNA damage‐induced senescence). Characterization of sEVs released by RPE cells was performed and markers of senescence were assessed.Results: RPE cells subjected to replicative exhaustion or ionizing radiation showed higher H2AX foci accumulation compared to p9 cells. Ionizing radiation significantly increased nucleus area and deformation, with a similar trend at p21 and p31. Levels of p21, critical for establishing senescence, increased at p31 and showed a tendency to rise in ionizing radiation‐exposed cells. Levels of p16, maintaining the senescence phenotype, also increased in ionizing radiation‐exposed cells and tended to rise at p21 and p31. Activities of active MMP‐2 and ‐9 increased in the conditioned medium of senescent cells, with MMP‐2 activity correlating with higher protein levels. sEVs from senescent cell‐conditioned medium showed significant increases in concentration and mean size compared to p9 cells.Conclusions: We successfully induced senescence in ARPE19 cells, creating a valuable model to investigate the role of sEVs in spreading senescence to neighboring cells, especially in the context of AMD onset.Support: FCT, Portugal: 2020.04811.BD; PEst UIDB/04539/Base/2020 and UIDP/04539/Programatico/2020.European Union's Horizon 2020 Teaming project MIA‐Portugal (no 857524) and CHAngeing – Connected Hubs in Ageing: Healthy Living to Protect Cerebrovascular Function (no 101087071), funded by Excellence Hubs Call – Horizon‐ WIDERA‐2022‐ACCESS‐04‐01.