Abstract
Diabetic retinopathy (DR) is a leading cause of blindness, yet its molecular mechanisms are unclear. Extracellular vesicles (EVs) contribute to dysfunction in DR, but the characteristics and functions of vitreous EVs are unclear. This study investigated the inflammatory properties of type 2 diabetic (db) vitreous EVs. EVs isolated from the vitreous of db and non-db donors were used for nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), immunogold staining, Western blotting, and proteomic analysis by mass spectrometry. Intracellular uptake of vitreous EVs by differentiated macrophages was evaluated using ExoGlow membrane labeling, and the impact of EVs on macrophage (THP-1) activation was assessed by cytokine levels using RT-qPCR. NTA and TEM analysis of db and non-db vitreous EVs showed non-aggregated EVs with a heterogeneous size range below 200 nm. Western blot detected EV markers (Alix, Annexin V, HSP70, and Flotillin 1) and an upregulation of Cldn5 in db EVs. While the db EVs were incorporated into macrophages, treatment of THP-1 cells with db EVs significantly increased mRNA levels of TNFα and IL-1β compared to non-db EVs. Proteomic and gene enrichment analysis indicated pro-inflammatory characteristics of db EVs. Our results suggest a potential involvement of EC-derived Cldn5+ EVs in triggering inflammation, offering a novel mechanism involved and presenting a possible therapeutic avenue for DR.