Characterisation of vitreous derived small extracellular vesicles in diabetic retinopathy

Diabetic retinopathy (DR) is a progressive retinal disease driven by microvascular dysfunction and inflammation, ultimately leading to vision loss. Early detection is essential for preventing irreversible damage; however, the asymptomatic nature of early-stage DR limits the effectiveness of current screening methods. Emerging evidence indicates that inflammatory processes, particularly activation of the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome, precede clinical manifestations. Small extracellular vesicles (sEVs), which mediate intercellular communication, may serve as biomarkers by carrying NLRP3-associated proteins. This study aims to investigate the role of sEVs in DR-related inflammation. Post-mortem vitreous was obtained from donors with a confirmed DR diagnosis and compared to samples from donors with no known ocular pathology. Additionally, vitrectomy samples from DR patients were compared to samples from donors who had undergone vitrectomy for macular holes or epiretinal membranes. The concentration and size of sEVs as well as the expression of EV- and inflammasome-associated cargo markers were evaluated in both sample types using nanoparticle tracking analysis and western blotting, respectively. The results showed that the sEV concentration was higher in post-mortem and vitrectomy samples from DR patients/donors compared to controls. Furthermore, DR sEVs contained higher levels of inflammasome-related proteins than controls, indicative of increased NLRP3 inflammasome activation. Finally, we demonstrated that vitreous sEVs contained RPE-65, a protein of retinal origin, suggesting that vitreous sEVs may be derived from the retina. These findings contribute to the growing knowledge of the role of sEVs in chronic diseases and suggest that they could serve as viable biomarkers for DR.