Extracellular Vesicles from the Vitreous Humor Diabetic Patients Contain Claudin-5 and Exhibit Inflammatory Properties

Abstract

Purpose : Diabetic retinopathy (DR) is a leading cause of irreversible blindness in working-age adults, yet the molecular mechanisms underlying its pathogenesis remain elusive. Our laboratory has shown that Claudin-5 (Cldn5, a vital endothelial cell (EC) tight junction protein) is elevated in the experimental models of DR. Extracellular vesicles (EVs) play a key role in cellular damage and dysfunction and are implicated in DR, but the characteristics and functions of vitreous EVs in DR and the presence of Cldn5 remain unclear. The present study investigated the presence of Cldn5 in vitreous EVs and their inflammatory properties.

Methods : Vitreous samples from deidentified diabetic (15-30 years duration) and non-diabetic donors were procured from the National Disease Research Interchange and used for EV isolation. Nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), immunogold staining, western blotting, and proteomic analysis by mass spectrometry were employed for characterization. The impact of EVs on macrophage (THP-1) activation was assessed by cytokine levels using RT-qPCR. Statistical analyses utilized GraphPad Prism 10.

Results : NTA analysis revealed diverse sub-populations in diabetic EV (db EV) and non-diabetic EV (non-db EV) in vitreous samples. TEM images confirmed non-aggregated EVs with a heterogeneous size range below 200 nm. Western blot analyses detected EV markers (Alix, Annexin V, HSP70, and Flotillin 1) and an upregulation of Cldn5 in db EVs (N=5; p<0.01), while VE-Cadherin was undetected. Immunogold labeling demonstrated increased Cldn5 localization in db EVs compared to non-db EVs. Treatment of THP-1 cells with db-EVs significantly increased mRNA levels of proinflammatory cytokines (TNFα and IL-1β) compared to non-db EVs (n=5; p<0.01). Proteomic analysis showed higher levels of pro-inflammatory agents in db EVs, including adapter proteins (HSP-90α/β, HSP-70, Protein 14-3-3α/β/ε/θ, and endoplasmic reticulum chaperone BiP), several metabolic enzymes, histone nucleosomal proteins, and other proteins (Clathrin heavy chain 1, Arrestin-C, Recoverin, Ubiquitin-like modifier-activating enzyme-1, Peroxiredoxin-1) compared to non-db EVs.

Conclusions : Our results suggest a potential involvement of EC-derived Cldn5 in triggering inflammation, offering a novel mechanism involved and presenting a possible therapeutic avenue for DR.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.