Background Diabetic retinopathy (DR) is a leading cause of vision loss in patients with diabetes mellitus due to damage of the retinal microvasculature, primarily involving retinal endothelial cells (HRECs) and pericytes (HRPs). HRPs regulate vascular stability, while HRECs maintain the blood-retina barrier. Dysfunction in these cells leads to retinal vascular leakage, pathologic neovascularization, and vision loss. Small extracellular vesicles (sEVs) are membrane bound nanoparticles that mediate intercellular signaling and have been implicated in DR-related vascular damage. While sEVs contribute to oxidative stress and angiogenesis, the role of retinal pericyte-derived sEVs on retinal endothelial dysfunction under diabetic stress remains unclear. This study investigates the impact of pericyte-derived sEVs on endothelial barrier integrity, metabolism, and angiogenic activity.

Methods HRPs were cultured in conditioned media under high glucose and hypoxic conditions as a diabetic condition, or mannitol and normoxic conditions as a control. The HRP cell medium was then collected after 48 hours, and the sEVs were obtained using the EXODUS exosome isolation system. sEV size distribution and concentration was assessed using a nanoparticle tracking analyzer (NTA) instrument which measured sEV hydrodynamic diameter and concentration. sEV morphology was confirmed using transmission electron microscopy (TEM). HRECs were then treated with the HRP derived sEVs, and functional assays were conducted. Trans-endothelial electrical resistance (TEER) was measured with cell-covered electrodes and endothelial cell permeability was measured with fluorescently labeled tracer dye diffusion across an HREC monolayer on a Transwell insert. Additional assays included MTT-based metabolic activity and scratch wound migration to assess angiogenesis.

Results Nanoparticle tracking analysis showed no significant differences in sEV size or concentration between the two conditions. Transmission electron microscopy showed no differences in sEV structure and morphology between the two conditions. However, sEVs from diabetic-stressed HRPs significantly reduced HREC trans-endothelial resistance (TEER) and increased HREC permeability in Transwell assays compared to the control. This suggests compromised barrier integrity in retinal blood vessels. At lower sEV doses, metabolic assays showed higher HREC viability in response to control HRP-derived sEVs compared to high glucose HRP-derived sEVs. Cell migration assays revealed that HRECs treated with diabetic stress HRP sEVs exhibited faster growth, consistent with angiogenesis and neovascularization in DR.

Conclusion These findings suggest that sEVs from HRPs under diabetic stress promote retinal vascular permeability and endothelial dysfunction, contributing to DR pathogenesis. Targeting sEV release could provide a novel therapeutic approach for preventing or treating DR. Further research is needed to elucidate the mechanisms involved and potential clinical benefits.