Small extracellular vesicles derived from human retinal pericytes under high glucose and hypoxia conditions promote endothelial cell dysfunction invitro.

PURPOSE: Diabetic retinopathy (DR) is a medical complication of diabetes in which damage occurs to the retina. DR involves microvascular dysfunction of the retina, with impaired communication between pericytes and endothelial cells (EC) contributing to disease progression. This study investigated how small extracellular vesicles (sEV) released by human retinal pericytes (HRP) under diabetes-like stress conditions (high glucose (HG) + hypoxia) influence EC function.

METHODS

HRP were cultured under HG + hypoxia and mannitol (control) conditions. sEV were isolated using differential ultracentrifugation and characterized using nanoparticle tracking analysis and transmission electron microscopy. Human retinal endothelial cells (HREC) were treated with HRP-derived sEV, and assessed for metabolic activity (MTT), barrier integrity (electric cell-impedance sensing), permeability (Transwell assay), migration (scratch assay), and angiogenic potential (tube formation). Protein expression was evaluated using Western blot and immunofluorescence staining. Proteomic profiles were performed using mass spectrometry and bioinformatics analyses.

RESULTS

HRP sEV from diabetes-like stress conditions and control conditions showed no differences in size, concentration, or morphology. HREC uptake of HRP sEV was efficient and comparable across the two conditions. However, HRP sEV from diabetes-like stress conditions impaired HREC metabolic activity and barrier function while increasing permeability, migration, and angiogenesis. Conversely, HRP sEV from control conditions enhanced barrier integrity and metabolism without affecting permeability or angiogenesis. Proteomic analysis identified 86 sEV proteins that are differentially abundant between the two conditions. These proteins are enriched in pathways involved in extracellular matrix remodeling, inflammation, signaling, and metabolism.

CONCLUSIONS

HRP-derived sEV from HG + hypoxia conditions elicit endothelial dysfunctions relevant to DR pathology, in contrast to sEV from control conditions. The functional and proteomic alterations by diabetes-like stress suggest a mechanistic role for pericyte-derived sEV in DR progression and provide insights into potential therapeutic targets.