Proteomics analysis of small extracellular vesicles derived from human retinal Müller glial cells under high glucose...

Diabetic retinopathy (DR) is a leading cause of vision loss and is characterized by early dysfunction of the retinal neurovascular unit (NVU), preceding neurodegenerative and vascular pathology. Müller glial cells are central regulators of retinal homeostasis, but the contribution of Müller glia-derived small extracellular vesicles (sEVs) to intercellular communication during hyperglycemic stress remains incompletely understood. Here, we examined the effects of high-glucose (HG) challenge on sEV release and protein cargo in the immortalized human Müller glial cell line MIO-M1. Cells were exposed to 35 mM D-glucose or an osmotic control. sEVs were isolated from cell culture media and characterized by nanoparticle tracking analysis, transmission electron microscopy, and immunoblotting. Quantitative proteomic profiling was performed using data-independent acquisition mass spectrometry. HG exposure did not significantly change sEV size distribution or particle yield but changed the proteomic profiles of both Müller glia cells and their secreted sEVs. Differentially abundant proteins were enriched in pathways and functional classes related to RNA binding and processing, protein translation, cytoskeletal organization, carbon metabolism, lysosomal function, and immune/inflammatory signaling. These findings provide a new proteomic resource and identify candidate pathways through which HG stress may modify Müller glia cellular and sEV protein profiles. Because the study used an immortalized cell line, a short-term in vitro exposure, and did not directly test the effects of HG-modified sEVs on recipient retinal cells, the functional and in-vivo relevance of these proteomic changes requires further investigation.