Abstract
Purpose :
Müller cells, pivotal glial cells in the retina, have been implicated in the pathogenesis of diabetic retinopathy (DR), yet the mechanisms are unclear. This study focuses on characterizing extracellular vesicles (EVs) or exosomes derived from retinal Müller cells and their proteome under diabetic conditions.
Methods :
Human retinal Müller cells (MC: MIO-M1) were cultured under normal glucose (NG: mannitol) and high glucose (HG: 35 mM) for 3 days. EVs were isolated through differential ultracentrifugation and characterized using nanoparticle tracking analysis (NTA) and transmission electron microscopy. Liquid chromatography-tandem mass spectrometry characterized the proteome of cells and EVs. Western blots (WB) and flow cytometry validated differentially expressed proteins (DEPs) between NG and HG conditions, and CD9-GFP reporter mice confirmed EV secretion by MCs.
Results :
The EVs’ physical features, particle sizes, concentrations, and morphology are not different between NG/ HG conditions: The mean particle sizes are 143.17 ± 32.25 nm for NG and 148.69 ± 28.26 nm for HG (p=0.18; n = 11). The average concentrations are 1.98E+09 ± 2.12E+09 particles/ml for HG and 2.18E+09 ± 3.43E+09 particles/ml for NG (p=0.17, n=11). The TEM images of HG and NG are similar. Proteomics analysis identified and characterized the proteome of MCs and secreted EVs. Using a protein FDR (false discovery rate) of <1% 7,946 proteins were identified for cells and 2,605 for EVs, with 2,155 in common, 450 only in EVs, and 5,831 only in cells. Compared to NG control, HG upregulates 102 proteins for cells and 92 for EVs and downregulates 218 proteins for cells and 824 for EVs. The EV marker proteins HSP70/90, CD9, CD81, CD63, Syntenin-1, and ESCRT complex I/III were significantly downregulated under HG conditions. Among the DEPs in EVs, ten RNA-binding proteins were selected for validation: ARF3, RBMX, SLP1, UBB, SMARCA2, EIF1AY, 4BPA, SNRPG, AHCYL1, CBX6. The HG-induced downregulation of EVs CD81, CBX6, SMARCA2, and AHCYL1 was validated by WB. Further validation and function studies for EV proteins and in-vivo studies are ongoing.
Conclusions :
Diabetes-induced abnormal EV secretion and disrupted protein cargos from Müller cells may contribute to DR development.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.