Image_2_Comparison of the extracellular vesicle proteome between glaucoma and non-glaucoma trabecular meshwork cells.tif

IntroductionExtracellular matrix (ECM) materials accumulate in the trabecular meshwork (TM) tissue of patients with glaucoma, which is associated with a decrease in aqueous humor outflow and therefore an increase in intraocular pressure. To explore a potential mechanism for ECM regulation in the TM, we purified extracellular vesicles (EVs) from conditioned media of differentiated TM cells in culture isolated from non-glaucomatous and glaucomatous human donor eyes. MethodsEVs were purified using the double cushion ultracentrifugation gradient method. Fractions containing EV markers CD9 and TSG101 were analyzed using nanoparticle tracking analysis to determine their size and concentration. We then determined their proteomic cargo by mass spectrometry and compared protein profiles of EVs between normal and glaucomatous TM cells using PANTHER. Key protein components from EV preparations were validated with Western blotting. ResultsResults showed changes in the percentage of ECM proteins associated with EVs from glaucomatous TM cells compared to non-glaucomatous TM cells (5.7% vs 13.1% respectively). Correspondingly, we found that two ECM-related cargo proteins found across all samples, fibronectin and EDIL3 were significantly less abundant in glaucomatous EVs (DiscussionOverall, these data establish that ECM materials are prominent proteomic cargo in EVs from TM cells, and their binding to EVs is diminished in glaucoma.