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W Michael Dismuke, W Daniel Stamer; Trabecular meshwork exosomes enhance cellular collagen uptake. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3025.
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© ARVO (1962-2015); The Authors (2016-present)
Extracellular nanovesicles called exosomes have an unknown function in the anterior chamber of the eye. Exosomes released from human trabecular meshwork (TM) cells contain collagen and collagen binding proteins. Since extracellular matrix turnover and homeostasis prominently impacts outflow facility, here we test the hypothesis that TM cell derived exosomes affect collagen uptake by TM cells.
Confluent human TM cell monolayers grown in 1% FBS DMEM were serum starved overnight. Cells remained in serum-free media or were treated with 10% FBS (exosome depleted) for 2.5 hours. Media was collected, and exosomes were prepared from the conditioned media by serial ultracentrifugation steps. Protein composition of exosomes was determined by western blot. To assay collagen uptake, human TM cells were seeded in 96 wells plates with media containing FITC conjugated bovine collagen I and exosomes (prepared from either stimulated or unstimulated TM cells) were added. After 16hr incubation, media was removed, cells were washed with PBS and internalized FITC fluorescence was quantified with a plate reader.
Human TM cells stimulated with FBS release exosomes displaying the opsonin milk fat globule-EGF factor 8 (MFG-E8), also known as lactadherin. Regardless of stimulation, exosome marker proteins annexin A2 and 6 were present on TM exosomes in equal amounts. Collagens I and XII were present in preparations of MFG-E8 positive exosomes but absent in exosomes from unstimulated cells. TM cells incubated with MFG-E8 positive exosomes showed a 39±11% (mean±SEM) increase in FITC-collagen I uptake compared to exosome-free controls (p=0.0435, n=5). Surprisingly, MFG-E8 negative exosomes also increased uptake of FITC collagen I 28±9% (mean±SEM) versus exosome-free controls (p=0.083, n=5).
Data demonstrate that TM cells rapidly release exosomes bound to the opsonin protein MFG-E8 in response to external stimuli. Released exosomes co-purify with collagen and increase the rate of collagen internalization by TM cells. These findings suggest a novel role for TM derived exosomes in ECM homeostasis in the aqueous humor outflow pathway and intraocular pressure regulation.
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