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W. Michael Dismuke, W Daniel Stamer; A role for exosomes in steroid induced ECM accumulation in the TM. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© 2017 Association for Research in Vision and Ophthalmology.
Human trabecular meshwork (TM) explants release extracellular nanovesicles (EVs) called exosomes. However, the function(s) of these exosomes in the conventional outflow pathway is unclear. Corticosteroids, like dexamethasone (DEX) alter the turnover of extracellular matrix (ECM) materials in the TM, including increasing the secretion of fibronectin (Fn). TM exosomes bind Fn and increase the cellular uptake of collagen. Hence, we hypothesize that DEX treatment of the TM alters the properties and protein cargo of its exosomes.
TM from whole globes or corneal rims was dissected and cultured in 1% exosome-free FBS or 0.1x B27 supplemented DMEM. For DEX treatments the TM from a single eye was split into two equal portions, one half untreated and the other treated with 100nM DEX. Exosomes were prepared from conditioned media by ultracentrifugation or precipitation and characterized by western blot, density gradients and nanoparticle tracking analysis. For pulldown assays, extracellular proteins were labeled with NHS-SS-biotin and exosomes were prepared after 2.5hr incubation in serum free media. Biotinylated proteins on exosomes were captured with streptavidin beads and identified by LC-MS/MS. For exosome/heparan sulfate(HS)/Fn binding assays, bound Fn was determined by dot blot.
Human TM explants released EVs with the size, proteins and density consistent with exosomes. DEX treatment did not significantly alter the mean and modal size of the exosomes or the amount released. In the TM, DEX treatment increased secretion of Fn yet exosomes from DEX treated TM explants had a significantly lower amount of Fn bound than controls. A pulldown assay identified annexinA2 (AnxA2) as the exosomal protein binding Fn. Consistent with reduced Fn binding, exosomes from DEX treated TM explants had significantly less AnxA2 than controls. AnxA2 does not bind Fn directly, yet AnxA2 and Fn bind HS. We tested whether Fn binds AnxA2 coated exosomes via HS. At 100pg/ml, HS significantly increased exosomes-Fn binding while 50U/ml heparanase II abolished Fn binding ability.
Human TM explants release exosomes. Treatment of TM explants with DEX alters the sorting of protein cargo to the exosomes which, in turn, alters their adhesive properties. Fn is considered a “master organizer” of the ECM and we show exosomes bind Fn via AnxA2 and HS. This novel mechanism for Fn binding likely plays an important role in ECM homeostasis.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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