Regulation of Myocilin-Associated Exosome Release from Human Trabecular Meshwork Cells

PURPOSE. The goal of the present study was to determine whether the release of exosomes containing MYOC from trabecular meshwork (TM) cells is constitutive or regulated. METHODS. Conditioned media from TM cells were analyzed for MYOC-associated exosomes after treatment with IFN-gamma, porcine aqueous humor, dexamethasone, or a calcium ionophore in cells pretreated with dexamethasone. Aqueous humor was tested whole or fractionated by size exclusion filters. Exosomes from conditioned media were purified by differential centrifugation. Proteins in whole, exosome, and soluble fractions were separated by SDS-PAGE and analyzed for MYOC content by Western blot and densitometry. RESULTS. Although treatment of TM cells with IFN-gamma increased the appearance of extracellular MYOC-associated exosomes, results were not significantly different from those of control (P = 0.13). In contrast, treatment with dexamethasone increased the appearance of MYOC in the exosome fraction by 376% (P < 0.01). The increase in MYOC-associated exosomes caused by dexamethasone was enhanced by an additional 379% after short-term exposure to ionomycin (P < 0.05). When cultured in media containing aqueous humor, MYOC-associated exosomes increased 514% over control (P < 0.01). Such an increase was diminished in cells treated with aqueous humor that was first passed through a 3-kDa or a 30-kDa, but not a 100-kDa, size exclusion filter. CONCLUSIONS. The appearance of MYOC-associated exosomes in conditioned media from human TM cells is regulated by a corticosteroid, a calcium ionophore, and a component of aqueous humor, suggesting that TM cells respond to environmental cues by releasing MYOC-associated exosomes. ( Invest Ophthalmol Vis Sci. 2009; 50: 1313-1318) DOI:10.1167/iovs.08-2326