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A more recent version of this article appeared on March 1, 2009
 (Investigative Ophthalmology and Visual Science. )
© 2008 by The Association for Research in Vision and Ophthalmology, Inc.
doi:10.1167/iovs.08-2326
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Article

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

Emely A Hoffman 1, Kristin M Perkumas 1, Lindsey Highstrom 2, and W. Daniel Stamer 3*

1 Ophthalmology and Vision Science, The University of Arizona, Tucson, Arizona, United States
2 Pharmacology, The University of Arizona, Tucson, Arizona, United States
3 Department of Ophthalmology, University of Arizona, 655 N. Alvernon Way, Suite 108, Tucson, Arizona, 85711-1824, United States

* To whom correspondence should be addressed. E-mail: dstamer{at}eyes.arizona.edu.


  Abstract

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 {gamma}-interferon, 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: While treatment of TM cells with {gamma}- interferon increased the appearance of extracellular MYOC-associated exosomes, results were not significantly different than control (p=0.13). In contrast, treatment with dexamethasone increased appearance of MYOC in the exosome fraction by 376% (p<0.01). The increase in MYOC-associated exosomes due to dexamethasone was enhanced by an additional 379% following 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 3kD or 30kD, but not a 100 kD 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 queues by releasing MYOC-associated exosomes.

Key Words: aqueous flow, glaucoma pharmacology, intraocular pressure, genetic diseases, trabecular meshwork






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