Myocilin (MYOC) is a protein with a broad expression pattern, but unknown function. MYOC associates with intracellular structures that are consistent with secretory vesicles, however, in most cell types studied, MYOC is limited to the intracellular compartment. In the trabecular meshwork, MYOC associates with intracellular vesicles, but is also found in the extracellular space. The purpose of the present study was to better understand the mechanism of extracellular transport of MYOC in trabecular meshwork cells. Using a biochemical approach, we found that MYOC localizes intracellularly to both the cytosolic and particulate fractions. When intracellular membranes were separated over a linear sucrose gradient, MYOC equilibrated in a fraction less dense than traditional secretory vesicles and lysosomes. In pulse-labeling experiments that followed nascent MYOC over time, the characteristic doublet observed for MYOC by SDS-PAGE did not change, even in the presence of brefeldin A; indicating that MYOC is not glycosylated and is not released via a traditional secretory mechanism. When conditioned media from human trabecular meshwork cells were examined, both native and recombinant MYOC associated with an extracellular membrane population having biochemical characteristics of exosomes, and containing the major histocompatibility complex class II antigen, HLA-DR. The association of MYOC with exosome-like membranes appeared to be specific, on the extracellular face, and reversible. Taken together, data suggest that MYOC appears in the extracellular space of trabecular meshwork cells by an unconventional mechanism, likely associated with exosome-like vesicles. Myocilin (MYOC),1 also known as trabecular meshwork inducible glucocorticoid response protein, is an acidic 504-amino acid protein. Structurally, MYOC contains at least two folding domains, an N-terminal coiled-coil and a C-terminal globular domain with significant homology to an olfactomedin module present in several different proteins (1, 2). Mammalian proteins with the olfactomedin module localize to different compartments of the secretory pathway, although little is known about the function of these proteins or the olfactomedin module (3-10).Despite a broad expression pattern (1, 2, 7, 11-13), the function of MYOC remains unknown and its cellular distribution ambiguous. For example, MYOC has been observed in various cell types to associate with structures that are part of the secretory pathway, including endoplasmic reticulum, Golgi apparatus, and intracellular vesicles. Conversely, MYOC has also been reported to associate with mitochondria and cytoplasmic filaments (11, 14 -17). Even more unusual, MYOC appears to be secreted by some cell types, but not by others (2, 18). Thus, MYOC is expressed by retinal ganglion cells, photoreceptors, and retinal pigment epithelium, but is not found extracellularly in the retina nor in conditioned medium of retinal pigment cells in culture (2,18,19). In contrast, MYOC is found in conditioned medium of trabecular meshwork (TM) ...

To better understand the role of exosomes in the trabecular meshwork (TM), the site of intraocular pressure control, the exosome proteome from primary cultures of human TM cell monolayers was analyzed. Exosomes were purified from urine and conditioned media from primary cultures of human TM cell monolayers and subjected to two dimensional HPLC separation and MS/MS analyses using the MudPIT strategy. Spectra were searched against a human protein database using Sequest. Protein profiles were compared to each other and the Exocarta database and the presence of specific protein markers confirmed by Western blot analyses of exosomes from aqueous humor and human TM cell strains (n=5) that were untreated, or exposed to dexamethasone and/or ionomycin. TM cell exosomes contained 108 of the 143 most represented exosome proteins in ExoCarta, including previously characterized markers such as membrane organizing and tetraspanin proteins. Several cell-specific proteins in TM exosomes were identified including myocilin, emilin-1 and neuropilin-1. All TM exosome proteins had flotation densities on sucrose gradients and release responses to ionomycin typical for exosomes. Taken together, TM exosomes have a characteristic exosome protein profile plus contain unique proteins, including the glaucoma-causing protein, myocilin; suggesting a role for exosomes in the control of intraocular pressure.

Advances in haploidentical bone marrow transplantation (h-BMT) have drastically broadened the treatment options for patients requiring BMT. The possibility of significantly reducing the complications resulting from graft-versus-host disease (GvHD) with the administration of post-transplant cyclophosphamide (PT-CY) has substantially improved the efficacy and applicability of T cell-replete h-BMT. However, higher frequency of disease recurrence remains a major challenge in h-BMT with PT-CY. There is a critical need to identify novel strategies to prevent GvHD while sparing the graft-versus-leukaemia (GvL) effect in h-BMT. To this end, we evaluated the impact of bendamustine (BEN), given post-transplant, on GvHD and GvL using clinically relevant murine h-BMT models. We provide results indicating that post-transplant bendamustine (PT-BEN) alleviates GvHD, significantly improving survival, while preserving engraftment and GvL effects. We further document that PT-BEN can mitigate GvHD even in the absence of Treg. Our results also indicate that PT-BEN is less myelosuppressive than PT-CY, significantly increasing the number and proportion of CD11b+Gr-1hi cells, while decreasing lymphoid cells. In vitro we observed that BEN enhances the suppressive function of myeloid-derived suppressor cells (MDSCs) while impairing the proliferation of T- and B-cells. These results advocate for the consideration of PT-BEN as a new therapeutic platform for clinical implementation in h-BMT.

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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-γ, 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-γ 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.