Emely A. Hoffman

Extracellular Trafficking of Myocilin in Human Trabecular Meshwork Cells

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.

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.

Role of Aquaporin-1 in Trabecular Meshwork Cell Homeostasis during Mechanical Strain

Aquaporin-1 (AQP1) channels are expressed by trabecular meshwork (TM) and Schlemms canal cells of the conventional outflow pathway where fluid movement is predominantly paracellular, suggesting a non-canonical role for AQP1. We hypothesized that AQP1 functions to protect TM cells during periods of mechanical strain. To test this idea, primary cultures of confluent human TM cells on Bioflex membranes were exposed to static and cyclic stretch for 8 and 24h using the Flexcell system. AQP1 expression in TM cells was assessed by SDS-PAGE and Western blot using anti-AQP1 IgGs. AQP1 protein bands were analyzed using densitometry and normalized to beta-actin expression. Cell damage was monitored by measuring lactate dehydrogenase (LDH) and histone deacetylase appearance in conditioned media. Recombinant expression of AQP1 in TM cell cultures was facilitated by transduction with adenovirus. Results show that AQP1 expression significantly increased 2-fold with 10% static stretch and 3.5-fold with 20% static stretch at 8h (n=4, p<0.05) and 24h (n=6, p<0.05). While histone deacetylase levels were unaffected by treatments, release of LDH from TM cells was the most profound at the 20% static stretch level (n=4, p<0.05). Significantly, cells were refractory to the 20% static stretch level when AQP1 expression was increased to near tissue levels. Analysis of LDH release with respect to AQP1 expression revealed an inverse linear relationship (r(2)=0.7780). Taken together, AQP1 in human TM appears to serve a protective role by facilitating improved cell viability during conditions of mechanical strain.

Unique Response Profile of Trabecular Meshwork Cells to the Novel Selective Glucocorticoid Receptor Agonist, GW870086X

PURPOSE Glucocorticoid (GC)-induced glaucoma is an undesirable side effect of traditional GCs. Ocular hypertension responsible for GC-induced glaucoma is due to alterations in conventional outflow homeostasis. The present study evaluates a novel selective GC receptor agonist (SEGRA), GW870086X, in two different in vitro models of the human conventional outflow pathway. METHODS Primary cultures of human trabecular meshwork (TM) cell monolayers were treated with dexamethasone (DEX), prednisolone (PRED), or GW870086X for 5 days and then assayed for cellular expression and secretion of fibronectin, myocilin, tissue plasminogen activator (tPA), and/or matrix metalloproteinase-2 (MMP2). In parallel, TM cell monolayers on permeable filters treated for 5 days with GCs were assayed for changes in hydraulic conductivity. RESULTS All three GCs increased fibronectin and myocilin secretion in a concentration-dependent manner (P < 0.05). In addition, DEX increased cellular fibronectin and both DEX and PRED significantly increased cellular myocilin (P < 0.0001), while GW870086X did neither. Interestingly, DEX and PRED significantly decreased tPA expression (P ≤ 0.01), while GW870086X had the opposite effect and increased tPA expression in a concentration-dependent manner (P = 0.01). For MMP2, only DEX treatment consistently decreased secretion (P < 0.01). In a functional assay, only PRED treatment significantly decreased hydraulic conductivity of TM cell monolayers (P < 0.05). CONCLUSIONS All three GCs induced differential responses from TM cells. While the novel SEGRA GW870086X increases fibronectin and myocilin secretion similar to two traditional GCs, effects on the matrix degradation enzymes MMP2 and tPA differed significantly, suggesting that GW870086X favors matrix turnover. Consequently, effects on conventional outflow homeostasis may also be dissimilar.

Post-transplant bendamustine reduces GvHD while preserving GvL in experimental haploidentical bone marrow transplantation

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.

Pak2 regulates myeloid-derived suppressor cell development in mice

Myeloid-derived suppressor cells (MDSCs) are CD11b+Gr1+ cells that induce T-cell hyporesponsiveness, thus impairing antitumor immunity. We have previously reported that disruption of Pak2, a member of the p21-activated kinases (Paks), in hematopoietic stem/progenitor cells (HSPCs) induces myeloid lineage skewing and expansion of CD11bhighGr1high cells in mice. In this study, we confirmed that Pak2-KO CD11bhighGr1high cells suppressed T-cell proliferation, consistent with an MDSC phenotype. Loss of Pak2 function in HSPCs led to (1) increased hematopoietic progenitor cell sensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling, (2) increased MDSC proliferation, (3) decreased MDSC sensitivity to both intrinsic and Fas-Fas ligand-mediated apoptosis, and (4) promotion of MDSCs by Pak2-deficient CD4+ T cells that produced more interferon γ, tumor necrosis factor α, and GM-CSF. Pak2 disruption activated STAT5 while downregulating the expression of IRF8, a well-described myeloid transcription factor. Together, our data reveal a previously unrecognized role of Pak2 in regulating MDSC development via both cell-intrinsic and extrinsic mechanisms. Our findings have potential translational implications, as the efficacy of targeting Paks in cancer therapeutics may be undermined by tumor escape from immune control and/or acceleration of tumorigenesis through MDSC expansion.

Bendamustine with total body irradiation limits murine graft-versus-host disease in part through effects on myeloid-derived suppressor cells

Graft-versus-host disease (GVHD) remains a significant challenge in allogeneic hematopoietic cell transplantation (HCT). An underinvestigated strategy to reduce GVHD is the modification of the preparative conditioning regimen. In the present study, we aimed to evaluate GVHD associated with bendamustine (BEN) conditioning in conjunction with total body irradiation (TBI) as an alternative to the standard myeloablative regimen of cyclophosphamide (CY) and TBI. We demonstrate that BEN-TBI conditioning, although facilitating complete donor chimerism, results in significantly less GVHD compared with CY-TBI. In BEN-TBI-conditioned mice, suppressive CD11b+Gr-1high myeloid cells are increased in the blood, bone marrow, spleen, and intestines. When Gr-1high cells are depleted before transplantation, the beneficial effects of BEN-TBI are partially lost. Alternatively, administration of granulocyte colony-stimulating factor, which promotes CD11b+Gr-1+ myeloid cell expansion, is associated with a trend toward increased survival in BEN-TBI-conditioned mice. These findings indicate a potential role of myeloid-derived suppressor cells in the mechanism by which BEN allows engraftment with reduced GVHD. BEN-TBI conditioning may present a safer alternative to CY-TBI conditioning for allogeneic HCT.