PurposeCanonical Wnt signaling is associated with glaucoma pathogenesis and intraocular pressure (IOP) regulation. Our goal was to gain insight into the influence of non-pigmented ciliary epithelium (NPCE)-derived exosomes on Wnt signaling by trabecular meshwork (TM) cells. The potential impact of exosomes on Wnt signaling in the ocular drainage system remains poorly understood.MethodsExosomes isolated from media collected from cultured NPCE cells by differential ultracentrifugation were characterized by dynamic light scattering (DLS), tunable resistive pulse sensing (TRPS), and nanoparticle tracking analysis (NTA), sucrose density gradient migration and transmission electron microscopy (TEM). The cellular target specificity of the NPCE-derived exosomes was investigated by confocal microscopy-based monitoring of the uptake of DiD-labeled exosomes over time, as compared to uptake by various cell lines. Changes in Wnt protein levels in TM cells induced by NPCE exosomes were evaluated by Western blot.ResultsExosomes derived from NPCE cells were purified and detected as small rounded 50–140 nm membrane vesicles, as defined by DLS, NTA, TRPS and TEM. Western blot analysis indicated that the nanovesicles were positive for classic exosome markers, including Tsg101 and Alix. Isolated nanoparticles were found in sucrose density fractions typical of exosomes (1.118–1.188 g/mL sucrose). Using confocal microscopy, we demonstrated time-dependent specific accumulation of the NPCE-derived exosomes in NTM cells. Other cell lines investigated hardly revealed any exosome uptake. We further showed that exosomes induced changes in Wnt signaling protein expression in the TM cells. Western blot analysis further revealed decreased phosphorylation of GKS3β and reduced β-catenin levels. Finally, we found that treatment of NTM cells with exosomes resulted in a greater than 2-fold decrease in the level of β-catenin in the cytosolic fraction. In contrast, no remarkable difference in the amount of β-catenin in the nuclear fraction was noted, relative to the control.ConclusionsThe data suggest that NPCE cells release exosome-like vesicles and that these nanoparticles affect canonical Wnt signaling in TM cells. These findings may have therapeutic relevance since canonical Wnt pathway is involved in intra-ocular pressure regulation. Further understanding of NPCE-derived exosome-responsive signaling pathways may reveal new targets for pharmacological intervention within the drainage system as a target for glaucoma therapy.

Primary open-angle glaucoma is a leading cause of irreversible blindness, often associated with increased intraocular pressure. Extracellular vesicles (EVs) carry a specific composition of proteins, lipids and nucleotides have been considered as essential mediators of cell-cell communication. Their potential impact for crosstalk between tissues responsible for aqueous humour production and out-flow is largely unknown.The study objective was to investigate the effects of EVs derived from non-pigmented ciliary epithelium (NPCE) primary cells on the expression of Wnt proteins in a human primary trabecular meshwork (TM) cells and define the mechanism underlying exosome-mediated regulation that signalling pathway. Consistent with the results in TM cell line, EVs released by both primary NPCE cells and NPCE cell line showed diminished pGSK3β phosphorylation and decreased cytosolic levels of β-catenin in primary TM cells. At the molecular level, we showed that NPCE exosome treatment downregulated the expression of positive GSKβ regulator-AKT protein but increased the levels of GSKβ negative regulator-PP2A protein in TM cells. NPCE exosome protein analysis revealed 584 miRNAs and 182 proteins involved in the regulation of TM cellular processes, including WNT/β-catenin signalling pathway, cell adhesion and extracellular matrix deposition. We found that negative modulator of Wnt signalling miR-29b was abundant in NPCE exosomal samples and treatment of TM cells with NPCE EVs significantly decreased COL3A1 expression. Suggesting that miR-29b can be responsible for decreased levels of WNT/β-catenin pathway. Overall, this study highlights a potential role of EVs derived from NPCE cells in modulating ECM proteins and TM canonical Wnt signalling. K E Y W O R D S exosomes, extracellular matrix, extracellular vesicles, non-pigmented ciliary epithelium, primary open-angle glaucoma, trabecular meshwork, Wnt | 4647 LERNER Et aL.

The importance of extracellular vesicles (EVs) as signaling mediators has been emphasized for several pathways with only limited data regarding their role as protective messages during oxidative stress (OS). The ocular drainage system is unique by being continuously exposed to OS and having a one-way flow of the aqueous humor carrying EVs taking role in glaucoma disease. Here, we aimed to examine the ability of EVs derived from the non-pigmented ciliary epithelium (NPCE)—the aqueous humor producing cells exposed to OS—to deliver protecting messages to the trabecular meshwork (TM)—the aqueous humor draining cells—a process with significance to the pathophysiology of glaucoma disease. EVs extracted from media of NPCE cells exposed to non-lethal OS and their unstressed control were incubated with TM cells. The effects of EVs derived from oxidative stressed cells on the activation of the nuclear factor erythroid 2-related factor 2-Kelch-like ECH-associated protein 1 (Nrf2-Keap1), a major OS pathway, and of the Wnt pathway, known for its role in primary open-angle glaucoma, were evaluated. EVs derived from oxidized NPCE cells significantly protected TM cells from direct OS. The TM cells uptake of EVs from oxidized NPCE and their cytosolic Nrf2 levels were significantly higher at 8 h post-exposure. EVs derived from oxidized NPCE cells significantly attenuated Wnt protein expression in TM cells and activated major antioxidant genes as measured by qRT-PCR. TM cells exposed to EVs derived from oxidized NPCE cells exhibited significantly lower OS and higher super oxide dismutase and catalase activity. Finally, we were able to show that carbonylated proteins and products of oxidized protein are presented in significantly higher levels in EVs derived from oxidized NPCE cells, supporting their suggested role in the signaling process. We hypothesize that these findings may have implications beyond understanding the pathophysiology of glaucoma disease and that transmitting signals that activate the antioxidant system in target cells represent a broad response common to many tissues communication.

PurposeIt is assumed that the non-pigmented ciliary epithelium plays a role in regulating intraocular pressure via its neuroendocrine activities. To test this hypothesis, we investigated the effect on a human trabecular meshwork (TM) cell line (NTM) of co-culture with a human non-pigmented ciliary epithelium cell line (ODM-2).MethodsThe cellular cross-talk between ODM-2 and NTM cells was studied in a co-culture system in which the two cell types were co-cultured for 5 to 60min or 2, 4 and 8h and then removed from the co-culture and analyzed. Analyses of the ERK and p38 mitogen-activated protein kinase (MAPK) pathways and of the activity of TM phosphatases and matrix metalloproteins (MMPs) were performed. Acid and alkaline phosphatase activity was determined by the DiFMUP (6, 8-difluoro-4-methylumbelliferyl phosphate) assay. MMP levels were determined by gelatin zymography.ResultsExposure of NTM cells to ODM-2 cells led to the activation of the MAPK signal transduction pathways in NTM cells within 5min of co-culture. Phosphorylation of ERK1/ERK2 and p38 peaked at 10 and 15min and then decreased over time. Interaction between ODM-2 and NTM cells promoted the expression of MMP-9 in the NTM cells after 4h of co-culture.ConclusionsOur findings provide support for the hypothesis that crosstalk does indeed take place between ODM-2 and NTM cells. Future studies should be designed to determine the relationship between the MMP system, MAPK kinases and phosphatases. Manipulation of these signaling molecules and the related NTM signal transduction pathways may provide targets for developing improved treatments for glaucoma.