Date Published: February 27, 2017
Publisher: Public Library of Science
Author(s): Natalie Lerner, Sofia Avissar, Elie Beit-Yannai, Masaru Katoh.
Canonical 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.
Exosomes 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.
Exosomes 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.
The 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.
Aqueous humor, previously considered as a nutrient supplier and waste product disposal from the ocular front section , is now accepted as a mediator of cell-cell communication. Proteomic analysis demonstrated that hundreds of protein are present in AH  and that their composition changes under pathological conditions . The presence of active factors in the ocular drainage system suggests that they assume an active role in drainage system homeostasis and pathology. Still, which tissue releases these active factors and what is their specific target remain open questions [6,7,19].
In the present study, we characterized NPCE derived EV and studied their effects on the Wnt signaling in TM cells. According to our in-vitro results we suggest that EV originated within the NPCE can influence the trabecular meshwork cadherin’s via reduction of phospho-GSK3 and β-catenin expression. This pathway is proposed to be part of the drainage system homeostasis.