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NATALIE LERNER; Trabecular meshwork Wnt signaling pathway is altered by non-Pigmented ciliary epithelium derived exosomes.. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3014. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Canonical Wnt signaling is associated with glaucoma pathogenesis and intraocular pressure (IOP) regulation. The potential role of exosomes on Wnt signaling in ocular drainage system remains poorly understood. Our goal is to provide insights into the influence of non-Pigmented ciliary epithelium (NPCE) derived exosomes on Trabecular meshwork (TM) cells Wnt signaling.
We isolated exosomes from media collected from cultured human NPCE cell by differential ultracentrifugation. The exosomes were characterized with Dynamic Light Scattering (DLS), Tunable Resistive Pulse Sensing (TRPS), Nanoparticle tracking analysis (NTA), Sucrose sedimentation and Transmission electron microscopy (TEM). The cellular target specificity of the NPCE exosomes was investigated using DiD-labeled exosomes incubated with human TM cells as compared to various cell lines and the uptake of exosomes was monitored by confocal microscopy along time. Wnt protein changes in TM cells induced by NPCE exosomes were evaluated using Western Blot technique.
Exosomes derived from NPCE cells were purified and detected as small rounded membrane vesicles within a range of 50-140 nm 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 the sucrose density fractions typical for exosomes (1.118-1.188g/mL). Using confocal microscopy we demonstrated time-dependent specific accumulation of the NPCE labeled exosomes in NTM cells. We further showed that exosomes induce changes in Wnt signaling proteins expression in the TM cells. Western blot analysis revealed decreased phosphorylation of GKS3β protein and reduced β-catenin protein levels. We found that treatment of the NTM cells with exosomes resulted in a greater than 2-fold decrease in the cytosolic fraction of β-catenin. In contrast, no remarkable difference in the β-catenin nuclear fraction was identified as compared to 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 IOP regulation. Further studies on exosomes in glaucoma may provide new therapeutic strategies.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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