title: Extracellular vesicles mediate signaling between the aqueous humor producing and draining cells in the ocular system.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Clinical Biochemistry and Pharmacology Department, The Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
authors.forename: Natalie
authors.initials: N
authors.lastname: Lerner
authors.affiliation: Clinical Biochemistry and Pharmacology Department, The Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
authors.forename: Sofia
authors.initials: S
authors.lastname: Avissar
authors.affiliation: Clinical Biochemistry and Pharmacology Department, The Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
authors.forename: Elie
authors.identifier: http://orcid.org/0000-0001-9347-822X
authors.initials: E
authors.lastname: Beit-Yannai
mesh_terms: D001082:Aqueous Humor / Q000166:cytology*; D002135:Calcium-Binding Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D002924:Ciliary Body / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D055354:Exosomes / Q000378:metabolism; D000067128:Extracellular Vesicles / Q000378:metabolism*; D005123:Eye / Q000166:cytology*; D005901:Glaucoma / Q000378:metabolism / Q000628:therapy*; D006801:Humans; D007429:Intraocular Pressure; D008027:Light; D053758:Nanoparticles / Q000737:chemistry; D012542:Scattering, Radiation; D015398:Signal Transduction; D013395:Sucrose / Q000737:chemistry; D014129:Trabecular Meshwork / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D060449:Wnt Signaling Pathway; D051176:beta Catenin / Q000378:metabolism; D000099177:Tsg101 Protein
chemical_list: D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D013395:Sucrose; D014157:Transcription Factors; D051176:beta Catenin; D000099177:Tsg101 Protein; C117020:PDCD6IP protein, human