Extracellular vesicles mediate signaling between the aqueous humor producing and draining cells in the ocular system

Abstract

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.

Extracellular vesicles have variable dose‐dependent effects on cultured draining cells in the eye

“…The mechanism of uptake of NPCE‐derived EV by NTM cells is yet unknown, and is presently assumed to be related to partially specific diffusion or uptake , as opposed to specific EVs recognition by a membrane transporter . The effect of NPCE‐derived EVs on NTM cells in our study was specific to the expression of Wnt signalling pathway components.…”

“…Data obtained by ImageStream analysis supported the entry of the EVs as fast as 8 hrs of incubation. In our previous report, 4 hrs of NPCE incubation at X1 resulted in a beginning of EVs accumulation within the NTM target cell by 4 hrs, reaching a maximum at 12 hrs .…”

“…Other biological effects including related gene expression and downstream MMPs activity were found to exhibit a general dose–response effect. A trend in the gene expression changes detected as early as 2 hrs of incubation was reported to be significant at low EVs amount (X1) following 4 hrs of EVs incubation with the NTM cells , earlier to the pGSK3β and β‐catenin changes at 8 hrs of EVs incubation. We hypothesize that a direct effect like a ligand binding to the Wnt receptor on NTM cells might result in the gene expression changes while the protein expression changes at 8 hrs of EVs incubation can reflect endocytosis processes.…”

Extracellular vesicle‐mediated crosstalk between NPCE cells and TM cells result in modulation of Wnt signalling pathway and ECM remodelling

“…Previously, we provided evidence that NPCE cells secrete EVs that were internalized specifically by TM cells in culture. In addition, we demonstrated that incubation of TM cells with NPCE‐derived EVs induced a significant decrease in the expression of the Wnt signalling pathways proteins and genes in human NPCE and TM cell lines …”

Altered expression of exosomal miRNAs from primary human trabecular meshwork cells induced by transforming growth factor-β2

“…Variable intraocular cells can secrete exosomes into the AH, and numerous molecules, particularly miRNAs, have been shown to change in response to ocular disease or injury. Recent study shows that non-pigmented ciliary epithelium cell (NPCE)-derived exosomes could affect canonical Wnt signaling in TM cells, a pathway involved in IOP regulation[53]. Thus, we suspect that the changes of exosomal miRNAs mediate cell-cell communication and play a major role in the crosstalk between TM cells and the macro−/microenvironment.The miRNA-miRNA interaction network showed that during the occurrence and development of diseases.…”