Cross-Talk between Ciliary Epithelium and Trabecular Meshwork Cells In-Vitro: A New Insight into Glaucoma

<div><p>Purpose</p><p>It 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).</p><p>Methods</p><p>The 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 60<b> </b>min or 2, 4 and 8<b> </b>h 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.</p><p>Results</p><p>Exposure of NTM cells to ODM-2 cells led to the activation of the MAPK signal transduction pathways in NTM cells within 5<b> </b>min of co-culture. Phosphorylation of ERK1/ERK2 and p38 peaked at 10 and 15<b> </b>min and then decreased over time. Interaction between ODM-2 and NTM cells promoted the expression of MMP-9 in the NTM cells after 4<b> </b>h of co-culture.</p><p>Conclusions</p><p>Our 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.</p></div

ERK1/2 activation in NTM cells co-cultured with ODM-2 cells for 5, 10, 15, 30, 60 min.

<p>Total protein was extracted from NTM cells. Phosphorylated and total ERK1/2 proteins were analyzed by Western blot. (A) Representative western blot film. (B) A significant increases in ERK activation in NTM cells following co-culture with ODM-2 cells. The bar graph represents the means ± SD of three independent experiments performed in duplicates. One-way ANOVA (***p<0.001, **p<0.01,*p<0.05).</p

p38 activation in NTM cells co-cultured with ODM-2 cells for 5, 10, 15, 30, 60 min.

<p>Total protein was extracted from NTM cells. Phosphorylated and total p38 proteins were analyzed by Western blot. (A) Representative western blot film. (B) A significant increases in p38 activation in NTM cells following co-culture with ODM-2 cells. The bar graph represents the means ± SD of three independent experiments performed in duplicates. One-way ANOVA (**p<0.01).</p

Schematic description of the co-culture model.

<p>Sterile round glass cover slips were placed in six-well plates, and seeded with NTM cell. The cultures were incubated for 24<b> </b>h to allow the NTM cells to attach to the cover slips. In parallel, ODM-2 as the tested cells, HCM and NTM cells as controls were seeded in 60-mm cell culture dishes. After 24<b> </b>h, two cover slips with their adhered NTM cells were introduced into each cell type culture dish.</p

Alkaline and acid phosphatase activity of NTM cells following co-culture with ODM-2 cells for 5, 10, 15, 30 or 60 min.

<p>NTM:NTM co-culuure was used as controls. (A) Alkaline phosphatase and (B) acid phosphatase activity determined by the DiFMUP assay. The quantity of DiFMU is expressed as phosphate liberated, since equivalent amounts of DiFMU and phosphate are generated in the reaction. Means of two independent experiments performed in triplicate; one-way ANOVA (***p<0.001).</p

Real-time qRT-PCR was performed to examine the mRNA expression levels (mean ± sem) of canonical WNT/β-CATENIN signaling components, including the Wnt transducers Axin2 (A), β-catenin (C), and the Wnt transcription factors LEF1 (B).

<p>Data were normalized to the average mRNA level of GADPH. Statistical difference is indicated by asterisks (*P < 0.05,).</p

Confocal microscopy of cellular uptake of DiD-labeled exosomes by different cell lines.

<p>DiD-labeled (white) NPCE-derived exosomes were incubated for 12 h with NTM, HCM, RPE, SKOV-3, LANCaP, CEND and MCF-7 cells. After incubation, the cells were washed, fixed and labeled using Alexa Fluor 488-labeled anti-α-tubulin antibodies (green) to mark the cytoskeleton and mounted in mounting medium containing DAPI (blue). Images were acquired using a confocal microscope. Scale bar: 30 μm.</p

Quantitative cellular uptake of DiD-labeled exosomes by different cell lines.

<p>Fluorescence intensity analysis of DiD-labeled NPCE-derived exosomes entry into each cell type after a 12 h incubation, by Easy-Quant. *P<0.05, **P<0.01, ***P<0.001.</p

Uptake of NPCE-secreted exosomes by NTM recipient cultured cells within 32 h.

<p>(A) Representative confocal images of NPCE-derived exosomes internalized by NTM cells. Purified exosomes were labeled with the dye DiD (white) and added to cells of the NTM line (20 μg per 1.5x10⁷cells). After incubation with exosomes for the indicated times, the cells were stained with an α-tubulin monoclonal antibody (green), DAPI (blue) and visualized by confocal microscopy. (B) The graph presents exosome dynamics by spot count of internalized labeled exosomes. Data are average ± SD of three independent experiments. A minimum of 350 cells were analyzed for each sample. *P<0.05, **P<0.01, ***P<0.001.</p

Characterization of exosomes isolated from NPCE cell culture supernatants.

<p>Size distribution of NPCE cell-derived exosomes as measured by (A) nanoparticle tracking analysis, (B) and tunable resistive pulse sensing (TPRS). (C) Cyto-TEM image of purified NPCE-derived exosomes. Scale bar: 100 nm. (D) Western blot analysis of exosomes floated on a continuous sucrose density gradient. 20 μg of exosomal proteins were loaded onto a continuous sucrose density gradient. Fractions collected from the gradient were separated by SDS-PAGE and probed for the exosomes markers Alix and TSG101. (E) Coomassie brilliant blue–stained SDS polyacrylamide gel after separation of 15 μg of total cell lysates or exosomal proteins from NPCE cells. (F) Exosomes antibody array marker analysis with Exo-Check, representative results.</p