TGFβ1, MMPs and cytokines profiles in ocular surface: Possible tear biomarkers for pseudoexfoliation

Abstract

Purpose Pseudoexfoliation (PXF) is a unique form of glaucoma characterized by accumulation of exfoliative material in the eyes. Changes in tear profile in disease stages may give us insights into molecular mechanisms involved in causing glaucoma in the eye. Methods All patients were categorized into three main categories; pseudoexfoliation (PXF), pseudoexfoliation glaucoma (PXG) and cataract, which served as control. Cytokines, transforming growth factor β1 (TGFβ1), matrix metalloproteases (MMPs) and fibronectin (FN1) were assessed with multiplex bead assay, enzyme-linked immunosorbent assay (ELISA), gelatin zymography, and immunohistochemistry (IHC) respectively in different ocular tissues such as tears, tenon’s capsule, aqueous humor (AH) and serum samples of patients with PXF stages. Results We found that TGFβ1, MMP-9 and FN1 protein expression were upregulated in tears, tenon’s capsule and AH samples in PXG compared to PXF, though the MMP-9 protein activity was downregulated in PXG compared with control or PXF. We have also found that in PXG tears sample the fold change of TGF-α (Transforming Growth Factor-α), MDC (Macrophage Derived Chemokine), IL-8 (Interleukin-8), VEGF (Vascular Endothelial Growth Factor) were significantly downregulated and the levels of GM-CSF (Granulocyte Macrophage Colony Stimulating Factor), IP-10 (Interferon- γ produced protein-10) were significant upregulated. While in AH; IL-6 (Interleukin-6), IL-8, VEGF, IFN-a2 (Interferon- α2), GRO (Growth regulated alpha protein) levels were found lower and IL1a (Interleukin-1α) level was higher in PXG compared to PXF. And in serum; IFN-a2, Eotaxin, GM-CSF, Fractalkine, IL-10 (Interleukin-10), IL1Ra (Interleukin-1 receptor antagonist), IL-7 (Interleukin-7), IL-8, MIP1β (Macrophage Inflammatory Protein-1β), MCP-1 (Monocyte Chemoattractant Protein-1) levels were significantly upregulated and PDGF-AA (Platelet Derived Growth Factor-AA) level was downregulated in the patients with PXG compared to PXF. Conclusions Altered expression of these molecules in tears may therefore be used as a signal for onset of glaucoma or for identifying eyes at risk of developing glaucoma in PXF.

Primary Human Trabecular Meshwork Model for Pseudoexfoliation

“…Cultures that reached 50–70% confluence in flasks were serum starved for 24 h, followed by treatment with TGF-β1 (T7039, Sigma Aldrich, St. Louis, MO, USA) at different concentrations (3 ng/mL, 5 ng/mL, 10 ng/mL, 15 ng/mL) for 24 h, 36 h, 48 h, 72 h, and 96 h while one set, that was left untreated, was used as control. Cell viability in control and treated cells was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, St. Louis, MO, USA) assay using a previously described procedure [ 14 ]. Cells were serum starved for 24 h and then treated with or without TGF-β1 (3 ng/mL, 5 ng/mL, 10 ng/mL, 15 ng/mL) for 24 h, 36 h, 48 h, 72 h, and 96 h. Absorbance at 570 nm was measured on an EPOCH microplate reader (BioTek, Winooski, VT, USA).…”

“…Enzymatic activity of MMP-9 was examined by substrate gelatin zymography as described previously [ 14 ]. Protein quantification was done by Bradford assay and equal quantity of supernatant media protein from control and treated HTM cells were separated on 10% SDS-PAGE gels containing 0.1% gelatin.…”

“…Immunoblotting was done by a method as described previously [ 14 ]. Protein quantification was done by Bradford assay and equal quantity of protein was loaded onto each lane.…”

“…These pro-inflammatory cytokines have been shown to favor and create a pro-fibrotic tissue milieu [ 9 , 10 , 11 , 12 ] and with oxidative stress they are known to play a role in PXF and PXG [ 12 , 13 ]. In our previous study, we found elevated TGF-β1 levels in the aqueous humor of patients that increased with disease stages, with maximal levels being found in eyes with ocular hypertension and glaucoma [ 14 ]. We also found decreasing tear and aqueous MMP9 activity, with increased inflammatory cytokine levels, in severe PXG eyes compared to primary glaucoma [ 14 ], suggesting that the elevated TGF levels somehow were important for molecular events that causes the disease.…”

“…In our previous study, we found elevated TGF-β1 levels in the aqueous humor of patients that increased with disease stages, with maximal levels being found in eyes with ocular hypertension and glaucoma [ 14 ]. We also found decreasing tear and aqueous MMP9 activity, with increased inflammatory cytokine levels, in severe PXG eyes compared to primary glaucoma [ 14 ], suggesting that the elevated TGF levels somehow were important for molecular events that causes the disease. We also found serum levels to parallel the levels in the aqueous and tears with several key miRNA driven by TGF-β1 to be upregulated in eyes with ocular hypertension (OHT) and PXG [ 15 ].…”