Primary Human Trabecular Meshwork Model for Pseudoexfoliation

The lack of an animal model or an in vitro model limits experimental options for studying temporal molecular events in pseudoexfoliation syndrome (PXF), an age related fibrillopathy causing trabecular meshwork damage and glaucoma. Our goal was to create a workable in vitro model of PXF using primary human TM (HTM) cell lines simulating human disease. Primary HTM cells harvested from healthy donors (n = 3), were exposed to various concentrations (5 ng/mL, 10 ng/mL, 15 ng/mL) of transforming growth factor-beta1 (TGF-β1) for different time points. Morphological change of epithelial–mesenchymal transition (EMT) was analyzed by direct microscopic visualization and immunoblotting for EMT markers. Expression of pro-fibrotic markers were analyzed by quantitative RT-PCR and immunoblotting. Cell viability and death in treated cells was analyzed using FACS and MTT assay. Protein complex and amyloid aggregate formation was analyzed by Immunofluorescence of oligomer11 and amyloid beta fibrils. Effect of these changes with pharmacological inhibitors of canonical and non-canonical TGF pathway was done to analyze the pathway involved. The expression of pro-fibrotic markers was markedly upregulated at 10 ng/mL of TGF-β1 exposure at 48–72 h of exposure with associated EMT changes at the same time point. Protein aggregates were seen maximally at these time points that were found to be localized around the nucleus and in the extracellular matrix (ECM). EMT and pro-fibrotic expression was differentially regulated by different canonical and non-canonical pathways suggesting complex regulatory mechanisms. This in vitro model using HTM cells simulated the main characteristics of human disease in PXF like pro-fibrotic gene expression, EMT, and aggregate formation.</jats:p

Global and Comparative Proteome Signatures in the Lens Capsule, Trabecular Meshwork, and Iris of Patients With Pseudoexfoliation Glaucoma

Pseudoexfoliation (PXF) is characterized by the accumulation of the exfoliative material in the eye and high rates of blindness if left untreated. Pseudoexfoliation glaucoma (PXG) is generally diagnosed too late due to its asymptomatic nature, necessitating the development of new effective screening tools for the early diagnosis of the disease. Thus, the increasing prevalence of this disease due to an aging population has demanded the identification of suitable biomarkers for the early detection of the disease or detection of the onset of glaucoma in the eyes with PXF. We applied a proteomics strategy based on a high-throughput screening method for the determination of proteins involving PXF and PXG pathogenesis. The lens capsule (LC), iris, and trabecular meshwork (TM) samples with PXF and PXG were taken by surgical trabeculectomy, and control samples were taken from the donor corneal buttons obtained from the institutional eye bank to characterize the proteome profile. Peptides from the LC were analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). The protein of interest and cytokine/chemokine profiles were verified using immunohistochemistry and the bio-plex kit assay, respectively. There were a total of 1433 proteins identified in the human LC, of which 27 proteins were overexpressed and eight proteins were underexpressed in PXG compared with PXF. Overexpressed proteins such as fibromodulin, decorin, lysyl oxidase homolog 1, collagen alpha-1(I) chain, collagen alpha-3(VI) chain, and biglycan were the major components of the extracellular matrix (ECM) proteins involved in cell-matrix interactions or ECM proteoglycans and the assembly and cross-linking of collagen fibrils. The ECM composition and homeostasis are altered in glaucoma. Thus, quantitative proteomics is a method to discover molecular markers in the eye. Monitoring these events can help evaluate disease progression in future studies.</jats:p

Primary Human Trabecular Meshwork Model for Pseudoexfoliation

The lack of an animal model or an in vitro model limits experimental options for studying temporal molecular events in pseudoexfoliation syndrome (PXF), an age related fibrillopathy causing trabecular meshwork damage and glaucoma. Our goal was to create a workable in vitro model of PXF using primary human TM (HTM) cell lines simulating human disease. Primary HTM cells harvested from healthy donors (n = 3), were exposed to various concentrations (5 ng/mL, 10 ng/mL, 15 ng/mL) of transforming growth factor-beta1 (TGF-&beta;1) for different time points. Morphological change of epithelial&ndash;mesenchymal transition (EMT) was analyzed by direct microscopic visualization and immunoblotting for EMT markers. Expression of pro-fibrotic markers were analyzed by quantitative RT-PCR and immunoblotting. Cell viability and death in treated cells was analyzed using FACS and MTT assay. Protein complex and amyloid aggregate formation was analyzed by Immunofluorescence of oligomer11 and amyloid beta fibrils. Effect of these changes with pharmacological inhibitors of canonical and non-canonical TGF pathway was done to analyze the pathway involved. The expression of pro-fibrotic markers was markedly upregulated at 10 ng/mL of TGF-&beta;1 exposure at 48&ndash;72 h of exposure with associated EMT changes at the same time point. Protein aggregates were seen maximally at these time points that were found to be localized around the nucleus and in the extracellular matrix (ECM). EMT and pro-fibrotic expression was differentially regulated by different canonical and non-canonical pathways suggesting complex regulatory mechanisms. This in vitro model using HTM cells simulated the main characteristics of human disease in PXF like pro-fibrotic gene expression, EMT, and aggregate formation

Table4_Global and Comparative Proteome Signatures in the Lens Capsule, Trabecular Meshwork, and Iris of Patients With Pseudoexfoliation Glaucoma.DOCX

Pseudoexfoliation (PXF) is characterized by the accumulation of the exfoliative material in the eye and high rates of blindness if left untreated. Pseudoexfoliation glaucoma (PXG) is generally diagnosed too late due to its asymptomatic nature, necessitating the development of new effective screening tools for the early diagnosis of the disease. Thus, the increasing prevalence of this disease due to an aging population has demanded the identification of suitable biomarkers for the early detection of the disease or detection of the onset of glaucoma in the eyes with PXF. We applied a proteomics strategy based on a high-throughput screening method for the determination of proteins involving PXF and PXG pathogenesis. The lens capsule (LC), iris, and trabecular meshwork (TM) samples with PXF and PXG were taken by surgical trabeculectomy, and control samples were taken from the donor corneal buttons obtained from the institutional eye bank to characterize the proteome profile. Peptides from the LC were analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). The protein of interest and cytokine/chemokine profiles were verified using immunohistochemistry and the bio-plex kit assay, respectively. There were a total of 1433 proteins identified in the human LC, of which 27 proteins were overexpressed and eight proteins were underexpressed in PXG compared with PXF. Overexpressed proteins such as fibromodulin, decorin, lysyl oxidase homolog 1, collagen alpha-1(I) chain, collagen alpha-3(VI) chain, and biglycan were the major components of the extracellular matrix (ECM) proteins involved in cell-matrix interactions or ECM proteoglycans and the assembly and cross-linking of collagen fibrils. The ECM composition and homeostasis are altered in glaucoma. Thus, quantitative proteomics is a method to discover molecular markers in the eye. Monitoring these events can help evaluate disease progression in future studies.</p

Image1_Global and Comparative Proteome Signatures in the Lens Capsule, Trabecular Meshwork, and Iris of Patients With Pseudoexfoliation Glaucoma.TIF

Pseudoexfoliation (PXF) is characterized by the accumulation of the exfoliative material in the eye and high rates of blindness if left untreated. Pseudoexfoliation glaucoma (PXG) is generally diagnosed too late due to its asymptomatic nature, necessitating the development of new effective screening tools for the early diagnosis of the disease. Thus, the increasing prevalence of this disease due to an aging population has demanded the identification of suitable biomarkers for the early detection of the disease or detection of the onset of glaucoma in the eyes with PXF. We applied a proteomics strategy based on a high-throughput screening method for the determination of proteins involving PXF and PXG pathogenesis. The lens capsule (LC), iris, and trabecular meshwork (TM) samples with PXF and PXG were taken by surgical trabeculectomy, and control samples were taken from the donor corneal buttons obtained from the institutional eye bank to characterize the proteome profile. Peptides from the LC were analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). The protein of interest and cytokine/chemokine profiles were verified using immunohistochemistry and the bio-plex kit assay, respectively. There were a total of 1433 proteins identified in the human LC, of which 27 proteins were overexpressed and eight proteins were underexpressed in PXG compared with PXF. Overexpressed proteins such as fibromodulin, decorin, lysyl oxidase homolog 1, collagen alpha-1(I) chain, collagen alpha-3(VI) chain, and biglycan were the major components of the extracellular matrix (ECM) proteins involved in cell-matrix interactions or ECM proteoglycans and the assembly and cross-linking of collagen fibrils. The ECM composition and homeostasis are altered in glaucoma. Thus, quantitative proteomics is a method to discover molecular markers in the eye. Monitoring these events can help evaluate disease progression in future studies.</p

Table2_Global and Comparative Proteome Signatures in the Lens Capsule, Trabecular Meshwork, and Iris of Patients With Pseudoexfoliation Glaucoma.DOCX

Pseudoexfoliation (PXF) is characterized by the accumulation of the exfoliative material in the eye and high rates of blindness if left untreated. Pseudoexfoliation glaucoma (PXG) is generally diagnosed too late due to its asymptomatic nature, necessitating the development of new effective screening tools for the early diagnosis of the disease. Thus, the increasing prevalence of this disease due to an aging population has demanded the identification of suitable biomarkers for the early detection of the disease or detection of the onset of glaucoma in the eyes with PXF. We applied a proteomics strategy based on a high-throughput screening method for the determination of proteins involving PXF and PXG pathogenesis. The lens capsule (LC), iris, and trabecular meshwork (TM) samples with PXF and PXG were taken by surgical trabeculectomy, and control samples were taken from the donor corneal buttons obtained from the institutional eye bank to characterize the proteome profile. Peptides from the LC were analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). The protein of interest and cytokine/chemokine profiles were verified using immunohistochemistry and the bio-plex kit assay, respectively. There were a total of 1433 proteins identified in the human LC, of which 27 proteins were overexpressed and eight proteins were underexpressed in PXG compared with PXF. Overexpressed proteins such as fibromodulin, decorin, lysyl oxidase homolog 1, collagen alpha-1(I) chain, collagen alpha-3(VI) chain, and biglycan were the major components of the extracellular matrix (ECM) proteins involved in cell-matrix interactions or ECM proteoglycans and the assembly and cross-linking of collagen fibrils. The ECM composition and homeostasis are altered in glaucoma. Thus, quantitative proteomics is a method to discover molecular markers in the eye. Monitoring these events can help evaluate disease progression in future studies.</p

Table1_Global and Comparative Proteome Signatures in the Lens Capsule, Trabecular Meshwork, and Iris of Patients With Pseudoexfoliation Glaucoma.DOC

Pseudoexfoliation (PXF) is characterized by the accumulation of the exfoliative material in the eye and high rates of blindness if left untreated. Pseudoexfoliation glaucoma (PXG) is generally diagnosed too late due to its asymptomatic nature, necessitating the development of new effective screening tools for the early diagnosis of the disease. Thus, the increasing prevalence of this disease due to an aging population has demanded the identification of suitable biomarkers for the early detection of the disease or detection of the onset of glaucoma in the eyes with PXF. We applied a proteomics strategy based on a high-throughput screening method for the determination of proteins involving PXF and PXG pathogenesis. The lens capsule (LC), iris, and trabecular meshwork (TM) samples with PXF and PXG were taken by surgical trabeculectomy, and control samples were taken from the donor corneal buttons obtained from the institutional eye bank to characterize the proteome profile. Peptides from the LC were analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). The protein of interest and cytokine/chemokine profiles were verified using immunohistochemistry and the bio-plex kit assay, respectively. There were a total of 1433 proteins identified in the human LC, of which 27 proteins were overexpressed and eight proteins were underexpressed in PXG compared with PXF. Overexpressed proteins such as fibromodulin, decorin, lysyl oxidase homolog 1, collagen alpha-1(I) chain, collagen alpha-3(VI) chain, and biglycan were the major components of the extracellular matrix (ECM) proteins involved in cell-matrix interactions or ECM proteoglycans and the assembly and cross-linking of collagen fibrils. The ECM composition and homeostasis are altered in glaucoma. Thus, quantitative proteomics is a method to discover molecular markers in the eye. Monitoring these events can help evaluate disease progression in future studies.</p

Table3_Global and Comparative Proteome Signatures in the Lens Capsule, Trabecular Meshwork, and Iris of Patients With Pseudoexfoliation Glaucoma.DOCX

Pseudoexfoliation (PXF) is characterized by the accumulation of the exfoliative material in the eye and high rates of blindness if left untreated. Pseudoexfoliation glaucoma (PXG) is generally diagnosed too late due to its asymptomatic nature, necessitating the development of new effective screening tools for the early diagnosis of the disease. Thus, the increasing prevalence of this disease due to an aging population has demanded the identification of suitable biomarkers for the early detection of the disease or detection of the onset of glaucoma in the eyes with PXF. We applied a proteomics strategy based on a high-throughput screening method for the determination of proteins involving PXF and PXG pathogenesis. The lens capsule (LC), iris, and trabecular meshwork (TM) samples with PXF and PXG were taken by surgical trabeculectomy, and control samples were taken from the donor corneal buttons obtained from the institutional eye bank to characterize the proteome profile. Peptides from the LC were analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). The protein of interest and cytokine/chemokine profiles were verified using immunohistochemistry and the bio-plex kit assay, respectively. There were a total of 1433 proteins identified in the human LC, of which 27 proteins were overexpressed and eight proteins were underexpressed in PXG compared with PXF. Overexpressed proteins such as fibromodulin, decorin, lysyl oxidase homolog 1, collagen alpha-1(I) chain, collagen alpha-3(VI) chain, and biglycan were the major components of the extracellular matrix (ECM) proteins involved in cell-matrix interactions or ECM proteoglycans and the assembly and cross-linking of collagen fibrils. The ECM composition and homeostasis are altered in glaucoma. Thus, quantitative proteomics is a method to discover molecular markers in the eye. Monitoring these events can help evaluate disease progression in future studies.</p

Table5_Global and Comparative Proteome Signatures in the Lens Capsule, Trabecular Meshwork, and Iris of Patients With Pseudoexfoliation Glaucoma.XLSX

Pseudoexfoliation (PXF) is characterized by the accumulation of the exfoliative material in the eye and high rates of blindness if left untreated. Pseudoexfoliation glaucoma (PXG) is generally diagnosed too late due to its asymptomatic nature, necessitating the development of new effective screening tools for the early diagnosis of the disease. Thus, the increasing prevalence of this disease due to an aging population has demanded the identification of suitable biomarkers for the early detection of the disease or detection of the onset of glaucoma in the eyes with PXF. We applied a proteomics strategy based on a high-throughput screening method for the determination of proteins involving PXF and PXG pathogenesis. The lens capsule (LC), iris, and trabecular meshwork (TM) samples with PXF and PXG were taken by surgical trabeculectomy, and control samples were taken from the donor corneal buttons obtained from the institutional eye bank to characterize the proteome profile. Peptides from the LC were analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). The protein of interest and cytokine/chemokine profiles were verified using immunohistochemistry and the bio-plex kit assay, respectively. There were a total of 1433 proteins identified in the human LC, of which 27 proteins were overexpressed and eight proteins were underexpressed in PXG compared with PXF. Overexpressed proteins such as fibromodulin, decorin, lysyl oxidase homolog 1, collagen alpha-1(I) chain, collagen alpha-3(VI) chain, and biglycan were the major components of the extracellular matrix (ECM) proteins involved in cell-matrix interactions or ECM proteoglycans and the assembly and cross-linking of collagen fibrils. The ECM composition and homeostasis are altered in glaucoma. Thus, quantitative proteomics is a method to discover molecular markers in the eye. Monitoring these events can help evaluate disease progression in future studies.</p

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

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. </jats:sec