[PXG]). These were compared to patients undergoing cataract surgery (controls) for this cross-sectional study. Functional activities of tear MMP-9 and MMP-2 were analyzed by gelatin zymography. Tenon's capsules (n ¼ 15) were harvested from the inferior quadrant in those undergoing cataract surgery and protein expression of MMP-9 was analyzed by immunohistochemistry (IHC). Hydrogen peroxide (H 2 O 2 ) stress-induced effects on in vitro activities of MMP-9 in human trabecular meshwork (HTM) cells were analyzed.RESULTS. The MMP-9 activity in tears was increased significantly in POAG, (n ¼ 27), PACG (n ¼ 24), and PXF (n ¼ 40) eyes compared to controls (n ¼ 35), and was increased significantly in eyes with glaucoma compared to moderate/severe glaucoma (P < 0.001). The MMP-9 expression was significantly lower in PXG (n ¼ 22) eyes. Immunohistochemistry of Tenon's capsule revealed increased expression of MMP-9 in primary glaucoma eyes. Increased MMP-9 activity was seen in in vitro by gelatin zymography and was confirmed by Western and immunofluorescent assay on HTM upon 800 and 1000 lM H 2 O 2 -induced stress for 2 to 3 hours with approximately 80% cell death. CONCLUSIONS.Increased tear MMP-9 activity in early glaucoma and pseudoexfoliation syndrome suggesting activation of extracellular matrix (ECM) degradation can be used as a tear-based predictive biomarker. Decreased expression in advanced stages suggests exhaustion of the degradation response.

To investigate the microRNA (miRNA) profile in patients with different stages of pseudoexfoliation (PXF). Methods: Peripheral blood of patients with PXF (naïve to medical therapy and with no systemic disease/drugs) with ocular hypertension (OHT) and pseudoexfoliation glaucoma (PXG) was evaluated in triplicate for miRNA profiling using polymerase chain reaction (PCR) arrays. Those identified in the discovery stage were validated with evaluation of serum transforming growth factor-β1 (TGF-β1) levels by ELISA. The downstream targets of TGF-β1 and unfolded protein response (UPR) were analyzed using reverse transcriptionquantitative polymerase chain reaction (RT-qPCR). Predicted targets of the identified miRNA and KEGG pathway analysis were done using miRbase and DIANA tools mirPathv3.1. Results: We found hsa-miR-122-5p, hsa-miR-124-3p and hsa-miR-424-5p to be upregulated in PXG targeting 3 specific pathways namely TGF-β1, fibrosis/ECM and proteoglycan metabolism with common effectors like SMAD/3/2. The unfolded protein response (UPR) genes were significantly downregulated in all stages of PXF suggesting this as the key mechanism for protein aggregates in PXF syndrome. Serum TGF-β1 was significantly upregulated as disease progressed to later stages in PXG. This elevation in advanced stages was associated with significantly differential expression of downstream pathways and fibrotic genes in OHT compared to PXG predominantly through the SMAD3, a canonical pathway marker. Conclusion: Circulatory miRNA differentially regulating TGF-β1 and downstream targets including UPR genes may be the key mechanisms for glaucoma onset in PXF.

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.

PurposeTo evaluate different clinical variants of pseudoexfoliation syndrome and their risk of developing ocular hypertension (OHT) or glaucoma (PXG)DesignCross sectional hospital based study.SettingAll patients seen at glaucoma services of a tertiary eye care center in east India.MethodsElectronic medical records search of hospital database including consecutive new and old cases seen during April 2013 to March 2015 was done to retrieve case sensitive words including pseudoexfoliation, PXF, PEX, PXG and pseudoexfoliative glaucoma over any part of the clinical electronic sheet of the patient. All demographic and clinical details including laterality, the pattern of deposits, need for medicines and disc damage at presentation was compared in eyes with radial pigmentary, classical or combined forms of PXF phenotypes.ResultsOf 110313 PXF patients seen during the period of 2013–2015, a total of 2297 eyes of 1150 PXF patients were identified including 525 unilateral PXF (meaning a total of 1775 PXF eyes with 625 patients having bilateral disease, n = 1250 eyes, other clinically normal eye, n = 522) at presentation. Of 525 unilateral PXF eyes, 105 had OHT and 131 had glaucoma while bilateral cases had more >50% (675 eyes of 1250 eyes) with glaucoma. Glaucoma with significant changes in IOP with or without disc damage was seen in 32% of pigmentary and 39% of classical PXF forms with eyes with combined forms of PXF having around 50% with glaucoma at presentation compared to other forms, p<0.001.ConclusionDifferent phenotypic variants of PXF in this Indian cohort was associated with 30–50% risk of OHT or glaucoma respectively. Adequate care is required while examining the pattern of PXF in each case to prognosticate each patient/eye.

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.

The key differences in cell death mechanisms in the trabecular meshwork (TM) in adult moderate and severe primary glaucoma remain still unanswered. This study explored key differences in cell death mechanisms in the trabecular meshwork (TM) in adult moderate and severe primary glaucoma. Design: In-vitro laboratory study on surgical specimens and primary cell lines. Methods: Select cell death-related proteins differentially expressed on mass spectrometric analysis in ex-vivo dissected TM specimens patients with severe adult primary open-angle (POAG) or angle-closure glaucoma (PACG) compared to controls (cadaver donor cornea) were validated for temporal changes in cell death-related gene expression on in-vitro primary human TM cell culture after 48 hours (moderate) or 72 hours (severe) oxidative stress with H 2 O 2 (400-1000 uM concentration). These were compared with histone modifications after oxidative stress in human TM (HTM) culture and peripheral blood of patients with moderate and severe glaucoma. Results: Autophagy-related proteins seemed to be the predominant cell-death mechanism over apoptosis in ex-vivo dissected TM specimens in severe glaucoma. Analyzing HTM cell gene expression at 48 hours and 72 hours of oxidative stress, autophagy genes were upregulated at 48-72 hours of exposure in contrast to apoptosis-related genes, showing downregulation at 72 hours. There was associated increased expression of H3K14ac in HTM after 72 hours of oxidative stress and in peripheral blood of severe POAG and PACG. Conclusion: A preference of autophagy over apoptosis may underlie stage transition from moderate to severe glaucoma in the trabecular meshwork or peripheral blood, which may be tightly regulated by epigenetic modulators.

PurposeProstaglandin analogues (PGA’s) are the mainstay and first line of treatment in current glaucoma practise. Though latanoprost and bimatoprost are the most commonly used PGA’s with minimal side effects at lower concentrations like bimaotoprost 0.01%, direct comparison of their cytokine/MMP profile in tears has not been evaluated earlier. The study intends to ascribe PGA to the upregulation of MMPs, Cytokines and Chemokines mediating varied pathways to result in side effects of the drugs.MethodsTear sample collection was done from outer canthus of 30 eyes of 30 patients (primary open angle glaucoma (n = 26 and n’ = 20), normal tension glaucoma (n = 4 and n’ = 10), in latanoprost (n) 0.005% and bimatoprost (n’) 0.01% group respectively, with a mean age of 62±10.5 years) on >6 months of PGA use using Tear floTM Schirmer filter strip. Tear samples from 30 eyes of 30 cataract patients without drug treatment were used as the control. Gelatinolytic activity of MMP-9 and MMP-2 were examined by substrate gelatine zymography MMP-1 and TIMP-1 concentrations from tears samples with PGAs were evaluated by ELISA while cytokine concentration in the eluted tears was evaluated using a convenient bioplex kit assay (Milliplex MAP kit, HCYTMAG-60K-PX41, Millipore, Massachusetts, United States). The mean duration of use of PGA in both groups did not differ significantly (median 1.3 years in bimatoprost and 1.1 years in latanoprost eyes, p = 0.6).ResultsThe tear MMP-9 expression was higher in eyes receiving latanoprost while the MMP-2 expression was higher in eyes receiving bimatoprost with MMP1 protein levels being higher in the former. Latanoprost treated eyes had marginally elevated tear cytokines involved in tissue remodelling while bimatoprost eyes showed elevated cytokines regulating allergic pathways.ConclusionDifferential cytokine and MMP expression indicates differential signalling pathways mediating different cellular effects (evident as clinical and side effects) with the two drugs which can be explored further.

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.