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      Proteomic analysis of aqueous humor from patients with primary open angle glaucoma

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          Abstract

          Purpose

          Primary open angle glaucoma (POAG) is a leading cause of irreversible blindness on a global level. Researchers have yet to specify the exact mechanisms of POAG; the respective relationships between POAG and elevated intraocular pressure (IOP), as well as optic neuropathy, remain particularly unclear. It is known, however, that the expression profile for some proteins in the aqueous humor (AH) changes in some diseases, and that AH changes play important roles in elevated IOP. To identify the possible roles of these AH proteins in POAG, a proteomic analysis of the AH compositions of POAG patients’ eyes was performed and compared with those derived from paired, non-POAG cataract (control) eyes.

          Methods

          We used Bradford’s method to determine total protein concentration in AH, and analyzed separation profiles via two-dimensional (2D) gel electrophoresis. We used silver stain to determine gel proteins, and analyzed separation profiles to assess spot density differences between POAG and non-POAG patients. These gel spots were isolated and identified via mass spectrometry. Prostaglandin H2 D-isomerase (PGDS) in AH were analyzed by western Blotting.

          Results

          There was no significant difference between the total protein concentration in AH of POAG patients and that in AH of non-POAG patients. A total of seven spots were increased in 2D gels from POAG patients. The spots were derived from PGDS, caspase 14 precursor, transthyretin, cystain C, albumin precursor, and tranferrin. And PGDS in AH from patients was more than from controls.

          Conclusions

          The protein composition in AH was significantly different in POAG patients versus non-POAG patients. The identified proteins could be a potential biomarker for POAG and may play a role in the mechanisms of elevated IOP and optic neuropathy in POAG.

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          Most cited references 25

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          Aqueous humor levels of vascular endothelial growth factor and pigment epithelium-derived factor in polypoidal choroidal vasculopathy and choroidal neovascularization.

          To determine the aqueous levels of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) in patients with active polypoidal choroidal vasculopathy (PCV) and choroidal neovascularization (CNV) secondary to age-related macular degeneration (AMD) and pathologic myopia. Prospective, comparative control study. Aqueous humors were collected from 32 eyes of 32 patients for either active PCV or CNV. Among them, 11 eyes had active and symptomatic PCV, 12 eyes had active CNV secondary to AMD, and nine eyes had active CNV of pathologic myopia. Levels of VEGF and PEDF were determined by commercially available enzyme-linked immunosorbent assay kits. A group of 10 aqueous samples from 10 patients who underwent cataract surgery without other ocular or systemic diseases comprised the controls. VEGF concentrations in aqueous humor were markedly increased in patients with PCV, CNV of AMD, and CNV of myopia when compared with the controls (analysis of variance [ANOVA], P < .001). VEGF levels in eyes with PCV were, however, significantly lower than those of exudative AMD (P = .045). The PEDF levels were also significantly different among the groups (ANOVA, P = .001), and we observed increased levels in PCV, CNV of AMD, and CNV of myopia. VEGF and PEDF factors were coexpressed and increased with positive correlation in aqueous humor of eyes with active PCV. The different levels of both factors in eyes of PCV and AMD might suggest distinct clinical entities or different angiogenesis courses between PCV and AMD.
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            Oxidative stress markers in aqueous humor of glaucoma patients.

            Oxidative stress and antioxidant status in eye tissues may be associated with glaucomatous damage. The aim of this study was to establish the antioxidant status of aqueous humor of patients with primary open-angle glaucoma. For this purpose the authors measured the total reactive antioxidant potential (TRAP) and the activities of the antioxidant enzymes superoxide dismutase (SOD), catalase, and glutathione peroxidase. Case control study. Aqueous humor was obtained at the time of surgery from 24 patients with glaucoma and 24 cataract patients; TRAP was measured by chemiluminescence. Activities of the antioxidant enzymes were measured spectrophotometrically. Superoxide dismutase activity was determined by inhibition of the rate of adrenochrome formation at 480 nm. Catalase activity was evaluated by decrease of H(2)O(2) absorbance at 240 nm. Glutathione peroxidase (GPx) activity was determined following nicotinamide adenine dinucleotide phosphate oxidation at 340 nm. Total reactive antioxidant potential value of the cataract group was 124 +/- 5 micromol/l Trolox. This value was significantly decreased, by 64%, in glaucoma patients. An increase of 57% in SOD activity was observed in glaucoma patients when compared with cataract patients (41.7 +/- 2.7 U SOD/ml). Glutathione activity was threefold higher in glaucoma patients than in the cataract group (6.1 +/- 0.6 U/ml). No significant changes were found in catalase levels. Oxidative stress may lead to an induction of antioxidant enzymes and contribute to TRAP decrease. Superoxide dismutase, GPx activities, and TRAP may be useful oxidative stress markers in aqueous humor of glaucoma patients.
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              TGFbeta2-induced changes in human trabecular meshwork: implications for intraocular pressure.

              Transforming growth factor (TGF)-beta2 levels are elevated in glaucomatous human aqueous humor. TGFbeta is a cytokine that alters extracellular matrix (ECM) metabolism, and excess ECM has been proposed to increase aqueous outflow resistance in the trabecular meshwork (TM) of glaucomatous eyes. This study was undertaken to investigate effects of TGFbeta2 on secretion of fibronectin and the protease inhibitor plasminogen activator inhibitor (PAI)-1 from human TM cell cultures and perfused human ocular anterior segments. Total RNA was isolated from pooled human TM cell monolayers and used for a gene microarray expression analysis. Supernatants from treated human TM cells were analyzed by ELISA for fibronectin or PAI-1 content. TGFbeta2 effects on intraocular pressure (IOP) were evaluated in a perfused organ culture model using human anterior segments, and eluates were analyzed for fibronectin and PAI-1 content. Overnight treatment of TM cells with TGFbeta2 upregulated multiple ECM-related genes, such as PAI-1. TGFbeta2 also increased secretion of both fibronectin and PAI-1 from TM cells. TGFbeta2 effects on TM cells were blocked by inhibitors of the TGFbeta type I receptor. In perfused human anterior segments, TGFbeta2 treatment elevated IOP and increased eluate fibronectin and PAI-1 content. TGFbeta2 effects on IOP may be transduced by TGFbeta type-I receptor-mediated changes in TM secretion of ECM-related factors such as fibronectin and PAI-1. Modulation of TGFbeta2-induced changes in the ECM may provide a novel and viable approach to the management of glaucoma.
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                Author and article information

                Journal
                Mol Vis
                MV
                Molecular Vision
                Molecular Vision
                1090-0535
                2010
                18 December 2010
                : 16
                : 2839-2846
                Affiliations
                [1 ]Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmology & Visual Sciences Key Laboratory, Beijing, China
                [2 ]Laborotary of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
                [3 ]Beijing Tongren Eye Center, Beijing Tongren Hospital, Beijing Institute of Ophthalmology, Capital Medical University, Beijing Ophthalmology & Visual Sciences Key Laboratory, Beijing, China
                Author notes

                The first two authors contributed equally to the work

                Correspondence to: Prof. Ningli Wang, Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmology & Visual Sciences Key Laboratory, Beijing, China; Phone: +86 10-58269920; FAX: +86 10-58269920; email: wningli@ 123456trhos.com
                Article
                304 2010MOLVIS0189
                3012650
                21203405
                Copyright © 2010 Molecular Vision.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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