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Anterior segment alterations and comparative aqueous humor proteomics in the buphthalmic rabbit (an American Ophthalmological Society thesis).

ABSTRACT: PURPOSE: To use an integrated proteohistologic approach to gain insight into the anterior segment alterations in the buphthalmic rabbit. METHODS: Eyes from 2- and 5-year-old buphthalmic and normal rabbits (n=20) were studied histologically. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of aqueous humor (AH) was used to determine differential protein expression between animal groups. Western blot and immunohistochemistry were performed on selected differentially expressed proteins identified by LC-MS/MS. RESULTS: The buphthalmic rabbits manifested a mild clinical phenotype with typical angle anomalies that appeared progressive by histology. Significantly thickened Descemet's membrane (DM) and anterior lens capsule in all buphthalmic rabbits showed increased fibronectin and collagen-IV immunolabeling. LC-MS/MS applying stringent filtering criteria revealed significant differential expression of several AH proteins in these rabbits. The protein of interest in the 2-year-old group was histidine-rich glycoprotein, and those in the 5-year-old group included alpha-2-HS-glycoprotein, clusterin, apolipoprotein E, interphotoreceptor retinoid-binding protein, transthyretin, cochlin, gelsolin, haptoglobin, hemopexin, and beta-2 microglobulin. The proteomic data for selected proteins was validated by Western blot and immunohistochemistry. A wide range of functional groups were affected by the altered AH proteins. These included extracellular matrix modulation, regulation of apoptosis, oxidative stress, and protein transport. CONCLUSIONS: Multiple anterior segment alterations were histologically identified in the buphthalmic rabbits that showed progressive changes with age. The differentially expressed AH proteins in these rabbits suggest a multifunctional role for AH in modulating pathologic changes in DM, anterior lens capsule, and the angular meshwork in these animals.

SUBMITTER: Edward DP

PROVIDER: S-EPMC3259673 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Anterior segment alterations and comparative aqueous humor proteomics in the buphthalmic rabbit (an American Ophthalmological Society thesis).

Edward Deepak P DP Bouhenni Rachida R

Transactions of the American Ophthalmological Society 20111201

<h4>Purpose</h4>To use an integrated proteohistologic approach to gain insight into the anterior segment alterations in the buphthalmic rabbit.<h4>Methods</h4>Eyes from 2- and 5-year-old buphthalmic and normal rabbits (n=20) were studied histologically. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of aqueous humor (AH) was used to determine differential protein expression between animal groups. Western blot and immunohistochemistry were performed on selected differentially expressed ...[more]

PMID: 22253484

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Project description:PurposeMutations of keratoepithelin (KE) and myocilin (MYOC) have been linked to certain types of inherited corneal stromal dystrophy and open-angle glaucoma, respectively. In this study, the feasibility of using small interfering RNAs (siRNAs) to suppress the expression of keratoepithelin and myocilin and their capabilities to reduce the related cytotoxic effects caused by mutant myocilins were investigated.MethodscDNAs of human KE gene and myocilin gene were amplified by polymerase chain reaction and subcloned into pEGFP-N1 to construct respective plasmids, KEpEGFP and MYOCpEGFP, to produce fluorescence-generating fusion proteins. Short hairpin RNAs (shRNAs) were generated from an RNA polymerase III promoter-driven vector (pH1-RNA). Transformed HEK293 and trabecular meshwork (TM) cells were cotransfected via liposomes with either KEpEGFP or MYOCpEGFP and respective shRNA-generating plasmids to evaluate the suppression efficacy of shRNAs. Suppression of KE-EGFP by KE-specific shRNAs was evaluated by fluorescence microscopy and Western blotting. Suppression of MYOC-EGFP by myocilin-specific shRNAs was quantified with UN-SCAN-IT software on digitized protein bands of Western blots. A BiP promoter-driven luciferase reporter assay was used to evaluate the stress response of TM cells induced by misfolded mutant myocilins.ResultsTwo KE-specific shRNAs that effectively suppressed the expression of KE-EGFP in HEK293 cells were identified. One shRNA (targeting the coding sequence starting at 1528bp of KE) reduced the expression of KE-EGFP approximately by 50%, whereas the other shRNA (targeting the 3'-UTR region of KE) suppressed greater than 80% of the expression. Cotransfection of MYOCpEGFP and various shRNA-generating plasmids targeting different regions of myocilin (containing amino acid residues R76, E352, K423, or N480 associated with inherited glaucoma) showed effective reduction of MYOC-EGFP, ranging from 78% to 90% on average. The activation of BiP gene (as a stress response induced by mutant myocilins) in transformed TM cells was significantly reduced when mutant myocilin proteins were suppressed by myocilin-specific shRNAs.ConclusionsKE- or myocilin-specific shRNAs could effectively suppress the expression of recombinant KE or myocilin proteins and the related cytotoxicity of mutant myocilins. RNA interference may have future therapeutic implications in suppressing these genes.

Dataset Information

Anterior segment alterations and comparative aqueous humor proteomics in the buphthalmic rabbit (an American Ophthalmological Society thesis).

Publications

Anterior segment alterations and comparative aqueous humor proteomics in the buphthalmic rabbit (an American Ophthalmological Society thesis).

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