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U. V. Jurkunas, D. L. Harris, K. Colby, N. C. Joyce; Differential Expression of Peroxiredoxins in Fuchs’ Endothelial Dystrophy. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2709.
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Previously, proteomic analysis of the human corneal endothelial cell layer and Descemet’s membrane (HCEC-DM) complex established a dataset of normally expressed proteins. The abundant expression of various isoforms of peroxiredoxin (Prx) unveiled the presence in HCEC-DM of a potent antioxidant scavenging system that was previously described in neuronal tissue. The purpose of these studies was to evaluate the differential expression of peroxiredoxin in corneal endothelium vs. epithelium/stroma and to compare the levels of expression between normal and Fuchs’ Endothelial Dystrophy (FED) HCEC-DM.
FED corneal buttons were removed during transplantation and normal corneas were obtained from the New England Tissue Bank. HCEC-DM complex was dissected from stroma and epithelium and proteins were extracted using previously established methods. Stroma/ epithelium was cut in pieces and the proteins were extracted by the identical protocol. Protein concentration was determined by modified Bio-Rad assay. Proteins were loaded onto 10% Bis-Tris NuPAGE gels. Peptides were transferred to a polyvinylidene difluoride (PVDF) membrane and nonspecific binding was blocked with 5% dry non-fat milk in PBS for 1 hour. Membranes were incubated overnight at 4°C with mouse monoclonal anti-peroxiredoxin diluted 1:1000 in blocking solution. Blots were rinsed, reblocked and exposed for 1 hour to horseradish peroxidase (HRP)-conjugated donkey anti-mouse IgG diluted 1:20,000 to control protein load. Peptides were detected with a chemiluminescent substate. The images were analyzed with NIH Image software, and protein content was normalized according to beta-actin content.
Corneal endothelium showed 4-fold increase in expression of Prx 5 compared to stroma/epithelium. FED HCEC-DM showed a 2-fold increase in Prx 3 expression and 1.4-fold increase in Prx 5 compared to normals.
Differential expression of Prx in HCEC-DM as compared to stroma/epithelium points to the unique and potent antioxidant system that appears to differentiate endothelium from the other corneal cells. Increased expression of these antioxidant proteins in FED cells signifies cellular response to oxidative stress. The ability to combat oxidative stress might be dysregulated in FED, pointing to the pathogenesis of this dystrophy.
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