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Laura A. Hecker, Michael P. Fautsch, Sanjay V. Patel; Identification of Differentially Expressed Proteins in Descemet Membrane and Endothelium of Corneas with Fuchs Endothelial Dystrophy. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6599.
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© ARVO (1962-2015); The Authors (2016-present)
The molecular mechanisms responsible for the pathogenesis of Fuchs endothelial corneal dystrophy are unknown. In this study, we identified proteins that were differentially expressed in Descemet membrane and endothelium of normal corneas and corneas with Fuchs dystrophy.
Human Descemet membrane with endothelium was isolated from 16 corneas of 15 female subjects with Fuchs dystrophy, and the tissue was pooled into 4 samples matched by age (range, 58-77 years). Descemet membrane with endothelium was also harvested from 12 normal corneas of 8 female donors (range, 63-74 years) and was pooled into 4 samples that were age-matched to the Fuchs dystrophy samples. Protein was extracted and digested with trypsin. Peptides were labeled with iTRAQ multiplex reagents (Applied Biosystems, CA, USA), and were combined and fractionated by strong cation exchange chromatography. Peptides were analyzed by liquid chromatography-tandem mass spectrometry and the data were searched using Protein Pilot software (Applied Biosystems). Identified proteins were considered to be differentially expressed if their average iTRAQ sample ratios were <0.67 or >1.5 with a p-value <0.05. One of the proteins identified by iTRAQ was selected for verification by Western blotting.
161 proteins with 2 or more peptide matches were identified in normal and Fuchs Descemet membrane-endothelium. Seven of these proteins were differentially expressed (2 decreased, 5 increased) in at least 3 of the 4 sample pairs. The 7 proteins are involved in cellular metabolic processes (alpha-enolase, phosphoglycerate kinase 1, apolipoprotein E), cell growth (HTRA1), cell proliferation (C-type lectin domain family 11 member A), apoptosis (clusterin), and cell adhesion (collagen alpha-1(XII) chain). Western blotting for clusterin confirmed the iTRAQ results.
Protein expression differences exist between Fuchs and normal Descemet membrane-endothelium. These differences may alter the corneal endothelial cell response to stress, the energy state of the cell, or extracellular matrix interactions, and might allude to mechanisms underlying Fuchs dystrophy.
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