Decreased Expression of Peroxiredoxins in Fuchs' Endothelial Dystrophy

Fuchs’ endothelial dystrophy (FED) is the most common cause of endogenous endothelial dysfunction and is the third most common indication for corneal transplants performed in the U.S.1 Despite the fact that this dystrophy was first described more that 100 years ago, the primary etiology of the endothelial cell degeneration is not known.2 In early stages, the dystrophy manifests by the formation of corneal guttae or dysregulated deposition of wide-spaced collagen between human corneal endothelial cells (HCEC) and Descemet’s membrane (DM) with concomitant changes in HCEC shape, size and density.3–5 Excessive deposition of collagen VIII has been noted in FED Descemet’s membrane and mutations in collagen VIII have been identified in familial, young-onset cases of FED.6 In the later stages of the disease, the progressive loss of Na+-K+-ATPase pump sites is associated with the inability of the endothelium to maintain corneal deturgescence, leading to corneal edema. 7 Recently, nuclear labeling and mRNA analysis techniques showed that FED endothelial cell death occurs via apoptosis.8–10 In other organ systems where cellular apoptosis is accompanied by abnormal extracellular matrix deposition, such as amyloid plaques in Alzheimer’s disease or drusen in age-related macular degeneration, a strong causal factor for cell death is oxidative stress due to excessive generation of reactive oxygen species (ROS).11 There is mounting evidence that oxidative stress induces damage to corneal endothelium in FED.12 Previous studies by Buddi et al.13 evaluated the relative amounts of cytotoxic byproducts of ROS in FED corneas. Although most of the differences between normal and FED were noted in the corneal epithelium, increased amounts of nitrotyrosine, an ROS byproduct, were also noted in the FED endothelium. From the genetic standpoint, Gottsch et al.14 found decreased transcript levels of the anti-oxidant glutathione S-transferase-pi in FED via serial analysis of gene expression. Initial studies from this laboratory used 2-D gel electrophoresis, MALDI-TOF protein identification, and Western blot analysis to compare protein expression between FED and normal corneal endothelium. This analysis revealed a number of protein differences, one of which was marked over-expression of clusterin in FED endothelium.15 Clusterin is a protein that protects against oxidative stress-induced cellular apoptosis. The current studies have further investigated the differential expression of proteins in FED with a particular focus on proteins with anti-oxidant properties. Specifically, MALDI-TOF analysis of normal gels at 15 to 30 kDa range (pI 6.0 to 9.0) identified the expression of a novel class of anti-oxidants, peroxiredoxins. We then investigated whether there is a difference in expression patterns of peroxiredoxins between normal and FED endothelial specimens. In the previous 2-D gel studies, proteins were separated in the first dimension using a linear pH 3–10 gradient. In the current studies, we changed the technique of isoelectric focusing by employing nonlinear gradient IPG strips to expand the region of the gel around neutral pH, thus promoting better separation of proteins that have isoelectric points in this area. Peroxiredoxins (Prx) function by removing cellular hydrogen peroxide. Six isotypes of Prx (1–6) have been identified in mammals. The subfamily of Prx 1–4 contains two conserved active site cysteine (Cys) residues, which use thioredoxin as an intermediate electron donor. Prx-5 also has two conserved active Cys residues, but does not possess a 40 amino acid residue segment on the C-terminus and is the smallest isoform. Prx-6 contains only one conserved Cys residue. 16–18 Since different isoforms of Prx proteins have different cellular functions, we compared the relative expression of Prx isoforms in normal and FED endothelium by both software analysis of 2-D gel patterns and by Western blotting. Real-time PCR was used to confirm the proteomic results by comparing the relative mRNA levels of Prx-2 between the normal and diseased HCEC. We also used Western blots to compare the relative expression of Prx isoforms in normal corneal endothelium and in epithelial/stromal tissues.