Publications by Year: 2008

2008
Jurkunas UV, Rawe I, Bitar MS, Zhu C, Harris DL, Colby K, Joyce NC. Decreased expression of peroxiredoxins in Fuchs' endothelial dystrophy. Invest Ophthalmol Vis Sci. 2008;49 (7) :2956-63.Abstract
PURPOSE: To compare the relative expression of peroxiredoxin (Prx) proteins in normal human corneal endothelium with endothelium in corneas affected by Fuchs' endothelial dystrophy (FED) and between normal human endothelium and epithelial/stromal tissue. METHODS: Human corneal endothelial cell-Descemet's membrane (HCEC-DM) complexes from normal and FED corneal buttons were dissected from the epithelium/stroma. For proteomic analysis, HCEC-DM protein extracts were separated by using two-dimensional gel electrophoresis. Relative differences in protein spot density was analyzed. Proteins of interest, including Prx isoforms, were identified by MALDI-TOF (matrix-assisted desorption ionization-time of flight) mass spectrometry. Western blot analysis compared the relative expression of Prx isoforms in normal and FED endothelium and between normal endothelium and normal epithelium/stroma. Expression of Prx-2 mRNA was compared by using real-time PCR. RESULTS: Proteomic analysis identified differences in the relative expression of Prx isoforms between normal and FED endothelium. Western blot analysis confirmed that expression of Prx-2, -3, and -5 was significantly decreased (P < 0.05) in FED cells. Normal HCECs expressed significantly (P < 0.05) higher levels of Prx-2 and -3 than did the epithelium/stroma. Expression of Prx-5 was not significantly different (P > 0.05) in the endothelium versus the epithelium/stroma. Real-time PCR analysis revealed that Prx-2 mRNA was significantly decreased (P = 0.027) in FED samples. CONCLUSIONS: Prx proteins were identified in human corneal endothelium. The fact that Prx-2 and -3 were expressed at significantly higher levels in HCEC-DM compared with the epithelium/stroma reflects the different physiologic activities of individual corneal cell types. Significantly decreased expression of Prx-2, -3, and -5 in FED may suggest an alteration in the ability of endothelial cells to withstand oxidant-induced damage and may be closely related to the pathogenesis of this disease.
Jurkunas UV, Bitar MS, Rawe I, Harris DL, Colby K, Joyce NC. Increased clusterin expression in Fuchs' endothelial dystrophy. Invest Ophthalmol Vis Sci. 2008;49 (7) :2946-55.Abstract
PURPOSE: To investigate the differential expression of the glycoprotein clusterin/apoJ (CLU) in normal and Fuchs' endothelial dystrophy (FED) corneal endothelium and to compare the expression of various forms of CLU in normal and FED tissue. METHODS: FED and pseudophakic bullous keratopathy (PBK) corneal buttons were removed during transplantation, and normal corneas were obtained from tissue banks. Human corneal endothelial cells and Descemet's membrane (HCEC-DM) complex was dissected from the stroma. Proteins were separated on 2-D gels and subjected to comparative proteomic analysis. Relative expression of presecretory CLU (pre-sCLU), secretory (s)CLU, and nuclear (n)CLU were compared between normal and FED HCEC-DM by Western blot analysis. Expression of CLU mRNA was compared by using RT-PCR. Subcellular localization of CLU was compared in corneal wholemounts from normal eyes and eyes with FED by immunocytochemistry followed by confocal microscopy. RESULTS: Proteomic analysis revealed an apparent increase in CLU expression in FED HCEC-DM compared with the normal control. Western blot analysis demonstrated that pre-sCLU protein expression was 5.2 times higher in FED than in normal samples (P = 3.52E-05), whereas the mature form modified for secretion (sCLU) was not significantly elevated (P = 0.092). Expression of nCLU protein was significantly elevated in FED (P = 0.013). RT-PCR analysis revealed that CLU mRNA was significantly increased (P = 0.002) in FED samples, but not in PBK samples. CLU also had a distinctive localization in FED samples with enhanced intracellular staining around the guttae and in the nuclei of endothelial cells. CONCLUSIONS: CLU expression is markedly elevated in FED-affected tissue, pointing to a yet undiscovered form of dysregulation of endothelial cell function involved in FED pathogenesis.