Evidence of oxidative stress in the pathogenesis of fuchs endothelial corneal dystrophy

Abstract

Fuchs endothelial corneal dystrophy (FECD) is a progressive, blinding disease characterized by corneal endothelial (CE) cell apoptosis. Corneal transplantation is the only measure currently available to restore vision in these patients. Despite the identification of some genetic factors, the pathophysiology of FECD remains unclear. In this study, we observed a decrease in the antioxidant response element-driven antioxidants in FECD corneal endothelium. We further demonstrated that nuclear factor erythroid 2-related factor 2, a transcription factor known to bind the antioxidant response element and activate antioxidant defense, is down-regulated in FECD endothelium. Importantly, we detected significantly higher levels of oxidative DNA damage and apoptosis in FECD endothelium compared with normal controls and pseudophakic bullous keratopathy (iatrogenic CE cell loss) specimens. A marker of oxidative DNA damage, 8-hydroxy-2'-deoxyguanosine, colocalized to mitochondria, indicating that the mitochondrial genome is the specific target of oxidative stress in FECD. Oxidative DNA damage was not detected in pseudophakic bullous keratopathy corneas, whereas it colocalized with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells in FECD samples. Ex vivo, oxidative stress caused characteristic morphological changes and apoptosis of CE, suggestive of findings that characterize FECD in vivo. Together, these data suggest that suboptimal nuclear factor erythroid 2-related factor 2-regulated defenses may account for oxidant-antioxidant imbalance in FECD, which in turn leads to oxidative DNA damage and apoptosis. This study provides evidence that oxidative stress plays a key role in FECD pathogenesis.

Figure 1

Relative expression of antioxidant and oxidative stress-related genes detected by PCR array in FECD and normal corneal endothelium. A: Scatter plot shows the distribution of the fold-changes in mRNAs between five normal and five FECD samples relative to the housekeeping genes B2M, ribosomal protein L13a, and β-actin. Bold lines represent a twofold change set as a threshold of up- and down-regulation. The middle line represents a fold-change of 1. Circles: black, more than twofold overexpressed genes; gray, gene expression less than twofold change; white, more than twofold change in underexpressed genes in FECD compared with normal. B: The data obtained from the PCR array are summarized in a scheme of the enzymatic antioxidant systems that reduce superoxide radical (O₂⁻) and H₂O₂ to water. There are three forms of SOD, the main enzymes responsible for scavenging superoxide radical. PRDX5, catalase, and glutathione peroxidases are the primary enzymes responsible for scavenging H₂O₂. TXNRD1 catalyzes the regeneration of peroxiredoxins. Glutathione reductase, glutathione transferase, and glutathione synthetase are the components of the glutathione peroxidase system. The other antioxidant systems that scavenge H₂O₂ are MT3, cytoglobin (CYGB), and albumin (ALB). The arrows indicate which genes are overexpressed or underexpressed in FECD CE as compared with normal CE. Boldfaced genes had statistically significant fold changes in FECD CE compared with normal.

Figure 2

Decreased Nrf2 protein levels in FECD compared with normal endothelium. A: Western blot analysis of Nrf2 production in FECD and normal (Nl) corneal endothelial samples. Mouse kidney extract was used as a positive control (C). Bands were detected at the appropriate molecular weight of 57 kDa. β-actin was used for normalization of protein loading. B: Densitometric analysis of Nrf2 expression in CE. Data are means ± SEM of six FECD and six normal samples. Nrf2 protein levels were significantly decreased in FECD CE compared with normal controls; *P = 0.03. C: Real-time PCR analysis showed a decrease in HO-1 expression in FECD compared with normal. Results were expressed as fold-changes and normalized to B2M mRNA expression from five FECD and normal samples. *P = 0.04. D: Real-time PCR analysis did not detect a difference in Nrf1 expression between five FECD and five normal endothelial samples. E: Confocal images of corneal endothelium from FECD and normal patients were taken after immunolocalization of Nrf2 (green). TOPRO-3 was used for nuclei staining (blue). Negative control was incubated with secondary antibody only. Original magnification, ×400 with 2 zoom. Asterisks indicate the characteristic guttae of FECD CE; multiple guttae shown in the image.

Figure 3

Increased oxidative DNA damage in FECD compared with normal CE and its colocalization with mitochondria. A: High-sensitivity ELISA was used to detect average concentration of 8-OHdG, an oxidative DNA damage marker, per nanogram DNA from patients with FECD and normal subjects. Data are means ± SEM of five FECD and five normal samples. The level of 8-OHdG in FECD CE was statistically significantly higher than in normal CE; *P = 0.006. B: In vivo confocal microscopy photographs of corneal endothelium from normal controls and patients with FECD. In FECD, the dark areas represent corneal guttae. C: Confocal images of normal (top row) and FECD CE (bottom row) in whole mounts of corneal tissue with endothelium side up. Representative images were taken after staining of mitochondria with MitoTracker Red, a mitochondrion-specific stain (red; first column), and immunolocalization of 8-OHdG (green; second column). Images of negative controls incubated with only secondary antibody are shown in the right column. TOPRO-3 was used for nuclei staining (blue). Overlay of the three channels shows colocalization of MitoTracker and 8-OHdG (fourth column) in FECD. Asterisks indicate the characteristic guttae of FECD CE. Original magnification, ×400 with 5 zoom (first column) and 8 zoom.

Figure 4

Effect of H₂O₂ treatment on the level of Nrf2 mRNA expression and oxidative DNA damage in HCECi. Corneal endothelial cells were treated with H₂O₂ (200 μmol/L) for 2 hours in three independent experiments. A: Real-time PCR analysis showed a decrease in Nrf2 expression after treatment with H₂O₂ (+H₂O₂) compared with nontreated HCECi (control). Results are expressed as fold-changes and normalized to B2M mRNA expression. B: High-sensitivity ELISA detected an increase in the average level of oxidized DNA (8-OHdG) in +H₂O₂-treated cells compared with control. Data are means ± SEM; *P < 0.05.

Figure 5

Colocalization of apoptosis and oxidative DNA damage in FECD compared with normal and PBK specimens. A: Corneal endothelium attached to its native basement membrane from normal (top), FECD (middle), and PBK (bottom) donors was labeled with TUNEL (red), anti-8-OHdG (green), and TOPRO-3 (blue). Colocalization of TUNEL and anti-8-OHdG antibodies is detected in CE cells from FECD specimens but not from PBK and normal corneas. Asterisks indicate the characteristic guttae of FECD CE. Original magnifications, ×600 and ×400 with 8 zoom. B: Corneal endothelial cell density is significantly lower in FECD and PBK specimens. The percentage of TUNEL-positive cells is higher in FECD. C: Densitometric analysis of CE labeled with anti-8-OHdG antibody indicates a significant increase in oxidative damage in FECD but not PBK compared with normal controls. Data are means ± SEM of four of each normal, FECD, and PBK samples; *P < 0.05 compared with normal CE.

Figure 6

Effect of H₂O₂ treatment on CE morphology. A: In vivo confocal microscopy photographs of corneal endothelium from normal controls and patients with FECD. Normal endothelium exhibits regularly shaped hexagonal CE cells. In FECD, the CE cell mosaic is interrupted by guttae (arrowheads) and exhibits variable size (polymegethism) and variable shape (pleomorphism). B: Mice corneal buttons were treated with H₂O₂-DMEM (0 to 100 μmol/L) for 30 minutes. Confocal images of the whole mount corneas with CE cell junctions detected by ZO-1 (white) localization. C: Automated cell analysis did not detect a change in CE cell density with increasing H₂O₂ concentrations, but the level of polymegethism (measured by coefficient of variation) and pleomorphism (measured by the number of hexagonal cells) was significantly altered after treatment with H₂O₂ at 50 μmol/L or greater concentrations. Data are means ± SD and are representative of four independent experiments; *P < 0.05, compared with untreated controls.

Figure 7

The effect of H₂O₂ on CE apoptosis and mitochondrial membrane potential ex vivo. Confocal images of whole mounts of mice corneal endothelium with detection of early apoptosis by annexin-V (green; Ann⁺/PI⁻) and late apoptosis by annexin-V and propidium iodide (red; Ann⁺/PI⁺). Endothelial cell junctions were detected by ZO-1 labeling (blue; A–D). Low-dose H₂O₂ (1 μmol/L, 37°C) induced early apoptosis after 60 minutes (B) and 90 minutes (C), and late apoptosis after 2 hours (D and E) compared with controls (A). Concurrent changes in staining with MitoTracker Red (red) were present at 60 minutes (G), 90 minutes (H), and 2 hours after the treatment (I and J). Cell nuclei were detected by TO-PRO-3 (blue) stain (F–I). Controls were incubated in DMEM only at 37°C for 0 to 12 hours, and no significant changes were detected (A and F). Results shown in E and J are means ± SD and are representative of four independent experiments; *P < 0.05, compared with untreated controls; *P < 0.01, compared with untreated controls.

Figure 8

Diagram of the pathogenesis of FECD. Endogenous and exogenous oxidative stress combined with genetic factors and postmitotic arrest of CE may lead to corneal edema seen in FECD since it causes oxidant-antioxidant imbalance, oxidative mitochondrial DNA damage, apoptosis, and CE morphological changes.