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

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.

Bilateral nummular infiltrates: An uncommon presentation of Candida keratitis.

PURPOSE: We present a case of misdiagnosed fungal keratitis due to a bilateral nummular presentation. OBSERVATIONS: A 41-year-old female patient, contact lens wearer, presented initially at an optometrist with acute bilateral blurred vision and photophobia. She was found on exam to have multiple round stromal infiltrates bilaterally. She did not have significant ocular surface issues prior and had no history of topical steroid use. Adenovirus testing was negative, and she was started on a topical antibiotic-steroid combination. She did not improve and was referred for further evaluation. We initially suspected a herpetic infection and began treatment with oral antivirals. Cultures came back positive for Candida albicans in the right eye. She was very photophobic and cultures were unable to adequately be obtained from the left eye. She was switched to topical voriconazole drop in both eyes and gradually improved with excellent visual outcome. Urogenital cultures were negative. Contact lens use was discontinued through the course of treatment. CONCLUSIONS: Bilateral Candida keratitis is rare and has not been reported in a nummular pattern and in a patient without significant ocular surface issues or chronic use of steroid drops. Differential diagnosis of nummular keratitis mostly includes viral and inflammatory conditions. This case highlights the need to stay alert to a possible fungal etiology and a potential risk of using topical steroids at initial presentation of nummular keratitis.

The Correlation Between Dry Eyes, Anxiety and Depression: The Sicca, Anxiety and Depression Study.

PURPOSE: To determine whether improvement in the severity of dry eye disease (DED) symptoms correlates with improvement in anxiety and depression. METHODS: This prospective interventional case series recruited 45 adults with evidence of DED. Patients were administered the University of North Carolina Dry Eye Management Scale (DEMS), Generalized Anxiety Disorder 7-item scale (GAD-7), and Personal Health Questionnaire Depression Scale (PHQ-8) to evaluate the severity of DED symptoms, anxiety, and depression, respectively. Standard of care treatment was provided for patients for 3 to 6 months, followed by re-administration of the DEMS, GAD-7, and PHQ-9 surveys. Statistical analysis was performed to assess the relationships between changes in survey scores. RESULTS: Participants had a mean age of 65.5 (SD, 13.3) years, and 37 (84.6%) were women. Seven patients were lost to follow-up. DEMS and GAD-7 significantly improved from 5.8 ± 1.8 to 4.6 ± 0.2.2 (P = 0.01) and from 5.6 ± 5.5 to 3.3 ± 4.6 (P = 0.05), respectively. Changes in DEMS correlated with changes in PHQ-8 (ρ = 0.3 P = 0.05), but not with changes in GAD-7 (ρ = 0.2 P = 0.3). Changes in DEMS correlated with changes in both PHQ-8 and GAD-7 in the subgroup of patients without prior depression or anxiety diagnosis (ρ = 0.6, P = 0.002; ρ = 0.4, P = 0.02). A multivariate analysis showed that the relationship between DEMS, PHQ-8, and GAD-7 was independent of a prior diagnosis of depression or anxiety and of the presence of comorbidities. CONCLUSIONS: There is a significant correlation between the severity of DED and symptoms of depression and anxiety. Effective DED treatment could have a positive impact on the symptoms of depression and anxiety.

Decline in DJ-1 and decreased nuclear translocation of Nrf2 in Fuchs endothelial corneal dystrophy.

PURPOSE: This study sought to determine factors involved in nuclear factor erythroid 2-related factor 2 (Nrf2) regulation and their response to oxidative stress in Fuchs endothelial corneal dystrophy (FECD) and normal corneal endothelial cells (CECs). METHODS: FECD corneal buttons were obtained from transplantations and normal human corneas from tissue banks. Oxidative stress was induced by tert-butyl hydroperoxide (tBHP). Protein and mRNA levels of Nrf2, DJ-1, p53, and Kelch-like ECH-associated protein1 (Keap1) were investigated using Western blotting and real-time PCR. Immunoprecipitation was used to detect levels of oxidized DJ-1 protein and Cullin 3- (Cul3)-regulated degradation of DJ-1 in immortalized FECD (FECDi) and normal CEC (HCECi) cell lines. Nrf2 subcellular localization was assessed by immunocytochemistry. RESULTS: Nrf2 protein stabilizer, DJ-1, decreased significantly in FECD CECs compared with normal, whereas Nrf2 protein repressor, Keap1, was unchanged at baseline but increased under oxidative stress. Under oxidative stress, normal CECs upregulated DJ-1 protein synthesis, whereas FECD CECs did not. DJ-1 decline correlated with increased DJ-1 oxidative modification and carbonylation in FECDi as compared with HCECi. Increased labeling of immunoprecipitated DJ-1 protein with anti-Cul3 antibody indicated enhanced DJ-1 degradation in FECDi as compared with HCECi. Following tBHP treatment, Nrf2 translocated from cytoplasm to nuclei in normal CECs, whereas Nrf2 nuclear localization was not observed in FECD. CONCLUSIONS: Decreased levels of DJ-1 in FECD at baseline and under oxidative stress correlate with impaired Nrf2 nuclear translocation and heightened cell susceptibility to apoptosis. Targeting the DJ-1/Nrf2 axis could yield a mechanism to slow CEC degeneration in FECD.

Decreased expression of peroxiredoxins in Fuchs' endothelial dystrophy.

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.

Increased clusterin expression in Fuchs' endothelial dystrophy.

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.