Abstract
Purpose: :
To compare the expression of antioxidant and oxidative stress-related genes in normal human corneal endothelium to endothelium of Fuchs endothelial corneal dystrophy (FECD), and to assess the in vivo levels of oxidant-induced DNA damage in the normal and diseased corneal endothelium.
Methods: :
Nine pairs of FECD and normal specimens were sex and age matched. Corneal endothelial cell-Descemet’s membrane (HCEC-DM) complexes were dissected from the stroma, and RNA or DNA was extracted. Gene expression studies were performed using a focused PCR array that contains genes involved in oxidative stress. Relative changes in mRNA level were calculated, and cut-off values were set at ≤0.5 fold and ≥2 fold for considering a gene to be underexpressed or overexpressed, respectively. Oxidative DNA damage was determined by measuring the level of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in normal and FECD HCEC-DM by a competitive ELISA assay.
Results: :
Out of 57 oxidative stress-related genes, 11 transcripts were downregulated, and 3 transcripts were upregulated in FECD compared with normal tissue. Of the downregulated genes, 6 were antioxidant genes (metallothionein 3 [MT3], superoxide dismutase [SOD] 2 and 3, and peroxiredoxin [Prx] 2, 5, and 6), 3 were oxidative stress responsive genes (angiopoietin-like 7, dual specificity phosphatase 1, and glyceraldehyde-3-phosphate dehydrogenase [GAPDH]), and 2 were reactive oxygen species metabolism genes (BCL2/adenovirus E1B 19kDa interacting protein 3 [BNIP3])andnitric oxide synthase 2A). The transcriptional decrease of Prx genes corroborated our previous proteomic data on Prx downregulation. Upregulated transcripts were glutathione reductase (GSR), epoxide hydrolase 2, and albumin. SOD2, Prx5, BNIP3, and GSR had a known mitochondrial location and involvement in the intrinsic apoptotic pathway. ELISA analysis revealed that the average level of 8-OHdG DNA lesions in FECD was 8-fold higher compared to normal endothelium (p=0.006).
Conclusions: :
This study identified a transcriptional modulation of antioxidant and oxidative stress responsive genes and a concomitant accumulation of oxidized DNA lesions in FECD endothelium. These findings suggest that dysregulation of the antioxidant profile can be directly linked to FECD endothelial cell oxidative damage and potentially apoptosis.
Keywords: cornea: endothelium • oxidation/oxidative or free radical damage • antioxidants