ARTICLE |
Correspondence to: Donald J. Brown, Ophthalmology Research, CedarsSinai Medical Center, D-5069, 8700 Beverly Blvd., Los Angeles, CA 90048. E-mail: brown2@cshs.org
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Summary |
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This study localized malondialdehyde (MDA, a toxic byproduct of lipid peroxidation), nitrotyrosine [NT, a cytotoxic byproduct of nitric oxide (NO)], and nitric oxide synthase isomers (NOS) in normal and diseased human corneas. Normal corneas (n=11) and those with clinical and histopathological diagnoses of keratoconus (n=26), bullous keratopathy (n=17), and Fuchs' endothelial dystrophy (n=12) were examined with antibodies specific for MDA, NT, eNOS (constitutive NOS), and iNOS (inducible NOS). Normal corneas showed little or no staining for MDA, NT, or iNOS, whereas eNOS was detected in the epithelium and endothelium. MDA was present in all disease groups, with each group displaying a distinct pattern of staining. NT was detected in all keratoconus and approximately one half of Fuchs' dystrophy corneas. iNOS and eNOS were evident in all the diseased corneas. Keratoconus corneas showed evidence of oxidative damage from cytotoxic byproducts generated by lipid peroxidation and the NO pathway. Bullous keratopathy corneas displayed byproducts of lipid peroxidation but not peroxynitrite (MDA but not NT). Conversely, Fuchs' dystrophy corneas displayed byproducts of peroxynitrite with little lipid peroxidation (NT >> MDA). These data suggest that oxidative damage occurs within each group of diseased corneas. However, each disease exhibits a distinctive profile, with only keratoconus showing prominent staining for both nitrotyrosine and MDA. These results suggest that keratoconus corneas do not process reactive oxygen species in a normal manner, which may play a major role in the pathogenesis of this disease.
(J Histochem Cytochem 50:341351, 2002)
Key Words: peroxynitrite, nitric oxide, nitrotyrosine, keratoconus, bullous keratopathy, Fuchs' endothelial dystrophy, malondialdehyde, lipid peroxidation
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Introduction |
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THE CORNEA is a transparent avascular tissue that allows transit of incident light to the more posterior ocular structures. Therefore, this tissue is constantly exposed to a wide spectrum of light, including the ultraviolet (UV) range. UV exposure is a well-documented environmental stress factor that generates free radicals and reactive oxygen species detrimental to most cells and tissues (
The most common indications for corneal transplantation are keratoconus and bullous keratopathy (
Recently, several lines of evidence have emerged suggesting that corneal components involved in protection against oxidative damage are altered in these disorders. ALDH3 is reportedly abnormal in keratoconus corneas (
Lipid peroxidation occurs in response to elevated levels of ROS with the liberation of reactive aldehydes, such as malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE) (
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In this study we examined over 90 corneas for evidence of oxidative damage. Using antibodies to detect MDA (lipid peroxidation), NOS isoenzymes (nitric oxide production), and NT (peroxynitrite), we demonstrated that (a) NT appeared differentially in diseased corneas (keratoconus > Fuchs' dystrophy > bullous keratopathy > normal), (b) eNOS could be found in both normal and diseased corneas, but iNOS was found only in the diseased corneas, (c) in keratoconus corneas at the site of Bowman's layer breaks, positive staining for NT correlated with the appearance of iNOS, and (d) MDA was also differentially associated with diseased corneas: keratoconus > bullous keratopathy > Fuchs' dystrophy > normal. These observations provide direct evidence that oxidative damage occurs in common human corneal disorders. Importantly, the distinct MDA and NT staining patterns for each disorder may give valuable clues to the underlying mechanisms involved in their pathogenesis.
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Materials and Methods |
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Archived formalin-fixed, paraffin-embedded corneas with confirmed clinical and histological diagnoses of keratoconus (n=26), bullous keratopathy (n=17), and Fuchs' endothelial dystrophy (n=12) were obtained for this retrospective analysis. Eleven normal corneas with no histopathological changes were also included in this study. Five-micrometer sections were mounted on poly-L-lysine coated slides. After deparaffinization and hydration, sections were incubated and permeabilized in 0.1% aqueous saponin at room temperature (RT) for 60 min and then rinsed three times in PBS. Endogenous peroxidase activity was blocked in all tissues by a 5-min incubation in 3% hydrogen peroxide (Sigma Chemical; St Louis, MO). The sections were incubated overnight at 4C with the following antibodies: (a) rabbit polyclonal anti-iNOS (the inducible isoform of NOS), raised to a 21-kD protein fragment corresponding to amino acids 9611144 of mouse macrophage iNOS (#N32030; Transduction Laboratories, San Diego, CA); (b) mouse monoclonal anti-eNOS (endothelial NOS), raised to a 20-kD protein fragment corresponding to amino acids 10301209 of human eNOS (#N30020, Transduction Laboratories); (c) mouse monoclonal antibody to nitrotyrosine (clone 1A6; Upstate Biotechnology, Lake Placid, NY) or rabbit polyclonal antibody to nitrotyrosine (raised against nitrated keyhole limpet hemocyanin; Upstate Biotechnology), and (d) rabbit anti-MDA antiserum (#MDA 11-C; Alpha Diagnostics, San Antonio, TX). This was followed by incubation (60 min, RT) in biotinylated anti-mouse or anti-rabbit secondary antibodies (Vector Laboratories; Burlingame, CA). The immunolabeled sites were visualized using an avidinbiotinperoxidase kit (Vector) with diaminobenzidine tablets (Sigma) used at the development stage. All sections were allowed to develop for 15 min, then immediately washed and counterstained with Mayer's hematoxylin (Sigma).
Corneas with fungal keratitis were used as a positive control for NT, iNOS, and eNOS. Negative controls included omission of the primary antibody and equimolar concentrations of purified IgG from normal rabbit serum (for the polyclonal primary antibodies). These samples were routinely negative. To confirm the specificity of the staining for nitrotyrosine the antibody was incubated with 10 mM nitrotyrosine as previously described (
The sections were examined and photographed with Kodak Ektachrome film using an Olympus BH-2 microscope. The developed prints were digitized using a MicroTek scanner and printed with an Epson Photo printer. Statistical analysis was performed using Fisher's exact test. p<0.05 was considered significant.
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Results |
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Lipid peroxidation occurs in response to elevated levels of ROS. This can lead to cell membrane destruction with the liberation of reactive aldehydes, such as malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE) (
Normal human corneas generally lacked MDA staining in any of the tissue layers (Fig 2A and Fig 2B; Table 1). The single case showing trace staining with this antibody is shown in Fig 2A. Bullous keratopathy corneas consistently had a positively stained epithelium, with strong diffuse staining in areas of subepithelial fibrosis (SEF: Fig 2C). The posterior collagenous layer (also referred to as the retrocorneal fibrous membrane) was also strongly positive for MDA in these corneas (Fig 2D). About two thirds of Fuchs' dystrophy corneas stained with MDA in the epithelium (Fig 2E) but were consistently negative in the posterior cornea, including the abnormal guttae associated with Descemet's membrane (Fig 2F). Keratoconus corneas were strongly stained for MDA in the epithelium and in areas of stroma adjacent to breaks in Bowman's layer (Fig 2G and Fig 2H). These observations indicate that, in these disease states, the cornea accumulates byproducts of lipid peroxidation.
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Nitric oxide, produced by oxidative deamination of L-arginine by NOS, serves as a mediator in diverse and complex cellular processes throughout the eye (
Specific patterns staining for NT were evident in the keratoconus (Fig 3G and Fig 3H) and Fuchs' endothelial dystrophy (Fig 3E) corneas, whereas minimal or no staining was seen in the normal (Fig 3A and Fig 3B) or bullous keratopathy corneas (Fig 3C and Fig 3D). It was interesting that bullous keratopathy corneas, which stained well for MDA in areas of subepithelial fibrosis and the posterior collagenous layer, lacked any detectable staining for NT. In keratoconus corneas, NT staining was extensive throughout the epithelial layer and extended into the stroma adjacent to disruptions in Bowman's layer (Fig 3G and Fig 3H). NT staining was completely blocked by pre-incubation of the antibody with 10 mM nitrotyrosine, thus confirming the specificity of the positive staining (Fig 4B). In addition, pretreatment of tissue sections with sodium hydrosulfite (to reduce nitrotyrosine to aminotyrosine) led to either markedly reduced staining or no staining at all (Fig 4C).
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Because NT staining suggested that the diseased corneas produced peroxynitrite, it was of interest to determine whether these corneas also produced NO locally. Therefore, each cornea was stained for the presence of both eNOS and iNOS. Immunoreactivity for eNOS was noted in normal corneas in the epithelium and endothelium but was not apparent in stromal cells (Fig 5A and Fig 5B). All other corneas stained clearly with this antibody in both the epithelial and endothelial layers of the tissue (Fig 5C5H). It was notable that keratoconus corneas displayed eNOS staining in stromal cells in areas adjacent to breaks in Bowman's layer (Fig 5G) and bullous keratopathy in posterior stromal cells (Fig 5D). In contrast to eNOS, iNOS antibody staining was absent or minimal in the normal corneas (Fig 6A). All the diseased corneas had increased iNOS staining in the epithelium compared to normal corneas (Fig 6A6C and Fig 6E). In bullous keratopathy, cells throughout the cornea showed weak but detectable staining (Fig 6C and Fig 6D), which was similar to the eNOS pattern. Fuchs' dystrophy was notable in that the cells near guttae (Fig 6F) were stained for iNOS. Again, keratoconus corneas displayed the most intense staining of all the corneas (Fig 6B) with all cell layers showing staining.
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The staining patterns for iNOS and NT appeared to correlate with each other. To more carefully assess this relationship, serial sections of keratoconus cases were stained for these antigens and examined. As shown in Fig 7, areas staining well with NT (Fig 7A) corresponded to areas enriched for iNOS staining (Fig 7B).
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Discussion |
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Because the cornea absorbs approximately 80% of the incident ultraviolet B (UVB) light, there is a potential for generating significant amounts of free radicals and ROS. We undertook this study to look for evidence of oxidative damage in common human corneal disorders. Because there are multiple pathways of ROS/free radical cytotoxicity, we probed for the byproducts of two prominent pathways, MDA (lipid peroxidation) and NT (peroxynitrite formation).
Malondialdehyde (MDA)
Lipid peroxidation results from UV-induced oxidative destruction of cell membranes and the formation of cytotoxic aldehydes. These aldehydes can result in altered enzyme activities, inhibition of DNA/RNA/protein synthesis, and other damaging events (
Nitrotyrosine
Several biochemical processes have been implicated in the pathogenesis of keratoconus (
Nitric Oxide Synthase
The cornea contains all the biochemical pathways necessary for free radical generation as well as free radical quenching. Corneal cells are capable of expressing isoforms of the NOS, suggesting that significant quantities of NO are produced physiologically in the cornea (
This study provides information about the distribution of eNOS in normal and diseased corneas. In agreement with others (
Finding both eNOS and iNOS in the diseased corneas certainly suggests that higher amounts of NO are present within the tissue, but this may not translate into increased NT or oxidative stress byproducts. As mentioned previously, the majority of bullous keratopathy corneas had both eNOS and iNOS but lacked NT/peroxynitrite staining. Even Fuchs' dystrophy corneas consistently displayed these NOS isoenzymes, but only a few corneas showed NT staining. This suggests that, even with ongoing disease, human corneas can adequately process the free radicals/ROS so that the cytotoxic peroxynitrite byproduct was not formed. Keratoconus appeared to lack this capacity because all these corneas showed consistent NT staining that correlated with iNOS distribution (Fig 7).
The increased formation of nitrotyrosine (peroxynitrite) and MDA in keratoconus corneas (and, to a lesser extent, in Fuchs' dystrophy and bullous keratopathy corneas) suggests an important role of free radicals in their pathogenesis. Peroxynitrite-mediated tyrosine nitration as well as peroxynitrite itself can lead to altered biological functions (
The relationship between MDA, NT, and NOS isoenzymes has been examined in a rat iron-overload renal system (
More recently, alterations in the corneal enzymes ALDH3 and extracellular SOD, required to process and scavenge free radicals, have been reported in association with human corneal disease (
To conclude, we demonstrated unique patterns of NT/peroxynitrite and/or MDA distribution in keratoconus, bullous keratopathy, and Fuchs' endothelial dystrophy corneas. The excessive amounts of both NT and MDA in the keratoconus corneas support the hypothesis that oxidative damage plays an important role in this corneal disease. In addition, although bullous keratopathy patients and Fuchs' dystrophy patients often have similar clinical symptoms, their NT and MDA staining patterns are different from each other, with MDA correlating with fibrotic changes and NT correlating with iNOS distribution, suggesting that the pathophysiological pathways may not be identical.
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Acknowledgments |
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Supported by NIH EY06807 and EY10836, the Schoellerman Charitable Foundation, the Discovery Fund for Eye Research, the Skirball Molecular Ophthalmology Program, and the National Keratoconus Foundation.
The authors wish to thank Dr. Narsing Rao (Doheny Eye Institue, Los Angeles) for his helpful comments and provision of tissues.
Received for publication October 5, 2001; accepted October 10, 2001.
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