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PURPOSE: To test the hypothesis that neovascular age-related macular degeneration is related to oxidative stress involving the macular retinal pigment epithelium. This study investigated, as a function of age, levels of enzymes that defend tissues against oxidative stress in the macular retinal pigment epithelium of human eyes with this disease. METHODS: Surgical specimens of macular choroidal neovascular membranes from eyes with age-related macular degeneration and the macular regions of whole donor eyes with neovascular age-related macular degeneration or without evident ocular disease were studied by quantitative electron microscopic immunocytochemistry with colloidal gold–labeled second antibodies. Relative levels in retinal pigment epithelium cell cytoplasm and lysosomes were determined of five enzymes believed to protect cells from oxidative stress, as well as levels of the retinal pigment epithelium marker cytoplasmic retinaldehyde-binding protein, for comparison with the enzymes. RESULTS: Copper, zinc superoxide dismutase immunoreactivity increased and catalase immunoreactivity decreased with age in cytoplasm and lysosomes from macular retinal pigment epithelium cells of normal eyes and eyes with age-related macular degeneration. Cytoplasmic retinaldehyde-binding protein immunoreactivity showed no significant relationship to age or the presence of neovascular age-related macular degeneration. Glutathione peroxidase immunoreactivity was absent from human retinal pigment epithelium cells. Both heme oxygenase-1 and heme oxygenase-2 had highly significantly greater immunoreactivity in retinal pigment epithelium cell lysosomes than in cytoplasm, differing from the much greater cytoplasmic immunoreactivity of the other proteins studied. This immunoreactivity decreased with age, particularly in the lysosomes of retinal pigment epithelium cells from eyes with neovascular age-related macular degeneration. These decreases were of borderline significance ( P = .067 for heme oxygenase-1; P = .12 for heme oxygenase-2) when eyes with age-related macular degeneration were compared with normal eyes by multivariable logistic regression. CONCLUSIONS: The high heme oxygenase-1 and heme oxygenase-2 lysosomal antigen levels in macular retinal pigment epithelium cells of eyes with neovascular age-related macular degeneration suggest that oxidative stress causes a pathologic upregulation of these enzymes. Increased lysosomal disposal may indicate that the reparative functions of these enzymes are accompanied by deleterious effects, necessitating their rapid removal from the cell. The much higher heme oxygenase-1 and heme oxygenase-2 antigen levels in macular retinal pigment epithelium cells from younger individuals suggest that protective mechanisms against oxidation and, hence, presumably to the development of age-related macular degeneration, decrease with age.

Purpose To determine whether vascular endothelial growth factor and basic fibroblast growth factor, which may be critical mitogens for neovascularization, are present together in human retinal and choroidal neovascular membranes. Methods Light microscopic immunocytochemistry using antibodies against vascular endothelial growth factor, basic fibroblast growth factor, and several cellular “oemarker” proteins on frozen sections from three choroidal neovascular membranes from patients with age-related macular degeneration, seven surgically excised epiretinal membranes from patients with proliferative diabetic retinopathy, and six epiretinal membranes from patients with nonischemic proliferative retinopathies. Results All three choroidal neovascular membranes and all seven epiretinal membranes stained positive for vascular endothelial growth factor. Two choroidal neovascular membranes and six of the epiretinal membranes were positive for basic fibroblast growth factor. The same cells were often positive for both antigens. None of the epiretinal membranes from patients with nonischemic proliferative retinopathies were positive for either growth factor. Many of the cells that demonstrated growth factors were glial cells, vascular endothelial cells, and retinal pigment epithelial cells. Conclusions Colocalization of two growth factors in the same cells of ocular neovascular membranes suggests that more than one growth factor may contribute to pathologic angiogenesis. Growth factors in neovascular tissues are not localized exclusively in the vascular endothelium. Because expression of some growth factors is stimulated by hypoxia, their localization within choroidal neovascular membranes suggests that hypoxia may be an etiologic factor for choroidal as well as for retinal neovascularization.