| The purpose of this thesis research is to better understand the role of oxidative damage in age related macular degeneration (AMD), the most common cause of legal blindness in the elderly population. In angiogenesis studies, carboxyethylpyrrole (CEP) adducts, derived from free radical oxidation of docosahexaenoyl lipids, were found to stimulate neovascularization in chick chorioallantoic membrane and rat corneal micropocket assays. Monoclonal anti-CEP antibody was found to neutralize CEP stimulated neovascularization, however, anti-vascular endothelial growth factor antibody only partially neutralized vessel growth. Primary retinal pigment epithelial (RPE) cells and the human ARPE19 treated in vitro with CEP-dipeptide did not stimulate vascular endothelial growth factor secretion, suggesting CEP adducts follow a vascular endothelial growth factor independent pathway. Overall, these results suggest that CEP may play a role in choroidal neovascularization, and anti-CEP therapeutic modalities might be of value in limiting choroidal neovascularization in AMD. In retinal light damage studies, CEP, argpyrimidine, and nitrotyrosine immunoreactivities in rodent retina were found to be significantly greater after 4 h green light exposure compared with control animals maintained in the dark. These findings justify further consideration of protein modifications as mediators in the light-induced biochemical sequelae leading to photoreceptor cell death. Similar results were obtained with 6 h blue light exposure. CEP adducts and autoantibodies were lowered after pretreatment with drugs prior to blue light exposure. Because CEP adducts and autoantibodies are also elevated in human AMD plasma, experimental drugs that lowered retinal light damage may eventually become useful as therapeutics for dry AMD. In studies directed at characterizing RPE lipofuscin, lipofuscin granules were purified with proteinase K or with SDS. Purified and crude lipofuscin were found to be equally phototoxic to cultured RPE cells and to exhibit no difference in the content of A2E, isoA2E, all-trans-retinal dimer-phosphatidylethanolamine, CEP, iso[4]levuglandin E2-adducts, or nitrotyrosine. Notably the purified granules were found to contain very little protein that is degradable to amino acids (∼2% w/w), implicating the bulk of the lipofuscin granule, which is not protein, as the bioactive component in phototoxicity. In studies of a superoxide dismutase2 knockdown mouse, CEP and nitrotyrosine immunoreactivities were found to be significantly greater in the RPE compared with wild type animals. The superoxide dismutase2 knockdown mouse develops an AMD-like phenotype, supporting a role for oxidative damage in AMD.; Overall, this dissertation research provides mechanistic clues to the etiology and pathology of AMD. |
