Age-related accumulation of 3-nitrotyrosine and nitro-A2E in human Bruch's membrane

Abstract

Age-related macular degeneration (AMD) is a disease leading to severe visual loss and legal blindness in the elderly population. The pathophysiology of AMD is complex and may include genetic predispositions, accumulation of lipofuscin and drusen, local inflammation and neovascularization. Recently four independent research groups have identified a commonly inherited variant (Y402H) of the complement factor H gene in the genome from different groups of AMD patients. The Y402H variant of CFH significantly increases the risk of AMD and links the genetics of the disease with inflammation. During inflammation there is activation of inducible nitric oxide synthase and release of nitric oxide, which in principal could lead to non-enzymatic nitration within extracellular deposits and/or intrinsic extracellular matrix protein components of human Bruch's membrane. We have identified two biomarkers for non-enzymatic nitration in aged human Bruch's membrane, indicative of inflammation, that include 3-nitrotyrosine identified in Bruch's membrane preparations and nitrated A2E from the lipid soluble extract of the Bruch's membrane preparation. Approximately 30-40 times more A2E is observed in samples of the organic soluble extract of lipofuscin compared to the extract of Bruch's membrane. It is of interest to note that although A2E is a major constituent of RPE lipofuscin, nitrated A2E could not be detected in RPE extracts. We show here that nitro-A2E is a specific biomarker of nitrosative stress in Bruch's membrane and its concentration correlates directly with tissue age.