The Accumulation of Advanced Glycation End Products in the Retinal Pigment Epithelium and in Bruch’s membrane of human donors.

Abstract

Abstract: : Purpose:Advanced glycation endproducts (AGEs) have been shown to occur in the outer retina during aging and it is becoming clear that these adducts may have a role in age–related dysfunction of the RPE–Bruch’s membrane axis. The current study has investigated accumulation of defined AGEs in human RPE and Bruch’s membrane to determine a) if these adducts are targeted to intracellular compartments and b) if they show an age–related accumulation in Bruch’s membrane. Methods:Monolayers of ARPE–19 (1 month confluent) were exposed to AGE–albumin or CML–albumin and lysosomal accumulation determined by co–localisation of immunoreactivity and lysotracker probe using dual–laser confocal microscopy. In control and AGE–exposed cells, activity of the lysosomal enzyme cathepsin D was determined by an enzymatic assay. Concurrently, Bruch’s membrane/ choroid was dissected from donor eye cups (n=72, age–range 17–93) and AGE autofluorescence levels measured spectrophotometrically (Ex: 370 nm, Em:440 nm). AGEs were also analysed by HPLC and confocal Raman spectroscopy. Results:Immunoreactive AGEs co–localised to lysosomal compartments of ARPE–19 and they caused a significant decrease in cathepsin–D activity (p<0.05). Within Bruch’s membrane/choroid, autofluorescence increased in aged samples and HPLC profiles indicated the increasing presence of the defined AGE, pentosidine, in aged individuals. Immunohistochemical staining of eye cup sections (7 µm) identified high levels of CML, especially at the level of the Bruch’s membrane but also within the choroid. Raman spectral analyses of Bruch’s membrane demonstrated distinctive novel bands that varied significantly between young and aged individuals. Conclusions:The current study has shown that AGEs accumulate in the lysosomal complex of RPE were they may cause enzymatic defects. It has also shown the presence of defined AGE adducts within the Bruch’s membrane–choriod complex that change according to age. Accumulation of AGEs in cellular and extracellular components of the RPE–Bruch’s axis may contribute to age–related abnormalities.

Keywords: Bruch's membrane • aging • retinal pigment epithelium