Purchase this article with an account.
J.V. Glenn, K. Wu, M. Boulton, A.W. Stitt; Exposure of Retinal Pigment Epithelium (RPE) to Advanced Glycation Endproduct (AGE)–Modified Substrate in vitro Alters RPE Lysosomal Capacity and Leads to Increased Lipofuscin Accumulation . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2868.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Advanced glycation endproducts (AGEs) accumulate in Bruch’s membrane during aging and have been causally linked to age–related dysfunction of the RPE and age–related macular degeneration (AMD). While it is established that AGE–exposure alters RPE cell function in vitro, the current study exposes cells to AGE–modified substrate akin to what occurs in vivo in aging Bruch’s membrane. Using this model system, we have investigated lysosomal function in RPE to determine AGE–regulation of outer segment degradative pathways.
Primary human donor RPE and the human cell line ARPE–19 were cultured (0–28 days) on a synthetic basement membrane (BM) extract (MatrigelTM). RPE were exposed to native or AGE–modified BM (AGE–BM) to mimic AGE accumulation in aged Bruch’s membrane. Cells were fed oxidised photoreceptor outer segments (ox–POS) and levels of autofluorescent inclusions monitored by flow cytometry. Enzymatic activity was examined by a colorimetric haemoglobin assay. Gene expression studies determined whether lysosomal enzyme expression and oxidative effects were altered with exposure to "aged" Bruch’s membrane. Degradative state of the phagocytosed POS was assessed by transmission electron microscopy (TEM).
Extensive alteration of enzymatic activity (cathepsin D) was evident in ARPE–19 and primary RPE exposed to AGE–BM for more than 14 days post–confluency (p<0.05). POS loading of RPE grown on AGE–BM cells significantly down regulated enzymatic activity (p<0.01, n=3). Ultrastructural evaluation of RPE grown on AGE–BM and fed ox–POS exhibited increased presence of secondary lysosomes and deposition of electron–dense granular material within cell cytoplasm. Flow cytometry results identified significant increases in auto–fluorescent cell inclusions (p<0.01) with most significant differences occurring in AGE–BM in comparison to native BM controls. JC–1 flow cytometric analysis revealed no significant change in cell integrity or depolarization of the mitochondrial membrane. Significant down–regulation of lysosomal enzyme mRNAs occurred in RPE exposed to AGE–BM.
AGE–modification of the RPE substrate, akin to what occurs in the aging retina, has a substantial impact on RPE function, in particular lysosomal degradative capacity and subsequent lipofuscin accumulation. AGE formation at the RPE–Bruch’s membrane axis may play a significant role in age–related dysfunction at the outer retina.
This PDF is available to Subscribers Only