The potential role of amyloid β in the pathogenesis of age-related macular degeneration

Drusen are extracellular deposits that lie beneath the retinal pigment epithelium (RPE) and are the earliest signs of age-related macular degeneration (AMD). Recent proteome analysis demonstrated that amyloid β (Aβ) deposition was specific to drusen from eyes with AMD. To work toward a molecular understanding of the development of AMD from drusen, we investigated the effect of Aβ on cultured human RPE cells as well as ocular findings in neprilysin gene– disrupted mice, which leads to an increased deposition Aβ. The results showed that Aβ treatment induced a marked increase in VEGF as well as a marked decrease in pigment epithelium-derived factor (PEDF). Conditioned media from Aβ-exposed RPE cells caused a dramatic increase in tubular formation by human umbilical vein endothelial cells. Light microscopy of senescent neprilysin gene–disrupted mice showed an increased number of degenerated RPE cells with vacuoles. Electron microscopy revealed basal laminar and linear deposits beneath the RPE layer, but we did not observe choroidal neovascularization (CNV). The present study demonstrates that Aβ accumulation affects the balance between VEGF and PEDF in the RPE, and an accumulation of Aβ reproduces features characteristic of human AMD, such as RPE atrophy and basal deposit formation. Some other factors, such as breakdown of integrity of Bruch membrane, might be necessary to induce CNV of AMD. Drusen are extracellular deposits that lie beneath the retinal pigment epithelium (RPE) and are the earliest signs of age-related macular degeneration (AMD). Recent proteome analysis demonstrated that amyloid β (A β ) deposition was specific to drusen from eyes with AMD. To work toward a molecular understanding of the development of AMD from drusen, we investigated the effect of A β on cultured human RPE cells as well as ocular findings in neprilysin gene–disrupted mice, which leads to an increased deposition A β . The results showed that A β treatment induced a marked increase in VEGF as well as a marked decrease in pigment epithelium-derived factor (PEDF). Conditioned media from A β -exposed RPE cells caused a dramatic increase in tubular formation by human umbilical vein endothelial cells. Light microscopy of senescent neprilysin gene–disrupted mice showed an increased number of degenerated RPE cells with vacuoles. Electron microscopy revealed basal laminar and linear deposits beneath the RPE layer, but we did not observe choroidal neovascularization (CNV). The present study demonstrates that A β accumulation affects the for β peptides. Tube formation assay by . Experiments on tube formation were conducted in triplicate in 24-multiwell dishes using an angiogenesis kit (Kurabo) according to the manufacturer’s instructions. Briefly, the conditioned medium from A β -treated RPE cells was collected and diluted 1:1 with endothelial cell medium (Kurabo). HUVECs cocultured with human fibroblasts were cultured in the presence of the conditioned medium for 5 days. Media were changed every 2 days. Thereafter, dishes were washed with PBS and then fixed with 70% ethanol at 48°C. After the fixed cells were rinsed twice with PBS, cells were incubated with sheep anti-human vWF in PBS containing 10% FBS for 60 minutes. After washing 5 times with PBS containing 10% FBS, cells were incubated for 60 minutes with donkey anti-sheep IgG conjugated with horseradish peroxidase. Metal-enhanced DAB was used as a substrate, and the reaction yielded a dark reddish-brown insoluble product. Finally, the cells were washed 5 times with PBS and then viewed in an Olympus BHS system microscope (Olym-pus). image analyzer (in ) in and the results were subjected to statistical analysis.