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

Abstract

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 beta (Abeta) 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 Abeta on cultured human RPE cells as well as ocular findings in neprilysin gene-disrupted mice, which leads to an increased deposition Abeta. The results showed that Abeta treatment induced a marked increase in VEGF as well as a marked decrease in pigment epithelium-derived factor (PEDF). Conditioned media from Abeta-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 Abeta accumulation affects the balance between VEGF and PEDF in the RPE, and an accumulation of Abeta 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.

Figure 1

RT-PCR detection of APP, neprilysin, and β-secretase in human RPE cells. Human RPE cells were cultured in DMEM containing 10% FCS. After cellular confluence, total RNA was extracted from cultured RPE cells. The cDNA was synthesized from 2 μg of total RNA, and the reaction product was subjected to PCR amplification using specific primers for APP, neprilysin, and β-secretase.

Figure 2

Immunohistochemical detection of APP, neprilysin, and β-secretase in the RPE cell layer of albino ddY mice. Paraformaldehyde-fixed sections from eyeballs of male albino ddY mice were stained for APP (A), neprilysin (B), or β-secretase (C). Immune complexes were detected with DAB as a brown reaction product. Immunoreactivity for APP, neprilysin, and β-secretase was observed in the RPE cell layer. DAB staining was absent when nonimmune serum was substituted for the primary antibody (D). Scale bars: 20 μm (A–D). ONL, outer nuclear layer; OS, outer segment.

Figure 3

Concentration of immunoreactive VEGF in culture supernatants of RPE cells. Human RPE cells were incubated with various concentrations of Aβ (1∼25 μM), and VEGF protein expression in the culture supernatants was analyzed by ELISA after 24 hours of culture. Values are expressed as mean ± SEM. n = 3; *P < 0.05.

Figure 4

Effects of the conditioned media from RPE on in vitro angiogenesis. HUVECs cocultured with fibroblasts were incubated with a 1:1 mixture of the supernatant of RPE cells and endothelial cell culture media. After 10 days of culture, the cells were fixed and stained with vWF. Representative phase-contrast microscopic images are shown (A–D). The supernatant used was conditioned by RPE cells without exposure to Aβ (B) or with exposure to 25 μM Aβ_40–1 (C) or 25 μM Aβ_1–40 (D) for 24 hours. In A, 25 μM Aβ_1–40 alone was mixed with the endothelial cell culture media. (E) The total length of tube-like structures in HUVECs. The area of capillary growth was quantitated with an image analyzer in 8 different fields for each condition at ×10 magnification and statistically analyzed. Data are expressed as mean ± SEM. Asterisk indicates that the value is significantly greater than that of the corresponding control. n = 3; P < 0.01.

Figure 5

Light microscopy of H&E-stained sections of 27-month-old neprilysin-deficient (B and D) and age-matched wild-type (A and C) mice retinas. C and D show magnified views of A and B, respectively. (A and C) Morphology of normal retina from age-matched wild-type mice. (B and D) Morphology of senescent neprilysin-deficient mice showing degenerated cells with vacuoles in the RPE layer (arrowheads). Scale bars: 150 μm (A and B); 30 μm (C and D).

Figure 6

Fluorescent micrographs of paraformaldehyde-fixed sections from 27-month-old neprilysin-deficient (B and D) and age-matched wild-type mouse (A and C) retinas. The sections were treated with antibodies against VEGF (FITC: A and B) or antibodies against PEDF (Texas Red: C and D). Scale bars: 40 μm (A–D).

Figure 7

Electron micrographs of retinas from a 27-month-old neprilysin gene–disrupted mouse (A–C) and an age-matched wild-type mouse (D–F). (A) An RPE cell layer from the retina of a senescent neprilysin gene–disrupted mouse. Note the abundant cytoplasmic vacuoles among attenuated RPE cells. (B) Disruption of junction structures between RPE cells and substantial subepithelial deposits. (C) An electron micrograph of extensive deposits beneath RPE cells. (D) An RPE cell layer from the retina of a senescent wild-type mouse. RPE cell layer shows normal appearance. Cytoplasmic vacuoles among RPE cells are not obvious. (E and F) Basal infoldings of RPE cells are prominent, and no subepithelial deposits are observed. Scale bars: 1 μm (A–F).

Figure 8

Localization of Aβ in sub-RPE deposits from neprilysin gene–disrupted mice by indirect immunogold labeling. Cryostat sections were prepared from senescent neprilysin gene–disrupted mice and incubated with anti-Aβ antibody (4G8) and anti-mouse 10- to 15-nm gold conjugate (for scanning electron microscopy images) or 5- to 10-nm gold conjugate (for transmission electron microscopy images). Gold particles were present within the subretinal deposit (C and E) as well as in the cytoplasm of RPE cells (E, arrowheads). A–C are scanning electron microscopy images. B shows higher magnification of A, and C shows higher magnification of B. D and E are transmission electron microscopy images. E shows higher magnification of D. DE, subretinal deposit; BM, Bruch membrane; CH, choroid. Scale bars: 10 μm (A); 3.6 μm (B); 360 nm (C); 1 μm (D); and 0.1 μm (E).