Drusen complement components C3a and C5a promote choroidal neovascularization

Abstract

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in industrialized nations, affecting 30-50 million people worldwide. The earliest clinical hallmark of AMD is the presence of drusen, extracellular deposits that accumulate beneath the retinal pigmented epithelium. Although drusen nearly always precede and increase the risk of choroidal neovascularization (CNV), the late vision-threatening stage of AMD, it is unknown whether drusen contribute to the development of CNV. Both in patients with AMD and in a recently described mouse model of AMD, early subretinal pigmented epithelium deposition of complement components C3 and C5 occurs, suggesting a contributing role for these inflammatory proteins in the development of AMD. Here we provide evidence that bioactive fragments of these complement components (C3a and C5a) are present in drusen of patients with AMD, and that C3a and C5a induce VEGF expression in vitro and in vivo. Further, we demonstrate that C3a and C5a are generated early in the course of laser-induced CNV, an accelerated model of neovascular AMD driven by VEGF and recruitment of leukocytes into the choroid. We also show that genetic ablation of receptors for C3a or C5a reduces VEGF expression, leukocyte recruitment, and CNV formation after laser injury, and that antibody-mediated neutralization of C3a or C5a or pharmacological blockade of their receptors also reduces CNV. Collectively, these findings establish a mechanistic basis for the clinical observation that drusen predispose to CNV, revealing a role for immunological phenomena in angiogenesis and providing therapeutic targets for AMD.

Fig. 1.

C3a and C5a were present in human drusen. Representative images from a 91-year-old woman with AMD (A–G) and an 87-year-old man without AMD (H and I). Examples of hard drusen (asterisks) and Bruch membrane (black arrows) immunoreactivity (blue) to C3a (A and B) or C5a (D and E) in the eye of patients with AMD and lack of immunolabeling in neural retina, choroid, or intervening RPE in the eye of control (H and I). C3a (C) and C5a (F) staining in soft drusen deposits between RPE (brown pigmented cells) and Bruch membrane (black arrows). C5a is localized to vesicular structures (red arrows) in hard drusen (D). Lack of nonspecific labeling in the AMD eye demonstrated by omitting primary anticomplement anaphylatoxin antibody (G). (Scale bars, 10 μm.)

Fig. 2.

C3a and C5a up-regulated VEGF in vitro and in vivo. C3a (50 ng/ml) and C5a (50 ng/ml) up-regulated human RPE cell secretion of VEGF 8 h after stimulation compared with unstimulated (control) or PBS-stimulated cells (A). Intravitreous injection of C3a (square) or C5a (circle) in wild-type mice increased VEGF levels in the RPE/choroid (solid lines) but not in the neurosensory retina (dotted lines) in a dose-dependent fashion 4 h after injection compared to PBS injection (0 ng) (B). ∗, P < 0.05 compared with PBS. No difference in the fraction (%) of neutrophils (C) or macrophages (D) in the choroid 4 h after intravitreous injection of C3a or C5a was observed compared with PBS.

Fig. 3.

Laser injury-induced complement in wild-type mice. ELISA demonstrated increased levels of C3a and C5a in the RPE/choroid within 4 h of injury and a maximum at 12 h after injury. (A) ∗, P < 0.05 compared with PBS. C3 (B) and C5 (C) were deposited (red) in the proximity of RPE cells (green) in the area of injury 4 h after injury. Areas of complement colocalization with RPE cells appear yellow. No deposition of C3 (D) or C5 (E) was identified in unlasered areas. (Scale bars, 5 μm.) DAPI staining appears blue.

Fig. 4.

VEGF induction was diminished in C3aR^–/– and C5aR^–/– mice. VEGF levels in the RPE/choroid were significantly reduced in both knockout strains at 1 and 3 days after laser injury. ∗, P < 0.05 compared with wild-type mice.

Fig. 5.

Leukocyte recruitment to the choroid was diminished in C3aR^–/– and C5aR^–/– mice. The fraction (%) of neutrophils in the choroid, maximal at 1 day after injury (A), and of macrophages in the choroid, maximal at 3 days after injury (C), was significantly reduced in both knockout strains (B and D). ∗, P < 0.05 compared with wild-type (wt) mice.

Fig. 6.

Disruption of complement function inhibited CNV. Stacked confocal images (1-μm sections) of FITC–isolectin B4-labeled tissue within laser scars in wild-type (A), C3aR^–/– (B), and C5aR^–/– (C) mice demonstrate reduction in CNV volume in knockout animals (D). ∗, P < 0.05 compared with wild-type (wt) mice. (Scale bar, 100 μm.) CNV was reduced in wild-type mice treated with neutralizing anti-C3a or -C5a antibodies (2 μg) compared with isotype control antibodies (E) or with C3a receptor antagonist or C5a receptor antagonist (1 μg) compared with control peptide or PBS (F). ∗, P < 0.05 compared with control IgGs (E) or control peptide (F).