Drusen complement components C3a and C5a promote choroidal neovascularization.
Journal: 2006/April - Proceedings of the National Academy of Sciences of the United States of America
ISSN: 0027-8424
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.
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Proc Natl Acad Sci U S A 103(7): 2328-2333

Drusen complement components C3a and C5a promote choroidal neovascularization

+2 authors
*Department of Ophthalmology and Visual Sciences, University of Kentucky, Lexington, KY 40536;
Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109;
Department of Microbiology, University of Alabama, Birmingham, AL 35294;
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104;
Department of Ophthalmology and Visual Science, Moran Eye Center and Program in Human Molecular Biology and Genetics, Eccles Institute of Human Genetics, University of Utah, Salt Lake City, UT 84132; and
**Department of Ophthalmology, Medical College of Georgia, Augusta, GA 30912
To whom correspondence should be addressed. E-mail: ude.yku@2abmaj
Edited by Jeremy Nathans, Johns Hopkins University School of Medicine, Baltimore, MD, and approved December 22, 2005
M.N., B.J.R., E.S., and J.Z.B. contributed equally to this work.

Author contributions: B.K.A. and J.A. designed research; M.N., B.J.R., E.S., Y.C., J.Z.B., and J.A. performed research; J.V.S., S.R.B., J.D.L., Y.C., and K.Z. contributed new reagents/analytic tools; J.A. analyzed data; and B.J.R., S.R.B., J.D.L., B.K.A., and J.A. wrote the paper.

Edited by Jeremy Nathans, Johns Hopkins University School of Medicine, Baltimore, MD, and approved December 22, 2005
Received 2004 Nov 29

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.

Keywords: angiogenesis, inflammation, injury
Abstract

Age-related macular degeneration (AMD) is the leading cause of permanent vision loss among the elderly in many industrialized countries (1). The majority of vision loss due to AMD is a result of pathologic new blood vessels, termed choroidal neovascularization (CNV), invading the retina from the underlying choroid through fractures in Bruch membrane, the extracellular matrix between the choroid and the retinal pigmented epithelium (RPE). The earliest clinical hallmark of AMD is the appearance of drusen (2), localized lipoproteinaceous deposits between the RPE and Bruch membrane. Although their presence is an epidemiological risk factor for the development of CNV (3, 4), the mechanism of how, or whether, drusen provoke CNV remains undefined. Some investigators have suggested that drusen are epiphenomena, whereas others have claimed that drusen constituents act as a focal stimulus for inflammatory cells that secrete angiogenic molecules such as VEGF, and still others have suggested that drusen disturb RPE homeostasis by impairing transport across Bruch membrane (reviewed in ref. 5).

Recent work has demonstrated that complement components C3 and C5 are constituents of drusen in patients with AMD (69). Their presence, as well as that of the membrane-attack-complex (MAC) C5b-9 and other acute-phase reactant proteins in RPE cells overlying drusen, has fueled speculation that drusen biogenesis involves chronic inflammatory processes that either can trigger complement activation and formation of MAC acting to lyse RPE cells or disturb physiological homeostasis in RPE cells (9).

Recently, we described an animal model of AMD in aged Ccl2 and Ccr2 mice, which develop many salient pathological features seen in the human condition (10). Interestingly, RPE and choroidal deposits of C3 and C5 also are found in these mice at a young age. The inability of these mice, which are impaired in induced macrophage trafficking, to clear these complement deposits, is thought to promote the later development of CNV via up-regulation of RPE cell secretion of VEGF (10). These findings suggest a mechanistic link between deposition of complement components in drusen and the development of CNV.

There is growing evidence that complement components are more than mere mediators of innate immunity (11). To date, their influence on the molecular regulation of angiogenesis in vivo has not been fully elucidated. C3a and C5a are the bioactive fragments of C3 and C5 that effect biological responses through their receptors C3aR and C5aR. Because drusen predispose to and predate CNV, we sought to obtain direct evidence that C3a and C5a are present in drusen and play a role in CNV development. We studied their impact on VEGF expression and on the development of laser-induced CNV, an accelerated model of neovascular AMD that reproduces much of the pathology and immunophenotype of human CNV (reviewed in ref. 5), by using neutralizing antibodies against C3a or C5a, small-molecule antagonists of C3aR or C5aR, and mice deficient in C3aR or C5aR.

Acknowledgments

We thank R. J. C. Albuquerque, S. Joshi, R. King, N. Mezei, and D. W. Skufca for technical assistance; P. A. Pearson, (University of Kentucky, Lexington), A. M. Rao and G. S. Rao (Augusta, GA) for illuminating discusions; and A. A. Humbles and C. Gerard (Children’s Hospital, Harvard Medical School, Boston) for the generous gift of knockout mice. J.A. is supported by National Institutes of Health Grant EY15422, the Dennis W. Jahnigen Career Development Award administered by the American Geriatrics Society and funded by the John A. Hartford Foundation and Atlantic Philanthropies, the American Health Assistance Foundation, the E. Matilda Ziegler Foundation for the Blind, the International Retinal Research Foundation, the Macula Vision Research Foundation, and a Research to Prevent Blindness department challenge grant. E.S. is supported by a Fight for Sight postdoctoral fellowship. J.Z.B. and B.J.R. are supported by National Institute on Deafness and Other Communication Disorders/National Institutes of Health training grant 5T32DC000065. M.N. is supported by the Association for Research in Vision and Ophthalmology/Japan National Society for the Prevention of Blindness. K.Z. is supported by National Institutes of Health Grants EY14428, EY14448, P30EY014800, and GCRC M01-RR00064; the Foundation Fighting Blindness; the Ruth and Milton Steinbach Fund; the Ronald McDonald House Charities; the Macula Vision Research Foundation; the Knights Templar Eye Research Foundation; the Grant Ritter Fund; the American Health Assistance Foundation; the Karl Kirchgessner Foundation; the Val and Edith Green Foundation; and the Simmons Foundation. J.D.L. is supported by National Institutes of Health Grants GM62134 and GM69736. B.K.A. is supported by a Veterans Administration Career Development Award and the Knights Templar Eye Research Foundation.

Acknowledgments

Glossary

Abbreviations:

AMDage-related macular degeneration
CNVchoroidal neovascularization
C3/5complement components 3/5
C3a/5abioactive fragments of C3/5
C3aR/5aRCea/5a receptors
RPEretinal pigmented epithelium.
Glossary

Footnotes

Conflict of interest statement: J.A. is listed on a patent application filed by the University of Kentucky describing these findings.

This paper was submitted directly (Track II) to the PNAS office.

Footnotes

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