Role of Complement and Complement Membrane Attack Complex in Laser-Induced Choroidal Neovascularization 1 Puran S. Bora, 2 Jeong-Hyeon Sohn, Jose M. C. Cruz, Purushottam Jha, Hiroki Nishihori, Yali Wang, Sankaranarayanan Kaliappan, Henry J. Kaplan, and Nalini S. Bora Choroidal neovascularization (CNV), or choroidal angiogenesis, is the hallmark of age-related macular degeneration and a leading cause of visual loss after age 55. The pathogenesis of new choroidal vessel formation is poorly understood. Although inflammation has been implicated in the development of CNV, the role of complement in CNV has not been explored experimentally. A reliable way to produce CNV in animals is to rupture Bruch’s membrane with laser photocoagulation. A murine model of laser-induced CNV in C57BL/6 mice revealed the deposition of C3 and membrane attack complex (MAC) in the neovascular complex. CNV was inhibited by complement depletion using cobra venom factor and did not develop in C3 / mice. Anti-murine C6 Abs in C57BL/6 mice inhibited MAC formation and also resulted in the inhibition of CNV. Vascular endothelial growth factor, TGF-2, and -fibroblast growth factor were elevated in C57BL/6 mice after laser-induced CNV; complement depletion resulted in a marked reduction in the level of these angiogenic factors. Thus, activation of complement, specifically the formation of MAC, is essential for the development of laser- induced choroidal angiogenesis in mice. It is possible that a similar mechanism may be involved in the pathophysiology of other angiogenesis essential diseases. The Journal of Immunology, 2005, 174: 491– 497. A ge-related macular degeneration (AMD) 3 is the leading cause of visual loss in individuals over age 55. Two major clinical phenotypes of AMD are recognized—a nonexudative (dry) type and an exudative (wet) type. Although choroidal neovascularization (CNV), which causes exudative type AMD, occurs in a minority of patients with AMD, 10% of AMD cases are of the exudative type. CNV is responsible for the sudden and disabling loss of central vision (1–5). CNV is a complex biological process and the pathogenesis of new choroidal vessel formation is not completely understood (6 – 11). Several factors, such as inflammation (3, 12–14), ischemia (13), and local production of angiogenic factors (15, 16), are thought to be important in the pathogenesis of CNV. Recent stud- ies have identified the macrophage as an important component of laser-induced CNV response (17–19). Although inflammation has been implicated in the development of CNV, the role of complement has not been explored. The com- plement system is a major component of innate immunity and plays a central role in the host defense against infection (20 –22). Membrane attack complex (MAC), the final product of the acti- vated complement cascade has been reported to release growth factors such as -fibroblast growth factor (-FGF), vascular en- dothelial growth factor (VEGF), and platelet-derived growth factor from various nucleated cells (23–26). These reports suggest that MAC-mediated release of growth factors from nucleated cells may be a pathogenic mechanism in angiogenesis. A reliable way to produce CNV in animals is to rupture Bruch’s membrane with laser photocoagulation (27, 28). In the present study, we used the mouse model of laser-induced CNV to investigate the role of the complement system, and particularly MAC formation in the cho- roidal angiogenesis. Materials and Methods Animals Male C57BL/6 mice (6 – 8 wk old), C3-deficient mice (C3 -/- ), and their wild-type control ((129  C57BL/6)F 1 ) were purchased from The Jackson Laboratory. This study was approved by the Institutional Animal Care and Use Committee of University of Louisville. Induction of CNV in mice Animals were divided into four groups. CNV was induced by laser pho- tocoagulation in C57BL/6 mouse (group 1; n = 10; complement sufficient) with the krypton red laser (50-m spot size; 0.05-s duration; 250 mW) as previously described by us (29, 30). Three laser spots were placed in each eye close to the optic nerve. In group 2, C57BL/6 mice (n = 10) were treated i.p. with 4 U of cobra venom factor (CVF; Quidel) 2 days before laser photocoagulation and every day after laser treatment. We refer to these animals as “complement depleted” throughout this paper. Group 3 had C3 -/- mice (n = 10), and group 4 consisted of 10 wild-type control ((129  C57BL/6)F 1 ) for the C3-deficient mice. Laser photocoagulation in all four groups was performed as described above. These experiments were repeated five times. Measurement of CNV and CNV lesions Seven days after laser treatment, all animals were perfused with 1 ml of PBS containing 50 mg/ml fluorescein-labeled dextran (FITC-dextran; av- erage molecular mass, 2  10 6 ; Sigma-Aldrich) and sacrificed. The eyes were harvested and fixed in 10% phosphate-buffered formalin, and retinal pigment epithelium (RPE)-choroid-scleral flat mounts were prepared as previously described (29, 30). RPE-choroid-scleral flat mounts were stained for elastin using a mAb specific for elastin (1.0 mg/ml; 1/200 di- lution; Sigma-Aldrich) followed by a Cy3-labeled secondary Ab (1.0 mg/ ml; 1/200 dilution; Sigma-Aldrich). The incidence and the size of CNV Department of Ophthalmology and Visual Sciences, Kentucky Lions Eye Center, University of Louisville, Louisville, KY 40202 Received for publication July 27, 2004. Accepted for publication October 8, 2004. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported in part by EY 014623 and EY 13335; Commonwealth of Kentucky Research Challenge Trust Fund; and Research to Prevent Blindness, Inc., New York. 2 Address correspondence and reprint requests to Dr. Puran S. Bora, Department of Ophthalmology and Visual Sciences, Kentucky Lions Eye Center, 301E Muhammad Ali Boulevard, University of Louisville, Louisville, KY 40202. E-mail address: psbora01@louisville.edu 3 Abbreviations used in this paper: AMD, age-related macular degeneration; CNV, choroidal neovascularization; MAC, membrane attack complex; -FGF, -fibroblast growth factor; VEGF, vascular endothelial growth factor; CVF, cobra venom factor; RPE, retinal pigment epithelium; F, forward; R, reverse; CH 50 , total complement hemolytic activity. 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