ABSTRACT
PURPOSE: Mononuclear phagocytes (MNPs) are present in neovascular age-related macular degeneration (nv AMD) which is also called choroidal neovascularization (CNV). The number and phenotype of the MNPs depend upon the local environment in the CNV and effect of nv AMD therapy. We investigated ocular cell infiltration and conditions that modulate angiogenesis in a laser-induced mouse CNV model. METHODS: We developed assays to quantify MNPs in our established mouse CNV model. One MNP assay quantified the number of subretinal cells peripheral to the CNV lesions. A second assay semiquantitatively assesses the number of MNPs localized to the CNV lesion. We used these assays to measure the effect of toll-like receptor-2 (TLR-2) activation, anti-vascular endothelial growth factor (VEGF) therapy, and chemokine (C-C motif) ligand 2 (Ccl2) genetic deletion on MNP infiltration after laser injury. RESULTS: Laser injury induced blood vessel growth and infiltration of MNPs. Systemic administration of a TLR-2 activating peptide increased laser-induced CNV area, MNP cell numbers, and MNP density over the CNV lesions. Systemic administration of a VEGF antibody reduced CNV area, while Ccl2 genetic deletion increased CNV area. Despite the change in amount of angiogenesis, MNP infiltration was, surprisingly, unchanged in these 2 conditions. CONCLUSIONS: MNP quantification provides biological insights for candidate AMD therapies. The number of infiltrating MNP cells does not correlate with the amount of laser-induced CNV area.
Subject(s)
Choroidal Neovascularization/metabolism , Phagocytes/metabolism , Angiogenesis Inhibitors/pharmacology , Animals , Choroidal Neovascularization/drug therapy , Choroidal Neovascularization/pathology , Disease Models, Animal , Female , Lasers , Mice , Mice, Inbred C57BL , Phagocytes/drug effects , Phagocytes/pathology , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Purpose: Genome-wide association studies suggest a role for the complement system in age-related macular degeneration (AMD). We characterized ocular complement activation and evaluated a complement factor D (FD) neutralizing antibody. Methods: Mice were treated with toll-like receptor (TLR) ligands, intravitreal injection (IVT), or corneal debridement. Levels of complement proteins and mRNA were measured. A FD neutralizing antibody was administered IVT into eyes of rabbits that were challenged with LPS (lipopolysaccharide) administered intravenously. Results: Levels of C3 and factor B (FB) mRNA and protein in the eye were increased following intraperitoneal injection of TLR4 ligand LPS. Increased levels of C3 and FB breakdown products were observed in both eye tissues and plasma. Complement activation products were markedly reduced in C3-/- and Cfb-/- mice challenged with LPS. Ocular complement levels were also elevated in mice treated systemically with TLR2 and -3 ligands, injured by IVT injection or corneal debridement, or even in normal aging. IVT administration of a complement FD neutralizing antibody in rabbits inhibited LPS-induced complement activation in the posterior segment of the eye, but not in the anterior segment of the eye or in plasma. Conclusions: Systemic TLR stimulation and eye tissue injury induced time-dependent alternative complement pathway activation in the eye. Ocular complement levels were also gradually elevated during aging. An anti-FD antibody IVT potently inhibited LPS-induced complement activation in the posterior segment of the eye. This study provides insights into the dynamic profile of ocular complement activation, which is valuable for complement research in eye diseases and for developing complement therapeutics for AMD.
