Neovascularization in a mouse model that simulates exudative macular degeneration is complement dependent.

Abstract

Abstract: : Purpose: To investigate the role of the complement system in the mouse model of laser–induced choroidal neovascularization (CNV). Methods: CNV was induced by laser photocoagulation in the C57BL/6 mouse with the krypton red laser. Three laser spots were placed in each eye of four different groups of mice (n=10): C57BL/6, C57BL/6 treated with Cobra Venom Factor (CVF), C3 –/– and wild–type control for C3 –/–. Mice were sacrificed on day 7, their eyes enucleated and RPE–choroidal–scleral flat mounts were stained with an anti–elastin (Cy–3 conjugated) antibody. The incidence and inhibition of CNV was determined by confocal microscopy. Flat mounts were also stained for C3 and membrane attack complex (MAC) using rabbit anti–mouse C3 and rabbit anti–mouse C9 respectively. The effect of complement on VEGF, TGFß–2 and ßFGF production was studied by RT–PCR and ELISA. Twenty laser spots were placed in each eye of complement sufficient and complement depleted (CVF treated) C57BL/6 mice and ten animals from each group were sacrificed at days 1, 3, 5 and 7 post laser treatment. RPE–choroid–scleral flat mounts harvested from the enucleated eyes were pooled separately for each time point for total RNA and protein extraction. Results: Complement depletion using CVF markedly inhibited laser–induced CNV in C57BL/6 mice. Additionally, laser induced CNV did not develop in C3 –/– mice. Increased deposition of C3 and MAC was observed in CNV lesions, particularly the RPE, of C57BL/6 mice and wild–type control for C3 –/– animals at days 1 and 3 post–laser treatment. However, at day 7 very little staining for C3 and MAC was observed in these animals. In contrast, no staining for C3 and MAC was observed in the laser spots in complement depleted as well as in C3 –/– mice at these time points. Low levels (both mRNA and protein) of VEGF, TGFß–2 and ßFGF were observed in the animals with depleted complement system at days 1, 3, 5 and 7 post–laser treatment. In contrast, the levels of these growth factors, which are thought to be important for the development of CNV, were elevated several folds in the animals with a functionally active complement system at days 3 and 5 and returned to basal levels at day 7 after laser treatment. Conclusions: Our results suggest that the presence and the activation of the complement system is essential for the development of laser induced choroidal angiogenesis in mice. These results further suggest that the production of angiogenic factors – VEGF, TGFß–2 and ßFGF – in laser induced CNV is temporally regulated and dependent on complement activation and MAC formation.

Keywords: age–related macular degeneration • laser • growth factors/growth factor receptors