Complement Activation by Alternative Pathway is Critical in the Development of Laser–Induced Choroidal Neovascularization

Abstract

Purpose: : The purpose of this study was to explore the role of classical, lectin and alternative pathways of complement activation in laser–induced choroidal neovascularization (CNV).

Methods: : CNV was induced by laser photocoagulation in C57BL/6 mouse with krypton red laser. Retinal pigment epithelium (RPE)–choroid–scleral flat mounts prepared from harvested eyes were stained for elastin and the incidence of CNV was determined by confocal microscopy. The size of the CNV complex was graded by morphometric analysis of the images obtained from confocal microscopy. In C57BL/6 mice classical, lectin and alternative pathways were blocked by tail vein injection of siRNA against C1q, C4 and factor B. CNV was also induced in C4–/– and C5 –/– mice. RPE–choroidal–scleral tissues were pooled separately for RT–PCR and Western blot analysis of growth factors.

Results: : C4 –/– mice developed CNV similar to their WT controls and inhibition of C1q by siRNA had no effect on the development of CNV. In contrast, CNV did not develop in C5 –/– mice and C57BL/6 mice treated with factor B siRNA. Inhibition of the alternative pathway by factor B siRNA resulted in decreased levels of angiogenic factors – VEGF and TGF–ß2.

Conclusions: : In conclusion, our results demonstrate that factor B–dependent alternative pathway activation plays a key role in laser–induced CNV in mouse. Thus, specific blockade of alternative pathway may represent a therapeutically relevant strategy to inhibit CNV.

Keywords: age-related macular degeneration • neovascularization • inflammation