Complement Factor–H Expression in Laser–Induced Choroidal Neovascularization and Its Regulation in RPE Cells

Abstract

Purpose: : To determine the pattern of complement factor–H (CFH) expression in laser–induced choroidal neovascularization (CNV) and its regulation in RPE cells in vitro.

Methods: : CNV was induced by laser photocoagulation in 129S3/SvJ mice. The mice were sacrificed and their eyes were enucleated two weeks after the photocoagulation. Immunohistochemical staining was performed on frozen sections of mouse retina using goat polyclonal anti–human CFH antibody. Passage 3 human fetal RPE cells were grown to confluence on chamber slides and 6–well plates, and adapted to serum–free conditions overnight. Recombinant human TNF–α (5, 10 ng/ml), CTGF (20, 50 ng/ml), VEGF (10, 20 ng/ml), advanced glycation endproducts (100, 200 µg/ml), S100B (100, 200 ng/ml) and HMGB1 (10, 20 ng/ml) were applied and incubated for 2 days. The expression of CFH in the stimulated RPE cells was analyzed by immunohistochemistry and Western blot.

Results: : CFH was expressed in the normal RPE monolayer. CFH expression was markedly increased within CNV lesions and adjacent RPE. VEGF and CTGF induced a strong upregulation of CFH immunoreactivity in cultured RPE, while CFH protein expression on Western blot was significantly increased by stimulation with VEGF, CTGF, S100B and HMGB1.

Conclusions: : CFH expression is increased in murine laser–induced CNV and upregulated in vitro by VEGF and other angiogenetic factors in RPE cells. The results suggest that CFH may play an important role in the development of CNV.

Keywords: age-related macular degeneration • gene/expression • cytokines/chemokines