Nicotine Receptor Antagonist Reduces the Size of Laser-Induced CNV in a Mouse Model and Inhibits VEGF-Receptor-2 Phosphorylation in vitro

Abstract

Purpose: : Anti-VEGF therapy is established as treatment for wet AMD, but new therapies are needed to increase efficacy, response rate or treatment interval. Nicotine is a risk factor for developing AMD and has angiogenic properties in vitro and in vivo. Kiuchi et al. (IOVS 2008) have demonstrated that topical application of a nicotine receptor antagonist (Mecamylamine) reduces the size of choroidal neovascularisation (CNV). This study compares the effects of an intravitreal VEGF inhibitor (Anti- Murine VEGF 500-P131) with intravitreal Mecamylamine in the mouse model of laser-induced CNV and investigates possible interactions of nicotine and VEGF signalling.

Methods: : In C57BL/6J mice CNV was induced by laser. 10min after laser either 0.5mg/ml anti-murine VEGF 500-P131 (Peprotech Inc.), mecamylamine (0.5mg/ml) or BSS was injected into the vitreous cavity. Two week after the procedure mice were sacrificed. After overnight fixation, choroid flatmounts were prepared. CNVs were visualized by fluorescence labelling (Lecitin FITC) and were quantified by a digital imaging system connected to a fluorescence microscope. The CNV lesion size was detemined with Image Tool® and averaged for each eye. The resulting arbitrary units were compared using a student’s t-test. The possible influence of nicotine and Mecamylamine on VEGFR-2 phosphorylation and down- stream signalling was studied by western blot on Human Umbilical Vein Endothelial Cells (HUVEC) and Late Outgrow Edothelial Progenitor Cells (OECs) isolated from peripheral blood of AMD patients

Results: : Intravitreal injection of Anti-Murine VEGF 500-P131 resulted in a significant change in size of CNVs (from 511876 ± 167889 in the control group to 369124 ± 135034 in the group of Anti-VEGF therapy (p=0,04)). The size of the CNVs in the Mecamylamine group decreased to 358144 ± 51081 (p=0,02).Western blot analysis demonstrated that nicotine caused VEGFR-2 phosphorylation in HUVEC and OECs whereas Mecamylamine significantly inhibited the baseline, VEGF- and nicotine-induced VEGFR-2 activation.

Conclusions: : Anti-VEGF treatment reduces the size of laser-induced CNV. Intravitreal application of nicotine receptor antagonist Mecamylamine was similary effective in inhibiting CNV. Therefore, nicotine receptor antagonists could be a promising new treatment for AMD. One of the underlying mechanisms may be, a result of transactivation of VEGF receptor signalling.

Keywords: choroid: neovascularization • age-related macular degeneration • retinal neovascularization