Mecamylamine suppresses Basal and nicotine-stimulated choroidal neovascularization

Abstract

Purpose: Nicotinic acetylcholine receptors (nAChR) are best known for their role in neurotransmission, but they have recently been demonstrated on vascular endothelial cells. Acetylcholine is their endogenous ligand, but they are also stimulated by nicotine. By stimulating nAChR, nicotine promotes tumor angiogenesis as well as atherosclerotic plaque neovascularization. In this study, the authors investigated the role of nAChR in the pathogenesis of choroidal neovascularization (CNV).

Methods: The effect of the nonselective nAChR antagonist mecamylamine was tested on human retinal and choroidal endothelial cells in vitro and in a murine model of CNV.

Results: Several nAChR isoforms were identified in retinal and choroidal microvascular endothelial cells, and the ability of these cells to form tubules when grown in growth factor-reduced basement membrane matrix and supplemented with VEGF was suppressed by the nAChR antagonist mecamylamine. Supplementation of the drinking water of mice with nicotine increased the size of CNV lesions at Bruch membrane rupture sites, an effect that was blocked by subcutaneous administration of mecamylamine (50 mg/kg/d) by an osmotic pump. In the absence of nicotine, CNV formation was suppressed by the infusion of 50 mg/kg/d mecamylamine or by topical application 0.1 or 1% mecamylamine to the cornea.

Conclusions: These data suggest that endogenous activation of nAChR promotes CNV and that activation of nAChR by nicotine may contribute to the increased incidence of CNV seen in smokers with age-related macular degeneration (AMD). Topically administered mecamylamine could provide an appealing new treatment approach for CNV.

Figure 1. Expression of mRNA for nAChR in endothelial cells

Histogram showing nAChR subunit mRNA levels normalized to the level of 18S RNA in HDMVEC, HCEC and HREC determined by real time RT-PCR. Each bar represents the mean (± SEM) calculated from three experimental values. All mammalian α and β nAChR subunits except α₄ were expressed in each of the endothelial cell types, but there were differences among the cell types in the level of expression of various isoforms.

Figure 2. Western analyses for nAChR expression in endothelial cells

Western blots confirmed the expression of α₅, α₇, β₂ and β₃ in retinal, choroidal, and dermal microvascular endothelial cells.

Figure 3. Inhibition of endothelial tube formation in Matrigel by mecamylamine

HDMVECs were treated with increasing concentrations of mecamylamine, plated (10⁴ cells/well) on growth factor-depleted Matrigel, and after 15 minutes, mecamylamine (1–100 nM) was added to the cells. Following a 30 minute incubation with mecamylamine, VEGF (100 ng/ml) was added. Cells were allowed to grow for 24 hours at 37°C and tube lengths were measured using the formation (A), those containing mecamylamine appeared to have fewer and shorter tubes (B). The mean (±SD) tube length per well calculated from 3 wells revealed a reduction in mean tube length in wells containing mecamylamine (C).

Figure 4. Mecamylamine blocks VEGF-induced stimulation of ocular endothelial cell tube formation

Human retinal endothelial cells (HREC) or human choroidal endothelial cells were seeded in Matrigel-coated wells to which were added media alone (controls), media containing 10 ng/ml of VEGF, or both 10 ng/ml of VEGF and 1 mM mecamylamine (MEC). Bars represent the mean (±SE) tube length per well calculated from three wells for each condition. **p<0.05; p,0.005 by ANOVA with Dunnett’s correction for multiple corrections

Figure 5. Mecamylamine blocks nicotine-induced stimulation of choroidal neovascularization

Six week old C57BL/6 mice were given 100 µg/ml of nicotine in their drinking water and had implantation of an osmotic pump that released mecamylamine or vehicle subcutaneously After 2 days, these mice and a control group of 3 mice that did not receive any nicotine, had rupture of Bruch’s membrane at 3 locations in each eye. Fourteen days after laser, the mice were perfused with fluorescein-labeled dextran and the area of choroidal neovascularization at Bruch’s membrane rupture sites was measured by image analysis. Compared to choroidal neovascularization lesions in untreated mice that did not receive nicotine (A), the lesions appeared larger in mice that received nicotine and had implantation of pumps that released only vehicle (B), but not in mice that received nicotine and mecamylamine (C). Measurement of the area of choroidal neovascularization by image analysis confirmed that nicotine caused a significant increase in lesion size that was completely blocked by Mecamylamine (D). *p=0.006470142; **p=0.007367749 by ANOVA with Dunnett’s adjustment for multiple comparisons

Figure 6. Mecamylamine suppresses choroidal neovascularization in the absence of nicotine

Six week old C57BL/6 mice had plasma drawn and then had implantation of an osmotic pump that released Mecamylamine subcutaneously. After 2 days, mice had rupture of Bruch’s membrane at 3 locations in each eye and after 14 days a plasma sample drawn, mice were perfused with fluorescein-labeled dextran, and the area of choroidal neovascularization at Bruch’s membrane rupture sites was measured by image analysis. Compared to control mice, there was a significant reduction in the area of CNV at rupture sites in mice treated with mecamylamine. *p=0.0048 by ANOVA with the Dunnett’s adjustment for multiple comparisons

Figure 7. Mecamylamine delivered topically to the eye suppresses choroidal neovascularization (CNV)

Six week old C57BL/6 mice were divided into 3 groups and started on twice a day administration of eye drops containing 0.1% or 1% mecamylamine, or vehicle with investigators masked with respect to treatment groups. Two hours after the first drop, Bruch’s membrane was ruptured with laser photocoagulation in 3 locations in each eye. After 1 or 2 weeks of treatment, mice were perfused with fluorescein-labeled dextran. When the code was broken, it was found that mice perfused after 1 week of treatment with 0.1% (A) or 1% (C) mecamylamine had smaller areas of CNV than mice treated vehicle (B). Image analysis confirmed that compared to mice treated with vehicle the area of CNV was significantly smaller in mice treated with either dose of mecamylamine (D). The results were very similar in mice treated with drops for 2 weeks after laser and then perfused (E–H).