Attenuation of choroidal neovascularization by β(2)-adrenoreceptor antagonism

Abstract

Objectives: To determine whether β-adrenergic blockade inhibits choroidal neovascularization (CNV) in a mouse model of laser-induced CNV and to investigate the mechanism by which β-adrenoreceptor antagonism blunts CNV.

Design: Mice were subjected to laser burns, inducing CNV, and were treated with daily intraperitoneal injections of propranolol hydrochloride. Neovascularization was measured on choroidal-scleral flat mounts using intercellular adhesion molecule 2 immunofluorescence staining. The effect of β-adrenoreceptor signaling on expression of vascular endothelial growth factor (VEGF) was investigated using primary mouse choroidal endothelial cells (ChECs) and retinal pigment epithelial (RPE) cells. These cells were incubated with β-adrenoreceptor agonists and/or antagonists and assayed for Vegf messenger RNA and protein levels.

Setting: University of Wisconsin School of Medicine and Public Health.

Participants: Wild-type 6-week-old female C57BL/6j mice.

Main outcome measures: Inhibition of CNV after propranolol treatment and Vegf messenger RNA and protein expression after treatment with β-adrenoreceptor agonists and antagonists.

Results: Propranolol-treated mice demonstrated a 50% reduction in laser-induced CNV. Treatment with norepinephrine bitartrate stimulated Vegf messenger RNA expression and protein secretion in ChECs and RPE cells. This effect was blocked by β2-adrenoreceptor antagonism and mimicked by β2-adrenoreceptor agonists.

Conclusions: Attenuation of CNV is achieved by β-adrenergic blockade. The β2-adrenoreceptors regulate VEGF expression in ChECs and RPE cells.

Clinical relevance: Antagonists of β-adrenoreceptors are safe and well tolerated in patients with glaucoma and cardiovascular disease. Thus, blockade of β-adrenoreceptors may provide a new avenue to inhibit VEGF expression in CNV.

Figure 1

Propranolol suppresses CNV. A–B: Representative images of vehicle- and propranolol-treated choroidal-sclera flat mounts stained with ICAM-2. C: Quantitative analysis of vehicle (veh)- and propranolol-treated eyes. Propranolol reduced CNV by 50% (** p< 0.01, N=20 control versus 13 propranolol).

Figure 2

Quantitative PCR assessment of VEGF (A) and all three β-adrenoreceptors (B–D) in ChEC and RPE cells. Data are normalized to RpL 13A housekeeping gene expression (** p<0.01, *** p<0.001).

Figure 3

Norepinephrine stimulates VEGF. A,C: RPE (N=6) and ChECs (N=7) were treated for 2 hours with vehicle or 10 μM norepinephrine (NE). B,D: VEGF secretion after 24 hours of treatment (N=6 for both, * p<0.05, ** p<0.01).

Figure 4

Propranolol prevents norepinephrine-stimulated VEGF mRNA expression. RPE (A) and ChECs (B) were pre-incubated with 1 μM propranolol for 30 minutes followed by incubation with 10 μM norepinephrine for 2 hours (N=5 for both, *** p<0.001).

Figure 5

β2-Adrenoreceptors drive VEGF. A–B: Cells were pre-incubated with 1 μM β1 or 100 nM β2 and β3 antagonists (N=4 for both). C–D: RPE (N=3) and ChECs (N=5) were incubated with 100 nM β1 and β2 or 1 μM β3 agonists.