Propranolol inhibition of β-adrenergic receptor does not suppress pathologic neovascularization in oxygen-induced retinopathy

Abstract

Purpose: Retinopathy of prematurity (ROP) is a leading cause of blindness in children and is, in its most severe form, characterized by uncontrolled growth of vision-threatening pathologic vessels. Propranolol, a nonselective β-adrenergic receptor blocker, was reported to protect against pathologic retinal neovascularization in a mouse model of oxygen-induced retinopathy (OIR). Based on this single animal study using nonstandard evaluation of retinopathy, clinical trials are currently ongoing to evaluate propranolol treatment in stage 2 ROP patients who tend to experience spontaneous disease regression and are at low risk of blindness. Because these ROP patients are vulnerable premature infants who are still in a fragile state of incomplete development, the efficacy of propranolol treatment in retinopathy needs to be evaluated thoroughly in preclinical animal models of retinopathy and potential benefits weighed against potential adverse effects.

Methods: Retinopathy was induced by exposing neonatal mice to 75% oxygen from postnatal day (P) 7 to P12. Three routes of propranolol treatment were assessed from P12 to P16: oral gavage, intraperitoneal injection, or subcutaneous injection, with doses varying between 2 and 60 mg/kg/day. At P17, retinal flatmounts were stained with isolectin and quantified with a standard protocol to measure vasoobliteration and pathologic neovascularization. Retinal gene expression was analyzed with qRT-PCR using RNA isolated from retinas of control and propranolol-treated pups.

Results: None of the treatment approaches at any dose of propranolol (up to 60 mg/kg/day) were effective in preventing the development of retinopathy in a mouse model of OIR, evaluated using standard techniques. Propranolol treatment also did not change retinal expression of angiogenic factors including vascular endothelial growth factor.

Conclusions: Propranolol treatment via three routes and up to 30 times the standard human dose failed to suppress retinopathy development in mice. These data bring into question whether propranolol through inhibition of β-adrenergic receptors is an appropriate therapeutic approach for treating ROP.

Figure 1.

Oral gavage of propranolol does not protect against OIR. (a) Representative images of retinal whole mounts isolated from P17 mice with induced retinopathy treated with propranolol or vehicle control daily from P12 to P16 (2 mg/kg/day). Retinas were stained with isolectin B₄ (red) to visualize vasculature. Quantifications of vasoobliteration (VO) and neovascularization (NV) are highlighted. Scale bar: 1000 μm. (b) Quantification of VO and (c) NV as percentage of total retinal area. N.S., not significant.

Figure 2.

IP injection of propranolol does not suppress pathologic neovascularization in OIR. (a) Representative images of retinal whole mounts isolated from P17 mice with induced retinopathy treated with propranolol or vehicle control daily from P12 to P16 (2 or 20 mg/kg/day). Retinas were stained with isolectin B₄ (red) to visualize vasculature. Quantifications of VO and NV are highlighted. Scale bar: 1000 μm. (b) Quantification of VO and (c) NV as percentage of total retinal area. N.S., not significant. ***P < 0.001.

Figure 3.

SC injection of propranolol fails to protect against retinopathy in OIR. (a) Representative images of retinal whole mounts isolated from P17 mice with induced retinopathy treated with propranolol (SC) or vehicle control daily from P12 to P16 (20 mg/kg/day). Retinas were stained with isolectin B₄ (red) to visualize vasculature. Quantifications of VO and NV are highlighted. Scale bar: 1000 μm. (b) Quantification of VO and (c) NV as percentage of total retinal area. N.S., not significant.

Figure 4.

Propranolol fails to suppress OIR and does not affect angiogenic factors in OIR. (a) Representative images of retinal whole mounts isolated from P17 mice with induced retinopathy treated with propranolol (SC) or vehicle control three times per day from P12 to P16 (60 mg/kg/day). Retinas were stained with isolectin B₄ (red) to visualize vasculature. Quantifications of VO and NV are highlighted. Scale bar: 1000 μm. (b) Quantification of VO and (c) NV as percentage of total retinal area. (d) Retinal expression of angiogenic factors (Vegf, Epo, Ang1, and Ang2) in OIR mice treated with propranolol (SC, 60 mg/kg/day) or control were quantified with RT-qPCR. (e) Protein levels of retinal VEGF from OIR mice treated with propranolol (SC, 60 mg/kg/day) or control were measured with ELISA assay. N.S., not significant.

Figure 5.

Retinal expression of three β-adrenergic receptors in OIR: (a) β-adrenergic receptor 1 (ADRb1), (b) β-adrenergic receptor 2(ADRb2), and (c) β-adrenergic receptor 3(ADRb3). Total retinal RNA was isolated from P15 and P17 mice with induced OIR or control mice raised in room air. Gene expression was measured with RT-qPCR and normalized to housekeeping gene cyclophilin A expression.

Figure 6.

Effect of propranolol on human retinal microvascular endothelial cell culture (HRMEC). (a, b) Expression of two VEGF receptors (VEGFR1 and VEGFR2) is not affected with varying concentrations of propranolol treatment (0–50 μM, 48 hours). (c) Propranolol treatment (48 hours) suppresses HRMEC proliferation at high concentrations (>10 μM) as measured by MMT assay. *P < 0.05, **P < 0.01.