Propranolol inhibits cavernous vascular malformations by β1 adrenergic receptor antagonism in animal models

Abstract

Propranolol, a pleiotropic β-adrenergic blocker, has been anecdotally reported to reduce cerebral cavernous malformations (CCMs) in humans. However, propranolol has not been rigorously evaluated in animal models, nor has its mechanism of action in CCM been defined. We report that propranolol or its S(-) enantiomer dramatically reduced embryonic venous cavernomas in ccm2 mosaic zebrafish, whereas R-(+)-propranolol, lacking β antagonism, had no effect. Silencing of the β1, but not β2, adrenergic receptor mimicked the beneficial effects of propranolol in a zebrafish CCM model, as did the β1-selective antagonist metoprolol. Thus, propranolol ameliorated cavernous malformations by β1 adrenergic antagonism in zebrafish. Oral propranolol significantly reduced lesion burden in 2 chronic murine models of the exceptionally aggressive Pdcd10/Ccm3 form of CCM. Propranolol or other β1-selective antagonists may be beneficial in CCM disease.

Keywords: Mouse models; Stroke; Vascular Biology; endothelial cells.

Figure 1. Propranolol and S-(-)-propranolol, but not R-(+)-propranolol, rescues embryonic CVP dilation in ccm2 CRISPR embryos.

At the 1-cell stage, Tg(fli1:EGFP; gata1:DsRED) embryos, expressing EGFP in endothelial cells and DsRED in red blood cells, were injected with cas9 mRNA and gRNAs targeting ccm2. At 24 hpf, the ccm2 CRISPR embryos were treated with 100 μM propranolol, S-(-)-propranolol, or R-(+)-propranolol. The CVP was scanned at 30 hpf (A–D), 36 hpf (E–H), and 2 days post fertilization (dpf) (I–L). Whereas R-(+)-propranolol–treated (C, G, and K) and untreated CRISPR embryos (D, H, and L) displayed abnormal intussusceptions within the dorsal vein and lack of a ventral vein, racemic propranolol–treated (A, E, and I) and S-(-)-propranolol–treated (B, F, and J) CRISPR embryos formed the primitive venous plexus by 30 hpf (A and B), a normal functional ventral vein by 36 hpf (E and F), and a mature caudal venous plexus by 2 dpf (I and J). Arrows indicate intussusceptions. Blue and yellow bars indicate the dorsal and ventral veins, respectively. The embryos shown in A–T are in the sagittal plane. Bright-field images revealed normal CVP development of racemic propranolol–treated (M) and S-(-)-propranolol–treated CRISPR embryos (N), whereas approximately 30% of R-(+)-propranolol–treated (O) and untreated (control) ccm2 CRISPR embryos (P) had dramatic CVP dilation. Approximately 10% of embryos in all 4 groups showed heart dilation (Q–T). Scale bars: 50 μm (A–L), 500 μm (M–P), and 200 μm (Q–T). Quantitation of ccm2 CRISPR embryos with cardiovascular defects (U) revealed that, compared with the untreated controls, significantly fewer propranolol-treated embryos harbored CVP dilation (****P < 0.0001), whereas a similar proportion exhibited heart dilation (P = 0.11). Quantitation of ccm2 CRISPR embryos with CVP dilation (V) showed that the S-(-)-propranolol enantiomer rescued CVP dilation (*P = 0.036), whereas the R-(+)-propranlol enantiomer failed to do so (P = 1.0) compared with untreated control embryos. A 2-tailed Fisher exact test was used for comparisons. Each replicate (colored bars) is presented with the total number. Black bars indicate the mean in U.

Figure 2. Loss of β1 adrenergic receptor function inhibits CVP dilation in ccm2 CRISPR embryos.

(A) One-cell-stage embryos were coinjected with ccm2 CRISPR and an adrb1 morpholino (MO) (A and C) to silence the β1 receptor or with a control morpholino (B and C) and imaged at 48 hpf. (A) β1 Adrenergic receptor–silenced embryos formed a normal CVP, whereas control morpholino embryos (B) showed dramatic dilation. The blue bar indicates the lumen of the dorsal vein, and the yellow bar indicates the ventral vein. Scale bars: 50 μm. (C) Quantification of the embryos in replicate experiments showed that CVP dilation in ccm2 CRISPR embryos was reversed by silencing of the β1 adrenergic receptor with an adrb1^–/– morpholino (****P < 0.0001), but not by silencing of the β2a adrenergic receptor with an adrb2a^–/– morpholino (P = 0.89). (D) Specificity of β adrenergic antagonists. The β1-specific antagonist metoprolol (400 μM) was added 24 hpf, and embryos were imaged 48 hpf. Metoprolol reversed CVP dilation (*P = 0.018) in comparison with untreated control embryos. The fraction of defective embryos for each replicate experiment is indicated by colored bars (C and D). The mean of the values from replicate experiments is indicated by thin black lines (C). The numbers above the colored bars indicate the sample size for each replicate experiment in each group (C and D). A 2-tailed Fisher’s exact test was used for comparisons.

Figure 3. Effect of propranolol in murine models of CCM disease.

Pdcd10^ECKO mice, treated with placebo (n = 5) or propranolol (n = 4), were challenged with increasing doses of isoproterenol (50–5000 pg) to determine whether the 50 mg/kg/day propranolol dose was sufficient to antagonize β-adrenergic receptors. Propranolol-treated mice showed a significant rightward shift in the dose response curve in (A) contractility (dP/dt max) and (B) heart rate (bpm) with increasing concentrations of isoproterenol. Data are expressed as the mean ± SEM. *P < 0.05 and **P < 0.01, by 2-way repeated-measures ANOVA with a post hoc independent samples t test. (C) Effect of propranolol on lesion burden in 2 Pdcd10 models. In the Pdcd10^ECKO model, propranolol (50 mg/kg/day) for 35 days (dose started on P21) significantly decreased lesion burden compared with placebo controls (P < 0.0001). One placebo-treated mouse was excluded as an outlier. In the Pdcd10^+/– Trp53^–/– model, propranolol (50 mg/kg/day) for 90 days (dose started on P21) significantly reduced lesion burden compared with placebo controls (P = 0.014). One placebo-treated mouse and 1 propranolol-treated mouse were excluded as outliers. Since the data were not normally distributed, the 2-sample Conover test was used. The mean (longer bars) and SEM (shorter lines above the mean bars) are indicated for each group. In both the Pdcd10^ECKO and Pdcd10^+/– Trp53^–/– models, the lesion burden was greater in the placebo-treated controls than in the propranolol-treated mice. (D) Representative micro-CT images of the brains of mice with a high lesion burden in the untreated groups and average mouse brains in the treated groups, showing the effect of propranolol primarily on animals with a high lesion burden. Propranolol did not affect survival in either the (E) Pdcd10^ECKO model (P = 0.59) or the (F) Pdcd10^+/– Trp53^–/– model (P = 0.76) compared with placebo controls. The log-rank (Mantel-Cox) test was used to determine significance.