Non–beta blocker enantiomers of propranolol and atenolol inhibit vasculogenesis in infantile hemangioma
Caroline T. Seebauer, Matthew S. Graus, Lan Huang, Alex McCann, Jill Wylie-Sears, Frank Fontaine, Tara Karnezis, David Zurakowski, Steven J. Staffa, Frédéric Meunier, John B. Mulliken, Joyce Bischoff, Mathias Francois
Caroline T. Seebauer, Matthew S. Graus, Lan Huang, Alex McCann, Jill Wylie-Sears, Frank Fontaine, Tara Karnezis, David Zurakowski, Steven J. Staffa, Frédéric Meunier, John B. Mulliken, Joyce Bischoff, Mathias Francois
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Research Article Angiogenesis Vascular biology

Non–beta blocker enantiomers of propranolol and atenolol inhibit vasculogenesis in infantile hemangioma

Abstract

Propranolol and atenolol, current therapies for problematic infantile hemangioma (IH), are composed of R(+) and S(–) enantiomers: the R(+) enantiomer is largely devoid of beta blocker activity. We investigated the effect of R(+) enantiomers of propranolol and atenolol on the formation of IH-like blood vessels from hemangioma stem cells (HemSCs) in a murine xenograft model. Both R(+) enantiomers inhibited HemSC vessel formation in vivo. In vitro, similar to R(+) propranolol, both atenolol and its R(+) enantiomer inhibited HemSC to endothelial cell differentiation. As our previous work implicated the transcription factor sex-determining region Y (SRY) box transcription factor 18 (SOX18) in propranolol-mediated inhibition of HemSC to endothelial differentiation, we tested in parallel a known SOX18 small-molecule inhibitor (Sm4) and show that this compound inhibited HemSC vessel formation in vivo with efficacy similar to that seen with the R(+) enantiomers. We next examined how R(+) propranolol alters SOX18 transcriptional activity. Using a suite of biochemical, biophysical, and quantitative molecular imaging assays, we show that R(+) propranolol directly interfered with SOX18 target gene trans-activation, disrupted SOX18-chromatin binding dynamics, and reduced SOX18 dimer formation. We propose that the R(+) enantiomers of widely used beta blockers could be repurposed to increase the efficiency of current IH treatment and lower adverse associated side effects.

Authors

Caroline T. Seebauer, Matthew S. Graus, Lan Huang, Alex McCann, Jill Wylie-Sears, Frank Fontaine, Tara Karnezis, David Zurakowski, Steven J. Staffa, Frédéric Meunier, John B. Mulliken, Joyce Bischoff, Mathias Francois

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Figure 3

R(+) atenolol inhibits hemangioma endothelial differentiation in vitro and vessel formation in vivo.

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R(+) atenolol inhibits hemangioma endothelial differentiation in vitro a...
(A) Atenolol and its purified R(+) enantiomer, both tested at 5 μM, inhibited endothelial differentiation of HemSCs isolated from 2 IH tumor specimens as effectively as did R(+) propranolol. R(+) propranolol served as a positive control for inhibition. The endothelial differentiation markers CD31 and VE-cadherin and the hemangioma endothelial markers NOTCH1, PlexinD1, and VEGFR1 under each treatment condition in 3 biological replicates, determined by qPCR, were standardized as previously described (76). The HemSC-to-endothelial differentiation assay was conducted 2 separate times with HemSC 167 and once with HemSC 165, providing 3 data points. Statistical significance was determined by 1-way ANOVA with Bonferroni’s post hoc test. P values are listed in Supplemental Figure 3. (B) HemSCs were pretreated with PBS, 10 μM atenolol, or 10 μM R(+) atenolol 24 hours before the experiment and were then suspended in Matrigel with PBS, 5 μM atenolol, or 5 μM R(+) atenolol and injected into nude mice, with 2 implants per mouse (see schematic in Figure 1A; n = 16 PBS-treated HemSCs, n = 8 atenolol-treated HemSCs, n = 8 R(+) atenolol–treated HemSCs). The mice were treated with 5 mg/kg atenolol, 5 mg/kg R(+) atenolol, or an equal volume of PBS twice a day. Matrigel implants harvested after 7 days are shown in the top panels of the images. Scale bars: 10 mm. Images also show H&E staining (middle panels) and anti–human CD31 staining (red, bottom panels), with nuclei counterstained with DAPI (blue). Scale bars: 100 μm. Data were collected for 2 implants in each of 4 mice, leading to an observation sample size of 8 per treatment group and 16 in the control group. (C) Quantification of vessel density based on H&E staining (middle panels) and anti–human CD31 staining (bottom panels) showed a significant reduction in vessel density in the implants of atenolol- and R(+) atenolol–treated mice versus implants of control mice. Statistical analysis was performed using 1-way ANOVA with Dunnett’s multiple-comparison test.