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 6

R(+) propranolol and R(+) atenolol inhibit IH vasculogenesis but not body weight or glucose levels.

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R(+) propranolol and R(+) atenolol inhibit IH vasculogenesis but not bod...
(A) HemSCs were pretreated with PBS or 10 μM treatment drug 24 hours before the experiment, suspended in Matrigel with PBS or 5 μM treatment drug, and injected into nude mice, with 2 implants/mouse [n = 10 PBS-treated mice, n = 8 propranolol-treated mice, n = 8 R(+) propranolol–treated mice, n = 10 R(+) atenolol–treated mice]. The mice were treated with 12.5 mg/kg propranolol, 12.5 mg/kg R(+) propranolol, 12.5 mg/kg R(+) atenolol, or an equal volume of PBS twice a day. Matrigel implants harvested after 7 days are displayed in the top panels of th images. The PBS control implants in A are also shown in Figure 3A, because the 5 mg/kg atenolol group shown in Figure 3B was run at the same time as the groups in A. Scale bars: 10 mm. Images show H&E staining (middle panels) and anti–human CD31 staining (red; bottom panels), with nuclei counterstained with DAPI (blue). Scale bars: 100 μm. (B) Quantification of vessel density based on H&E staining (A, middle panels) and anti–human CD31 staining (A, bottom panels) showed that R(+) atenolol was as effective as R(+) propranolol and propranolol in inhibiting vessel formation. Statistical significance was determined by 1-way ANOVA with Dunnett’s multiple-comparison test. P values are listed in the table in Supplemental Figure 5E. (C) Body weight and glucose levels were measured daily. Neither propranolol, R(+) propranolol, or R(+) atenolol affected body weight or glucose levels of nude mice. Data show the mean ± SD in all graphs. Data were collected for 2 implants in each mouse, leading to an observation sample size of 8 in the propranolol and R(+) propranolol treatment groups and 10 in the atenolol and PBS control groups.