Non–beta blocker enantiomers of propranolol and atenolol inhibit vasculogenesis in infantile hemangioma
Caroline T. Seebauer, … , Joyce Bischoff, Mathias Francois
Caroline T. Seebauer, … , Joyce Bischoff, Mathias Francois
Published December 7, 2021
Citation Information: J Clin Invest. 2022;132(3):e151109. https://doi.org/10.1172/JCI151109.
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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 2

R(+) propranolol does not affect HemSC to HemPericyte differentiation.

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R(+) propranolol does not affect HemSC to HemPericyte differentiation. (...
(A) HemSCs and HemECs (1:1) were suspended in Matrigel and injected into nude mice, with 2 implants/mouse (n = 8 mice). The mice were treated with 5 mg/kg R(+) propranolol or an equivalent volume of PBS twice a day. Matrigel implants harvested after 7 days are displayed in the top panel of the images. Scale bars: 10 mm. H&E staining showed similar vessel density in the implants of R(+) propranolol–treated mice compared with vessel density in the implants of control mice (middle panels). Scale bars: 100 μm. Anti–human CD31 staining (red) confirmed the similar number of blood vessels in R(+) propranolol–treated mice and control mice (bottom panels). Nuclei were counterstained with DAPI (blue). Scale bars: 100 μm. P values were calculated using a 2-tailed, unpaired Student’s t test. Data were collected for 2 implants in each of 4 mice, leading to an observation sample size of 8 per group. (B) Implant sections stained with UEA I (green) and anti-αSMA (red) showed similar pericyte coverage per vessel area in mice treated with PBS (n = 7 mice) or R(+) propranolol (n = 6 mice). Nuclei were counterstained with DAPI (blue). Scale bars: 100 μm. P values were calculated by 2-tailed, unpaired Student’s t test. Only implants showing vessel formation were used for further analysis [n = 7 PBS implants; n = 6 R(+) propranolol implants]. Graphs show quantification of vessels/mm2 in the H&E-stained sections (top), human CD31+ vessels/mm2 (middle), and pericytes/vessel area (bottom). (C) qPCR showed that treatment with propranolol or its R(+) enantiomers (10 μM) did not affect the expression of pericyte markers (calponin, PDGFRβ, and αSMA) in HemSCs cocultured with HemECs. Coculturing was conducted for 5 days: CD31+ cells were removed by magnetic beads before RNA extraction of the CD31– cells as shown in the schematic. DAPT (10 μM) served as a positive control. Data from 3 independent experiments were plotted. Statistical significance was determined by 1-way ANOVA with Dunnett’s multiple-comparison test. P values can be found in Supplemental Figure 2C. Data in all graphs show the mean ± SD.