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R-propranolol is a small molecule inhibitor of the SOX18 transcription factor in a rare vascular syndrome and hemangioma

• Authors : Overman, Jeroen; Fontaine, Frank; Wylie-Sears, Jill ...

• Source: eLife ; volume 8 ; ISSN 2050-084X

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Figure 3. The R(+) enantiomer of propranolol and SOX18 small molecule inhibitor halt infantile hemangioma stem cell differentiation. ; (A) Infantile) hemangioma tissue section stained for SOX18 (red), Ki67 (green), CD31 (orange), D2-40 (pink) and DAPI (blue) reveals the presence of SOX18 expression in a large subset of hemangioma endothelial cells (arrows). (B) Schematic representation of infantile hemangioma stem cell (HemSC) endothelial differentiation assay. VEGF-B stimulates HemSC to differentiate into hemangioma endothelial cells (HemEC). This differentiation process is inhibited by propranolol, the R(+) enantiomer of propranolol, and by SOX18 small molecule inhibitor Sm4 (all at 5 uM). (C) VEGF-B treatment of HemSC from four different infantile hemangiomas resulted in increased CDH5 (an endothelial cell marker), SOX18 and ADBR2 (β2 adrenergic receptor) mRNA. Means and standard deviations are shown. (D) The effects of SOX18 inhibitor Sm4, its scaffold aspirin as a negative control, propranolol and its purified R(+) and S(-) enantiomers on HemSC-to-HemEC differentiation from two infantile hemangioma patients. Endothelial differentiation markers, CD31 and CDH5 and hemangioma endothelial markers NOTCH1, PLXND1 and VEGFR1 under each treatment condition in four biological replicates, determined by qPCR, were standardized as described (Willems et al., 2008). Means and standard deviations are shown. Statistical analysis in 3C and 3D was done using one-way ANOVA, Fisher Tests, and two-tailed two independent sample T-Tests.

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Figure 3—figure supplement 1. SOX18 expression in infantile hemangioma-derived cells. ; Human ECFC served as a positive control for SOX18. Hemangioma-derived GLUT1-negative ECs, HemSC, GLUT1-positively selected ECs, and pericytes were analysed for SOX18 mRNA expression by qPCR analysis.

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Author response: R-propranolol is a small molecule inhibitor of the SOX18 transcription factor in a rare vascular syndrome and hemangioma

• Authors : Overman, Jeroen; Fontaine, Frank; Wylie-Sears, Jill

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Figure 3—figure supplement 2. SOX18 inhibitor (Sm4) and propranolol block HemSC to EC differentiation. ; (A) VEGF-B treatment of HemSC from four different infantile hemangiomas resulted in increased CD31 and CDH5 (endothelial cell markers), and hemangioma endothelial markers NOTCH1, PLXND1 and VEGFR1, determined by qPCR. Inclusion of SOX18 inhibitor (Sm4) or propranolol blocked the VEGF-B induced increases in these markers. Results from eight biological replicates were standardized as described (Willems et al., 2008). Means and standard deviations are shown. All P values were < 0.001 for Control versus DMSO (VEGF-B treated); DMSO versus Sm4 and DMSO versus propranolol. (B–D) VEGFR2 protein and phosphorylation were not affected by pre-treatment with propranolol, R + enantiomer or S(-) enantiomer (each tested at 5 uM). (B) Human endothelial colony forming cells (ECFC) were pre-treated for one hour with drug as indicated, stimulated with 25 ng/ml VEGF-A for 5 min, lysed and analysed by Western blotting. (C) HemSC were induced to differentiate for 5 days with VEGF-B; control indicates cells in differentiation media without VEGF-B. Differentiated cells were pre-treated for one hour with drugs as indicated, stimulated with 25 ng/ml VEGF-A for 5 min, and lysed for VEGFR2 Western blotting. (D) HemSCs were treated as in B, cell lysates were immunoprecipitated with anti-VEGFR2, followed by Western blotting for phosphorylated VEGFR2 and total VEGFR2. B-D, M indicates lane with molecular weight standards.

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Decision letter: R-propranolol is a small molecule inhibitor of the SOX18 transcription factor in a rare vascular syndrome and hemangioma

• Authors : Koh, Gou Young; Ostergaard, Pia;

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VEGFR-2 conformational switch in response to ligand binding

• Authors : Sarabipour, Sarvenaz; Ballmer-Hofer, Kurt; Hristova, Kalina ...

• Source: eLife ; volume 5 ; ISSN 2050-084X

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