β-adrenergic receptor antagonists inhibit vasculogenesis of embryonic stem cells by downregulation of nitric oxide generation and interference with VEGF signalling

The beta-adrenoceptor antagonist Propranolol has been successfully used to treat infantile hemangioma. However, its mechanism of action is so far unknown. The hypothesis of this research was that beta-adrenoceptor antagonists may interfere with endothelial cell differentiation of stem cells. Specifically, the effects of the non-specific beta-adrenergic receptor (beta-adrenoceptor) antagonist Propranolol, the beta(1)-adrenoceptor-specific antagonist Atenolol and the beta(2)-adrenoceptor-specific antagonist ICI118,551 on vasculogenesis of mouse embryonic stem (ES) cells were investigated. All three beta-blockers dose-dependently downregulated formation of capillary structures in ES cell-derived embryoid bodies and decreased the expression of the vascular cell markers CD31 and VE-cadherin. Furthermore, beta-blockers downregulated the expression of fibroblast growth factor-2 (FGF-2), hypoxia inducible factor-1 alpha (HIF-1 alpha), vascular endothelial growth factor 165 (VEGF(165)), VEGF receptor 2 (VEGF-R2) and phospho VEGF-R2, as well as neuropilin 1 (NRP1) and plexin-B1 which are essential modulators of embryonic angiogenesis with additional roles in vessel remodelling and arteriogenesis. Under conditions of beta-adrenoceptor inhibition, the endogenous generation of nitric oxide (NO) as well as the phosphorylation of endothelial nitric oxide synthase (eNOS) was decreased in embryoid bodies, whereas an increase in NO generation was observed with the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP). Consequently, vasculogenesis of ES cells was restored upon treatment of differentiating ES cells with beta-adrenoceptor antagonists in the presence of NO donor. In summary, our data suggest that beta-blockers impair vasculogenesis of ES cells by interfering with NO generation which could be the explanation for their anti-angiogenic effects in infantile hemangioma.