NOTCH3 regulates stem-to-mural cell differentiation in infantile hemangioma.

Infantile hemangioma (IH) is a vascular tumor that begins with rapid vascular proliferation shortly after birth, followed by vascular involution in early childhood. We have found that NOTCH3, a critical regulator of mural cell differentiation and maturation, is expressed in hemangioma stem cells (HemSCs), suggesting that NOTCH3 may function in HemSC-to-mural cell differentiation and pathological vessel stabilization. Here, we demonstrate that NOTCH3 is expressed in NG2+PDGFRß+ perivascular HemSCs and CD31+GLUT1+ hemangioma endothelial cells (HemECs) in proliferating IHs and becomes mostly restricted to the αSMA+NG2loPDGFRßlo mural cells in involuting IHs. NOTCH3 knockdown in HemSCs inhibited in vitro mural cell differentiation and perturbed αSMA expression. In a mouse model of IH, NOTCH3 knockdown or systemic expression of the NOTCH3 inhibitor, NOTCH3 Decoy, significantly decreased IH blood flow, vessel caliber, and αSMA+ perivascular cell coverage. Thus, NOTCH3 is necessary for HemSC-to-mural cell differentiation, and adequate perivascular cell coverage of IH vessels is required for IH vessel stability.

Propranolol Targets Hemangioma Stem Cells via cAMP and Mitogen-Activated Protein Kinase Regulation.

UNLABELLED: Infantile hemangiomas (IHs) are the most common vascular tumor and arise from a hemangioma stem cell (HemSC). Propranolol has proved efficacious for problematic IHs. Propranolol is a nonselective ß-adrenergic receptor (ßAR) antagonist that can lower cAMP levels and activate the mitogen-activated protein kinase (MAPK) pathway downstream of ßARs. We found that HemSCs express ß1AR and ß2AR in proliferating IHs and determined the role of these ßARs and the downstream pathways in mediating propranolol's effects. In isolated HemSCs, propranolol suppressed cAMP levels and activated extracellular signal-regulated kinase (ERK)1/2 in a dose-dependent fashion. Propranolol, used at doses of <10(-4) M, reduced cAMP levels and decreased HemSC proliferation and viability. Propranolol at ≥10(-5) M reduced cAMP levels and activated ERK1/2, and this correlated with HemSC apoptosis and cytotoxicity at ≥10(-4) M. Stimulation with a ßAR agonist, isoprenaline, promoted HemSC proliferation and rescued the antiproliferative effects of propranolol, suggesting that propranolol inhibits ßAR signaling in HemSCs. Treatment with a cAMP analog or a MAPK inhibitor partially rescued the HemSC cell viability suppressed by propranolol. A selective ß2AR antagonist mirrored propranolol's effects on HemSCs in a dose-dependent fashion, and a selective ß1AR antagonist had no effect, supporting a role for ß2AR signaling in IH pathobiology. In a mouse model of IH, propranolol reduced the vessel caliber and blood flow assessed by ultrasound Doppler and increased activation of ERK1/2 in IH cells. We have thus demonstrated that propranolol acts on HemSCs in IH to suppress proliferation and promote apoptosis in a dose-dependent fashion via ß2AR perturbation, resulting in reduced cAMP and MAPK activation. SIGNIFICANCE: The present study investigated the action of propranolol in infantile hemangiomas (IHs). IHs are the most common vascular tumor in children and have been proposed to arise from a hemangioma stem cell (HemSC). Propranolol, a nonselective ß-adrenergic receptor (ßAR) antagonist, has proven efficacy; however, understanding of its mechanism of action on HemSCs is limited. The presented data demonstrate that propranolol, via ßAR perturbation, dose dependently suppresses cAMP levels and activated extracellular signal-regulated kinase 1/2. Furthermore, propranolol acts via perturbation of ß2AR, and not ß1AR, although both receptors are expressed in HemSCs. These results provide important insight into propranolol's action in IHs and can be used to guide the development of more targeted therapy.