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.

Growth Hormone Induces Recurrence of Infantile Hemangiomas After Apparent Involution: Evidence of Growth Hormone Receptors in Infantile Hemangioma.

Infantile hemangiomas (IHs) are the most common benign tumor of infancy, characterized by a natural history of early proliferation in the first months of life to eventual involution during childhood, often with residual fibrofatty tissue. Once involution has been achieved, IHs do not typically recur. We present two cases of exogenous growth hormone therapy resulting in the recurrence of IHs in late childhood, supported by radiological, immunohistochemical, in vitro, and in vivo evidence.

VEGFR-1 blockade disrupts peri-implantation decidual angiogenesis and macrophage recruitment.

BACKGROUND: Angiogenesis and macrophage recruitment to the uterus are key features of uterine decidualization; the progesterone-mediated uterine changes that allow for embryo implantation and initiation of pregnancy. In the current study, we characterized the expression of vascular endothelial growth factor receptor-1 (VEGFR-1) in macrophages and endothelial cells of the peri-implantation uterus and determined if VEGFR-1 function is required for decidual angiogenesis, macrophage recruitment, and/or the establishment of pregnancy. METHODS: Expression of VEGFR-1 in uterine endothelial cells and macrophages was determined with immunohistochemistry. To assess the effect of continuous VEGFR-1 blockade, adult female mice were given VEGFR-1 blocking antibody, MF-1, every 3 days for 18 days. After 6 doses, females were mated and a final dose of MF-1 was given on embryonic day 3.5. Endothelial cells and macrophages were quantified on embryonic day 7.5. Pregnancy was analyzed on embryonic days 7.5 and 10.5. RESULTS: F4/80(+) macrophages are observed throughout the stroma and are abundant adjacent to the endometrial lumen and glands prior to embryo implantation and scatter throughout the decidua post implantation. VEGFR-1 expression is restricted to the uterine endothelial cells. F4/80(+) macrophages were often found adjacent to VEGFR-1(+) endothelial cells in the primary decidual zone. Continuous VEGFR-1 blockade correlates with a significant reduction in decidual vascular and macrophage density, but does not affect embryo implantation or maintenance of pregnancy up to embryonic day 10.5. CONCLUSIONS: We found that VEGFR-1 functions in both decidual angiogenesis and macrophage recruitment to the implantation site during pregnancy. VEGFR-1 is expressed by endothelial cells, however blocking VEGFR-1 function in endothelial cells results in reduced macrophage recruitment to the uterus. VEGFR-1 blockade did not compromise the establishment and/or maintenance of pregnancy.