Elisa Boscolo

Suppressed NFAT-dependent VEGFR1 expression and constitutive VEGFR2 signaling in infantile hemangioma

Infantile hemangiomas are localized and rapidly growing regions of disorganized angiogenesis. We show that expression of vascular endothelial growth factor receptor-1 (VEGFR1) in hemangioma endothelial cells (hemECs) and hemangioma tissue is markedly reduced compared to controls. Low VEGFR1 expression in hemECs results in VEGF-dependent activation of VEGFR2 and downstream signaling pathways. In hemECs, transcription of the gene encoding VEGFR1 (FLT1) is dependent on nuclear factor of activated T cells (NFAT). Low VEGFR1 expression in hemECs is caused by reduced activity of a pathway involving β1 integrin, the integrin-like receptor tumor endothelial marker-8 (TEM8), VEGFR2 and NFAT. In a subset of individuals with hemangioma, we found missense mutations in the genes encoding VEGFR2 (KDR) and TEM8 (ANTXR1). These mutations result in increased interactions among VEGFR2, TEM8 and β1 integrin proteins and in inhibition of integrin activity. Normalization of the constitutive VEGFR2 signaling in hemECs with soluble VEGFR1 or antibodies that neutralize VEGF or stimulate β1 integrin suggests that local administration of these or similar agents may be effective in hemangioma treatment.

Multipotential stem cells recapitulate human infantile hemangioma in immunodeficient mice

Infantile hemangioma is a benign endothelial tumor composed of disorganized blood vessels. It exhibits a unique life cycle of rapid postnatal growth followed by slow regression to a fibrofatty residuum. Here, we have reported the isolation of multipotential stem cells from hemangioma tissue that give rise to hemangioma-like lesions in immunodeficient mice. Cells were isolated based on expression of the stem cell marker CD133 and expanded from single cells as clonal populations. The CD133-selected cells generated human blood vessels 7 days after implantation in immunodeficient mice. Cell retrieval experiments showed the cells could again form vessels when transplanted into secondary recipients. The human vessels expressed GLUT-1 and merosin, immunodiagnostic markers for infantile hemangioma. Two months after implantation, the number of blood vessels diminished and human adipocytes became evident. Lentiviral expression of GFP was used to confirm that the hemangioma-derived cells formed the blood vessels and adipocytes in the immunodeficient mice. Thus, when transplanted into immunodeficient mice, hemangioma-derived cells recapitulated the unique evolution of infantile hemangioma--the formation of blood vessels followed by involution to fatty tissue. In summary, this study identifies a stem cell as the cellular origin of infantile hemangioma and describes for what we believe is the first time an animal model for this common tumor of infancy.

CORTICOSTEROID SUPPRESSION OF VEGF-A IN INFANTILE HEMANGIOMA-DERIVED STEM CELLS

BACKGROUND Corticosteroids are commonly used to treat infantile hemangioma, but the mechanism of action of this therapy is unknown. We investigated the effect of corticosteroids in a previously described in vivo model of infantile hemangioma and in cultured hemangioma-derived cells. METHODS We tested hemangioma-derived stem cells for vasculogenic activity in vivo after implantation into immune-deficient (nude) mice. We studied dexamethasone treatment of both the cells before implantation and the mice after implantation. We also tested hemangioma-derived stem cells for expression of vascular endothelial growth factor A (VEGF-A) in vitro and studied the inhibition of VEGF-A expression, using short hairpin RNA (shRNA) in vivo and in vitro. RESULTS Systemic treatment with dexamethasone led to dose-dependent inhibition of tumor vasculogenesis in the murine model. Pretreatment of hemangioma-derived stem cells in vitro before implantation also inhibited vasculogenesis. Dexamethasone suppressed VEGF-A production by hemangioma-derived stem cells in vitro but not by hemangioma-derived endothelial cells or human umbilical-vein endothelial cells. Silencing VEGF-A in hemangioma-derived stem cells reduced vasculogenesis in vivo. VEGF-A was detected in hemangioma specimens in the proliferating phase but not in the involuting phase and was shown by immunostaining to reside outside of vessels. Corticosteroid treatment suppressed other proangiogenic factors in hemangioma-derived stem cells, including urokinase plasminogen activator receptor, interleukin-6, monocyte chemoattractant protein 1, and matrix metalloproteinase 1. CONCLUSIONS In a murine model, dexamethasone inhibited the vasculogenic potential of stem cells derived from human infantile hemangioma. The corticosteroid also inhibited the expression of VEGF-A by hemangioma-derived stem cells, and silencing of VEGF-A expression in these cells inhibited vasculogenesis in vivo.

Rapamycin Suppresses Self-Renewal and Vasculogenic Potential of Stem Cells Isolated from Infantile Hemangioma

Infantile hemangioma (IH) is a common childhood vascular tumor. Although benign, some hemangiomas cause deformation and destruction of features or endanger life. The current treatments, corticosteroid or propranolol, are administered for several months and can have adverse effects for the infant. We designed a high-throughput screen to identify FDA-approved drugs that could be used to treat this tumor. Rapamycin, an mTOR inhibitor, was identified based on its ability to inhibit proliferation of a hemangioma-derived stem cell population, human vasculogenic cells we had previously discovered. In vitro and in vivo studies show that Rapamycin reduces the self-renewal capacity of the hemangioma stem cells, diminishes differentiation potential, and inhibits the vasculogenic activity of these cells in vivo. Longitudinal in vivo imaging of blood flow through vessels formed with hemangioma stem cells shows that Rapamycin also leads to regression of hemangioma blood vessels, consistent with its known anti-angiogenic activity. Finally, we demonstrate that Rapamycin-induced loss of stemness can work in concert with corticosteroid, the current standard therapy for problematic hemangioma, to block hemangioma formation in vivo. Our studies reveal that Rapamycin targets the self-renewal and vascular differentiation potential in patient-derived hemangioma stem cells and suggests a novel therapeutic strategy to prevent formation of this disfiguring and endangering childhood tumor.

JAGGED1 Signaling Regulates Hemangioma Stem Cell–to–Pericyte/Vascular Smooth Muscle Cell Differentiation

Objective—The aim of our study is to determine the cellular and molecular origin for the pericytes in infantile hemangioma (IH) and their functional role in the formation of pathological blood vessels. Methods and Results—Here we show that IH-derived stem cells (HemSCs) form pericyte-like cells. With in vitro studies, we demonstrate that HemSC-to-pericyte differentiation depends on direct contact with endothelial cells. JAGGED1 expressed ectopically in fibroblasts was sufficient to induce HemSCs to acquire a pericyte-like phenotype, indicating a critical role for JAGGED1. In vivo, we blocked pericyte differentiation with recombinant JAGGED1, and we observed reduced formation of blood vessels, with an evident lack of pericytes. Silencing JAGGED1 in the endothelial cells reduced blood vessel formation and resulted in a paucity of pericytes. Conclusion—Our data show that endothelial JAGGED1 controls HemSC-to-pericyte differentiation in a murine model of IH and suggests that pericytes have a fundamental role in formation of blood vessels in IH.

Rapamycin improves TIE2-mutated venous malformation in murine model and human subjects.

Venous malformations (VMs) are composed of ectatic veins with scarce smooth muscle cell coverage. Activating mutations in the endothelial cell tyrosine kinase receptor TIE2 are a common cause of these lesions. VMs cause deformity, pain, and local intravascular coagulopathy, and they expand with time. Targeted pharmacological therapies are not available for this condition. Here, we generated a model of VMs by injecting HUVECs expressing the most frequent VM-causing TIE2 mutation, TIE2-L914F, into immune-deficient mice. TIE2-L914F-expressing HUVECs formed VMs with ectatic blood-filled channels that enlarged over time. We tested both rapamycin and a TIE2 tyrosine kinase inhibitor (TIE2-TKI) for their effects on murine VM expansion and for their ability to inhibit mutant TIE2 signaling. Rapamycin prevented VM growth, while TIE2-TKI had no effect. In cultured TIE2-L914F-expressing HUVECs, rapamycin effectively reduced mutant TIE2-induced AKT signaling and, though TIE2-TKI did target the WT receptor, it only weakly suppressed mutant-induced AKT signaling. In a prospective clinical pilot study, we analyzed the effects of rapamycin in 6 patients with difficult-to-treat venous anomalies. Rapamycin reduced pain, bleeding, lesion size, functional and esthetic impairment, and intravascular coagulopathy. This study provides a VM model that allows evaluation of potential therapeutic strategies and demonstrates that rapamycin provides clinical improvement in patients with venous malformation.

VEGFR-1 mediates endothelial differentiation and formation of blood vessels in a murine model of infantile hemangioma.

Vascular endothelial growth factor receptor-1 (VEGFR-1) is a member of the VEGFR family, and binds to VEGF-A, VEGF-B, and placental growth factor. VEGFR-1 contributes to tumor growth and metastasis, but its role in the pathological formation of blood vessels is still poorly understood. Herein, we used infantile hemangioma (IH), the most common tumor of infancy, as a means to study VEGFR-1 activation in pathological vasculogenesis. IH arises from stem cells (HemSCs) that can form the three most prominent cell types in the tumor: endothelial cells, pericytes, and adipocytes. HemSCs can recapitulate the IH life cycle when injected in immuncompromised mice, and are targeted by corticosteroids, the traditional treatment for IH. We report here that VEGF-A or VEGF-B induces VEGFR-1-mediated ERK1/2 phosphorylation in HemSCs and promotes differentiation of HemSCs to endothelial cells. Studies of VEGFR-2 phosphorylation status and down-regulation of neuropilin-1 in the HemSCs demonstrate that VEGFR-2 and NRP1 are not needed for VEGF-A- or VEGF-B-induced ERK1/2 activation. U0216-mediated blockade of ERK1/2 phosphorylation or shRNA-mediated suppression of VEGFR-1 prevents HemSC-to-EC differentiation. Furthermore, the down-regulation of VEGFR-1 in the HemSCs results in decreased formation of blood vessels in vivo and reduced ERK1/2 activation. Thus, our study reveals a critical role for VEGFR-1 in the HemSC-to-EC differentiation that underpins pathological vasculogenesis in IH.

SOCS3 is an endogenous inhibitor of pathologic angiogenesis

Inflammatory cytokines and growth factors drive angiogenesis independently; however, their integrated role in pathologic and physiologic angiogenesis is not fully understood. Suppressor of cytokine signaling-3 (SOCS3) is an inducible negative feedback regulator of inflammation and growth factor signaling. In the present study, we show that SOCS3 curbs pathologic angiogenesis. Using a Cre/Lox system, we deleted SOCS3 in vessels and studied developmental and pathologic angiogenesis in murine models of oxygen-induced retinopathy and cancer. Conditional loss of SOCS3 leads to increased pathologic neovascularization, resulting in pronounced retinopathy and increased tumor size. In contrast, physiologic vascularization is not regulated by SOCS3. In vitro, SOCS3 knockdown increases proliferation and sprouting of endothelial cells costimulated with IGF-1 and TNFα via reduced feedback inhibition of the STAT3 and mTOR pathways. These results identify SOCS3 as a pivotal endogenous feedback inhibitor of pathologic angiogenesis and a potential therapeutic target acting at the converging crossroads of growth factor- and cytokine-induced vessel growth.

A switch in Notch gene expression parallels stem cell to endothelial transition in infantile hemangioma

BackgroundInfantile hemangioma (IH) is the most common benign tumor of infancy, yet its pathogenesis is poorly understood. Notch family members are known to play a role in vascular development during embryogenesis and postnatal tumor angiogenesis, yet the role of Notch signaling in the pathogenesis of IH has not been investigated. This study aims to survey Notch expression in IH.Materials and methodsRNA from resected hemangioma tissue and hemangioma-derived stem cells (HemSCs) and endothelial cells (HemECs) was used for gene expression analyses by real-time PCR. Results were confirmed with immunofluorescence for protein expression in tissue.ResultsReal-time PCR showed that Notch family gene expression in IH is distinct from placenta and skin. Notch3 is expressed in HemSCs, but not in HemECs, indicating Notch3 is downregulated as HemSCs differentiate into HemECs. Moreover, expression of endothelial-associated Notch proteins, Notch1, Notch4, and Jagged-1 are increased in involuting hemangiomas and HemECs, suggesting that as hemangioma progresses toward involution, it acquires more differentiated endothelium. A subset of cells stained double positive for Notch3 and CD31, pointing to a potential intermediate between the HemSC cellular differentiation into HemEC.ConclusionHemSCs have distinct Notch expression patterns from differentiated HemECs and from normal human endothelial cells. Notch3 is expressed in HemSCs, while Notch1, Notch4, and Jagged-1 have higher expression levels in HemECs. Notch3 was localized to the interstitial cells outside of the nascent vascular channels in proliferating IH tissue sections, but became more apparent in the perivascular cells in involuting IH. In summary, the pattern of Notch gene expression mirrors the progression from immature cells to endothelial-lined vascular channels (i.e., endothelial differentiation) that characterizes the growth and involution of IH.

IGF-2 and FLT-1/VEGF-R1 mRNA levels reveal distinctions and similarities between congenital and common infantile hemangioma.

Common infantile hemangioma appears postnatally, grows rapidly, and regresses slowly. Two types of congenital vascular tumors present fully grown at birth and behave differently from infantile hemangioma. These rare congenital tumors have been designated rapidly involuting congenital hemangioma (RICH) and noninvoluting congenital hemangioma (NICH). RICH and NICH are similar in appearance, location, and size, and have some overlapping histologic features with infantile hemangioma. At a molecular level, neither expresses glucose transporter-1, a diagnostic marker of infantile hemangioma. To gain further insight into the molecular differences and similarities between congenital and common hemangioma, we analyzed expression of insulin-like growth factor-2, known to be highly expressed in infantile hemangioma and VEGF-receptors, by quantitative real-time PCR, in three RICH and five NICH specimens. We show that insulin-like growth factor-2 mRNA was expressed in both RICH and NICH, at a level comparable with that detected in common hemangioma over 4 y of age. In contrast, mRNA levels for membrane-associated fms-like tyrosine-kinase receptor, also known as VEGF receptor-1, were uniformly increased in congenital hemangiomas compared with proliferating or involuting phase common hemangioma. These results provide the first evidence of the molecular distinctions and similarities between congenital and postnatal hemangioma.