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Background Propranolol, a nonselective β-adrenergic receptor antagonist, is approved by the U.S. Food and Drug Administration to treat problematic infantile hemangiomas, but a subset of patients experience treatment complications. Parents wary of long-term use and side effects consult plastic surgeons on surgical options or as a second opinion. Understanding the mechanism(s) of action of propranolol will allow plastic surgeons to better inform parents. Methods A systemic literature search was performed to query published translational and basic science studies on propranolol effects on infantile hemangiomas and cells derived from these lesions. Results In experimental studies, propranolol was antiproliferative and cytotoxic against hemangioma endothelial and stem cells and affected infantile hemangioma perivascular cell contractility. Propranolol inhibited migration, network formation, vascular endothelial growth factor A production, and vascular endothelial growth factor receptor 2 activation and down-regulated PI3K/AKT and mitogen-activated protein kinase signaling in hemangioma endothelial cells, but it increased ERK1/2 activity in hemangioma stem cells. At effective clinical doses, measured propranolol plasma concentration is 100 times higher than necessary for complete β-adrenergic receptor blockade, yet was 10 to 100 times less than required to induce hemangioma stem cell death. Conclusions Propranolol targets multiple cell types in infantile hemangiomas by means of β-adrenergic receptor-dependent and -independent mechanisms. Plasma concentration played a significant role. At clinically relevant doses, incomplete infantile hemangioma suppression may explain the rebound phenomenon and worsening ulceration, and propranolol off target effects may lead to commonly reported adverse effects, such as sleep and gastrointestinal disturbances. Propranolol limitations and complications underscore the importance of surgical treatment options in cases of rebound and severe adverse effects. Surgical intervention remains an important treatment choice when parents are hesitant to use propranolol.

Objective— Infantile hemangiomas (IHs) are the most common benign vascular neoplasms of infancy, characterized by a rapid growth phase followed by a spontaneous involution, or triggered by propranolol treatment by poorly understood mechanisms. LIN28/let-7 axis plays a central role in the regulation of stem cell self-renewal and tumorigenesis. However, the role of LIN28B/let-7 signaling in IH pathogenesis has not yet been elucidated. Approach and Results— LIN28B is highly expressed in proliferative IH and is less expressed in involuted and in propranolol-treated IH samples as measured by immunofluorescence staining and quantitative RT-PCR. Small RNA sequencing analysis of IH samples revealed a decrease in microRNAs that target LIN28B, including let-7, and an increase in microRNAs in the mir-498(46) cistron. Overexpression of LIN28B in HEK293 cells induced the expression of miR-516b in the mir-498(46) cistron. Propranolol treatment of induced pluripotent stem cells, which express mir-498(46) endogenously, reduced the expression of both LIN28B and mir-498(46) and increased the expression of let-7. Furthermore, propranolol treatment reduced the proliferation of induced pluripotent stem cells and induced epithelial–mesenchymal transition. Conclusions— This work uncovers the role of the LIN28B/let-7 switch in IH pathogenesis and provides a novel mechanism by which propranolol induces IH involution. Furthermore, it provides therapeutic implications for cancers in which the LIN28/let-7 pathway is imbalanced.

Infantile hemangiomas (IHs) are the most common vascular tumor and arise from a hemangioma stem cell (HemSC). Propranolol has proved efficacious against IHs. A selective β2-adrenergic receptor (AR) antagonist mirrored propranolol’s effects on HemSCs. These results show that propranolol acts on HemSCs in IH to suppress proliferation and promote apoptosis in a dose-dependent fashion via β2AR perturbation., 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 β-adrenergic receptor (βAR) antagonist that can lower cAMP levels and activate the mitogen-activated protein kinase (MAPK) pathway downstream of βARs. We found that HemSCs express β1AR and β2AR in proliferating IHs and determined the role of these βARs 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

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.

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+PDGFRβ+ perivascular HemSCs and CD31+GLUT1+ hemangioma endothelial cells (HemECs) in proliferating IHs and becomes mostly restricted to the αSMA+NG2loPDGFRβlo 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.

BACKGROUND Infantile hemangiomas (IHs) are the most common tumor of infancy, yet there are no Food and Drug Administration-approved therapeutics to date. Recently, the nonselective β-adrenergic-blocker propranolol has been shown to be a safe and effective means of treating IHs, although its mechanism has yet to be elucidated. We have previously demonstrated that propranolol induces early and incomplete adipogenesis in stem cells derived from hemangiomas. We hypothesize that propranolol promotes dysregulated adipogenesis via the improper regulation of adipogenic genes. METHODS Hemangioma stem cells (HemSCs) isolated from resected IH specimens were treated with adipogenic medium for 1 or 4 days in either propranolol or vehicle. Cell death was measured by the incorporation of annexin V and propidium iodide by flow cytometry. Adipogenesis was assessed by visualizing lipid droplet formation by Oil Red O staining. Proadipogenic genes C/EBPα, C/EBPβ, C/EBPδ, PPARδ, PPARγ, RXRα, and RXRγ were analyzed by quantitative reverse transcription and polymerase chain reaction. RESULTS Hemangioma stem cells treated with propranolol increased lipid droplet formation compared to vehicle-treated cells indicating increased adipogenesis. Cell death as measured by FACS analysis indicated that the propranolol-treated cells died due to necrosis and not apoptosis. During adipogenesis, transcript levels of PPARδ, PPARγ, C/EBPβ, and C/EBPδ were significantly increased (P

Infantile hemangiomas can cause significant morbidity during proliferation, yet there is no U.S. Food and Drug Administration-approved treatment. They are believed to form from hemangioma stem cells, which differentiate toward a hemangioma endothelial cell phenotype. Recently, propranolol has demonstrated effectiveness in treating complicated infantile hemangiomas. The authors hypothesize that propranolol facilitates their involution by altering cellular behavior in both hemangioma endothelial and stem cells.Hemangioma endothelial and stem cells were isolated from resected infantile hemangioma specimens. Cells were treated with 100 μM propranolol for 48 hours, and apoptosis was determined by the presence of annexin V antibody. Proliferation of stem and endothelial cells was assessed after treatment with 50 or 100 μM propranolol or vehicle, for 72 and 96 hours, respectively. Adipogenesis was induced in stem cells with and without propranolol. Pro-adipogenic genes PPARδ, PPARγ, C/EBPα, C/EBPβ, C/EBPδ, RXRα, and RXRγ were analyzed by quantitative polymerase chain reaction.Annexin V levels were increased in propranolol-treated endothelial cells but not in stem cells. Proliferation of stem and endothelial cells was inhibited by propranolol in a dose-dependent manner. Propranolol-treated stem cells demonstrated accelerated adipogenesis when compared with untreated controls. Transcript levels of C/EBPβ (p0.05), RXRγ (p0.05), and PPARγ (p0.02) were significantly increased when treated with 50 or 100 μM propranolol; and C/EBPδ (p0.05), RXRα (p0.05), and PPARδ (p0.01) transcripts were increased when treated with 100 μM propranolol. C/EBPα transcript levels remained unchanged at either dose.Propranolol increased apoptosis of hemangioma endothelial cells, but not stem cells, and accelerated adipogenesis of hemangioma stem cells. Thus, propranolol likely accelerates involution to fibrofatty residuum.

We present a case of a large congenital hemangioma (CH) on the neck causing cardiac failure and thrombocytopenia in a female neonate. A trial of medical therapy with corticosteroids and propranolol was attempted, but the patient ultimately underwent definitive treatment with embolization and surgical resection with a positive outcome. A review of the English language literature revealed 16 previously reported cases of CHs complicated by congestive heart failure. This series supports known demographic features of CHs, including a lack of gender discrepancy and a predilection to affect the head and neck. These CHs are rarely diagnosed in utero; most patients present with a mass at birth. Cardiac failure is identified prenatally or in the first days of life. A mild to moderate thrombocytopenia and coagulopathy, which is likely transient and distinct from classic Kasabach-Merritt phenomenon, accompanies many of these cases. There is a 30% associated mortality rate. Both medical and interventional treatment modalities have been reported. Steroids are the most commonly used medication, but without any clear benefit. We hypothesize that, based on its possible mechanisms of action,propranolol may be a more effective treatment for CHs requiring treatment. As surgical intervention may be necessary, we recommend a multidisciplinary approach to treating patients with problematic CHs.

Hemangiomas often leave contour deformities and scarring after involution. Surgical resection leaves permanent scars and may distort adjacent anatomy. Circular excision with purse-string closure results in the smallest possible scar and minimal distortion. Thirty-six patients with varying stages of hemangiomas underwent resection with purse-string closure. The dimensions of each hemangioma were measured at the time of excision. Dimensions of the scar at follow-up office visits were recorded. The final scar was calculated as a percentage of the original size of the lesion. Thirty-six patients underwent circular excision and purse-string closure of 39 hemangiomas. Thirty (83.3%) were girls and 6 (16.7%) were boys. The ages of the patients ranged from 5 months to 13 years, with a median age of 2.08. The most common locations were: scalp 9 (23.1%), cheek 9 (23.1%), forehead 5 (12.8%), and eyebrow 4 (10.3%). The median area of the hemangiomas was 491 mm 2 (range: 113-2826 mm 2 ). Follow-up measurements were available for 25 patients with 33 hemangiomas. Follow-up ranged from 1 to 22 months (median follow-up, 3 months). The median area of the purse-string scar was 70.7 mm 2 (range: 3.925-706.5 mm 2 ). The median percentage reduction in area of involvement was 80.6% (range: 24.2%-99.1%). When the hemangiomas were stratified into "large" and "small" hemangiomas, the median percentage reduction in the areas were 84.1% and 79.2%, respectively. There were 3 complications: 1 patient scratched open her incision (ultimately 76% of original size), and 1 toddler scraped her scalp against the carpet and dehisced the purse-string closure (final size, 43% of original size), and a second toddler fell and dehisced a scalp hemangioma (final size, 80% of original size). Traditional lenticular excision of hemangiomas results in increased scar length as compared with the original lesion. Circular excision and purse-string closure of defects results in a scar smaller than the original lesion. It is well tolerated and can be considered the default method in most anatomic locations.

Hemangiomas are the most common benign tumor of infancy, yet its pathogenesis and the mechanisms governing proliferation and involution are not well understood. It is believed that hemangiomas arise out of clonal, abnormal hemangioma endothelial cells (HemECs). The underlying anomaly of the HemEC is not known, although studies have shown that vascular endothelial growth factor (VEGF) and VEGF signaling may influence HemECs. Moreover, there are numerous subtypes of hemangiomas, with differences in natural history, potential for morbidity, and prognosis, and little is known how this relates to HemEC. The Notch signaling pathway is a highly conserved pathway across species from worms to mammals. Notch signaling has been shown to play a role during embryogenesis in directing vascular patterning and development and arterial and venous cell fate determination. Postnatally, it has been implicated in tumor angiogenesis in multiple malignancies. Notch signaling triggers tumor angiogenesis at least in part to stimulation by VEGF, thus establishing that there is a cross talk between the VEGF and Notch pathways. Given the presence of VEGF and its receptors in hemangiomas and known VEGF-Notch cross talk in tumor angiogenesis, the authors hypothesize that Notch signaling may contribute to hemangioma proliferation and involution. Preliminary studies of resected hemangioma specimens by reverse transcription polymerase chain reaction (RT-PCR) show that all 4 Notch receptors and 2 Notch ligands, Jagged1 and Delta-like ligand 4, are expressed by hemangiomas. These findings support a role for Notch in hemangiomas, meriting further analysis of the functional relevance of Notch signaling in hemangiomas.

Infantile hemangiomas (IHs) spontaneously involute, but some leave contour deformities necessitating surgical correction. There is a paucity of data reviewing predictive risk factors associated with a need for surgery to guide clinicians when counseling parents. Patients undergoing IH resection by a single surgeon from August 2004 to August 2011 were reviewed to determine patient (age, gender, birth history) and IH characteristics (size, location) associated with surgical intervention. Data were compared to published data from the Hemangioma Investigator Group (HIG). Statistical analysis was performed using Student's t-test, odds ratio, and logistic regression analysis. Out of 196 referred patients, 112 underwent surgery. There was a female preponderance (3.5:1). Two-thirds of patients (64.9%) first presented to the surgeon at ≤2 years of age, but most underwent surgery between 2 and 3 years (52.7%; average lag time, 11 months). 18 patients underwent surgery at ≤1 year of age. IH patients with preterm birth history had increased risk for needing surgical intervention (odds ratio 2.124, CI 1.31-3.44; p 0.0012). A majority (84.7%) of resected IHs were located on the head or neck, significantly higher than the distribution from the HIG data (62.2%; p 0.0001). Resected head and neck IHs were smaller than those below the neck (average, 8.85 cm(2) vs. 22.35 cm(2), p = 0.017). Preterm birth is associated with higher risk for requiring surgical intervention. IHs on the head and neck are more likely to be removed when compared to those below the neck, and at a smaller size threshold.

Infantile hemangiomas (IHs) belong to a family of lesions called vascular anomalies. Vascular anomalies are classified into either vascular tumors or vascular malformations, with the IH being the most common vascular tumor, affecting approximately 5% of infants (Frieden, Haggstrom et al 2005). Despite their prevalence, the origin and pathogenesis of IHs remains poorly understood. Clinically, IHs undergo a predictable course of rapid proliferation shortly after birth, followed by stabilization and involution throughout childhood. Occasionally, a fibrofatty residuum results after involution is complete. Despite its predictable clinical course, the regulatory mechanisms throughout different phases are only recently being elucidated (Frieden, Haggstrom et al. 2005). During the proliferative phase, IH can cause serious morbidity and even mortality. Rapidly proliferating hemangiomas can be dangerous as they have potential to ulcerate and bleed. While disorganized, IHs are high-flow lesions; occasionally, life-threatening bleeding can occur. The location of the IH can also be detrimental. Hemangiomas in the peri-orbital area can cause obstructive amblyopia and astigmatism, and airway hemangiomas can cause stridor and respiratory distress. Visceral hemangiomas of the liver can cause congestive heart failure, hepatomegaly, and anemia; the mortality rate with treatment is significant, up to 30% (Arneja and Mulliken, 2010; Bitar et al., 2005; Boon et al., 1996; Ceisler & Blei, 2003; Chamlin et al., 2007; Haggstrom et al., 2006a; Schwartz et al., 2006). In recent years, PHACE syndrome has been described and characterized. PHACE syndrome comprises a constellation of findings including Posterior fossa anomalies, large facial Hemangioma, Arterial anomalies, Cardiac abnormalities/aortic Coarctation, and Eye anomalies (Frieden et al., 1996). Infants with PHACE syndrome are at increased risk for strokes, neurological and cardiac consequences (Burrows et al., 1998; Drolet et al., 2006). Another syndrome featuring a large hemangioma over an area with aberrant underlying anatomical structures have also been described. PELVIS syndrome describes Perineal hemangioma, External genitalia malformations, Lipomyelomeningocele, Vesicorenal anomalies, Imperforate anus, and Skin tag (Girard et al., 2006). PHACE and PELVIS syndromes suggest that there may be an association between a cutaneous hemangioma and

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.

Summary Purpose Hemangiomas are the most common benign tumor of infancy, affecting females more than males. Lip hemangiomas are of particular concern because of their relatively increased risk to ulcerate during the proliferative period. Ulcerated hemangiomas of the lip can lead to increased scarring, loss of lip contour, and disfigurement. Most will require surgical correction to restore normal labial anatomy. Methods A retrospective chart review between 2004 and 2010 for surgically treated lip hemangiomas was performed. Demographic data, location of the hemangioma, age at operation, and number of operations were recorded. Two independent observers evaluated lip appearance post-operatively using 5-point scales to examine scar, symmetry, contour, and color, with 5 being excellent and 1 being poor. Results Between 2004 and 2010, eleven patients underwent surgical correction. Ten of the eleven were female. 18% (2/11) were ulcerated. One third (4/11) was in the upper lip and two-thirds (7/11) were in the lower lip. The mean age of the patients at the time of operation was 3.6 years (range, 14 months to 17 years). The average number of operations per patient was 1.6 (range, 1–3). The average scores for lip appearance after surgical correction ranged between 3.95 (good) for lip contour to 4.5 (good to excellent) for color. Conclusions Lip hemangiomas often require surgical correction. Treatment goals include restoration of normal lip contour and strategic placement of the incision. By taking advantage of the natural involution that occurs and careful planning, procedures can be staged to minimize distortion of the lip. Even lip hemangiomas that cross the vermilio-cutaneous (VC) junction can be excised and lip contour achieved without the need to extend scars beyond the VC junction.

Infantile 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.RNA 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.Real-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.HemSCs 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.

Infantile hemangioma is an endothelial tumor that grows rapidly after birth but slowly regresses during early childhood. Initial proliferation of hemangioma is characterized by clonal expansion of endothelial cells (ECs) and neovascularization. Here, we demonstrated mRNA encoding CD133-2, an important marker for endothelial progenitor cells (EPCs), predominantly in proliferating but not involuting or involuted hemangioma. Progenitor cells coexpressing CD133 and CD34 were detected by flow cytometry in 11 of 12 proliferating hemangioma specimens from children 3 to 24 months of age. Furthermore, in 4 proliferating hemangiomas, we showed that 0.14% to 1.6% of CD45– nucleated cells were EPCs that coexpressed CD133 and the EC marker KDR. This finding is consistent with the presence of KDR+ immature ECs in proliferating hemangioma. Our results suggest that EPCs contribute to the early growth of hemangioma. To our knowledge, this is the first study to show direct evidence of EPCs in a human vascular tumor.

CONCLUSION: Transplantation of DRCC after ionizing radiation exposure proved to be the most effective rescue therapy against acute radiation syndrome, as confirmed by 100% of recipients’ survival and expedited recovery of the hematopoietic system without developing GVHD. Our novel approach of DRCC transplantation may act as a bridging therapy supporting hematopoietic recovery and ameliorating injuries in patients exposed to the harmful effects of radiation. 51 NOTCH3 Regulates Hemangioma Stem Cell Differentiation and Alters the Vascular Phenotype in an Infantile Hemangioma Murine Model

Background: Infantile hemangiomas (IHs) are the most common benign tumor of infancy, yet their pathogenesis is poorly understood. IHs are believed to originate from a progenitor cell, the hemangioma stem cell (HemSC). Recent studies by our group showed that NOTCH proteins and NOTCH ligands are expressed in hemangiomas, indicating Notch signaling may be active in IHs. We sought to investigate downstream activation of Notch signaling in hemangioma cells by evaluating the expression of the basic HLH family proteins, HES/HEY, in IHs. Materials and Methods: HemSCs and hemangioma endothelial cells (HemECs) are isolated from freshly resected hemangioma specimens. Quantitative RT-PCR was performed to probe for relative gene transcript levels (normalized to beta-actin). Immunofluorescence was performed to evaluate protein expression. Co-localization studies were performed with CD31 (endothelial cells) and NOTCH3 (peri-vascular, non-endothelial cells). HemSCs were treated with the gamma secretase inhibitor (GSI) Compound E, and gene transcript levels were quantified with real-time PCR. Results: HEY1, HEYL, and HES1 are highly expressed in HemSCs, while HEY2 is highly expressed in HemECs. Protein expression evaluation by immunofluorescence confirms that HEY2 is expressed by HemECs (CD31+ cells), while HEY1, HEYL, and HES1 are more widely expressed and mostly expressed by perivascular cells of hemangiomas. Inhibition of Notch signaling by addition of GSI resulted in down-regulation of HES/HEY genes. Conclusions: HES/HEY genes are expressed in IHs in cell type specific patterns; HEY2 is expressed in HemECs and HEY1, HEYL, HES1 are expressed in HemSCs. This pattern suggests that HEY/HES genes act downstream of Notch receptors that function in distinct cell types of IHs. HES/HEY gene transcripts are decreased with the addition of a gamma-secretase inhibitor, Compound E, demonstrating that Notch signaling is active in infantile hemangioma cells.