Alvin Wong

Propranolol Accelerates Adipogenesis in Hemangioma Stem Cells and Causes Apoptosis of Hemangioma Endothelial Cells

Background: 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. Methods: Hemangioma endothelial and stem cells were isolated from resected infantile hemangioma specimens. Cells were treated with 100 &mgr;M 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 &mgr;M propranolol or vehicle, for 72 and 96 hours, respectively. Adipogenesis was induced in stem cells with and without propranolol. Pro-adipogenic genes PPAR&dgr;, PPAR&ggr;, C/EBP&agr;, C/EBP&bgr;, C/EBP&dgr;, RXR&agr;, and RXR&ggr; were analyzed by quantitative polymerase chain reaction. Results: 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/EBP&bgr; (p < 0.05), RXR&ggr; (p < 0.05), and PPAR&ggr; (p < 0.02) were significantly increased when treated with 50 or 100 &mgr;M propranolol; and C/EBP&dgr; (p < 0.05), RXR&agr; (p < 0.05), and PPAR&dgr; (p < 0.01) transcripts were increased when treated with 100 &mgr;M propranolol. C/EBP&agr; transcript levels remained unchanged at either dose. Conclusions: 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.

Expression of HES and HEY genes in infantile hemangiomas

BackgroundInfantile 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 MethodsHemSCs 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.ResultsHEY1, 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.ConclusionsHES/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.

Management of lip hemangiomas: Minimizing peri-oral scars☆

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.

Aberrant Lymphatic Endothelial Progenitors in Lymphatic Malformation Development

Lymphatic malformations (LMs) are vascular anomalies thought to arise from dysregulated lymphangiogenesis. These lesions impose a significant burden of disease on affected individuals. LM pathobiology is poorly understood, hindering the development of effective treatments. In the present studies, immunostaining of LM tissues revealed that endothelial cells lining aberrant lymphatic vessels and cells in the surrounding stroma expressed the stem cell marker, CD133, and the lymphatic endothelial protein, podoplanin. Isolated patient-derived CD133+ LM cells expressed stem cell genes (NANOG, Oct4), circulating endothelial cell precursor proteins (CD90, CD146, c-Kit, VEGFR-2), and lymphatic endothelial proteins (podoplanin, VEGFR-3). Consistent with a progenitor cell identity, CD133+ LM cells were multipotent and could be differentiated into fat, bone, smooth muscle, and lymphatic endothelial cells in vitro. CD133+ cells were compared to CD133− cells isolated from LM fluids. CD133− LM cells had lower expression of stem cell genes, but expressed circulating endothelial precursor proteins and high levels of lymphatic endothelial proteins, VE-cadherin, CD31, podoplanin, VEGFR-3 and Prox1. CD133− LM cells were not multipotent, consistent with a differentiated lymphatic endothelial cell phenotype. In a mouse xenograft model, CD133+ LM cells differentiated into lymphatic endothelial cells that formed irregularly dilated lymphatic channels, phenocopying human LMs. In vivo, CD133+ LM cells acquired expression of differentiated lymphatic endothelial cell proteins, podoplanin, LYVE1, Prox1, and VEGFR-3, comparable to expression found in LM patient tissues. Taken together, these data identify a novel LM progenitor cell population that differentiates to form the abnormal lymphatic structures characteristic of these lesions, recapitulating the human LM phenotype. This LM progenitor cell population may contribute to the clinically refractory behavior of LMs.

Propranolol promotes accelerated and dysregulated adipogenesis in hemangioma stem cells.

BackgroundInfantile hemangiomas (IHs) are the most common tumor of infancy, yet there are no Food and Drug Administration–approved therapeutics to date. Recently, the nonselective &bgr;-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. MethodsHemangioma 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/EBP&agr;, C/EBP&bgr;, C/EBP&dgr;, PPAR&dgr;, PPAR&ggr;, RXR&agr;, and RXR&ggr; were analyzed by quantitative reverse transcription and polymerase chain reaction. ResultsHemangioma 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 PPAR&dgr;, PPAR&ggr;, C/EBP&bgr;, and C/EBP&dgr; were significantly increased (P < 0.01) in propranolol-treated cells relative to control cells. In contrast, RXR&agr; and RXR&ggr; levels were significantly decreased (P < 0.05), and C/EBP&agr;, a gene required for terminal adipocyte differentiation, was strongly suppressed by propranolol when compared to vehicle-treated cells (P < 0.01). ConclusionsIn HemSCs, propranolol accelerated dysregulated adipogenic differentiation characterized by improper adipogenic gene expression. Consistent with accelerated adipogenesis, propranolol significantly increased the expression of the proadipogenic genes, PPAR&ggr;, C/EBP&bgr;, and C/EBP&ggr; compared to control. However, propranolol treatment also led to improper induction of PPAR&dgr; and suppression of C/EBP&agr;, RXR&agr;, and RXR&ggr;. Taken together these data indicate that propranolol promoted dysregulated adipogenesis and inhibited the HemSCs from becoming functional adipocytes, ultimately resulting in cell death. Understanding this mechanism behind propranolol’s effectiveness will be a vital factor in producing more effective therapies in the future.

Herpes simplex encephalitis following spinal ependymoma resection: case report and literature review

Herpes simplex encephalitis (HSE) is a rare complication of neurosurgical procedures but must be considered in early deterioration of the postoperative patient. This is the first report of HSE following spinal cord tumor resection. A 65-year-old woman had C2–C5 laminectomy for subtotal resection of intramedullary ependymoma. Six days postoperatively she developed fever, vomiting and rapid decline in mental status. Brain MRI revealed enhancement of left insular cortex. Polymerase chain reaction on cerebrospinal fluid (CSF) identified herpes simplex virus type 1 (HSV-1) as the causal agent. Twenty-one days of acyclovir led to improvement. Three subsequent admissions to neurological intensive care unit were required for deterioration in mental status, including pneumonia, hydrocephalus and deep vein thromboses. Ventriculoperitoneal shunt (VPS), tracheotomy, percutaneous intravenous central catheter (PICC) line and percutaneous endoscopic gastrostomy (PEG) were placed. She was discharged to skilled nursing home care. Acyclovir is effective therapy against HSV, though outcomes may be poor even in optimally treated cases. Empiric treatment must be started even in the absence of serologic evidence of HSV infection if suspicion for HSE is high.

Postoperative Prophylactic Antibiotic Use following Ventral Hernia Repair with Placement of Surgical Drains Reduces the Postoperative Surgical-Site Infection Rate.

Background: To help prevent complications after incisional ventral hernia repair, traditional teaching has recommended using closed suction drains. However, some studies suggest that there is an increased infection risk with use of surgical drains, and it is uncertain whether use of extended postoperative prophylactic antibiotics while drains remain in place helps prevent surgical-site infections. Evidence guiding surgeons’ clinical practice regarding antibiotic use following hernia repair is lacking. Methods: The authors sought to determine whether the use of extended postoperative antibiotic prophylaxis beyond standard Surgical Care Improvement Project guidelines with closed-suction surgical drain placement in incisional ventral hernia repair reduces the incidence of postoperative surgical-site infections. A retrospective review of 234 patients who underwent an incisional ventral hernia repair from 2003 to 2013 at a single institution was performed. Demographic, preoperative, operative, and postoperative data were collected and analyzed. Results: Extended postoperative prophylactic antibiotics significantly reduce the incidence of postoperative surgical-site infections (OR, 0.31; p < 0.01). The odds ratio, when stratified by Ventral Hernia Working Group Scale, was 0.63, 0.25, 0.30, and 0.13 (p < 0.001 by the Mantel-Haenszel combined test) for grades 1, 2, 3, and 4, respectively, but was not statistically significant for differences between hernia grades. However, as the hernia grade increased, the odds ratio tended to decrease, suggesting that extended prophylactic antibiotics may be more effective at decreasing the incidence of surgical-site infections at higher grades. Conclusion: Extended antibiotic prophylaxis reduces surgical site infection risk following complex ventral hernia repairs, and should be considered in all cases. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, III.

Infantile Hemangiomas: A Disease Model in the Study of Vascular Development, Aberrant Vasculogenesis and Angiogenesis

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

Abstract 18: NOTCH3 KNOCKDOWN IN HEMANGIOMA STEM CELLS ALTERS HEMANGIOMA FORMATION IN A MURINE MODEL

Background: Infantile hemangiomas (IHs) are thought to originate from hemangioma stem cells (HemSC), though IH pathogenesis is poorly understood. Our recent work showed that NOTCH3 is highly expressed in HemSCs and may play a role in IH progression. In cultured HemSCs, NOTCH3 knockdown perturbed adipogenesis and enhanced endothelial cell differentiation. Using a murine model of IH, we compared the development of IHs from control HemSCs (HemSC-scr) to HemSCs with Notch3 shRNA (HemSC-shN3).

Abstract 80: EXAMINING THE ROLE OF MACROPHAGES IN PROLIFERATING INFANTILE HEMANGIOMAS

Background: Infantile hemangiomas (IH) are a common benign endothelial cell tumor thought to be of stem cell origin (HemSC). Histological analysis indicates that IH consist of endothelial cells (HemEC) and associated macrophages. A role for macrophages in IH progression has been postulated, though their function has yet to be characterized. Our study investigates macrophages in hemangioma development using a murine IH model. This model can eventually be used to study the origin and function of IH-associated macrophages.