Alex Kitajewski

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.

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.

Abstract 108: Vascular Endothelial Growth Factor Signaling is affected by Changes in the β-Adrenergic Receptor Pathways in Hemangioma Stem Cells

78 lipoproteoplex by incorporating varying CSP:siRNA ratios into a standardized CLN and treated 3T3 fibroblasts to assess gene expression changes. Cellular internalization of fluorescent siRNA (siGLO) packaged in lipoproteoplex was visualized with fluorescent microscopy. In vivo, we applied lipoproteoplex with siGLO or siRNA to the skin of C57BL/6 mice. We then measured biodistribution via IVIS imaging, dermal penetration using histological sections, and gene expression in treated skin and internal organs 0-5 days post-treatment.

Abstract 2: NOTCH3 Regulates Mural Cell Differentiation of HemSCs.

PurPose: Infantile hemangiomas (IHs) are vascular hyperplasias with high flow and have been proposed to originate from hemangioma stem cells (HemSCs). Proper vessel maturation requires interactions between endothelial cells and their surrounding mural cells. IH pathology has been proposed to be partially due to defective mural cell function. Previous studies in our lab showed NOTCH3 inhibition in HemSC resulted in reduced blood flow in a murine IH model as detected by US Doppler. This correlated with reduced vessel diameter when compared to controls. Since Notch3 functions to regulate mural cell maturation, we determined if Notch3 has a role in mural cell differentiation of HemSCs.

Abstract 70: Propranolol Effects on Hemangiomas are Mediated via Distinct Pathways.

BaCkground: Wnt signaling is a critical pathway regulating craniofacial development, where dysregulation leads to orofacial clefts. Intracellular trafficking and secretion of wnt ligands is chaperoned by wntless (wls). In human, the WLS gene is on the short arm of the chromosome 1 (1p31.3) and several deletions of this region associated with craniofacial malformations have been reported. We hypothesize that the wls gene functions to modulate wnt signaling important in morphogenesis of the craniofacial skeleton.

135A: NOTCH SIGNALING TARGET GENES ARE EXPRESSED IN INFANTILE HEMANGIOMAS

Background: Infantile hemangiomas (IH) are the most common benign tumor of infancy, yet its pathogenesis is still not well understood. Our laboratory has shown that Notch genes are expressed in hemangiomas. Moreover, Notch genes are differentially expressed. Endothelial associated Notch genes (Notch1, -4, and Notch ligand Jagged-1) are highly expressed in hemangioma endothelial cells (HemECs), whereas Notch3, a mural cell-associated Notch gene, is highly expressed in hemangioma stem cells (hemSCs) and negligible in HemECs. (Wu et al, Angiogenesis, in press). While Notch genes are known to play a role in cell fate determination and vascular development, the mechanisms by which this is achieved is poorly understood. One family of transcription factors the Hes/Hey genes are known to be activated regulated by Notch signaling. We hypothesize that specific Notch genes may be responsible for regulating expression of Hes/Hey genes in Infantile Hemangioma.