Volume 133, Number 3 (Supplement) • PSRC Abstract Supplement
3 Propranolol Accelerates Involution in a Murine Model of Infantile Hemangioma
Kyle J Glithero, MD; Naikhoba C O Munabi, BA; Ryan W England, BS; Alex Kitajewski, BS; Michelle Chang, BS; Jan K Kitajewski, PhD; Carrie J Shawber, PhD; June K Wu, MD Columbia University Medical Center, New York, NY
Naikhoba C O Munabi, BA; Ryan W England, BS; Kyle J Glithero, MD; Michelle M Chang, BS; Alex Kitajewski, BA; Jan K Kitajewski, PhD; Carrie J Shawber, PhD; June K Wu, MD Columbia University, New York, NY
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.
Purpose: Propranolol has shown efficacy in the treatment of problematic infantile hemangiomas (IHs). Propranolol achieves this in part via an anti-vasculogenic effect on hemangioma endothelial cells. Previous studies in our laboratory suggested that propranolol induces accelerated adipogenesis of hemangioma stem cells (HemSCs) in culture. The goal of this study is to investigate the effects of propranolol treatment in a murine model of IH. We hypothesize that systemic propranolol will accelerate involution by affecting both vasculogenesis and adipogenesis.
Methods: CD133+ HemSCs were isolated from resected hemangioma specimens. HemSC were transduced with lentivirus encoding a NOTCH3 shRNA (HemSC- shN3), an activated form of NOTCH3 (HemSC-N3IC) or a virus containing a scrambled sequence (HemSC-scr) serving as a control. HemSC-N3IC, HemSC-shN3 and HemSC-scr were grown in mural cell differentiation media. After two weeks, immunofluorescent staining was performed with antibodies against the mural cell markers, neuron-glial antigen 2 (NG2) and alpha smooth muscle actin (α-SMA). Fluorescence was quantified and averaged over multiple areas with ImageJ. Results: All CD133+ HemSC cell lines expressed low levels of NG2 at baseline (data not shown). When mural cell differentiation was induced, NOTCH3 knockdown upregulated NG2 compared to HemSC-scr. In contrast, NOTCH3 activation decreased NG2 expression. Expression of α-SMA was strongly upregulated in HemSC-scr and Hem-N3IC to a similar level in differentiation medium compared to cell maintained in growth medium (basal). α-SMA expression was unchanged from basal conditions in HemSC-shN3 cells. Conclusions: In HemSCs, perturbations in NOTCH3 level differential altered the expression of mural cell proteins NG2 and α-SMA. In mural cell differentiation conditions NOTCH3 activation strongly downregulated NG2, while having no effect on the expression of α-SMA. In contrast, NOTCH3 inhibition modestly increased NG2 levels, and suppressed the upregulation of α-SMA. Thus, precise regulation of NOTCH3 signaling levels maybe necessary for proper mural cell differentiation of HemSCs.
Methods: HemSCs isolated from human specimens and suspended in matrigel were injected subcutaneously into immunodeficient mice. Twenty-four hours after injection, mice were continuously administered oral propranolol or vehicle solution. Ultrasound (US) Doppler imaging of matrigel implants was performed weekly to assess for vascular flow. After 3 weeks, the matrigel implants were harvested and H&Es performed. Selected sections were examined for vascular density, vessel diameter, cellularity, total adipose content and number of adipocytes at 20X magnification. Results: US doppler of implants detected the presence of blood flow in mice receiving vehicle solution at 2 weeks after implantation. In contrast, there was an absence of discernable flow to the propranolol treated mice at the 3-week endpoint. Histologically, propranolol-treated IHs showed a 33% decrease in vascular density and 50% reduction in vessel caliber when compared to controls (p < 0.0001). Total adipose content was unchanged between vehicle and treatment groups. However adipocyte size was decreased in the propranolol-treated implant resulting in a significant increase in adipocyte number (p < 0.05). Despite the increase in adipocyte number, overall cellularity of the propranolol-treated implant was statistically reduced when compared to control (p < 0.005). Conclusion: Propranolol treatment led to a loss of detectable blood flow, which correlated with decreased vessel density and caliber in a murine IH model. Propranolol treatment also resulted in increased adipocytes in the setting of decreased cellularity, which are hallmark characteristics of involuting IHs. These findings are consistent with published in vitro data and suggest that propranolol treatment accelerates involution of IH in this murine model.
www.PRSJournal.com
11
Friday, March 7, 2014
2 NOTCH3 Regulates Mural Cell Differentiation of HemSCs
3 Propranolol Accelerates Involution in a Murine Model of Infantile Hemangioma
Kyle J Glithero, MD; Naikhoba C O Munabi, BA; Ryan W England, BS; Alex Kitajewski, BS; Michelle Chang, BS; Jan K Kitajewski, PhD; Carrie J Shawber, PhD; June K Wu, MD Columbia University Medical Center, New York, NY
Naikhoba C O Munabi, BA; Ryan W England, BS; Kyle J Glithero, MD; Michelle M Chang, BS; Alex Kitajewski, BA; Jan K Kitajewski, PhD; Carrie J Shawber, PhD; June K Wu, MD Columbia University, New York, NY
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.
Purpose: Propranolol has shown efficacy in the treatment of problematic infantile hemangiomas (IHs). Propranolol achieves this in part via an anti-vasculogenic effect on hemangioma endothelial cells. Previous studies in our laboratory suggested that propranolol induces accelerated adipogenesis of hemangioma stem cells (HemSCs) in culture. The goal of this study is to investigate the effects of propranolol treatment in a murine model of IH. We hypothesize that systemic propranolol will accelerate involution by affecting both vasculogenesis and adipogenesis.
Methods: CD133+ HemSCs were isolated from resected hemangioma specimens. HemSC were transduced with lentivirus encoding a NOTCH3 shRNA (HemSC- shN3), an activated form of NOTCH3 (HemSC-N3IC) or a virus containing a scrambled sequence (HemSC-scr) serving as a control. HemSC-N3IC, HemSC-shN3 and HemSC-scr were grown in mural cell differentiation media. After two weeks, immunofluorescent staining was performed with antibodies against the mural cell markers, neuron-glial antigen 2 (NG2) and alpha smooth muscle actin (α-SMA). Fluorescence was quantified and averaged over multiple areas with ImageJ. Results: All CD133+ HemSC cell lines expressed low levels of NG2 at baseline (data not shown). When mural cell differentiation was induced, NOTCH3 knockdown upregulated NG2 compared to HemSC-scr. In contrast, NOTCH3 activation decreased NG2 expression. Expression of α-SMA was strongly upregulated in HemSC-scr and Hem-N3IC to a similar level in differentiation medium compared to cell maintained in growth medium (basal). α-SMA expression was unchanged from basal conditions in HemSC-shN3 cells. Conclusions: In HemSCs, perturbations in NOTCH3 level differential altered the expression of mural cell proteins NG2 and α-SMA. In mural cell differentiation conditions NOTCH3 activation strongly downregulated NG2, while having no effect on the expression of α-SMA. In contrast, NOTCH3 inhibition modestly increased NG2 levels, and suppressed the upregulation of α-SMA. Thus, precise regulation of NOTCH3 signaling levels maybe necessary for proper mural cell differentiation of HemSCs.
Methods: HemSCs isolated from human specimens and suspended in matrigel were injected subcutaneously into immunodeficient mice. Twenty-four hours after injection, mice were continuously administered oral propranolol or vehicle solution. Ultrasound (US) Doppler imaging of matrigel implants was performed weekly to assess for vascular flow. After 3 weeks, the matrigel implants were harvested and H&Es performed. Selected sections were examined for vascular density, vessel diameter, cellularity, total adipose content and number of adipocytes at 20X magnification. Results: US doppler of implants detected the presence of blood flow in mice receiving vehicle solution at 2 weeks after implantation. In contrast, there was an absence of discernable flow to the propranolol treated mice at the 3-week endpoint. Histologically, propranolol-treated IHs showed a 33% decrease in vascular density and 50% reduction in vessel caliber when compared to controls (p < 0.0001). Total adipose content was unchanged between vehicle and treatment groups. However adipocyte size was decreased in the propranolol-treated implant resulting in a significant increase in adipocyte number (p < 0.05). Despite the increase in adipocyte number, overall cellularity of the propranolol-treated implant was statistically reduced when compared to control (p < 0.005). Conclusion: Propranolol treatment led to a loss of detectable blood flow, which correlated with decreased vessel density and caliber in a murine IH model. Propranolol treatment also resulted in increased adipocytes in the setting of decreased cellularity, which are hallmark characteristics of involuting IHs. These findings are consistent with published in vitro data and suggest that propranolol treatment accelerates involution of IH in this murine model.
www.PRSJournal.com
11
Friday, March 7, 2014
2 NOTCH3 Regulates Mural Cell Differentiation of HemSCs
