Propranolol accelerates adipogenesis in hemangioma stem cells and causes apoptosis of hemangioma endothelial cells

Abstract

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 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.

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/EBPβ (p < 0.05), RXRγ (p < 0.05), and PPARγ (p < 0.02) were significantly increased when treated with 50 or 100 μM propranolol; and C/EBPδ (p < 0.05), RXRα (p < 0.05), and PPARδ (p < 0.01) transcripts were increased when treated with 100 μM propranolol. C/EBPα 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.

Figure 1

Ulcerated abdominal segmental hemangioma on a 6-month-old female patient. Note the typical red, raised, bosselated appearance of the superficial portion of the infantile hemangioma with ulcerating and bleeding areas, whereas the deeper areas, at the end of the forceps, consisted of fibrofatty tissue typically seen in an involuting or involuted IH.

Figure 2

ADRB1 (β1) and ADRB2 (β2) genes are expressed in HemECs and HemSCs by PCR. In both cell types, ADRB2 expression is greater than ADRB1 expression. PCR was done for 35 cycles and products run on a 1% agarose gel.

Figure 3

FACS analysis of HemECs and HemSCs treated with 100μM propranolol for 48 hours. (Top panel) Propranolol treatment of HemECs increased Annexin V levels on the cell surface. (Bottom panel) Annexin V levels were not altered in HemSCs, suggesting a lack of apoptotic response in HemSCs. Black line indicates control; gray line indicates propranolol treatment.

Figure 4

Effect of varying concentrations of propranolol on 96 hours of HemEC proliferation in EGM-2/20%-FBS. Propranolol significantly inhibits proliferation of HemECs in a dose-dependent manner. Data are representative of three independent experiments. *p < 0.05 relative to control. **p < 0.05 relative to 50μM propranolol.

Figure 5

Effect of varying concentrations of propranolol on 72 hours of HemSC proliferation in EGM-2/20%-FBS. Propranolol significantly inhibits proliferation of HemSCs in a dose-dependent manner. Data are representative of three independent experiments. *p < 0.05 relative to control. **p < 0.05 relative to 50μM propranolol.

Figure 6

HemSCs cultured in adipogenic media in the presence of vehicle (left) and 50μM propranolol (right) for four days and stained for presence of lipid vacuoles by Oil Red O staining. Note minimal presence of intracytoplasmic lipid vacuoles in control cells. By contrast, there was increased presence of lipid vacuoles in HemSCs exposed to propranolol. (Magnification 20×). Scale bar represents 10μM.

Figure 7

Treatment with either 50μM or 100μM of propranolol resulted in significantly increased transcript levels of PPARγ, C/EBPβ, and RXRγ, (top panel). Treatment with 100μM, but not 50μM, of propranolol resulted in increased transcript levels of PPARδ, C/EBPδ, and RXRα (middle panel). There were no changes in transcript levels of C/EBPα with either 50μM or 100μM of propranolol (bottom panel). *p<0.05, **p<0.01.