Propranolol Promotes Accelerated and Dysregulated Adipogenesis in Hemangioma Stem Cells

Abstract

Background Infantile hemangiomas are the most common tumor of infancy, yet there are no FDA-approved therapeutics to date. Recently, the non-selective beta-adrenergic-blocker propranolol has been shown to be a safe and effective means of treating infantile hemangiomas, though 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 isolated from resected infantile hemangioma 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. Pro-adipogenic genes C/EBPβ, 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 < 0.01) in propranolol-treated cells relative to control cells. In contrast, RXRα and RXRγ levels were significantly decreased (p < 0.05), and C/EBPα, a gene required for terminal adipocyte differentiation, was strongly suppressed by propranolol when compared to vehicle-treated cells (p < 0.01). Conclusions In hemangioma stem cells, propranolol accelerated dysregulated adipogenic differentiation characterized by improper adipogenic gene expression. Consistent with accelerated adipogenesis, propranolol significantly increased the expression of the pro-adipogenic genes, PPARγ , C/EBPβ and C/EBPγ compared to control. However, propranolol treatment also led to improper induction of PPARδ and suppression of C/EBPα, RXRα and RXRγ. Taken together this data indicates that propranolol promoted dysregulated adipogenesis and inhibited the hemangioma stem cells 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.

Propranolol Targets Hemangioma Stem Cells via cAMP and Mitogen-Activated Protein Kinase Regulation

“…We found that high‐dose propranolol rapidly induced HemSC apoptosis, with induction observed within 6 hours by Annexin V assay and 24 hours by caspase‐3 assay. This is in contrast to previous studies, in which it was shown that propranolol induced HemSC death by a nonapoptotic pathway [21, 23, 41]. The difference between the present study and others mostly likely resulted from the timing of the apoptosis assessment.…”

“…We have demonstrated that HemSCs in tissues express both β1AR and β2AR, but cultured HemSCs predominantly express β2AR. Propranolol has been suggested to target HemECs [17, 21, 47, 48] and hemangioma‐derived pericytes [19], and the present study and others [21–23, 41] have demonstrated that propranolol also targets HemSCs. Thus, propranolol mediates its effects by targeting multiple cell types in IHs.…”

“…Kum and Khan [23] assessed the propranolol effects on apoptosis after 72 hours of propranolol treatment. In contrast, we reported that surface Annexin V expression was not increased at 24 hours of treatment [21, 41]. Annexin V can only be detected in cells actively undergoing apoptosis; thus, we believe we did not assess apoptosis at the appropriate time, because most cells were already dead, confirmed by propidium iodide incorporation.…”

Review of topical beta blockers as treatment for infantile hemangiomas

“…Propranolol in vitro affects M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT hemogenic stem cells causing dysregulated adipogenesis and apoptosis. This may explain not only the early involution of lesions treated with propranolol, but also its inability to completely remove the fatty residua of hemangiomas (13) . It is still not understood whether propranolol only targets actively proliferating hemangioma endothelial cells or whether the hemangioma endothelial cells have a unique response to propranolol (52) .…”

Evidence and nuances of propranolol safety

“…Involution is maximal in over 90% of IHs by age 4 years, with well over half of untreated IHs leaving residual skin changes or scars . Evidence of the pluripotency of haemangioma stem cells to become haemangioma endothelial cells or adipocytes, and of the effects of propranolol on these populations continues to refine our understanding of the mechanism of propranolol …”