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Putative mechanism of action of propranolol on regression of infantile hemangiomas
Harvey Chim, MD, Erin Miller, BS, Christy Gliniak, MSc, Arun K. Gosain, MD, FACS.
Case Western Reserve University, Cleveland, OH, USA.

PURPOSE: Propranolol has been found to be effective in treatment of severe hemangiomas of infancy. However, its mechanism of action is as yet unknown. The aim of this study was to demonstrate a cytotoxic effect in vitro and determine possible etiologies of action of propranolol.
METHODS: Hemangioma endothelial cells at P3 or P4 were isolated from resected proliferative and involuting hemangiomas through explant culture followed by CD31 dynabead selection. Cells were exposed to varying concentrations of propranolol ranging from 0.3 uM to 300 uM for up to 4 days. RT-PCR was used to analyze mRNA expression for VEGF, FGF-2, MMP-2, MMP-9, MMP-1, FGF-2, IL6, MCP-1, HIF-1α, VEGF, VEGFR1 and VEGFR2. Secretion of VEGF, bFGF and MMP-2 and expression of HIF-1a was assayed. MTS assay, apoptosis caspase, endothelial cell migration and tubulogenesis assay with matrigel were performed. Phosphorylation of PI3/ Akt and ERK 1/2 was assayed at different propranolol concentrations. Flow cytometry for VEGF-R2 and FGF-R2 expression was performed. Quantification of microvessel density using subcutaneous nude mouse matrigel model was done with cells exposed to different propranolol concentrations prior to implantation. Animal experiments were repeated with unstimulated cells where different doses of propranolol were administered via IP injection to mice over a 7 day period. Experiments were repeated in proliferative and involuting hemangioma cells, and also in CD133+ and CD133- cells in vitro.
RESULTS: HIF-1α, ΜCP-1 and FGF-2 mRNA were downregulated with increasing propranolol concentration, with increasing dose dependent protein production. VEGF, MMP-2, MMP-9, MMP-1, VEGFR1 and VEGFR2 mRNA were upregulated with increased propranolol concentration, with decreased secretion of VEGF and MMP-2 protein. MTS assay showed dose dependent decreased cell proliferation with increased apoptosis at increasing propranolol doses. Migration assay showed stepwise decrease in migration at 30uM and 100uM propranolol. Tubulogenesis assay showed cessation of tubule formation at 300uM dosage for both proliferative and involuting cells, with shorter tubules in involuting cells. Total PI3/ Akt and ERK 1/2 expression was decreased with increasing propranolol. Phosphorylation of PI3/ Akt was proportionately decreased at higher propranolol concentrations of 200uM and 300uM. Flow cytometry showed upregulation of VEGF-R2 at 300uM concentration. No major differences were found between CD133+ and CD133- cells in their response to propranolol. Proliferative hemangioma cells displayed a delayed fall in VEGF and MMP-2 secretion after 4 days compared to involuting cells, with a fall after only 2 days of propranolol treatment. Quantification of microvessel density showed statistically significant decrease in density at IP injections of 10 mg/kg (Image 1B; white areas) and 2 mg/kg compared to control (Image 1A) mice.
CONCLUSION: These results suggest a mechanism of action for propranolol on hemangiomas through a block in secretion of VEGF and related angiogenesis proteins such as MMP-2, which act in combination with decreased PI3/ Akt phosphorylation to cause a direct cytotoxic effect, decreased tubulogenesis and endothelial cell migration and subsequent apoptosis.


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