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Propranolol Effects on Hemangiomas are Mediated via Distinct Pathways
Ryan W. England, BS, Naikhoba CO Munabi, BA, Kyle J. Glithero, MD, Alex Kitajewski, BS, Jan Kitajewski, PhD, Carrie J. Shawber, PhD, June K. Wu, MD.
Columbia University, New York, NY, USA.
Propranolol has clinical efficacy in the treatment of problematic infantile hemangiomas (IHs). We have shown that propranolol caused decreased cellular proliferation and cytotoxicity on hemangioma stem cells (HemSCs) in vitro. We hypothesize that these effects are mediated by disrupting downstream signals of the beta-adrenergic receptor, although which mediators are involved are not well understood. While uncommon, infants treated with propranolol can experience symptomatic bradycardia, hypotension, hypoglycemia, and even seizures. Therefore, understanding these pharmacologic effects of propranolol will help develop targeted therapy that can minimize these adverse effects.
CD133+ HemSCs isolated from resected IH specimens were treated over a 9-log scale with either propranolol hydrochloride or vehicle. Effects of non-selective beta-adrenergic blockade in HemSCs were assessed for cellular proliferation using a WST-8 cell counting kit, and cellular cytotoxicity using the fluorescence-based digital image microscopy system, DIMSCAN. The activation of mitrogen-activated protein kinase (MAPK) was measured by immunoblotting for phosphorylated MAPK after a 30-minute incubation with propranolol and normalized against total MAPK using ImageJ. Effects of beta-adrenergic blockade on downstream cAMP levels after isoprenaline stimulation were assessed using a LANCE Ultra cAMP kit. Student’s t-test and Prism were used for statistical analysis.
Beta-adrenergic blockade via treatment with propranolol resulted in a significant decrease in proliferation at a low dose of 1x10-9M (1pM; p<0.01) and continued in a dose-dependent manner until 1x10-4M (100uM; p<0.0001), whereby proliferation was completely inhibited. This coincided with the decrease in cAMP activity (Figure 1). At concentrations above 1x10-4M, propranolol became cytotoxic to HemSCs, with an LC50 of 158uM (R2=0.992; p<0.0001). Similarly, at higher cytotoxic doses of propranolol, HemSCs also showed a significant increase in MAPK activation (Figure 2) (LC50=55uM; R2=0.992), suggesting that propranolol is having an antagonistic effect at lower doses along the G-protein-cAMP pathway and an agonistic effect at the higher dosages, possibly via a G protein-independent pathway (Figure 3).
We have demonstrated that there are at least two distinct downstream pathways that are affected by beta-adrenergic receptor signaling. Cellular proliferation appears to be mediated via the G protein-dependent, cAMP-mediated pathway, whereas cellular cytotoxicity is affected by a G protein-independent, MAPK-mediated pathway. By further understanding the mechanism by which propranolol causes IH involution independent of the G protein pathway, therapies can be developed that may circumvent the adverse cardiovascular effects of beta-adrenergic antagonist treatments.
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