The Role Of β-adrenergic Receptor Signaling In The Proliferation And Apoptosis Of Hemangioma-derived Endothelial Cells

BackgroundInfantile hemangioma (IH), which is the most common infancy tumor, is a benign vascular neoplasm resulting from the abnormal proliferation of endothelial cells and angiogenesis. Although IH is usually harmless, approximately10%of IHs are destructive, disfiguring, and even vision-or life-threatening. Several therapeutic approaches for IHs have been suggested over the past decade, including hormone and interferon injection, lasers, embolisms, surgical operation. Systemic corticosteroids, which commence in the first few weeks of life, have been the gold standard of treatment for the prevention of IH growth. However, corticosteroids, while efficacious in many patients, have undesired side effects such as temporary growth retardation, an increased risk of infection, and psychological changes. Additionally, these drugs act by halting additional IH proliferation rather than inducing tumor shrinkage:thus, their usefulness is limited to the early proliferative phase.Since2008, an increasing number of studies have reported the exceptional effectiveness of propranolol, which is a well-known nonselective P-blocker, on IH in children. The authors described the rapid onset of the effect of propranolol treatment as an IH color change from red to purple within24hours and a softening of the lesions. This rapid action was especially dramatic in cases involving dyspnea, hemodynamic compromise, or palpebral occlusion. Another remarkable aspect of the results of propranolol treatment is that, not only was the IH growth stabilized, but the improvement continued until completed involution was achieved, leading to a considerable shortening of the natural courses of IH. This medication may be particularly useful in clinical practice to hasten involution as a replacement for early surgery or to lower the age at which timely surgery can be performed to maximize excision with minimal scaring. Additionally, propranolol has a well-documented safety and side effect profile, and there have been no cases of life-threatening complications resulting from direct propranolol exposure. Recently, a randomized controlled trial also demonstrated that propranolol is a safe and effective medication for treating IHs in patients aged9weeks to5years.However, despite the well-documented efficacy of propranolol in treating IH, very little is known about its mechanism of action. The potential explanations for the therapeutic effects of propranolol on IH include vasoconstriction, angiogenesis inhibition, and the induction of apoptosis in capillary endothelial cells. We hypothesized that one possible mechanism of propranolol-mediated IH treatment may be the modulation of the number of hemangioma-derived endothelial cell (HemECs). Accordingly, this study examined the mechanisms underlying the relationship between the P-adrenergic signaling pathway and the proliferation and apoptosis of HemECs. Part I Effects of propranolol on the proliferation and apoptosis of hemangioma-derived endothelial cellsBackground/Purpose Propranolol, a non-selective beta-blocker, has recently been introduced as a novel treatment modality for proliferating hemangiomas. However, the mechanism of action of this therapy is unknown. In this study, we investigated propranolol in the etiology of hemangiomas that are associated with the proliferation and apoptosis of hemangioma-derived endothelial cells (HemECs).Methods HemECs were isolated from freshly resected hemangioma specimens. We studied propranolol-treated HemECs in vitro. We measured the effect of propranolol on HemEC viability using the Cell Counting Kit-8(CCK-8) assay and proliferation and apoptosis using a BrdU labeling assay, annexin-V-fluorescein isothiocyanate/propidium iodide flow cytometry, and Hoechst33342fluorescent staining. We explored the potential mechanisms of propranolol-induced HemEC dysfunction using western blot analysis, a caspase assay kit, and real-time quantitative PCR.Results We observed that propranolol had inhibitory effects on the viability and proliferation of HemECs. HemEC apoptosis significantly increased with100μM propranolol treatment. We found that vascular endothelial growth factor (VEGF) expression was down-regulated by propranolol in a dose-dependent manner. We also demonstrated activation of the caspase cascade, including caspase-9and caspase-3of the intrinsic pathway, and an increased p53gene expression and Bax/Bcl-xL ratio in HemECs treated with100μM propranolol.Conclusions Overall, we obtained novel data that suggested that propranolol could inhibit HemEC proliferation and induce apoptosis. The effects were likely mediated through the suppression of VEGF expression, activation of caspase-9and caspase-3, up-regulation of the pro-apoptotic genes p53and Bax and down-regulation of the anti-apoptotic gene Bcl-xL. Part Ⅱ The role of β-adrenergic receptor signaling in the proliferation of hemangioma-derived endothelial cellsBackground/Purpose Infantile hemangioma (IH) is a benign vascular neoplasm that arises from the abnormal proliferation of endothelial cells and enhanced angiogenesis. Recently, propranolol has been found to be effective in the management of IH, suggesting that β-adrenergic receptors (β-ARs) may play an important role in the pathogenesis of IH.Methods In the present study, we investigated the β-adrenergic signaling that is associated with hemangioma-derived endothelial cell (HemEC) proliferation. Expression of the β1-and β2-ARS in HemECs was measured at the mRNA and protein levels by quantitative real-time PCR and Western blotting, respectively. We studied β-AR agonist or β-AR antagonist treated HemECs in vitro. We measured the effect of these agents on HemEC viability using the Cell Counting Kit-8(CCK-8) assay and proliferation using a BrdU labeling assay. We explored the potential mechanisms of P-ARs induced HemEC proliferation using western blot analysis and enzyme immunoassay method.Results The results showed that both β1-and β2-ARs were expressed in HemECs. Stimulation of the β-ARs by isoprenaline induced cell proliferation and elevation of second messenger cAMP levels. The proliferation-promoting action of isoprenaline was abolished by a β1-selective antagonist and was more effectively abolished by a β2-selective antagonist; the mechanism for the action of the antagonists was a G0/G1phase cell cycle arrest which was associated with decreased cyclin D1, CDK-4, CDK-6and phospho-Rb expression. Pretreatment of the cells with VEGFR-2or ERK inhibitors also prevented the isoprenaline-mediated proliferation of cells. In agreement with the involvement of β-ARs and VEGFR-2in the HemEC response, β-AR antagonists and the VEGFR-2inhibitor significantly attenuated isoprenaline-induced ERK phosphorylation. Moreover, treating the cells with isoprenaline markedly increased VEGF-A expression and VEGFR-2activity in a β2-AR-dependent manner.Conclusions We have demonstrated that the activation of the β-ARs in the ERK pathway may be important mechanisms in promoting HemEC growth. Furthermore, stimulation of the β-AR may transactivate VEGFR-2signaling and further increase HemEC proliferation. Part Ⅲ Autocrine VEGF-VEGFR-2signaling prevent apoptosis in hemangioma-derived endothelial cellsBackground/Purpose The VEGF-VEGFR-2pathway is aberrantly activated in wide range of tumor, including infantile hemangioma (IH), and is required to maintain the transformed phenotype of many of these tumors. VEGF-VEGFR-2signaling contributes to the transformed phenotype, in part, by preventing apoptosis in response to many different stimuli. However, it is unclear whether VEGF-VEGFR-2activation can lead to a general suppression of apoptosis in IH.Methods The HemECs and HUVECs were serum starvation for12to48h. Cell viability was measured by using the Cell Counting Kit-8(CCK-8) assay and apoptosis was measured by using a annexin-V-fluorescein isothiocyanate/propidium iodide flow cytometry, Hoechst33342and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. The expression of VEGF and PTEN, and activation of VEGFR-2was measured. The potential mechanisms of VEGF-VEGFR-2induced HemEC survival were investigated.Results We show here that VEGF-VEGFR-2signaling induced an autocrine signaling loop that inhibited PTEN and activated Akt in hemangioma-derived endothelial cells (HemECs) as compared with’normal’endothelial cells (HUVECs). The activation of Akt was necessary for VEGFR-2-induced protection against serum starvation-induced apoptosis in HemECs. Inhibition of VEGF-VEGFR-2signaling potently inducing PTEN expression while decreased Akt activity; and caused an increase in sensitivity to apoptosis in HemEcs. Moreover, activation of PI3K/Akt signaling which function as proapoptotic factors and direct downstream targets of VEGFR-2, was decreased by over-expression of PTEN, and thus lead to an increasing in cell apoptosis.Conclusions We demonstrated that up-regulated VEGF-VEGFR-2through PTEN/PI3K/Akt signaling may be important mechanisms in preventing apoptosis in HemECs.