Propranolol treatment of infantile hemangioma endothelial cells: A molecular analysis

Infantile hemangiomas (IHs) are non-malignant, largely cutaneous vascular tumors affecting approximately 5–10% of children to varying degrees. During the first year of life, these tumors are strongly proliferative, reaching an average size ranging from 2 to 20 cm. These lesions subsequently stabilize, undergo a spontaneous slow involution and are fully regressed by 5 to 10 years of age. Systemic treatment of infants with the non-selective β-adrenergic receptor blocker, propranolol, has demonstrated remarkable efficacy in reducing the size and appearance of IHs. However, the mechanism by which this occurs is largely unknown. In this study, we sought to understand the molecular mechanisms underlying the effectiveness of β blocker treatment in IHs. Our data reveal that propranolol treatment of IH endothelial cells, as well as a panel of normal primary endothelial cells, blocks endothelial cell proliferation, migration, and formation of the actin cytoskeleton coincident with alterations in vascular endothelial growth factor receptor-2 (VEGFR-2), p38 and cofilin signaling. Moreover, propranolol induces major alterations in the protein levels of key cyclins and cyclin-dependent kinase inhibitors, and modulates global gene expression patterns with a particular affect on genes involved in lipid/sterol metabolism, cell cycle regulation, angiogenesis and ubiquitination. Interestingly, the effects of propranolol were endothelial cell-type independent, affecting the properties of IH endothelial cells at similar levels to that observed in neonatal dermal microvascular and coronary artery endothelial cells. This data suggests that while propranolol markedly inhibits hemangioma and normal endothelial cell function, its lack of endothelial cell specificity hints that the efficacy of this drug in the treatment of IHs may be more complex than simply blockage of endothelial function as previously believed

Targeting of beta adrenergic receptors results in therapeutic efficacy against models of hemangioendothelioma and angiosarcoma.

Therapeutic targeting of the beta-adrenergic receptors has recently shown remarkable efficacy in the treatment of benign vascular tumors such as infantile hemangiomas. As infantile hemangiomas are reported to express high levels of beta adrenergic receptors, we examined the expression of these receptors on more aggressive vascular tumors such as hemangioendotheliomas and angiosarcomas, revealing beta 1, 2, and 3 receptors were indeed present and therefore aggressive vascular tumors may similarly show increased susceptibility to the inhibitory effects of beta blockade. Using a panel of hemangioendothelioma and angiosarcoma cell lines, we demonstrate that beta adrenergic inhibition blocks cell proliferation and induces apoptosis in a dose dependent manner. Beta blockade is selective for vascular tumor cells over normal endothelial cells and synergistically effective when combined with standard chemotherapeutic or cytotoxic agents. We demonstrate that inhibition of beta adrenergic signaling induces large scale changes in the global gene expression patterns of vascular tumors, including alterations in the expression of established cell cycle and apoptotic regulators. Using in vivo tumor models we demonstrate that beta blockade shows remarkable efficacy as a single agent in reducing the growth of angiosarcoma tumors. In summary, these experiments demonstrate the selective cytotoxicity and tumor suppressive ability of beta adrenergic inhibition on malignant vascular tumors and have laid the groundwork for a promising treatment of angiosarcomas in humans

Beta blockade inhibits malignant vascular tumor cell proliferation in a dose dependent manner.

<p>(<b>A</b>) The panel of malignant vascular tumor lines was subjected to a dose curve of propranolol over a 48 hour time course and changes in cell proliferation were quantified by counting the number of cells per vision field. (<b>B</b>) Representative images of sham or 100 µM propranolol treated SVR angiosarcoma cells after 48 hours. (<b>C</b>) The panel of malignant vascular tumor lines was grown on Alvetex polystyrene membranes for 48 hours, treated with sham or 25 µM propranolol, and cell density was accessed by quantifying positive Hoechst nuclear staining after 96 hours of treatment. (<b>D</b>) Differential interference contast (DIC) and fluorescence image overlays of sham or 25 µM propranolol treated SVR angiosarcoma cells in the Alvetex membranes 96 hours after treatment. (<b>E</b>) Cell cycle analysis of propidium iodide stained SVR angiosarcoma cells treated with sham or 100 µM propranolol for 24 hours. (<b>F</b>) Cell cycle profile quantification of malignant vascular tumor cells treated with sham or 100 µM propranolol for 24 hours. For all experiments, the data is the average +/− standard deviation for at least three biological replicates. Statistical significant was determined using Students t-test (p<0.05).</p

Three fold or greater changes in gene expression (p<0.05) after 24 hours of 100 mM propranolol in SVR angiosarcoma cells.

<p>Three fold or greater changes in gene expression (p<0.05) after 24 hours of 100 mM propranolol in SVR angiosarcoma cells.</p

Beta blockade induces apoptosis of malignant vascular tumor cells.

<p>(<b>A</b>) Apoptosis quantification of Hoechst 33342 and propidium iodide stained malignant vascular tumor cells after 24 hours treatment with sham or 100 µM propranolol. (<b>B</b>) Fluorescent images of SVR angiosarcoma cells stained with Hoechst 33342 and propidium iodide after 24 hours treatment with sham or 100 µM propranolol. (<b>C</b>) 3D rendering of representative SVR angiosarcoma cell nuclei after 24 hours treatment with sham or 100 µM propranolol. (<b>D</b>) qPCR analysis of a panel of 84 genes involved in apoptotic regulation. Shown are the 18 genes whose steady state mRNA expression levels were statistically altered 1.5 fold or more following 24 hours of 100 µM propranolol in SVR cells. (<b>E</b>) Immunofluorescent detection of cleaved caspase-3 in SVR angiosarcoma cells after 24 hours treatment with sham or 100 µM propranolol. (<b>F</b>) Western analysis detecting phospho-p38 MAPK and total p38 MAPK protein levels after 1 hour of sham or 100 µM propranolol treatment of SVR angiosarcoma cells. (<b>G</b>) Western blot detection of apoptotic protein levels. (<b>H</b>) Unstained SVR angiosarcoma cells were co-cultured with CellTracker Blue stained HDMVECs. The co-cultures were subjected to sham or 100 µM propranolol and DIC/fluorescent image overlays were obtained after 48 hours treatment. For all experiments, the data is the average +/− standard deviation for at least three biological replicates. Statistical significant was determined using Students t-test (p<0.05).</p

Beta blockade induces alterations in key cell cycle regulators and the Tie2 angiogenic regulator.

<p>(<b>A</b>) qPCR analysis of a panel of 84 genes involved in cell cycle regulation. Shown are the 25 genes whose mRNA expression levels were statistically altered 1.5 fold or more following 24 hours of 100 µM propranolol in SVR cells. The qPCR data is the average +/− standard deviation for at least three biological replicates. Statistical significant was determined using Students t-test (p<0.05). (<b>B</b>) Western blot detecting cell cycle regulatory protein levels in SVR cells subjected to 24 hours of sham or 100 µM propranolol. (<b>C</b>) Immunofluorescent staining for p21 (<i>red</i>) and p27 (<i>green</i>) in SVR cells subjected to 24 hours of sham or 100 µM propranolol. (<b>D</b>) SVR cells were grown in standard growth media (10% FBS) or serum starved overnight, treated as indicated with sham, 100 µM propranolol (24 hours), or 10 ng/ml angiopoietin-1 (2.5 minutes), and Western analysis detected the expression of phosphorylated Tie2, total Tie2, and actin.</p