Propranolol treatment of infantile hemangioma endothelial cells: A molecular analysis

Abstract

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.

Figure 1

β-adrenergic receptor expression on infantile hemangioma (IH) and normal endothelial cells. RT-PCR expression assays measuring the steady state levels of ADRB1, ADRB2, and ADRB3 mRNA in primary cultures of human infantile hemangioma endothelial cells (HemECs), human dermal microvascular endothelial cells (HDMVECs) and human coronary artery endothelial cells (HCAECs). Expression data are represented as the relative abundance of each transcript normalized to the GAPDH levels.

Figure 2

Propranolol decreases the proliferation of human infantile hemangioma endothelial cells (HemECs). (A) HemECs, human dermal microvascular endothelial cells (HDMVECs), and human coronary artery endothelial cells (HCAECs) were treated with a dose curve of propranolol (0 to 100 μM) and cell proliferation was measured by counting changes in the number of cells/defined vision field over a 48-h period. (B) Time lapse microscopy image of sham and 50 μM propranolol treated HemECs over a 48-h period. (C) DNA content analysis of propidium iodide stained HemECs treated with sham or 50 μM propranolol for 48 h. (D) Western blot analysis detecting the levels of phosphorylated and total vascular endothelial growth factor receptor-2 (p-VEGFR-2 and VEGFR-2, respectively) and the phosphorylated forms of p38 (p-p38), p44 (p-p44), p42 (p-p42), stress activated protein kinase (p-SAPK), c-jun N-terminal kinase (p-JNK), and activating transcription factor 4 (p-ATF4) in HemECs treated for 24 h with sham or 50 μM propranolol. Actin levels were used as a loading control. (E) Western blot analysis detecting the levels of cyclins, cyclin dependent kinases, and cyclin dependent kinase inhibitors in HemECs treated for 24 h with sham or 50 μM propranolol. Actin levels were used as a loading control. Prop, propranolol.

Figure 3

Propranolol does not induce apoptosis in human infantile hemangioma endothelial cells (HemECs) at its effective inhibitory concentration. (A) Confocal imaging of HemECs treated for 72 h with sham or 50 μM propranolol and subsequently co-stained with propidium iodide (PI) and Hoechst dye (blue, Hoechst-positive nuclei; pink, Hoechst-positive/PI-positive nuclei). (B) Quantification of PI-positive nuclei in HemECs, human dermal microvascular endothelial cells (HDMVECs), and human coronary artery endothelial cells (HCAECs) treated for 72 h with sham or 50 μM propranolol. (C) Western blot analysis detecting the levels of cleaved caspase-9 and -3 (cl-caspase-9 and cl-caspase -3, respectively). Actin levels were used as a loading control.

Figure 4

Propranolol disrupts HIHEC migration and actin cytoskeleton dynamics. (A) Confluent monolayers of human infantile hemangioma endothelial cells (HemECs) were scratch wounded and treated with sham or 50 μM propranolol. Progress of migration was monitored using time lapse photography over a period of 12 h. (B) Quantification of the speed (μm/h) of HemECs, human dermal microvascular endothelial cells (HDMVECs), and human coronary artery endothelial cells (HCAECs) treated with sham or propranolol from the time lapse images of the scratch assay. (C) Western blot analysis detecting the levels of the total and phophorylated (p-) forms of focal adhesion kinase (FAK), cofilin, ezrin/radixin/moesin (ERM), and myosin phosphatase-targeting subunit 1 (MYPT1) in HemECs treated with sham or 50 μM propranolol for 48 h. Actin levels were used as a loading control. (D) Confocal immunofluorescent imaging of sham or 50 μM propranolol-treated HemECs co-stained with Rhodamine conjugated phalloidin (red), DAPI (blue), and antibodies against phospho-FAK. Prop, propranolol.

Figure 5

Propranolol induces significant alterations in global gene expression of human infantile hemangioma endothelial cells (HemECs). (A) Correlation map comparing the significant gene expression changes (>2 fold gene expression alteration, p<0.05) as determined by microarray analysis between HemECs, human dermal microvascular endothelial cells (HDMVECs), and human coronary artery endothelial cells (HCAECs) treated with sham or 50 μM propranolol for 24 h. (B) RT-PCR confirmation of a subset of genes in HemECs whose expression was statistically altered in the microarray.