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.
Infantile hemangiomas (IHs) are the most common benign tumors in infancy affecting 5–10% of the population, and are largely composed of densely packed over-proliferating capillaries with high cellular density and the absence of an open lumen. These lesions are most prevalent in Caucasian children and are three times more common in female infants than male. The head and neck region is the most frequently involved area (60%), followed by the trunk (25%) and the extremities (15%), and these tumors exhibit a non-random distribution largely correlating with regions of embryological fusion (
Propranolol, which is administered systemically in pediatric patients with IHs, is a non-selective β-adrenergic receptor antagonist that blocks the action of epinephrine and norepinephrin. This drug has been shown to suppress angiogenesis via inhibition of proliferation, migration, barrier function, and induction of apoptosis in primary cultures of normal epithelial cells (
HemECs were previously isolated from proliferating-phase IH specimens collected from female infants (
RNA was isolated from cells using the Ambion Purelink Mini kit according to the manufacturer’s directions. qRT-PCR was performed on an ABI7900HT RT-PCR system using TaqMan assays with predesigned primer sets for the genes of interest (Invitrogen). All RT-PCR experiments were performed in triplicate. Data shown are the average RQ value ± standard deviation of 4 replicates.
Cell lysates were collected after 48 h treatment and subjected to SDS-PAGE on gradient (4–15%) gels and subsequently transferred to PVDF for western blotting. p-vascular endothelial growth factor receptor-2 (VEGFR-2) (Cell Signaling #2478), VEGFR-2 (Cell Signaling #2479), p-p38 (Cell Signaling #4511), p-p44/42 (Cell Signaling #4370), p-SAPK/JNK (Cell Signaling #4668), p-ATF2 (Cell Signaling #5112), actin (Santa Cruz #SC47778), cyclin A1 (Abcam #ab13337), cyclin A2 (Abcam #7956), cyclin B2 (Abcam #18250), cyclin D1 (Cell Signaling #2978), cyclin D2 (Cell Signaling #3741), cyclin D3 (Cell Signaling #2936), cyclin E1 (Cell Signaling #4129), p15 (Cell Signaling #4822), p21 (Cell Signaling #2947), p27 (Cell Signaling #3698), cleaved caspase-9 (Cell Signaling #9509), cleaved caspase-3 (Cell Signaling #9664), p-FAK (Cell Signaling #3283), p-cofilin (Cell Signaling #3313), cofilin (Cell Signaling #3318), p-ERM (Cell Signaling #3149), ERM (Cell Signaling #3142), p-MYPT1 (Cell Signaling #4563) and MYPT1 (Cell Signaling #2634) antibodies were used at a 1:1000 dilution, followed by incubation with 1:1000 HRP-conjugated anti-mouse or anti-rabbit antibodies (as appropriate). Proteins were detected using Supersignal West Dura Extended Duration Substrate (Thermo Scientific) and digitally captured using a GE Image Quant LAS4000 imaging system.
Cells were plated at subconfluent density and subjected to the indicated treatments for 48 h. Images from 5 independent areas were collected at 1-h intervals using a Nikon Biostation CT time lapse imaging robot. Changes in cell density were calculated every 24 h by counting the number of cells in the selected field of vision. Data shown represent the average of 5 independent areas ± the standard deviation.
Cells were treated as indicated, trypsinized, and fixed in 70:30 ethanol:phosphate-buffered saline overnight. Cells were then stained with 200 μg/ml ethidium bromide plus 20 μg/ml RNase A and incubated overnight. DNA content was analyzed using an Accuri C6 flow cytometer. Data shown are representative of at least 3 independent experiments. Quantitative analysis of DNA content was performed using CFlow Plus software (Accuri) and is the average of triplicate data points.
Cells were treated as indicated, stained for 10 min with 5 μg/ml Hoechst and 5 μg/ml propidium iodide, and washed 3 times in PBS. A Nikon C2SI scanning laser confocal microscope was used to image the red and blue channels. Percent apoptosis (A) was calculated by the following formula: A = (number of red cells/number of blue cells) x 100. The data presented is the average of triplicates.
Confluent cultures were treated as indicated, scratch wounded, and the progress of ‘healing’ of the wound was monitored using a Nikon Biostation CT time lapse imaging robot. Migration speed was calculated by monitoring the movement of the ‘wound’ toward its center at each hour over a 12-h period.
Cells were grown on glass coverslips, treated as indicated and fixed in 4% paraformaldehyde. Then, the coverslips were blocked in 5% bovine serum albumin plus 0.5% Tween-20, incubated with 1:200 of the p-FAK antibody and detected with fluorescently conjugated secondary antibodies. Actin microfilaments were detected by staining with Rhodamine-conjugated phalloidin, and cell nuclei were detected with 4′,6-diamidino-2-phenylindole (DAPI). Immunofluorescent images were captured as z-stacks using a Nikon C2SI scanning laser confocal microscope. Image analysis of cytoskeletal organization included calculating the actin stress fiber correlation and binarizing this correlation image to determine fiber lengths using the FiberScore algorithm (
Total RNA was amplified and biotin-labeled using Illumina TotalPrep RNA Amplification kit (Ambion). A total amount of 750 ng of biotinylated aRNA was then briefly heat-denatured and loaded onto expression arrays to hybridize overnight. Following hybridization, arrays were labeled with Cy3-streptavidin and imaged on the Illumina ISCAN. Intensity values were transferred to Agilent GeneSpring GX microarray analysis software and data were filtered based on the quality of each call. Statistical relevance was determined using ANOVA with a Benjamini Hochberg FDR multiple testing correction (p<0.05). Data were then limited by fold change analysis to statistically relevant data points demonstrating a 2-fold or more change in expression.
The presence of β-adrenergic receptors on normal human endothelial cells has been previously confirmed (
Despite the extensive use of propranolol, many of the mechanisms of action of this drug on IHs have been inferred from its effects on normal endothelial cells (
To determine whether this drug affects apoptosis, we treated HemECs, HDMVECs and HCAECs with 50 μM propranolol for 3 days. As a control we treated HemECs with 5 μM cisplatin for an equivalent amount of time. Cells were co-stained with propidium iodide (which only stains the nuclei of dead cells) and Hoechst dye (which stains the nuclei of both live and dead cells). Calculation of the apoptotic index from each treatment revealed that 50 μM propranolol did not induce apoptosis of any of the cell lines tested, while 5 μM cisplatin resulted in almost 100% apoptosis (
Several reports have presented mixed results for the role of β-adrenergic receptor signaling in wound healing and cell migration, with evidence that inhibition of this class of receptors delays (
Propranolol has been shown to affect the expression of cyclins across multiple cell types (
IHs as a whole are largely understudied considering the high prevalence of these lesions in children and the serious threat to health they pose in certain instances. To date, there remains a great deal of uncertainty as to the origin of these tumors, with evidence suggesting they may be caused by aberrant transplantation of placental endothelial cells (
The expression of β1- and β2-adrenergic receptors has been extensively studied in the cardiovascular system, with high expression occurring in cardiac myocytes and vascular smooth muscle cells (
There is evidence that VEGFR-2 phosphorylation is controlled by β-adrenergic signaling (
As propranolol appears to work with great efficacy against IHs, similar inhibitory effects could potentially be observed in other vascular tumors such as angiosarcomas and Kaposi’s sarcomas. Indeed, propranolol has been tested in preclinical and clinical models of malignant tumors, demonstrating good efficacy in the treatment of melanoma (
Support of this study was provided by a National Heart, Lung, and Blood Institute grant HL098931 and TTUHSC startup funds to BAB, a National Institute of Arthritis and Musculoskeletal and Skin Diseases grant (AR048564) to JB, a NASA EPSCoR award to NMSU, and internal support from NMSU to LB.
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.
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.
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.
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.
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.
Percentage of endothelial cells in each cell cycle phase.
Cells | Sham | Propranolol |
---|---|---|
HemECs | ||
G1 | 68±2.3 | 74±2.2 |
S | 8±0.6 | 5±0.3 |
G2/M | 24±2.7 | 21±1.1 |
HDMVECs | ||
G1 | 69±3.0 | 75±1.6 |
S | 8±4.1 | 4±0 |
G2/M | 22±3.9 | 20±4.5 |
HCAECs | ||
G1 | 71±1.4 | 76±1.7 |
S | 5±1.6 | 3±1.3 |
G2/M | 23±3.3 | 20±2.5 |
HemECs, human infantile hemangioma endothelial cells; HDMVECs, human dermal microvascular endothelial cells; HCAECs, human coronary artery endothelial cells.
Alterations in gene expression (fold-change) induced by propranolol treatment.
Gene symbol | Gene name | Accession no. | HIHEC | HDMVEC | HCAEC |
---|---|---|---|---|---|
3-Hydroxy-3-methylglutaryl-CoA synthase 1 | NM_002130.6 | 4.6 | 5.8 | 6.2 | |
Methylsterol monooxygenase 1, TV2 | NM_001017369.2 | 4.5 | 4.0 | 5.8 | |
Insulin induced gene 1 | NM_198336.2 | 4.3 | 4.6 | 4.9 | |
Low density lipoprotein receptor | NM_000527.4 | 3.6 | 3.8 | 3.9 | |
Mevalonate decarboxylase | NM_002461.1 | 3.6 | 5.7 | 3.4 | |
7-Dehydrocholesterol reductase | NM_001360.2 | 3.6 | 4.7 | 3.4 | |
Stearoyl-CoA desaturase | NM_005063.4 | 3.3 | 3.2 | 5.0 | |
Acetyl-CoA acetyltransferase 2 | NM_005891.2 | 3.2 | 4.6 | 5.2 | |
Lanosterol synthase | NM_002340.5 | 3.2 | 4.5 | 5.1 | |
Transmembrane 7 superfamily member 2 | NM_003273.2 | 3.0 | 4.8 | 5.2 | |
3-Hydroxy-3-methylglutaryl-CoA reductase | NM_000859.2 | 2.9 | 3.1 | 3.8 | |
Fatty acid synthase | NM_004104.4 | 2.8 | 4.6 | 4.3 | |
Methylsterol monooxygenase 1, TV1 | NM_006745.4 | 2.8 | 2.0 | 3.0 | |
Squalene epoxidase | NM_003129.3 | 2.7 | 2.6 | 3.6 | |
Pregnancy specific β-1-glycoprotein 4 | NM_002780.3 | 2.6 | 1.3 | 4.5 | |
24-Dehydrocholesterol reductase | NM_014762.3 | 2.5 | 2.4 | 3.3 | |
Farnesyl-diphosphate farnesyltransferase 1 | NM_004462.3 | 2.5 | 2.2 | 3.2 | |
Isopentenyl-diphosphate δ isomerase 1 | NM_004508.2 | 2.4 | 2.3 | 3.4 | |
Fatty acid desaturase 2 | NM_004265.2 | 2.4 | 4.2 | 2.8 | |
Niemann-Pick disease, type C1 | NM_000271.4 | 2.3 | 2.3 | 3.8 | |
Fructose-2,6-biphosphatase 4 | NM_004567.2 | 2.2 | 2.8 | 2.3 | |
Acyl-CoA synthetase family member 2, TV2 | NM_001076552.2 | 2.2 | 2.6 | 3.5 | |
Acyl-CoA synthetase family member 2, TV1 | NM_018677.3 | 2.2 | 2.7 | 3.2 | |
Emopamil binding protein | NM_006579.2 | 2.1 | 2.4 | 2.3 | |
LOC100129669 | XM_001713607.1 | 2.1 | 2.2 | 2.5 | |
Heme oxygenase (decycling) 1 | NM_002133.2 | 2.1 | 2.1 | 3.5 | |
Sterol-C5-desaturase-like | NM_006918.4 | 2.1 | 1.4 | 2.4 | |
NAD(P) dependent steroid dehydrogenase-like | NM_015922.2 | 2.1 | 2.3 | 2.2 | |
Purinergic receptor P2X, 4 | NM_002560.2 | 2.1 | 1.7 | 1.8 | |
Lipin 1 | NM_145693.1 | 2.0 | 1.6 | 2.9 | |
Placental growth factor | NM_002632.5 | 2.0 | 1.7 | 2.3 | |
Angiopoietin 2 | NM_001147.2 | 2.0 | 1.0 | 2.1 | |
LOC729010 | XR_042330.1 | 2.0 | 1.6 | 2.6 | |
Interleukin 8 | NM_000584.3 | −2.0 | −2.9 | −3.1 | |
Cyclin-dependent kinase 1 | NM_001786.4 | −2.0 | −1.8 | −2.1 | |
Tubulin, β 4B Ivb | NM_006088.5 | −2.0 | −1.4 | −2.5 | |
Pituitary tumor-transforming 1 | NM_004219.2 | −2.0 | −2.0 | −2.0 | |
Opa interacting protein 5 | NM_007280.1 | −2.0 | −2.3 | −2.1 | |
Cell division cycle associated 8 | NM_018101.3 | 2.0 | −1.4 | −2.3 | |
Transgelin | NM_003186.3 | −2.0 | −1.7 | −2.9 | |
Cyclin-dependent kinase inhibitor 3 | NM_005192.3 | −2.0 | −1.7 | −1.6 | |
Anillin, actin binding protein | NM_018685.2 | −2.0 | −1.8 | −1.7 | |
Holliday junction recognition protein | NM_018410.3 | −2.0 | −1.2 | −2.0 | |
PDZ binding kinase | NM_018492.2 | −2.0 | −1.6 | −2.5 | |
Ubiquitin-conjugating enzyme E2T (putative) | NM_014176.3 | −2.0 | −1.4 | −1.5 | |
6-Transmembrane epithelial antigen of the prostate 1 | NM_012449.2 | −2.0 | −1.8 | −1.8 | |
UBE2C | Ubiquitin-conjugating enzyme E2C, TV3 | NM_181800.1 | −2.0 | −1.9 | −2.8 |
CKS1B | CDC28 protein kinase regulatory subunit 1B | NM_001826.2 | −2.0 | −1.6 | −1.9 |
TACC3 | Transforming, acidic coiled-coil containing protein 3 | NM_006342.2 | −2.0 | −1.4 | −1.8 |
NCAPG | Non-SMC condensin I complex, subunit G | NM_022346.3 | −2.0 | −1.7 | −1.6 |
PCDH7 | Protocadherin 7 | NM_002589.2 | −2.0 | −1.2 | −2.2 |
FAM64A | Family with sequence similarity 64, member A | NM_019013.2 | −2.1 | −1.2 | −2.0 |
PRC1 | Protein regulator of cytokinesis 1 | NM_199413.1 | −2.1 | −1.5 | −2.3 |
MELK | Maternal embryonic leucine zipper kinase | NM_014791.3 | −2.1 | −1.8 | −2.1 |
TPX2 | TPX2, microtubule-associated | NM_012112.4 | −2.1 | −1.4 | −2.2 |
MCM4 | Minichromosome maintenance complex CMPT 4, TV2 | NM_182746.2 | −2.1 | −1.6 | −1.9 |
ZW10 interactor | NM_001005413.1 | −2.1 | −1.5 | −2.0 | |
Kinesin family member C1 | NM_002263.2 | −2.1 | −1.4 | −2.2 | |
Cell division cycle 20 | NM_001255.2 | −2.2 | −2.0 | −4.0 | |
Ubiquitin-conjugating enzyme E2C, TV6 | NM_181803.1 | −2.2 | −1.8 | −2.4 | |
Non-SMC condensin II complex, subunit G2 | NM_017760.5 | −2.2 | −1.3 | −1.4 | |
Serpin peptidase inhibitor, clade D, member 1 | NM_000185.3 | −2.2 | −1.8 | −2.6 | |
Cell division cycle 45 | NM_003504.3 | −2.2 | −1.8 | −3.0 | |
Ly1 antibody reactive | NM_017816.2 | −2.2 | −1.6 | −1.3 | |
Cyclin A1 | NM_003914.3 | −2.2 | −2.2 | −1.9 | |
Thyroid hormone receptor interactor 13 | NM_004237.3 | −2.2 | −1.9 | −2.6 | |
Myelin protein zero-like 2, TV2 | NM_144765.2 | −2.2 | −1.3 | −2.4 | |
Centrosomal protein 55kDa | NM_018131.4 | −2.2 | −2.0 | −2.1 | |
Chemokine (C-X-C motif) ligand 1 | NM_001511.3 | −2.2 | −2.5 | −2.8 | |
Cyclin B2 | NM_004701.3 | −2.2 | −2.2 | −2.3 | |
Kinesin family member 20A | NM_005733.2 | −2.2 | −1.9 | −2.5 | |
RAD51 associated protein 1 | NM_006479.4 | −2.2 | −1.6 | −2.2 | |
GINS complex subunit 2 | NM_016095.2 | −2.2 | −1.7 | −2.9 | |
Family with sequence similarity 83, member D | NM_030919.2 | −2.3 | −1.6 | −2.4 | |
KIAA0101 | NM_014736.4 | −2.3 | −2.0 | −2.3 | |
Discs, large (Drosophila) homolog-associated protein 5 | NM_014750.4 | −2.3 | −2.0 | −2.3 | |
Cyclin A2 | NM_001237.3 | −2.3 | −2.2 | −2.8 | |
LOC399943 | XM_934471.1 | −2.3 | −2.4 | −3.4 | |
Myelin protein zero-like 2, TV1 | NM_005797.3 | −2.3 | −2.7 | −2.2 | |
Topoisomerase II α 170kDa | NM_001067.3 | −2.3 | −2.4 | −2.6 | |
Ribonucleotide reductase M2 | NM_001034.3 | −2.3 | −1.5 | −2.8 | |
F-box protein 5 | NM_012177.3 | −2.4 | −1.9 | −2.6 | |
Cell division cycle associated 7 | NM_031942.4 | −2.4 | −1.9 | −2.5 | |
Minichromosome maintenance complex CMPT 4, TV1 | NM_005914.3 | −2.4 | −1.5 | −2.0 | |
MAD2 mitotic arrest deficient-like 1 | NM_002358.3 | −2.4 | −2.3 | −2.1 | |
Ubiquitin-like with PHD and ring finger domains 1 | NM_001048201.1 | −2.5 | −1.6 | −2.6 | |
Angiopoietin-like 4 | NM_139314.1 | −2.8 | −2.8 | −2.9 | |
Regulator of G-protein signaling 4 | NM_005613.5 | −3.0 | −3.8 | −3.8 | |
Interleukin 1 receptor-like 1 | NM_003856.2 | −3.2 | 3.4 | −5.2 |
HDMVEC, human dermal microvascular endothelial cells; HCAEC, human coronary artery endothelial cells.