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Gene expression analysis reveals marked differences in the transcriptome of infantile hemangioma endothelial cells compared to normal dermal microvascular endothelial cells

  • Jessica M Stiles 1
  • Rebecca K Rowntree 1
  • Clarissa Amaya 1
  • Dolores Diaz 1
  • Victor Kokta 2
  • Dianne C Mitchell 1
  • Brad A Bryan 1 g @

@ corresponding author,  & equal contributor

Vascular Cell. 2013; 5(1):6 | © Stiles et al

Received: 24 January  2013 | Accepted: 13 March  2013 | Published: 25 March  2013

Vascular Cell ISSN: 2045-824X


Author information Copyright & License
  • 1Department of Biomedical Sciences - Paul L. Foster School of Medicine, Texas Tech University Health Sciences CenterEl Paso, TX USA
  • 2Department of Pathology - CHU Sainte-Justine, University of MontrealMontreal, QC Canada



Infantile hemangiomas are benign vascular tumors primarily found on the skin in 10% of the pediatric population. The etiology of this disease is largely unknown and while large scale genomic studies have examined the transcriptomes of infantile hemangioma tumors as a whole, no study to date has compared the global gene expression profiles of pure infantile hemangioma endothelial cells (HEMECs) to that of normal human dermal microvascular endothelial cells (HDMVECs).


To shed light on the molecular differences between these normal and aberrant dermal endothelial cell types, we performed whole genome microarray analysis on purified cultures of HEMECs and HDMVECs. We then utilized qPCR and immunohistochemistry to confirm our microarray results.


Our array analysis identified 125 genes whose expression was upregulated and 104 genes whose expression was downregulated by greater than two fold in HEMECs compared to HDMVECs. Bioinformatics analysis revealed three major classifications of gene functions that were altered in HEMECs including cell adhesion, cell cycle, and arachidonic acid production. Several of these genes have been reported to be critical regulators and/or mutated in cancer, vascular tumors, and vascular malformations. We confirmed the expression of a subset of these differentially expressed genes (ANGPT2, ANTXR1, SMARCE1, RGS5, CTAG2, LTBP2, CLDN11, and KISS1) using qPCR and utilized immunohistochemistry on a panel of paraffin embedded infantile hemangioma tumor tissues to demonstrate that the cancer/testis antigen CTAG2 is highly abundant in vessel-dense proliferating infantile hemangiomas and with significantly reduced levels during tumor involution as vascular density decreases.


Our data reveal that the transcriptome of HEMECs is reflective of a pro-proliferative cell type with altered adhesive characteristics. Moveover, HEMECs show altered expression of many genes that are important in the progression and prognosis of metastatic cancers.


Infantile hemangiomas are benign tumors of vascular origin that affect approximately 10% of the pediatric population. These tumors are characterized by a rapid proliferation phase over the first 1–2 years of the child’s life, followed by a slow and steady decline over the next 5–7 years leading to the complete involution of the tumor mass. Approximately 90% of all infantile hemangiomas remain small and are best left alone to naturally involute. However in about 10% of the cases the tumors exhibit aggressive characteristics based on their size, location, number, etc. and must be actively treated to avoid patient disfigurement and/or mortality.

The etiology of infantile hemangiomas is largely unknown, particularly with regard to the cellular origin of the tumor. Circumstantial evidence suggests that these lesions are of aberrant placental origin as evidenced by upregulated Glut1 expression [1], and some labs have ventured to hypothesize that they may be formed from metastatic invasion of placenta-derived chorangioma cells [2]. Indeed, transcriptional profiling of human placenta, infantile hemangioma, and eight normal and diseased vascularized tissues suggests that high transcriptome similarity is shared between placenta and hemangioma tissues, more so than any of the other tissues tested [3]. Global gene expression analysis of infantile hemangioma tumors has been previously performed by two labs. Ritter et al. [4] utilized microarray analysis on whole tumors and identified immune regulators and indoleamine 2,3 dioxygenase as key regulators of infantile hemangioma involution. Calicchio et al. [5] utilized laser capture microdissection and genome-wide transcriptional profiling of vessels from proliferating and involuting hemangiomas. The authors strongly associated proliferating hemangioma vessels with increased expression of genes involved in endothelial-pericyte interactions and neuronal/vascular patterning, and involuting hemangiomas with chronic inflammatory mediators and angiogenic inhibitors. Given the high density of tightly associated pericytes in infantile hemangiomas and the inevitable collateral capture of intraluminal white cells, fibroblasts, mast cells, and perivascular collagen with laser microdissection, these data represent changes from numerous cell types within the infantile hemangioma tumor, but are not reflective specifically of the aberrant endothelial cells which contribute to disease. While these genomics studies have provided great mechanistic insight into the etiology and progression of the disease, they have not addressed the unique differences between abnormal infantile hemangioma endothelial cells and the normal dermal endothelial cells that are resident in the surrounding skin area of the patient. Understanding these differences could identify targetable pathways that could be exploited to preferentially block hemangioma growth and spread, but spare normal endothelial cells.

To date, no direct whole genome comparison of pure cultures of human dermal microvascular endothelial cells (HDMVECs) and infantile hemangioma endothelial cells (HEMECs) has been reported. To address this, we performed whole genome microarray profiling of the gene expression alterations between low passage pure cultures of HEMECs and HDMVECs. We identified a number of transcriptional alterations that are likely to contribute to the aggressive phenotype of infantile hemangiomas and that could potentially be utilized in immunotherapy against particularly aggressive hemangiomas tumors.

Materials and methods

Cell culture and chemicals

The HEMEC cell line was previously isolated from a proliferating-phase infantile hemangioma specimen collected from a female infant and generously donated to us by Joyce Bischoff (Harvard Medical School) [6]. The primary culture of neonatal HDMVECs was purchased from ATCC. Both cell lines were cultured as previously reported [7]. For all experiments, cell lines were used at <5 passages.

Proliferation assay

Cells were plated at equivalent sub-confluent densities and maintained in a Nikon Biostation CT time lapse imaging station. Cell proliferation was measured by counting cells per vision field from 5 independent areas over a 96 hour time course. Data presented is the average of the counts plus or minus the standard deviation. Student’s t-test was used to evaluate statistical significance. Data with p<0.05 was considered significant.

Migration assay

Confluent cultures were scratch wounded and the progress of “wound healing” was monitored using a Nikon Biostation CT time lapse imaging station over a 9 hour period. Data presented is the average migration speed plus or minus the standard deviation. Student’s t-test was used to evaluate statistical significance (p<0.05). Data with p<0.05 was considered significant.


Cells were plated onto collagen type I coated glass coverslips, fixed in 4% paraformaldehyde, and incubated with antibodies against phospho-focal adhesion kinase (p-FAK; 1:1000; Cell Signaling #3283), rhodamine conjugated phalloidin (1:350; Cytoskeleton Inc.), or DAPI and imaged via a Nikon Eclipse Ti laser scanning confocal microscope.

Microarray analysis

Total RNA was amplified and biotin-labeled using Illumina TotalPrep RNA Amplification Kit (Ambion). 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 was filtered based on quality of each call. Statistical relevance was determined using ANOVA with a Benjamini Hochberg FDR multiple testing correction (p-value < 0.05). Data were then limited by fold change analysis to statistically relevant data points demonstrating a 2-fold or more change in expression. Pathway analysis was performed using Metacore software. The microarray data from this experiment is publically available on the Gene Expression Omnibus (GEO Accession #GSE43742).

Quantitative real time PCR analysis

RNA was isolated from cells using the Ambion Purelink Minikit 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.


Paraffinized infantile hemangioma tissues were labeled with CTAG2 antibody (1:200, Santa Cruz Biotechnology #sc99243) and quantified using Alkaline Phosphatase detection (CellMarque). Positive and negative controls from breast carcinoma tissues were stained with CTAG2 antibody or sham, respectively. Use of de-identified human tissues was approved by the Texas Tech University Health Sciences Center Institutional Review Board for the Protection of Human Subjects (IRB E13029). Waiver of informed consent was approved by IRB.

Results and discussion

A comparison of the proliferation and migration rates of HEMECs and HDMVECs under standard growth conditions revealed no significant difference between normal and hemangioma endothelial cell types, however HEMECs grown under reduced serum conditions (0.5% fetal bovine serum) exhibited an approximately 30% increase in proliferation and an approximately 18% increase in migration relative to HDMVECs grown under the same conditions (Figure 1A & B). This suggests the higher serum concentrations were likely masking any phenotypic advantage attributed to the HEMECs. Moreover, it indicates the proliferative and migratory capacity of HEMECs are unique from that observed in HDMVECs and agrees with earlier reports suggesting advantages in these areas for HEMECs [6]. Comparisons of fluorescent images of the actin cytoskeleton and active focal adhesion complexes obtained with confocal microscopy revealed that HDMVECs display primarily peripheral membrane localized p-FAK, indicating sites of cellular attachment to the extracellular matrix (ECM) (Figure 1C). In contrast, p-FAK localization in HEMECs was observed along the entirety of the actin stress fibers, suggesting cellular adhesion to its substrate is markedly altered in HEMECs. Indeed, it has previously been reported that HEMECs display unique expression of genes involved in cellular adhesion [8].

Figure 1   ( Jump back to reading )

Figure 1 caption

Analysis of HDMVEC and HEMEC phenotypes. (A) Analysis of proliferation rates between HDMVECs and HEMECs over a 48 hr time course. (B) Analysis of the migration rates of HDMVECs and HEMECs nine hours after initial scratch from a micropipette. (C) Immunofluorescent imaging of actin (), p-FAK (), and nucleus (). (red asterisks for panels A &B represent statistically significant values [p<0.05] as determined by Student’s -test).

Whole genome microarray analysis reveals large scale alterations in gene expression between HEMECs and HDMVECs

Given the phenotypic differences observed between HEMECs and HDMVECs, we compared the global gene expression patterns between pure cultures of these cells using Illumina high density BeadArrays to elucidate which molecular factors are deregulated in HEMECs. Our array analysis identified 125 genes whose expression was upregulated and 104 genes whose expression was downregulated (2 fold or greater, p<0.05) in HEMECs compared to HDMVECs (Table 1). Metacore analysis of the 2 fold or greater gene expression changes revealed three major classifications of gene functions that are altered in HEMECs including cell adhesion (TIMP1, COL1A1, COL1A2, MMP1, MMP13, SERPINE2, COL4A6, LAMC2, MMP2, CD44, CAV1, CCL2, JAM3, CLDN11, LYVE1), cell cycle (CCND2, CDKN2A, CCNA1, NCAPD2), and arachidonic acid production (ACSL5, FAP, LIPG, PLA2G4C). Given the number of adhesion genes whose expression is altered in HEMECs compared to HDMVECs, it is no surprise that we observed altered subcellular localization of p-FAK in HEMECs (Figure 1C), reflecting a unique adhesive phenotype in these cells. Our data reflect altered cell cycle regulation in HEMECs, with a downregulation of CCND2 (cyclin D2) and CDKN2A (p16Ink4A) and a potent 6.6 fold increase in CCNA1 (cyclin A1), and these changes may contribute to the enhanced proliferation rates in HEMECs and the uncontrolled cell growth observed in infantile hemangiomas tumors. Alterations in the expression of genes involved in arachidonic acid production were unique in that this polyunsaturated fatty acid can serve as a lipid second messenger in the regulation of phospholipase-C and protein kinase-C signaling, is a key inflammatory intermediate, and can act as a vasodilator [9].

Table 1   ( Jump back to reading )

Fold changes in mRNA expression levels of genes in HEMECs compared to HDMVECs
Gene symbolGene nameAccession numberFC
CTAG2Cancer/testis antigen 2NM_020994.311.6
IL13RA2Interleukin 13 Receptor, alpha 2NM_000640.210.7
IFI27Interferon, alpha-inducible protein 27NM_005532.38.3
TPM2Tropomyosin 2 (beta)NM_213674.17.8
RPL14Ribosomal protein L14NM_001034996.16.6
CCNA1Cyclin A1NM_003914.36.6
RGS5G-protein signaling 5 regulatorNM_003617.36.0
FBN2Fibrillin 2NM_001999.35.9
D4S234EDNA segment on chromosome 4 (unique)NM_001040101.15.5
BST2Bone marrow stromal cell antigen 2NM_004335.25.1
QPCTGlutaminyl-peptide cyclotransferaseNM_012413.34.8
TNFSF4Tumor necrosis factor (ligand) superfamily, member 4NM_003326.34.6
RGS5Regulator of G-protein signaling 5NM_003617.34.6
SPOCK1Sparc/osteonectin, cwcv and kazal-like domains proteoglycan 1NM_004598.34.6
SNHG8Small nucleolar RNA host gene 8 (non-protein coding)NR_003584.34.6
ANTXR1Anthrax toxin receptor 1NM_032208.24.5
CHST1Carbohydrate sulfotransferase 1NM_003654.54.5
MPZL2Myelin protein zero-like 2NM_005797.34.4
HEY2Hairy/enhancer-of-spilt related with YRPW motif 2NM_012259.24.3
SLITRK4SLIT and NTRK-like family, member 4NM_173078.34.2
SHISA2Shisa homolog 2NM_001007538.14.0
LRRC17Leucine rich repeat containing 17, TV2NM_005824.23.9
NUDT11Nudix-type motif 11NM_018159.33.8
RNASE1Ribonuclease, Rnase A family, 1, TV1NM_198235.23.7
SERPINE2Serpin peptidase inhibitor, clade E, member 2NM_006216.33.6
LIPGLipase, endothelialNM_006033.23.4
PCSK5Proprotein convertase subtilisin/kexin type 5NM_006200.33.4
CXCR4Chmeokine (C-X-C motif) receptor 4, TV2NM_003467.23.2
TMEM200ATransmembrane protein 200ANM_052913.23.1
CXCR4Chemokine (C-X-C motif) receptor 4, TV1NM_001008540.13.1
RAB34RAB34, member RAS onogene familyNM_031934.53.0
DPYSL3Dihydropyrimidinase-like 3NM_001387.22.9
FBXL13F-box and leucine-rich repeat protein 13NM_145032.32.9
PNMA2Paraneoplastic Ma antigen 2NM_007257.52.9
NLGN1Neuroligin 1NM_014932.22.8
DDIT4DNA-damage-inducible transcript 4NM_019058.22.8
PFN2Profilin 2NM_053024.32.8
GABBR2Gamma-aminobutyric acid B receptor, 2NM_005458.72.8
MEIS2Meis homeobox 2NM_172315.22.7
PMEPA1Prostate transmembrane protein, androgen induced 1NM_199169.22.7
PLEK2Pleckstrin 2NM_016445.12.7
CARD11Caspase recruitment domain family, member 11NM_032415.42.6
SNORD13Small nucleolar RNA, C/D box 13, small nucleolar RNANR_003041.12.6
GFPT2Glutamine-fructoce-6-phosphate transaminase 2NM_005110.22.6
FAPFibroblast activation protein, alphaNM_004460.22.6
OCIAD2OCIA domain containing 2, TV2NM_152398.22.5
F2RL1Coagulation factor II receptor-like 1NM_005242.42.5
DSTYKDual serine/threonine and tyrosine protein kinaseNM_199462.22.5
NYNRINNYN domain and retroviral integrase containingNM_025081.22.5
COL8A1Collagen, type VIII, alpha 1NM_020351.32.5
LTBP2Latent transforming growth factor beta binding protein 2NM_000428.22.4
RNASE1Ribonuclease, Rnase A family, 1, TV3NM_198232.22.4
IFI27L2Interferon, alpha-inducible protein 27-like 2NM_032036.22.4
SOX4SRY (sex determining region Y)-box4NM_003107.22.4
LRRC17Leucine rich repeat containing 17, TV1NM_001031692.22.3
DSEDermatan sulfate epimeraseNM_013352.22.3
CD44CD44 molecule (Indian blood group), TV5NM_001001392.12.3
NT5DC25'-nucleotidase domain containing 2NM_022908.22.3
NPFFR2Neuropeptide FF receptor 2NM_004885.22.3
MEX3BMex-3 homolog BNM_032246.32.3
C1orf54Chromosome 1 open reading frame 54NM_024579.32.3
HDDC2HD domain containing 2NM_016063.22.3
CYB5ACytochrome b5 type ANM_001914.32.3
PIRPirin (iron binding nuclear protein)NM_001018109.22.3
GPR37G protein-coupled receptor 37NM_005302.22.3
PPAPDC1APhosphatidic acid phosphatase type 2 domain containing 1ANM_001030059.12.3
CD44CD44 molecule (Indian blood group), TV4NM_001001391.12.2
CTAG1ACancer/testis antigen 1ANM_139250.12.2
C4orf18Chromosome 4 open reading frame 18NM_016613.62.2
LDOC1Leucine zipper, down-regulated in cancer 1NM_012317.22.2
TGFBITransforming growth factor, beta-inducedNM_000358.22.2
COL5A2Collagen, type V, alpha 2NM_000393.32.2
NOX4NADPH oxidase 4NM_016931.32.2
TSHZ3Teashirt zinc finger homeobox 3NM_020856.22.2
FNDC3BFibronectin type III domain containing 3B, TV2NM_001135095.12.2
ADAM19ADAM metallopeptidase domain 19NM_033274.32.2
JAM3Junctional adhesion molecule 3NM_032801.42.1
CGNL1Cingulin-like 1NM_032866.42.1
COL4A6Collagen, type IV, alpha 6NM_001847.22.1
BMXBMX non-receptor tyrosine kinaseNM_001721.62.1
DUSP23Dual specificity phosphatase 23NM_017823.32.1
MMP2Matrix metallopeptidase 2NM_004530.42.1
NCAPD2Non-SMC condensin I complex, subunit D2NM_014865.32.1
CYBRD1Cytochrome b reductase 1, TV1NM_024843.22.1
FAM89AFamily with sequence similarity 89, member ANM_198552.22.1
GAS6Growth arrest-specific 6NM_000820.22.1
S100A13S100 calcium binding protein A13NM_001024211.12.1
SMARCE1SWI/SNF related, subfamily e, member 1NM_003079.42.1
LFNGO-fucosylpeptide 3-beta-N-acetylglucosaminyltransferaseNM_001040167.12.1
MTMR11Myotubularin related protein 11NM_181873.32.1
ITGA10Integrin, alpha 10NM_003637.32.1
PTGFRNProstaglandin F2 receptor negative regulatorNM_020440.22.0
LOC644936Actin, beta pseudogeneNR_004845.12.0
CPS1Carbamoyl-phosphate synthase 1, mitochonfrialNM_001875.42.0
C18orf56Chromosome 18 open reading frame 56NM_001012716.22.0
ADAAdenosine deaminaseNM_000022.22.0
NETO2Neuropilin and tolliod-like2NM_018092.42.0
STC2Stanniocalcin 2NM_003714.22.0
PRKAR1AProtein kinase, cAMP-dependent, regulatory, type I, alphaNM_002734.32.0
EGFLAMEGF-like, fibronectin type III and laminin G domainsNM_182801.22.0
SPECC1Sperm antigen with calponin homology, coiled-coil domains 1NM_001033555.22.0
FNDC3BFibronectin type III domain containing 3B, TV1NM_022763.32.0
THOC3THO complex 3NM_032361.22.0
COL5A1Collagen, type V, alpha 1NM_000093.32.0
LANCL1LanC lantibiotic synthetase component C-like 1NM_006055.22.0
OCIAD2OCIA domain containing 2, TV1NM_001014446.12.0
LRIG1Leucine-rich repeats and immunoglobulin-like domains 1NM_015541.22.0
HOXB2Homeobox B2NM_002145.32.0
TIMP1TIMP metallopeptidase inhibitor 1NM_003254.2−2.0
NAAAN-acylethanolamine acid amidaseNM_014435.3−2.0
MAOAMonoamine oxidase ANM_000240.2−2.0
KISS1KiSS metastasis-suppressorNM_002256.3−2.0
SLC25A22Solute carrier family 25, member 22NM_024698.5−2.0
NOSIPNitric oxide synthase interacting proteinNM_015953.3−2.0
COL1A2Collagen, type I, alpha 2NM_000089.3−2.0
ZDHHC14Zinc finger, DHHC-type containing 14NM_024630.2−2.0
HPCAL1Hippocalcin-like 1NM_134421.1−2.0
VLDLRVery low density lipoprotein receptorNM_001018056.1−2.0
BMP2Bone morphogenetic protein 2NM_001200.2−2.0
ABLIM1Actin binding LIM protein 1NM_006720.3−2.0
PIK3C2APhosphoinositide-3-kinase, class 2, alpha polypeptideNM_002645.2−2.0
IRF1Interferon regulatory factor 1NM_002198.2−2.0
MBPMyelin basic proteinNM_001025100.1−2.0
PRKAR1BProtein kinase, cAMP-dependent, regulatory type I, betaNM_002735.2−2.1
FAM101BFamily with sequence similarity 101, member BNM_182705.2−2.1
ERCC2DNA excision repair protein 2NM_000400.3−2.1
CCND2Cyclin D2NM_001759.3−2.1
HLA-BMajor histocompatibility complex, class I, BNM_005514.6−2.1
PDE2APhosphodiesterase 2A, cGMP-stimulatedNM_002599.4−2.1
AKAP12A kinase anchor protein 12NM_005100.3−2.1
CLEC2BC-type lectin domain family 2, member BNM_005127.2−2.1
S100A4S100 calcuim binding protein A4NM_019554.2−2.1
SLC30A3Solute carrier family 30, member 3NM_003459.4−2.2
PLIN2Perilipin 2NM_001122.3−2.2
IL32Interleukin 32NM_001012633.1−2.2
TIMM22Translocase of inner mitochondrial membrane 22 homologNM_013337.2−2.2
SYNMSynemin, intermediate filament proteinNM_015286.5−2.2
ADRB2Adrenergic, beta-2-, receptor surfaceNM_000024.5−2.2
PRR5Proline rich 5NM_001017529.2−2.2
CFIComplement factor INM_000204.3−2.3
COL1A1Collagen, type I, alpha 1NM_000088.3−2.3
CCL2Chemokine (C-C motif) ligand 2NM_002982.3−2.3
COL6A1Collagen, type VI, alpha 1NM_001848.2−2.3
GALNTL4GalNAc-T-like protein 4NM_198516.2−2.3
S100A3S100 calcuim binding protein A3NM_002960.1−2.4
ALDH1A1Aldehyde dehydrogenase 1 family, member A1NM_000689.4−2.4
TNFRSF14Tumor necosis factor receptor superfamily, member 14NM_003820.2−2.4
CAV1Caveolin 1NM_001753.4−2.4
LAMC2Laminin, gamma 2NM_005562.2−2.4
NOSTRINNitric oxide synthase traffickerNM_052946.3−2.4
CEACAM1Carcinoembryonic antigen-related cell adhesion molecule 1NM_001024912.2−2.4
CYYR1Cysteine/tyrosine-rich 1NM_052954.2−2.5
SLC22A23Solute carrier family 22, member 23NM_021945.5−2.5
ACSL5Acyl-CoA synthetase long-chain family member 5NM_016234.3−2.5
AADACArylacetamide deacetylaseNM_001086.2−2.6
COLEC12Collectin sub-family member 12NM_130386.2−2.6
RNASET2Ribonuclease T2NM_003730.4−2.6
PLA2G4CPhospholipase A2, group IVCNM_003706.2−2.6
SERPINB2Serpin peptidase inhibitor, clade B, member 2NM_002575.2−2.6
CETPCholesteryl ester transfer protein, plasmaNM_000078.2−2.7
PLA2G16Phospholipase A2, group XVINM_007069.3−2.7
TNFSF18Tumor necrosis factor superfamily, member 18NM_005092.3−2.8
CITED2Cbp/p300-interacting transactivator 2NM_006079.3−2.8
C10orf116Chromosome 10 open reading fame 116NM_006829.2−2.8
PROX1Prospero homeobox 1NM_002763.3−2.9
ZSCAN18Zinc finger and SCAN domain containing 18NM_023926.4−2.9
LEPREL1Leprecan-like 1NM_018192.3−2.9
CTSHCathepsin HNM_004390.3−2.9
KHDRBS3RNA-binding protein T-StarNM_006558.1−3.0
CDH11Cadherin 11, type 2, OB-cadherinNM_001797.2−3.1
DDIT4LDNA-damage-inducible transcript 4-likeNM_145244.3−3.2
NR5A2Nuclear receptor subfamily 5, group A, member 2NM_003822.3−3.3
ABCA3ATP-binding cassette, sub-family A, member 3NM_001089.2−3.3
MARCH2Membrane-associated ring finger 2NM_001005416.1−3.3
CDKN2ACyclin-dependent kinase inhibitor 2ANM_000077.4−3.3
MGPMatrix Gla proteinNM_000900.3−3.3
ALDH1A2Aldehyde dehydrogenase 1 family, member A2NM_170697.2−3.5
HOXB7Homeobox B7NM_004502.3−3.5
ANGPT2Angiopoietin 2NM_001147.2−3.5
GIMAP5GTPase, IMAP family member 5NM_018384.4−3.6
NDNNecdin homologNM_002487.2−3.8
TACSTD2Tumor associate calcuim signal transducer 2NM_002353.2−3.8
KRT19Keratin 19NM_002276.4−3.8
FAM174BFamily with sequence similarity 174, member BNM_207446.2−3.9
CECR1Cat eye syndrome chromosome region, candidate 1NM_177405.1−4.2
GPR116G protein-coupled receptor 116NM_015234.4−4.3
TNFRSF6BTumor necrosis factor superfamily, member 6b, decoyNM_032945.2−4.3
PIEZO2Piezo-type mechanosensitive ion channel component 2NM_022068.2−4.4
UCHL1Ubiquitin carboxyl-terminal esterase L1NM_004181.4−4.9
KBTBD11Kelch repeat and BTB domain containing 11NM_014867.2−5.3
HSD17B2Hydroxysteroid dehydrogenase 2NM_002153.2−8.4
LYVE1Lymphatic vessel endothelial hyaluronan receptor 1NM_006691.3−8.8
GYPCGlycophorin CNM_016815.3−22.6
MMP1Matrix metallopeptidase 1NM_002421.3−25.8
FABP4Fatty acid binding protein 4, adipocyteNM_001442.2−28.1
CLDN11Claudin 11NM_005602.5−36.9

We confirmed a small subset of these gene expression changes utilizing qPCR, revealing equivocal trends in gene expression between the microarray and qPCR data for ANGPT2, ANTXR1, SMARCE1, RGS5, CTAG2, LTBP2, CLDN11, and KISS1 (Table 2). Each of these genes has been firmly established to play critical roles in regulating angiogenesis and/or tumor progression [1017]. Missense mutations in ANTXR1 have been reported in several infantile hemangiomas and contribute to the constitutive VEGFR2 signaling associated with these tumors [18]. Mutations and signaling aberrations in Tie2, the cognate receptor for ANGPT2, play central roles in the development of various vascular disorders [19, 20]. ANGPT2 has previously been shown to be down-regulated in response to serum in HEMECs [19]. Interestingly, ANGPT2 expression is higher in HEMECs compared to normal placental endothelial cells and is increased in proliferative infantile hemangioma tumors relative to involuting ones [5]. Virtually undetectable in normal vasculature, RGS5 is greatly upregulated in the vasculature of solid tumors and may have the potential to serve as a tumor biomarker [12]. The downregulation of the metastasis suppressor KISS1 that we observed in HEMECs may partially explain the locally aggressive properties of infantile hemangiomas, as this gene encodes an angiogenic suppressor [16, 21]. Moreover, the expression of KISS1 is markedly reduced in aggressive metastatic melanomas and breast cancers, and this loss of expression contributes to the metastatic phenotype of these cells [17, 22]. It is intriguing that such genes (particularly the cancer-specific genes) are aberrantly expressed in HEMECs, and undoubtedly their deregulation could potentiate aberrant vascular tumor states. As it has been proposed that infantile hemangiomas may be derived from motile placental-derived chorangioma cells [2], future genomics analysis should compare the transcriptomes of each tumor type to identify if aberrant expression of tumor-related genes is shared between the tissues.

Table 2   ( Jump back to reading )

qPCR confirmation of a subset of gene expression changes in HEMECs compared to HDMVECs
GeneExpression Δ
RGS592.4 ± 11.2
CTAG239.9 ± 4.8
SMARCE14.4 ± 1.4
LTBP23.3 ± 0.5
ANGPT2−2.1 ± 0.3
KISS1−2.5 ± 0.4
ANTXR1−2.8 ± 0.4
CLDN11−10.0 ± 0.9

Overexpression of the CTAG2 cancer/testis antigen in a panel of infantile hemangioma tumors

In our microarray analysis, the cancer/testis antigen CTAG2 displayed the highest upregulation of mRNA expression in HEMECs compared to the HDMECs. This gene, whose function is completely unknown, has been shown to be significantly increased in several metastatic cancers, and is actively being researched as a target of immune therapy for aggressive cancers [2329]. If CTAG2 is preferentially upregulated in infantile hemangiomas, it is possible that treatment of disfiguring or life threatening infantile hemangioma tumors could employ immune therapy against this antigen. Furthermore, CTAG2 is reported to be a target for antigen-specific T-cells in patients with various metastatic tumors [29, 30]. A recent study has shown that nearly half of the patients with spontaneous CTAG2-specific CD4(+) T cell responses had circulating CTAG2-specific antibodies that recognized epitopes located in the C-terminal portion of CTAG2 [30]. As involution of infantile hemangiomas is believed to be due in part to an immune mediated attack on the tumor itself [4], it is possible that T-cell targeting of the overexpressed CTAG2 protein could contribute to this process. We confirmed our microarray data at the protein level by performing immunohistochemistry on a panel of 16 paraffin embedded infantile hemangioma tumors representing both the proliferating and involuting stages of the disease and 4 normal neonatal dermal tissues. A limited amount of CTAG2 expression was observed in the normal dermal tissues (a few nerve cells and bundles present staining, whereas the fibroblasts and collagen fibers are negative), and despite this gene being coined a “cancer/testis specific antigen”, analysis of publically available microarray datasets suggests this gene is expressed at a low level across a large number of tissues ( and it has been reported in the literature to be expressed in the placenta and ovary [31]. In proliferating tumors (composed of densely proliferating endothelial cells), we observed intense CTAG2 staining in the endothelial cells for all sections analyzed (Figure 2). In contrast, involuting tumors (marked by substantial adipocyte deposits—a characteristic of the later stages in the development of this tumor [32]) exhibited significantly reduced levels of CTAG2 staining. As Calicchio et al. did not detect significant differences in CTAG2 expression between microdissected endothelial cells from proliferating and involuting infantile hemangiomas and the staining intensity of individual blood vessels appears relatively constant between proliferating and involuting hemangiomas, we suspect that the reduced CTAG2 staining in involuting tumors is most likely due to reductions in tumor vascular density but not changes in gene transcription.

Figure 2   ( Jump back to reading )

Figure 2 caption

Detection of CTAG2 protein levels in infantile hemangioma tissues. Proliferating and involuting infantile hemangioma tissues as well as normal neonatal foreskin tissues were cut from paraffin blocks, incubated with antibodies against CTAG2, and detected using alkaline phosphatase staining (). Immunohistochemistry () controls included incubations without CTAG2 antibody () and with CTAG2 antibody () in thin sections from metastatic breast cancer. All images were obtained at 100X total magnification.


Our data indicate that global transcriptional expression patterns are markedly unique between pure cultures of HDMVECs and HEMECs with major alterations in cell cycle, adhesion, and arachidonic acid metabolism genes. Though considered benign, HEMECs showed surprising aberrant regulation in the expression of several genes involved in tumor progression. Our finding that CTAG2 is highly expressed in infantile hemangiomas may lead to the development of immune-mediated therapies against infantile hemangiomas.

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  1. North PE, Waner M, Mizeracki A, Mihm MC.  GLUT1: a newly discovered immunohistochemical marker for juvenile hemangiomas. Hum Pathol. 2000;31:11-22.
  2. Mihm MC, Nelson JS.  Hypothesis: the metastatic niche theory can elucidate infantile hemangioma development. J Cutan Pathol. 2010;37(Suppl 1):83-87.
  3. Barnes CM, Huang S, Kaipainen A, Sanoudou D, Chen EJ, Eichler GS, Guo Y, Yu Y, Ingber DE, Mulliken JB, Beggs AH, Folkman J, Fishman SJ.  Evidence by molecular profiling for a placental origin of infantile hemangioma. Proc Natl Acad Sci USA. 2005;102:19097-19102.
  4. Ritter MR, Moreno SK, Dorrell MI, Rubens J, Ney J, Friedlander DF, Bergman J, Cunningham BB, Eichenfield L, Reinisch J, Cohen S, Veccione T, Holmes R, Friedlander SF, Friedlander M.  Identifying potential regulators of infantile hemangioma progression through large-scale expression analysis: a possible role for the immune system and indoleamine 2,3 dioxygenase (IDO) during involution. Lymphat Res Biol. 2003;1:291-299.
  5. Calicchio ML, Collins T, Kozakewich HP.  Identification of signaling systems in proliferating and involuting phase infantile hemangiomas by genome-wide transcriptional profiling. Am J Pathol. 2009;174:1638-1649.
  6. Boye E, Yu Y, Paranya G, Mulliken JB, Olsen BR, Bischoff J.  Clonality and altered behavior of endothelial cells from hemangiomas. J Clin Invest. 2001;107:745-752.
  7. Stiles J, Amaya C, Pham R, Rowntree RK, Lacaze M, Mulne A, Bischoff J, Kokta V, Boucheron LE, Mitchell DC, Bryan BA.  Propranolol treatment of infantile hemangioma endothelial cells: A molecular analysis. Exp Ther Med. 2012;4:594-604.
  8. Khan ZA, Melero-Martin JM, Wu X, Paruchuri S, Boscolo E, Mulliken JB, Bischoff J.  Endothelial progenitor cells from infantile hemangioma and umbilical cord blood display unique cellular responses to endostatin. Blood. 2006;108:915-921.
  9. Pfister SL, Gauthier KM, Campbell WB.  Vascular pharmacology of epoxyeicosatrienoic acids. Adv Pharmacol. 2010;60:27-59.
  10. Fagiani E, Christofori G.  Angiopoietins in angiogenesis. Cancer Lett. 2013;328:18-26.
  11. Chaudhary A, St Croix B.  Selective blockade of tumor angiogenesis. Cell Cycle. 2012;11:2253-2259.
  12. Silini A, Ghilardi C, Figini S, Sangalli F, Fruscio R, Dahse R, Pedley RB, Giavazzi R, Bani M.  Regulator of G-protein signaling 5 (RGS5) protein: a novel marker of cancer vasculature elicited and sustained by the tumor's proangiogenic microenvironment. Cell Mol Life Sci. 2012;69:1167-1178.
  13. Garcia-Pedrero JM, Kiskinis E, Parker MG, Belandia B.  The SWI/SNF chromatin remodeling subunit BAF57 is a critical regulator of estrogen receptor function in breast cancer cells. J Biol Chem. 2006;281:22656-22664.
  14. Lethe B, Lucas S, Michaux L, De Smet C, Godelaine D, Serrano A, De Plaen E, Boon T.  LAGE-1, a new gene with tumor specificity. Int J Cancer. 1998;76:903-908.
  15. Wessells H, Sullivan CJ, Tsubota Y, Engel KL, Kim B, Olson NE, Thorner D, Chitaley K.  Transcriptional profiling of human cavernosal endothelial cells reveals distinctive cell adhesion phenotype and role for claudin 11 in vascular barrier function. Physiol Genomics. 2009;39:100-108.
  16. Cho SG, Yi Z, Pang X, Yi T, Wang Y, Luo J, Wu Z, Li D, Liu M.  Kisspeptin-10, a KISS1-derived decapeptide, inhibits tumor angiogenesis by suppressing Sp1-mediated VEGF expression and FAK/Rho GTPase activation. Cancer Res. 2009;69:7062-7070.
  17. Mitchell DC, Stafford LJ, Li D, Bar-Eli M, Liu M.  Transcriptional regulation of KiSS-1 gene expression in metastatic melanoma by specificity protein-1 and its coactivator DRIP-130. Oncogene. 2007;26:1739-1747.
  18. Jinnin M, Medici D, Park L, Limaye N, Liu Y, Boscolo E, Bischoff J, Vikkula M, Boye E, Olsen BR.  Suppressed NFAT-dependent VEGFR1 expression and constitutive VEGFR2 signaling in infantile hemangioma. Nat Med. 2008;14:1236-1246.
  19. Yu Y, Varughese J, Brown LF, Mulliken JB, Bischoff J.  Increased Tie2 expression, enhanced response to angiopoietin-1, and dysregulated angiopoietin-2 expression in hemangioma-derived endothelial cells. Am J Pathol. 2001;159:2271-2280.
  20. London NR, Whitehead KJ, Li DY.  Endogenous endothelial cell signaling systems maintain vascular stability. Angiogenesis. 2009;12:149-158.
  21. Ramaesh T, Logie JJ, Roseweir AK, Millar RP, Walker BR, Hadoke PW, Reynolds RM.  Kisspeptin-10 inhibits angiogenesis in human placental vessels ex vivo and endothelial cells in vitro. Endocrinology. 2010;151:5927-5934.
  22. Mitchell DC, Abdelrahim M, Weng J, Stafford LJ, Safe S, Bar-Eli M, Liu M.  Regulation of KiSS-1 metastasis suppressor gene expression in breast cancer cells by direct interaction of transcription factors activator protein-2alpha and specificity protein-1. J Biol Chem. 2006;281:51-58.
  23. Odunsi K, Jungbluth AA, Stockert E, Qian F, Gnjatic S, Tammela J, Intengan M, Beck A, Keitz B, Santiago D, Williamson B, Scanlan MJ, Ritter G, Chen YT, Driscoll D, Sood A, Lele S, Old LJ.  NY-ESO-1 and LAGE-1 cancer-testis antigens are potential targets for immunotherapy in epithelial ovarian cancer. Cancer Res. 2003;63:6076-6083.
  24. Zeng G, Aldridge ME, Wang Y, Pantuck AJ, Wang AY, Liu YX, Han Y, Yuan YH, Robbins PF, Dubinett SM, deKernion JB, Belldegrun AS.  Dominant B cell epitope from NY-ESO-1 recognized by sera from a wide spectrum of cancer patients: implications as a potential biomarker. Int J Cancer. 2005;114:268-273.
  25. Kan T, Yamasaki S, Kondo K, Teratani N, Kawabe A, Kaganoi J, Meltzer SJ, Imamura M, Shimada Y.  A new specific gene expression in squamous cell carcinoma of the esophagus detected using representational difference analysis and cDNA microarray. Oncology. 2006;70:25-33.
  26. Shao Y, Sun ZY, Sun SW, Zhao Y, Sin WY, Yuan YH, Simpson AJ, Old LJ, Sang XT, Mao YL, Xie Y, Huang JF, Zhao HT.  Identification and expression analysis of novel LAGE-1 alleles with single nucleotide polymorphisms in cancer patients. J Cancer Res Clin Oncol. 2008;134:495-502.
  27. Andrade VC, Vettore AL, Felix RS, Almeida MS, Carvalho F, Oliveira JS, Chauffaille ML, Andriolo A, Caballero OL, Zago MA, Colleoni GW.  Prognostic impact of cancer/testis antigen expression in advanced stage multiple myeloma patients. Cancer Immun. 2008;8:2-.
  28. Wang XY, Chen HS, Luo S, Zhang HH, Fei R, Cai J.  Comparisons for detecting NY-ESO-1 mRNA expression levels in hepatocellular carcinoma tissues. Oncol Rep. 2009;21:713-719.
  29. Pollack SM, Li Y, Blaisdell MJ, Farrar EA, Chou J, Hoch BL, Loggers ET, Rodler E, Eary JF, Conrad EU, Jones RL, Yee C.  NYESO-1/LAGE-1s and PRAME are targets for antigen specific T cells in chondrosarcoma following treatment with 5-Aza-2-deoxycitabine. PLoS One. 2012;7:e32165-.
  30. Kudela P, Sun Z, Fourcade J, Janjic B, Kirkwood JM, Maillere B, Zarour HM.  Epitope hierarchy of spontaneous CD4+ T cell responses to LAGE-1. J Immunol. 2011;186:312-322.
  31. Kalejs M, Erenpreisa J.  Cancer/testis antigens and gametogenesis: a review and "brain-storming" session. Cancer Cell Int. 2005;5:4-.
  32. Kleiman A, Keats EC, Chan NG, Khan ZA.  Evolution of hemangioma endothelium. Exp Mol Pathol. 2012;93:264-272.
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