Open Access

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

  • Jessica M Stiles1,
  • Rebecca K Rowntree1,
  • Clarissa Amaya1,
  • Dolores Diaz1,
  • Victor Kokta2,
  • Dianne C Mitchell1 and
  • Brad A Bryan1Email author
Vascular Cell20135:6

https://doi.org/10.1186/2045-824X-5-6

Received: 24 January 2013

Accepted: 13 March 2013

Published: 25 March 2013

Abstract

Background

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).

Methods

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.

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.

Conclusion

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.

Introduction

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.

Immunofluorescence

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.

Immunohistochemistry

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

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 (red), p-FAK (green), and nucleus (blue). (red asterisks for panels A &B represent statistically significant values [p<0.05] as determined by Student’s t-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

Fold changes in mRNA expression levels of genes in HEMECs compared to HDMVECs

Gene symbol

Gene name

Accession number

FC

CTAG2

Cancer/testis antigen 2

NM_020994.3

11.6

IL13RA2

Interleukin 13 Receptor, alpha 2

NM_000640.2

10.7

IFI27

Interferon, alpha-inducible protein 27

NM_005532.3

8.3

TPM2

Tropomyosin 2 (beta)

NM_213674.1

7.8

RPL14

Ribosomal protein L14

NM_001034996.1

6.6

CCNA1

Cyclin A1

NM_003914.3

6.6

RGS5

G-protein signaling 5 regulator

NM_003617.3

6.0

FBN2

Fibrillin 2

NM_001999.3

5.9

D4S234E

DNA segment on chromosome 4 (unique)

NM_001040101.1

5.5

BST2

Bone marrow stromal cell antigen 2

NM_004335.2

5.1

QPCT

Glutaminyl-peptide cyclotransferase

NM_012413.3

4.8

TNFSF4

Tumor necrosis factor (ligand) superfamily, member 4

NM_003326.3

4.6

RGS5

Regulator of G-protein signaling 5

NM_003617.3

4.6

SPOCK1

Sparc/osteonectin, cwcv and kazal-like domains proteoglycan 1

NM_004598.3

4.6

SNHG8

Small nucleolar RNA host gene 8 (non-protein coding)

NR_003584.3

4.6

ANTXR1

Anthrax toxin receptor 1

NM_032208.2

4.5

CHST1

Carbohydrate sulfotransferase 1

NM_003654.5

4.5

MPZL2

Myelin protein zero-like 2

NM_005797.3

4.4

HEY2

Hairy/enhancer-of-spilt related with YRPW motif 2

NM_012259.2

4.3

SLITRK4

SLIT and NTRK-like family, member 4

NM_173078.3

4.2

SHISA2

Shisa homolog 2

NM_001007538.1

4.0

LRRC17

Leucine rich repeat containing 17, TV2

NM_005824.2

3.9

NUDT11

Nudix-type motif 11

NM_018159.3

3.8

RNASE1

Ribonuclease, Rnase A family, 1, TV1

NM_198235.2

3.7

SERPINE2

Serpin peptidase inhibitor, clade E, member 2

NM_006216.3

3.6

LIPG

Lipase, endothelial

NM_006033.2

3.4

PCSK5

Proprotein convertase subtilisin/kexin type 5

NM_006200.3

3.4

LPXN

Leupaxin

NM_004811.2

3.3

CXCR4

Chmeokine (C-X-C motif) receptor 4, TV2

NM_003467.2

3.2

TMEM200A

Transmembrane protein 200A

NM_052913.2

3.1

CXCR4

Chemokine (C-X-C motif) receptor 4, TV1

NM_001008540.1

3.1

RAB34

RAB34, member RAS onogene family

NM_031934.5

3.0

DPYSL3

Dihydropyrimidinase-like 3

NM_001387.2

2.9

FBXL13

F-box and leucine-rich repeat protein 13

NM_145032.3

2.9

PNMA2

Paraneoplastic Ma antigen 2

NM_007257.5

2.9

LOC440354

LOC440354

NR_002473.2

2.9

NLGN1

Neuroligin 1

NM_014932.2

2.8

DDIT4

DNA-damage-inducible transcript 4

NM_019058.2

2.8

PFN2

Profilin 2

NM_053024.3

2.8

GABBR2

Gamma-aminobutyric acid B receptor, 2

NM_005458.7

2.8

MEIS2

Meis homeobox 2

NM_172315.2

2.7

PMEPA1

Prostate transmembrane protein, androgen induced 1

NM_199169.2

2.7

LOC647307

LOC647308

XR_039752.1

2.7

PLEK2

Pleckstrin 2

NM_016445.1

2.7

CARD11

Caspase recruitment domain family, member 11

NM_032415.4

2.6

SNORD13

Small nucleolar RNA, C/D box 13, small nucleolar RNA

NR_003041.1

2.6

GFPT2

Glutamine-fructoce-6-phosphate transaminase 2

NM_005110.2

2.6

FAP

Fibroblast activation protein, alpha

NM_004460.2

2.6

OCIAD2

OCIA domain containing 2, TV2

NM_152398.2

2.5

F2RL1

Coagulation factor II receptor-like 1

NM_005242.4

2.5

DSTYK

Dual serine/threonine and tyrosine protein kinase

NM_199462.2

2.5

LOC649497

LOC649498

XM_938576.1

2.5

LOC654194

LOC654195

XM_942669.1

2.5

NYNRIN

NYN domain and retroviral integrase containing

NM_025081.2

2.5

LOC387763

LOC387764

XM_941665.2

2.5

COL8A1

Collagen, type VIII, alpha 1

NM_020351.3

2.5

MGC39900

MGC39901

XM_936687.1

2.4

LTBP2

Latent transforming growth factor beta binding protein 2

NM_000428.2

2.4

RNASE1

Ribonuclease, Rnase A family, 1, TV3

NM_198232.2

2.4

IFI27L2

Interferon, alpha-inducible protein 27-like 2

NM_032036.2

2.4

SOX4

SRY (sex determining region Y)-box4

NM_003107.2

2.4

LRRC17

Leucine rich repeat containing 17, TV1

NM_001031692.2

2.3

DSE

Dermatan sulfate epimerase

NM_013352.2

2.3

CD44

CD44 molecule (Indian blood group), TV5

NM_001001392.1

2.3

LOC100131139

LOC100131140

XR_037336.1

2.3

CBS

Systathionine-beta-synthase

NM_000071.2

2.3

NT5DC2

5'-nucleotidase domain containing 2

NM_022908.2

2.3

NPFFR2

Neuropeptide FF receptor 2

NM_004885.2

2.3

LOC100129685

LOC100129686

XM_001723814.1

2.3

LXN

Latexin

NM_020169.3

2.3

MEX3B

Mex-3 homolog B

NM_032246.3

2.3

C1orf54

Chromosome 1 open reading frame 54

NM_024579.3

2.3

HDDC2

HD domain containing 2

NM_016063.2

2.3

LOC648823

LOC648824

XM_943477.1

2.3

CYB5A

Cytochrome b5 type A

NM_001914.3

2.3

PIR

Pirin (iron binding nuclear protein)

NM_001018109.2

2.3

GPR37

G protein-coupled receptor 37

NM_005302.2

2.3

PPAPDC1A

Phosphatidic acid phosphatase type 2 domain containing 1A

NM_001030059.1

2.3

CD44

CD44 molecule (Indian blood group), TV4

NM_001001391.1

2.2

LOC100131905

LOC100131906

XR_039334.1

2.2

CTAG1A

Cancer/testis antigen 1A

NM_139250.1

2.2

C4orf18

Chromosome 4 open reading frame 18

NM_016613.6

2.2

LDOC1

Leucine zipper, down-regulated in cancer 1

NM_012317.2

2.2

TGFBI

Transforming growth factor, beta-induced

NM_000358.2

2.2

COL5A2

Collagen, type V, alpha 2

NM_000393.3

2.2

NOX4

NADPH oxidase 4

NM_016931.3

2.2

TSHZ3

Teashirt zinc finger homeobox 3

NM_020856.2

2.2

FNDC3B

Fibronectin type III domain containing 3B, TV2

NM_001135095.1

2.2

KIT

V-kit

NM_001093772.1

2.2

ADAM19

ADAM metallopeptidase domain 19

NM_033274.3

2.2

JAM3

Junctional adhesion molecule 3

NM_032801.4

2.1

CGNL1

Cingulin-like 1

NM_032866.4

2.1

COL4A6

Collagen, type IV, alpha 6

NM_001847.2

2.1

BMX

BMX non-receptor tyrosine kinase

NM_001721.6

2.1

DUSP23

Dual specificity phosphatase 23

NM_017823.3

2.1

MMP2

Matrix metallopeptidase 2

NM_004530.4

2.1

NCAPD2

Non-SMC condensin I complex, subunit D2

NM_014865.3

2.1

CYBRD1

Cytochrome b reductase 1, TV1

NM_024843.2

2.1

FAM89A

Family with sequence similarity 89, member A

NM_198552.2

2.1

GAS6

Growth arrest-specific 6

NM_000820.2

2.1

S100A13

S100 calcium binding protein A13

NM_001024211.1

2.1

SMARCE1

SWI/SNF related, subfamily e, member 1

NM_003079.4

2.1

LOC643977

LOC643978

XM_932991.1

2.1

LFNG

O-fucosylpeptide 3-beta-N-acetylglucosaminyltransferase

NM_001040167.1

2.1

MTMR11

Myotubularin related protein 11

NM_181873.3

2.1

ITGA10

Integrin, alpha 10

NM_003637.3

2.1

PTGFRN

Prostaglandin F2 receptor negative regulator

NM_020440.2

2.0

LOC644936

Actin, beta pseudogene

NR_004845.1

2.0

CPS1

Carbamoyl-phosphate synthase 1, mitochonfrial

NM_001875.4

2.0

C18orf56

Chromosome 18 open reading frame 56

NM_001012716.2

2.0

ADA

Adenosine deaminase

NM_000022.2

2.0

NETO2

Neuropilin and tolliod-like2

NM_018092.4

2.0

DKFZp761P0423

DKFZp761P0424

XM_291277.4

2.0

STC2

Stanniocalcin 2

NM_003714.2

2.0

PRKAR1A

Protein kinase, cAMP-dependent, regulatory, type I, alpha

NM_002734.3

2.0

EGFLAM

EGF-like, fibronectin type III and laminin G domains

NM_182801.2

2.0

SPECC1

Sperm antigen with calponin homology, coiled-coil domains 1

NM_001033555.2

2.0

FNDC3B

Fibronectin type III domain containing 3B, TV1

NM_022763.3

2.0

THOC3

THO complex 3

NM_032361.2

2.0

COL5A1

Collagen, type V, alpha 1

NM_000093.3

2.0

LANCL1

LanC lantibiotic synthetase component C-like 1

NM_006055.2

2.0

OCIAD2

OCIA domain containing 2, TV1

NM_001014446.1

2.0

LRIG1

Leucine-rich repeats and immunoglobulin-like domains 1

NM_015541.2

2.0

HOXB2

Homeobox B2

NM_002145.3

2.0

TIMP1

TIMP metallopeptidase inhibitor 1

NM_003254.2

−2.0

NAAA

N-acylethanolamine acid amidase

NM_014435.3

−2.0

MAOA

Monoamine oxidase A

NM_000240.2

−2.0

MYOF

Myoferlin

NM_013451.3

−2.0

KISS1

KiSS metastasis-suppressor

NM_002256.3

−2.0

SLC25A22

Solute carrier family 25, member 22

NM_024698.5

−2.0

NOSIP

Nitric oxide synthase interacting protein

NM_015953.3

−2.0

COL1A2

Collagen, type I, alpha 2

NM_000089.3

−2.0

ZDHHC14

Zinc finger, DHHC-type containing 14

NM_024630.2

−2.0

HPCAL1

Hippocalcin-like 1

NM_134421.1

−2.0

VLDLR

Very low density lipoprotein receptor

NM_001018056.1

−2.0

LOC730525

LOC730525

XM_001126202.1

−2.0

BMP2

Bone morphogenetic protein 2

NM_001200.2

−2.0

ABLIM1

Actin binding LIM protein 1

NM_006720.3

−2.0

PIK3C2A

Phosphoinositide-3-kinase, class 2, alpha polypeptide

NM_002645.2

−2.0

IRF1

Interferon regulatory factor 1

NM_002198.2

−2.0

MBP

Myelin basic protein

NM_001025100.1

−2.0

PRKAR1B

Protein kinase, cAMP-dependent, regulatory type I, beta

NM_002735.2

−2.1

FAM101B

Family with sequence similarity 101, member B

NM_182705.2

−2.1

ERCC2

DNA excision repair protein 2

NM_000400.3

−2.1

CCND2

Cyclin D2

NM_001759.3

−2.1

HLA-B

Major histocompatibility complex, class I, B

NM_005514.6

−2.1

SYBU

Syntabulin

NM_001099743.1

−2.1

PDE2A

Phosphodiesterase 2A, cGMP-stimulated

NM_002599.4

−2.1

AKAP12

A kinase anchor protein 12

NM_005100.3

−2.1

CLEC2B

C-type lectin domain family 2, member B

NM_005127.2

−2.1

S100A4

S100 calcuim binding protein A4

NM_019554.2

−2.1

FST

Follistain

NM_013409.2

−2.2

SLC30A3

Solute carrier family 30, member 3

NM_003459.4

−2.2

PLIN2

Perilipin 2

NM_001122.3

−2.2

IL32

Interleukin 32

NM_001012633.1

−2.2

LOC100128252

LOC100128253

XM_001725603.1

−2.2

TIMM22

Translocase of inner mitochondrial membrane 22 homolog

NM_013337.2

−2.2

SYNM

Synemin, intermediate filament protein

NM_015286.5

−2.2

LOC729985

LOC729986

XM_001131964.1

−2.2

ADRB2

Adrenergic, beta-2-, receptor surface

NM_000024.5

−2.2

KIAA1274

KIAA1274

NM_014431.2

−2.2

PRR5

Proline rich 5

NM_001017529.2

−2.2

LOC387841

LOC387842

XM_932678.1

−2.3

CFI

Complement factor I

NM_000204.3

−2.3

LOC646836

LOC646837

XM_001718162.1

−2.3

COL1A1

Collagen, type I, alpha 1

NM_000088.3

−2.3

CCL2

Chemokine (C-C motif) ligand 2

NM_002982.3

−2.3

COL6A1

Collagen, type VI, alpha 1

NM_001848.2

−2.3

LOC201651

LOC201652

XR_017321.2

−2.3

GALNTL4

GalNAc-T-like protein 4

NM_198516.2

−2.3

S100A3

S100 calcuim binding protein A3

NM_002960.1

−2.4

ALDH1A1

Aldehyde dehydrogenase 1 family, member A1

NM_000689.4

−2.4

TNFRSF14

Tumor necosis factor receptor superfamily, member 14

NM_003820.2

−2.4

CAV1

Caveolin 1

NM_001753.4

−2.4

LAMC2

Laminin, gamma 2

NM_005562.2

−2.4

NOSTRIN

Nitric oxide synthase trafficker

NM_052946.3

−2.4

CEACAM1

Carcinoembryonic antigen-related cell adhesion molecule 1

NM_001024912.2

−2.4

CYYR1

Cysteine/tyrosine-rich 1

NM_052954.2

−2.5

SLC22A23

Solute carrier family 22, member 23

NM_021945.5

−2.5

ACSL5

Acyl-CoA synthetase long-chain family member 5

NM_016234.3

−2.5

AADAC

Arylacetamide deacetylase

NM_001086.2

−2.6

COLEC12

Collectin sub-family member 12

NM_130386.2

−2.6

KIAA1324L

KIAA1324-like

NM_152748.3

−2.6

RNASET2

Ribonuclease T2

NM_003730.4

−2.6

NXN

Nucleoredoxin

NM_022463.4

−2.6

PLA2G4C

Phospholipase A2, group IVC

NM_003706.2

−2.6

SERPINB2

Serpin peptidase inhibitor, clade B, member 2

NM_002575.2

−2.6

CETP

Cholesteryl ester transfer protein, plasma

NM_000078.2

−2.7

PLA2G16

Phospholipase A2, group XVI

NM_007069.3

−2.7

TNFSF18

Tumor necrosis factor superfamily, member 18

NM_005092.3

−2.8

CITED2

Cbp/p300-interacting transactivator 2

NM_006079.3

−2.8

C10orf116

Chromosome 10 open reading fame 116

NM_006829.2

−2.8

PROX1

Prospero homeobox 1

NM_002763.3

−2.9

PALM

Paralemmin

NM_002579.2

−2.9

ZSCAN18

Zinc finger and SCAN domain containing 18

NM_023926.4

−2.9

LEPREL1

Leprecan-like 1

NM_018192.3

−2.9

CTSH

Cathepsin H

NM_004390.3

−2.9

KHDRBS3

RNA-binding protein T-Star

NM_006558.1

−3.0

CDH11

Cadherin 11, type 2, OB-cadherin

NM_001797.2

−3.1

DDIT4L

DNA-damage-inducible transcript 4-like

NM_145244.3

−3.2

GAPDHL6

GAPDHL7

XM_001726954.1

−3.2

NR5A2

Nuclear receptor subfamily 5, group A, member 2

NM_003822.3

−3.3

ABCA3

ATP-binding cassette, sub-family A, member 3

NM_001089.2

−3.3

MARCH2

Membrane-associated ring finger 2

NM_001005416.1

−3.3

CDKN2A

Cyclin-dependent kinase inhibitor 2A

NM_000077.4

−3.3

MGP

Matrix Gla protein

NM_000900.3

−3.3

ALDH1A2

Aldehyde dehydrogenase 1 family, member A2

NM_170697.2

−3.5

HOXB7

Homeobox B7

NM_004502.3

−3.5

EMCN

Endomucin

NM_016242.3

−3.5

ANGPT2

Angiopoietin 2

NM_001147.2

−3.5

GIMAP5

GTPase, IMAP family member 5

NM_018384.4

−3.6

NDN

Necdin homolog

NM_002487.2

−3.8

TACSTD2

Tumor associate calcuim signal transducer 2

NM_002353.2

−3.8

KRT19

Keratin 19

NM_002276.4

−3.8

FAM174B

Family with sequence similarity 174, member B

NM_207446.2

−3.9

CECR1

Cat eye syndrome chromosome region, candidate 1

NM_177405.1

−4.2

GPR116

G protein-coupled receptor 116

NM_015234.4

−4.3

TNFRSF6B

Tumor necrosis factor superfamily, member 6b, decoy

NM_032945.2

−4.3

PIEZO2

Piezo-type mechanosensitive ion channel component 2

NM_022068.2

−4.4

UCHL1

Ubiquitin carboxyl-terminal esterase L1

NM_004181.4

−4.9

KBTBD11

Kelch repeat and BTB domain containing 11

NM_014867.2

−5.3

LOC375295

LOC375296

XM_374020.4

−5.5

HSD17B2

Hydroxysteroid dehydrogenase 2

NM_002153.2

−8.4

LYVE1

Lymphatic vessel endothelial hyaluronan receptor 1

NM_006691.3

−8.8

PDPN

Podoplanin

NM_001006625.1

−15.8

GYPC

Glycophorin C

NM_016815.3

−22.6

MMP1

Matrix metallopeptidase 1

NM_002421.3

−25.8

FABP4

Fatty acid binding protein 4, adipocyte

NM_001442.2

−28.1

CLDN11

Claudin 11

NM_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

qPCR confirmation of a subset of gene expression changes in HEMECs compared to HDMVECs

Gene

Expression Δ

RGS5

92.4 ± 11.2

CTAG2

39.9 ± 4.8

SMARCE1

4.4 ± 1.4

LTBP2

3.3 ± 0.5

ANGPT2

−2.1 ± 0.3

KISS1

−2.5 ± 0.4

ANTXR1

−2.8 ± 0.4

CLDN11

−10.0 ± 0.9

p≤0.05 for all values.

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 (http://www.biogps.org) 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

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 (red). Immunohistochemistry (IHC) controls included incubations without CTAG2 antibody (negative control) and with CTAG2 antibody (positive control) in thin sections from metastatic breast cancer. All images were obtained at 100X total magnification.

Conclusion

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.

Declarations

Authors’ Affiliations

(1)
Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center
(2)
Department of Pathology, CHU Sainte-Justine, University of Montreal

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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.