Identification of Signaling Systems in Proliferating and Involuting Phase Infantile Hemangiomas by Genome-Wide Transcriptional Profiling
2009; Elsevier BV; Volume: 174; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2009.080517
ISSN1525-2191
AutoresMonica L. Calicchio, Tucker Collins, Harry P. Kozakewich,
Tópico(s)Renal and related cancers
ResumoInfantile hemangiomas are characterized by rapid capillary growth during the first year of life followed by involution during early childhood. The natural history of these lesions creates a unique opportunity to study the changes in gene expression that occur in the vessels of these tumors as they proliferate and regress. Here we use laser capture microdissection and genome-wide transcriptional profiling of vessels from proliferating and involuting hemangiomas to identify differentially expressed genes. Relative to normal placental vessels, proliferating hemangiomas were characterized by increased expression of genes involved in endothelial-pericyte interactions, such as angiopoietin-2 (ANGPT2), jagged-1 (JAG1), and notch-4 (NOTCH4), as well as genes involved in neural and vascular patterning, such as neuropilin-2 (NETO2), a plexin domain containing receptor (plexinC1), and an ephrin receptor (EPHB3). Insulin-like growth factor binding protein-3 (IGFBP3) was down-regulated in proliferating hemangiomas. Involuting hemangiomas were characterized by the expression of chronic inflammatory mediators, such as the chemokine, stromal cell-derived factor-1 (SDF-1), and factors that may attenuate the angiogenic response, such as a member of the Down syndrome critical region (DSCR) family. The identification of genes differentially expressed in proliferating and involuting hemangiomas in vivo will contribute to our understanding of this vascular lesion, which remains a leading cause of morbidity in newborn children. Infantile hemangiomas are characterized by rapid capillary growth during the first year of life followed by involution during early childhood. The natural history of these lesions creates a unique opportunity to study the changes in gene expression that occur in the vessels of these tumors as they proliferate and regress. Here we use laser capture microdissection and genome-wide transcriptional profiling of vessels from proliferating and involuting hemangiomas to identify differentially expressed genes. Relative to normal placental vessels, proliferating hemangiomas were characterized by increased expression of genes involved in endothelial-pericyte interactions, such as angiopoietin-2 (ANGPT2), jagged-1 (JAG1), and notch-4 (NOTCH4), as well as genes involved in neural and vascular patterning, such as neuropilin-2 (NETO2), a plexin domain containing receptor (plexinC1), and an ephrin receptor (EPHB3). Insulin-like growth factor binding protein-3 (IGFBP3) was down-regulated in proliferating hemangiomas. Involuting hemangiomas were characterized by the expression of chronic inflammatory mediators, such as the chemokine, stromal cell-derived factor-1 (SDF-1), and factors that may attenuate the angiogenic response, such as a member of the Down syndrome critical region (DSCR) family. The identification of genes differentially expressed in proliferating and involuting hemangiomas in vivo will contribute to our understanding of this vascular lesion, which remains a leading cause of morbidity in newborn children. Infantile hemangiomas are extremely common tumors, affecting ∼4 to 10% of all infants.1North PE Waner M Buckmiller L James CA Mihm MC Vascular tumors of infancy and childhood: beyond capillary hemangioma.Cardiovasc Pathol. 2006; 15: 302-317Abstract Full Text Full Text PDF Scopus (94) Google Scholar, 2Waner M North PE Scherer KA Frieden IJ Waner A Mihm MC The nonrandom distribution of facial hemangiomas.Arch Dermatol. 2003; 139: 869-875Crossref PubMed Scopus (238) Google Scholar, 3Mulliken JB Fishman SJ Burrows PE Vascular anomalies.Curr Probl Surg. 2000; 37: 517-584Abstract Full Text PDF PubMed Google Scholar The lesions have unique growth characteristics, typically being absent or barely noticeable at birth, followed by a period of rapid growth and subsequent involution. The proliferating phase is characterized by abundant immature endothelial cells and adjacent pericyte-like cells (Figure 1A), whereas the involuting phase is characterized by fewer and larger capillary-like vessels surrounded by connective tissue (Figure 1B). Recent studies provide some insights into the pathogenesis of these vascular tumors.4Brouillard P Vikkula M Genetic causes of vascular malformations.Hum Mol Genet. 2007; 16: R140-R149Crossref PubMed Scopus (195) Google Scholar First, hemangiomas are clonal lesions. Evidence of nonrandom X inactivation in endothelial cells was obtained from endothelial cells cultured from infantile hemangiomas.5Boye E Yu Y Pafranya G Mulliken JB Olsen BR Bischoff J Clonality and altered behavior of endothelial cells from hemangiomas.J Clin Invest. 2001; 107: 745-752Crossref PubMed Scopus (262) Google Scholar Similarly, evidence of clonality was seen in cells obtained directly from proliferating phase infantile hemangiomas.6Walter JW North PE Waner M Mizeracki A Blei F Walker JW Reinisch JF Marchuk DA Somatic mutation of vascular endothelial growth factor receptors in juvenile hemangiomas.Genes Chromosom Cancer. 2002; 33: 295-303Crossref PubMed Scopus (179) Google Scholar Although the vast majority of infantile hemangiomas occur sporadically,7Berg JN Walter JW Thisanagayam U Evans M Blei F Waner M Diamond AG Marchuk DA Porteous ME Evidence for loss of heterozygosity of 5q in sporadic haemangiomas: are somatic mutations involved in haemangioma formation?.J Clin Pathol. 2001; 54: 249-252Crossref PubMed Scopus (48) Google Scholar several kindreds have been identified to segregate as an autosomal dominant trait with high penetrance. Fibroblast growth factor receptor-4 (FGFR4), platelet-derived growth factor receptor-β (PDGFRB), and Flt-4 have been suggested as candidate genes for familial infantile hemangioma.8Walter JW Blei F Anderson JL Orlow SJ Speer MC Marchuk DA Genetic mapping of a novel familial form of infantile hemangioma.Am J Med Genet. 1999; 82: 77-83Crossref PubMed Scopus (147) Google Scholar Also, somatic missense mutations in the kinase insert of the vascular endothelial growth factor receptor-2 (VEGFR2 or FLK1/KDR) gene and the VEGFR3 (FLT4) gene have been found.6Walter JW North PE Waner M Mizeracki A Blei F Walker JW Reinisch JF Marchuk DA Somatic mutation of vascular endothelial growth factor receptors in juvenile hemangiomas.Genes Chromosom Cancer. 2002; 33: 295-303Crossref PubMed Scopus (179) Google Scholar Recently, in some individuals with infantile hemangioma, germline mutations have been identified in the genes encoding VEGFR2 (KDR) and TEM8 (ANTXR1). These mutations in infantile hemangioma result in low VEGFR1 expression, VEGF-dependent activation of VEGFR2 and its downstream signaling pathways, and endothelial proliferation.9Jinnin 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: 1236-1246Crossref PubMed Scopus (288) Google Scholar Second, endothelial progenitor cells, in small numbers, are found in proliferating hemangiomas. Early proliferating hemangiomas contain endothelial cells that express CD133 and CD34, markers for endothelial precursor cells, suggesting that the endothelial cells in proliferating hemangiomas may be arrested at an immature stage of vascular development.10Yu Y Flint AF Mulliken JB Wu JK Bischoff J Endothelial progenitor cells in infantile hemangioma.Blood. 2004; 103: 1373-1375Crossref PubMed Scopus (172) Google Scholar, 11Dadras SS North PE Bertoncini J Mihm MC Detmar M Infantile hemangiomas are arrested in an early developmental vascular differentiation state.Mod Pathol. 2004; 17: 1068-1079Crossref PubMed Scopus (98) Google Scholar Third, some aspects of hemangioma resemble that seen in the placenta. An unexpected set of tissue-specific markers co-expressed by both infantile hemangiomas at all stages of their evolution and placental vessels has suggested that the vasculature of the placenta and hemangioma may share a common cellular origin.12North PE Waner M Mizeracki A Mrak RE A unique microvascular phenotype shared by juvenile hemangiomas and human placenta.Arch Dermatol. 2001; 137: 559-570Crossref PubMed Google Scholar, 13North PE Waner M Brodsky MC Are infantile hemangiomas of placental origin?.Ophthalmology. 2002; 109: 223-224Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar Despite the number of studies on proliferating hemangioma, there is little information on the molecular mechanisms of involution. Once hemangiomas reach their maximum size, they spontaneously regress in a process that is characterized by endothelial apoptosis,14Iwata J Sonobe H Furihata M Ido E Ohtsuki Y High frequency of apoptosis in infantile capillary haemangioma.J Pathol. 1996; 179: 403-408Crossref PubMed Scopus (46) Google Scholar, 15Razon MJ Kraling BM Mulliken JB Bischoff J Increased apoptosis coincides with onset of involution in infantile hemangioma.Microcirculation. 1998; 5: 189-195Crossref PubMed Scopus (152) Google Scholar dilation of vascular lumina, flattening of endothelial cells, loss of mitotic activity, thickening of basement membranes, and drop-out of lesional capillaries (Figure 1B). The maturation process results in an involuting hemangioma that consists of large capillary-like vessels dispersed in fibroadipose tissue. Mesenchymal stem cells present in the tumor may give rise to the adipocyte-like cells found in the involuting phase hemangioma.16Yu Y Fuhr J Boye E Gyorffy S Soker S Atala A Mulliken JB Bischoff J Mesenchymal stem cells in adipogenesis in hemangioma involution.Stem Cells. 2006; 24: 1605-1612Crossref PubMed Scopus (104) Google Scholar The natural evolution of infantile hemangiomas creates a unique opportunity to study the changes in gene expression that take place as the vessels of these tumors proliferate and then regress. Initial expression analysis has been used to try and identify potential regulators of hemangioma progression. These studies identified factors such as insulin-like growth factor-2 (IGF-2) and several interferon inducible genes as potentially important regulators of hemangioma growth and involution.17Ritter MR Dorrell MI Edmonds J Friedlander SF Friedlander M Insulin-like growth factor 2 and potential regulators of hemangioma growth and involution identified by large-scale expression analysis.Proc Natl Acad Sci USA. 2002; 99: 7455-7460Crossref PubMed Scopus (143) Google Scholar, 18Ritter 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: 291-299Crossref PubMed Scopus (43) Google Scholar Additionally, molecular profiling suggested a placental origin for infantile hemangioma.19Barnés CM Huang S Kaipainen A Sanoudou D Chen EJ Eichler GS Guo TY Yu Y Ingber DE Mulliken JB Beggs AM Folkman J Fishman SJ Evidence by molecular profiling for a placental origin of infantile hemangioma.Proc Natl Acad Sci USA. 2005; 102: 19097-19102Crossref PubMed Scopus (150) Google Scholar However, none of these studies used genome wide arrays and typically involved preparing transcript from random samples of the resected tumor. To more precisely define patterns of gene expression in proliferating and involuting hemangiomas, we used laser capture microdissection (LCM) to selectively isolate the lesional vessels and avoid missing molecular events that are masked by studying random samples from the resected specimen.20Bonner RF Emmert-Buck M Cole K Pohida T Chuaqui R Goldstein S Liotta LA Laser capture microdissection: molecular analysis of tissue.Science. 1997; 278: 1481-1483Crossref PubMed Scopus (793) Google Scholar The gene profiling approach with LCM-captured vascular cells provided new insights into some of the signaling pathways that are associated with proliferating and involuting hemangiomas. Keeping in mind the age of the patient, cases were selected such that identification of genes involved in the early proliferative and early- and mid-involutive phases would be enriched, in an attempt not to miss molecular events. Infants with proliferating hemangioma were under the age of 12 months and children with involuting hemangioma were 2 to 7 years. To determine the pattern of gene expression in the hemangiomas, blocks containing formalin-fixed, paraffin-embedded lesions were screened for useable RNA. In an initial quality control step, blocks were screened for RNA quantity and quality using a quantitative real-time polymerase chain reaction (PCR) assay with primers designed to two amplicons on the β-actin gene, following the manufacturer's recommended protocol (Molecular Devices, Mountain View, CA). The 3′ amplicon is designed from a region ∼100 nucleotides and the 5′ amplicon ∼400 nucleotides from the poly A tail, respectively. The assumption in this screen is that the 3′ amplicon represents an indication of RNA quantity and a ratio of 3′:5′ amplicons represents a qualitative indicator of transcript length and transcribability. The samples represent the average status (i.e., length and transcribability) of other RNA molecules in the same block. This assay was performed on a Mx3000p real-time PCR thermal cycler (Stratagene, La Jolla, CA) using the Brilliant SYBR Green QPCR Master Mix (Stratagene). Dilutions of Human Universal RNA (Stratagene) are run from which a standard curve is used to estimate the amount of RNA in the sample. The assay measures the average β-actin cDNA length by quantification of the PCR product yield from the 3′ end and compares this yield to a relatively 5′ sequence. The following primer sequences are used: 3′ primers: HBAC1650: 5′-TCCCCCAACTTGAGATGTATGAAG-3′, HBAC1717: 5′-AACTGGTCTCAAGTCAGTGTACAGG-3′, and HBAC1355: 5′-ATCCCCCAAAGTTCACAATG-3′, and HBAC1472: 5′-GTGGCTTTAGGATGGCAAG-3′. cDNA generated from the uRNA was serially diluted with polyI (Sigma-Aldrich, St. Louis, MO) yielding a standard curve consisting of four standards: 100 ng, 10 ng, 1 ng, and 0.1 ng. cDNA generated during the first strand synthesis reaction served as the 100-ng standard. The standard curve of the 3′ primer set (HBAC1650, HBAC1717) was used to estimate the quantity of RNA in the sample. The ratio of the RNA yield obtained from both sets of PCR primers is the 3′/5′ ratio and was used as an indication of RNA quality. For example, if the cDNA contains both the 3′ and 5′ target sequences, the 3′/5′ ratio would be ∼1. Cross-linked or modified RNAs would have a ratio greater than 1. Based on this ratio, an estimation of the quality of RNA was made. Lesions from which RNAs yielded acceptable 3′ to 5′ ratios were selected for microdissection. Three blocks each (proliferating hemangioma and normal term placenta) and four blocks of involuting hemangioma were identified with useable RNA in the quality control screen. Multiple, serially sectioned formalin-fixed, paraffin-embedded sections were cut from the blocks at 7 μ thickness, deparaffinized, stained and dehydrated following the manufacturer's protocol (Paradise reagent system Kit, Molecular Devices). Lesional tissue was microdissected from PEN membrane glass slides (Molecular Devices) using both the UV cutting and IR capture lasers on the Veritas microdissection instrument (Molecular Devices). All attempts were made to capture only lesional vessels so that connective tissue including adipose tissue would be excluded. It was inevitable; however, that intraluminal white cells, some perivascular collagen, and occasional resident cells such as fibroblasts and mast cells would have been included in the samples. Still, these would have made a very minor contribution to the sample and resultant data. After microdissection, RNA was extracted from the captured cells on the cap by incubating dissected tissue in the RNA extraction buffer (Paradise reagent system kit, Molecular Devices) overnight at 50°C. RNA isolation was performed following the manufacturer's protocol using the MiraCol purification column as part of the Paradise reagent system kit. Samples were eluted in 12 μl of elution buffer. RNA was amplified according to the manufacturer's suggested protocol (Paradise reagent system kit). Briefly, first-strand synthesis was performed on each RNA yielding cDNAs that incorporate a single-stranded T7 promoter sequence. cDNAs generated in a second-strand synthesis reaction using exogenous primers were purified using the MiraCol purification column provided in the paradise reagent system kit. Amplified and purified cDNAs were submitted to the Partners Health Care Center for Personalized Genetic Medicine (Cambridge, MA) for labeling, quantification, and hybridization. Samples were labeled with biotinylated probes using the Bioarray high-yield transcription kit following the manufacturer's protocol (Enzo Biochemical, New York, NY). The concentration of the biotin-labeled cRNA was determined by UV absorbance using a Bio-Tek plate reader (Bio-Tek Instruments, Winooski, VT). In all cases, 20 μg of each biotinylated cRNA preparation was fragmented, assessed by gel electrophoresis, and placed in a hybridization cocktail containing hybridization controls as recommended by the manufacturer. Samples were then hybridized to the Affymetrix Human X3P GeneChip Array (Affymetrix Inc., Santa Clara, CA) at 45°C for 24 hours. Microarrays were washed and stained using the manufacturer's protocol for the Human X3P GeneChip Array on a Model 450 Fluidics station (Affymetrix Inc.). The Fluidics station process is controlled by the Affymetrix GeneChip Operating System (GCOS). The Affymetrix Human X3P GeneChip Array is designed for whole-genome expression profiling of RNA from formalin-fixed, paraffin-embedded samples. The target sequences on the X3P array are identical to those used for designing the Human Genome U133 Plus 2.0 GeneChip array, for a total of 47,000 transcripts with 61,000 probe sets, although the probes on the two types of arrays are significantly different. The probe selection region on the X3P array is restricted to the 300 bp at the most 3′ end of the transcripts. In contrast, the standard Affymetrix design selects probe sets within the region 600 bases proximal to the 3′ ends (Affymetrix Inc.). Images from the scanned chips were processed using an Affymetrix Model 7000 scanner with autoloader (Affymetrix Inc.). The Affymetrix GCOS v1.3 operating system (Affymetrix Inc.) controls the Model 7000 scanner and data acquisition functions. Image files were downloaded, imported, and analyzed using the GeneSifter statistical software package (Geospiza, Inc., Seattle, WA). Statistical t-test analysis was performed in which a pairwise comparison was made between the proliferating hemangioma versus placenta, proliferating hemangioma versus involuting hemangioma, and involuting hemangioma versus placenta (see Supplemental Tables S1, S2, and S3 at http://ajp.amjpathol.org). Data were normalized to the mean. Results were filtered more stringently by imposing a threshold cutoff of 3.0 or greater fold change in expression and a quality call of 1 (P) in all three replicates of at least one group. False-positives were reduced by applying the Benjamini and Hochberg correction coefficient. By plotting the relative intensities of each of the genes from both samples on the same graph, it is possible to directly compare the data (see Supplemental Figure S1 at http://ajp.amjpathol.org). Their similarity (mirror images) suggests that the procedure is reproducible. For example, box plots of the range of intensities of the two samples also show that the data from the two samples is comparable (data not shown). Pearson's correlation coefficients were calculated from the array data as previously described (see Supplemental Figure S2 at http://ajp.amjpathol.org). A one-way analysis of variance was performed on the three groups (proliferating hemangioma, involuting hemangioma, and placenta). See Supplemental Table S4 at http://ajp.amjpathol.org. Data were normalized to the mean, then filtered, by imposing a threefold threshold cutoff, a quality call of 1, and application of the Benjamini and Hochberg correction coefficient. GeneSifter uses Gene Ontology (GO) reports and z-scores to summarize the biological processes, molecular functions, or cellular components, as well as, the KEGG (Kyto Encyclopedia of Genes and Genomes) pathways21Kanehisa M Goto S Kawashima S Okuno Y Hattori M The KEGG resource for deciphering the genome.Nucleic Acids Res. 2004; 32: D277-D280Crossref PubMed Google Scholar associated with a gene list. The z-score is calculated by subtracting the expected number of genes in a GO term meeting the criterion from the observed number of genes and dividing by the SD of the observed number of genes.22Doniger SW Salomonis N Dahlquist KD Vranizan K Lawlor SC Conklin BR MAPPFinder: using Gene Ontology and GenMAPP to create a global gene-expression profile from microarray data.Genome Biol. 2003; 4: R7Crossref PubMed Google Scholarz-scores can then be used to identify GO terms that are significantly over- or under represented in a gene list. Expression of proteins generated by some of the differentially regulated genes was validated by immunohistochemical analysis. Formalin-fixed, paraffin-embedded tissue sections were mounted on microscope slides. Immunohistochemical staining was optimized using a Ventana Discovery XT automated immunohistochemistry slide processing platform according to the manufacturer's instructions (Ventana Medical Systems, Tucson, AZ). Following the closed loop assay development (CLAD) protocol (Ventana Medical Systems), antibodies were optimized using either the OmniMap or UltraMap DAB Anti-Mouse (HRP) or Anti-Rabbit (HRP) detection kits (Ventana Medical Systems). Antibodies used in this study are listed in Table 1. For each antibody, standard quality control procedures were undertaken to optimize antigen retrieval, primary antibody dilution, secondary antibody detection, and other factors for both signal and noise. Specificity for the antibodies was demonstrated by Western blot analysis.Table 1List of AntibodiesAntibodyManufacturerSourceConcentrationMIB-1DAKOMouse monoclonalPredilutePDGFRαSanta CruzRabbit polyclonal1:100ClusterinSanta CruzMouse monoclonal1:100SDF-1R&D SystemsMouse monoclonal1:100DSCR1L1AbgentRabbit polyclonal1:100 Open table in a new tab In an initial quality control step, blocks of formalin-fixed, and paraffin-embedded proliferating and involuting hemangiomas were screened for useable RNA by real-time PCR. Seven primary tumor blocks with useable RNA were identified from recent cases: three proliferating and four involuting hemangiomas, as well as three term placental specimens (see Supplemental Table S5 at http://ajp.amjpathol.org). LCM was used to isolate vessels from the blocks that had useable RNA. Gene expression profiles were measured in amplified RNA from laser-captured microdissected vessels using the recently developed Human Affymetrix GeneChip X3P array, which can be used for whole-genome expression profiling of formalin-fixed, paraffin-embedded samples.23Resnick MB Sabo E Meitner PA Kim SS Cho Y Kim HK Tavares R Moss SF Global analysis of the human gastric epithelial transcriptome altered by Helicobacter pylori eradication in vivo.Gut. 2006; 55: 1717-1724Crossref PubMed Scopus (66) Google Scholar Comparing proliferating hemangioma with placenta using pairwise analysis, 843 genes showed significant differential regulation (459 up-regulated and 384 down-regulated). The complete list of these genes is found in Supplementary Table S1 (available at http://ajp.amjpathol.org). In the pairwise comparison of proliferating and involuting hemangiomas, only 289 genes showed differential expression (116 up-regulated and 173 down-regulated). The complete list of these genes is found in Supplementary Table S2 (available at http://ajp.amjpathol.org). Differentially expressed genes were placed into biological process gene ontology categories and several highly represented gene ontology categories were found. Rather than presenting the entire data set, we will focus on just some of the interesting signaling systems that may be relevant to the biology of hemangiomas. Expression levels for selected genes (discussed below) were consistent, when reviewed case by case, for the proliferative and involutive phases and generally varied minimally when correlated with patient age within each group (see Supplemental Figure S3 at http://ajp.amjpathol.org). Not unexpectedly, proliferating hemangiomas express genes involved in cellular proliferation. For example, proliferating hemangiomas selectively express the gene for the antigen identified by the monoclonal antibody Ki-67 (MK167) (Figure 2, A–C). Expression of this gene is increased 3.5-fold, relative to levels found in the placenta. These findings confirm the observations of many other groups and help validate the gene expression analysis from formalin-fixed tissue. The first biological process category of differentially expressed genes in proliferating hemangioma, relative to placenta, were categories related to endothelial differentiation (z-score, 6.53), angiogenesis (z-score, 4.03), as well as blood vessel development and morphogenesis (z-score, 3.83). Some of the significantly up-regulated genes in these categories included established components of vascular growth factor signaling systems. For example angiopoietin-2 (ANGPT2 or Ang2) is increased 33.36-fold in proliferating hemangioma, relative to placenta. Ang2 is expressed at low levels in many normal adult tissues, but is strongly up-regulated at sites of active vessel remodeling, such as in tumors.24Zagzag D Hooper A Friedlander DR Chan W Holash J Weigland SJ Yancopoulos GD Grumet M In situ expression of angiogenesis in astrocytomas identifies angiopoietin-2 as an early marker of tumor angiogenesis.Exp Neurol. 1999; 159: 391-400Crossref PubMed Scopus (200) Google Scholar Jagged 1 (JAG1) (Figure 3A) and Notch 4 (NOTCH4) were increased 6.5- and 3.2-fold, respectively, relative to placenta (Table 2).Table 2Genes Identified in Proliferating Hemangioma Versus Placenta in the Endothelial Gene Ontology GroupGene descriptionGene symbolCluster IDPlacentaSEMPHSEMFold changeGenes up-regulated in proliferating hemangioma Angiopoietin 2ANGPT2Hs.5534840.0296±0.01550.9858±0.2653+78.6 Angiopoietin 2ANGPT2Hs.5534840.6157±0.40184.8394±0.7700+33.36 Matrix-remodeling associated 5DKFZp564I1922Hs.3694221.6427±0.329237.8485±5.9250+23.04 Insulin-like growth factor binding protein 7IGFBP7Hs.4798080.3566±0.16784.916±1.5634+13.78 Neuropilin and tolloid-like 2NETO2Hs.4440460.1026±0.02011.1525±0.3427+11.23 Neuropilin and tolloid-like 2NETO2Hs.4440460.1802±0.02200.9866±0.1610+5.48 Brain-specific angiogenesis inhibitor 3BAI3Hs.132610.187±0.02521.3583±0.2695+7.26 Plexin domain containing 1PLXDC1Hs.1250360.2664±0.09471.8975±0.4650+7.12 Plexin domain containing 1PLXDC1Hs.1250360.5977±0.11282.1092±0.0928+3.53 Jagged 1JAG1Hs.2240123.0387±0.390819.8124±4.8743+6.52 Endothelin receptor type AEDNRAHs.1837133.3445±1.198516.6182±3.0545+4.97 Intercellular adhesion molecule 2ICAM2Hs.4314601.1697±0.49594.7548±0.7278+4.06 Notch homolog 4NOTCH4Hs.4361000.958±0.22943.0168±0.5963+3.15 Stabilin 1STAB1Hs.3019892.1844±0.58726.8518±0.8059+3.14 EPH receptor B3EPHB3Hs.29130.8408±0.14702.6435±0.2397+3.14 Latrophilin 1LPHN1Hs.6546581.9484±42596.0487±0.4863+3.1 Natriuretic peptide receptor ANPR1Hs.4903300.3672±0.08991.1348±0.1405+3.09Genes down-regulated in proliferating hemangioma G protein-coupled receptor 37 (endothelin receptor type B-like)GPR37Hs.4060940.9995±0.24300.0327±0.0299−30.57 Insulin-like growth factor binding protein 3IGFBP3Hs.45023058.0815±8.29212.2601±0.1342−25.7 fms-related tyrosine kinase 1FLT1Hs.5076210.9922±0.17170.2611±0.0568−22.67 fms-related tyrosine kinase 1FLT1Hs.5076217.2929±0.37330.3216±0.0334−3.8 Platelet-derived growth factor receptor, α polypeptidePDGFRAHs.746150.8004±0.21680.0698±0.0159−19.94 Platelet-derived growth factor receptor, α polypeptidePDGFRAHs.7461521.6065±5.94011.0834±0.3965−11.47 Transforming growth factor, β receptor III (betaglycan, 300 kDa)TGBFGR3Hs.48239024.4882±7.41503.5753±1.1188−6.85 Endothelial differentiation, lysophosphatidic acid G-protein-coupled receptor, 2EDG2Hs.1266670.1337±0.02040.0219±0.0065−6.12 Insulin-like growth factor binding protein 5IGFBP5Hs.3699823.0338±0.82380.5067±0.2263−5.99 Endothelin receptor type BEDNRBHs.820025.4154±1.15970.9312±0.4842−5.82 Platelet-derived growth factor CPDGFCHs.1481622.2281±0.56250.5379±0.0195−5.28 Platelet-derived growth factor CPDGFCHs.1481621.7552±0.14430.3326±0.0759−4.14 Bone morphogenetic protein 4BMP4Hs.688793.2881±0.20850.581±0.1587−5.66 Angiopoietin-like 1ANGPTL1Hs.5559036.0324±0.15311.1551±0.2386−5.22 Vascular cell adhesion molecule 1VCAM1Hs.10922511.9634±2.67592.851±0.0521−4.2 Bone morphogenetic protein 5BMP5Hs.2966481.1733±0.02690.3078±0.1370−3.81 Insulin-like growth factor 1 receptorIGF1RHs.205731.1978±0.18450.3409±0.1461−3.51Data from the arrays is normalized to the mean and a t-test is performed. Differences greater than threefold were considered significant, as outlined in Supplemental Table S1 (available at http://ajp.amjpathol.org). Shown are average intensity values and standard errors of the mean. Open table in a new tab Data from the arrays is normalized to the mean and a t-test is performed. Differences greater than threefold were considered significant, as outlined in Supplemental Table S1 (available at http://ajp.amjpathol.org). Shown are average intensity values and standard errors of the mean. Other more novel genes involved in signal transduction are exp
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