Identification of Vascular Lineage-Specific Genes by Transcriptional Profiling of Isolated Blood Vascular and Lymphatic Endothelial Cells
2003; Elsevier BV; Volume: 162; Issue: 2 Linguagem: Inglês
10.1016/s0002-9440(10)63851-5
ISSN1525-2191
AutoresSatoshi Hirakawa, Young‐Kwon Hong, Natasha L. Harvey, Vivien Schacht, Kant Matsuda, Towia A. Libermann, Michael Detmar,
Tópico(s)Cancer Cells and Metastasis
ResumoIn mammals, the lymphatic vascular system develops by budding of lymphatic progenitor endothelial cells from embryonic veins to form a distinct network of draining vessels with important functions in the immune response and in cancer metastasis. However, the lineage-specific molecular characteristics of blood vascular versus lymphatic endothelium have remained poorly defined. We isolated lymphatic endothelial cells (LECs) and blood vascular endothelial cells (BVECs) by immunomagnetic isolation directly from human skin. Cultured LECs but not BVECs expressed the lymphatic markers Prox1 and LYVE-1 and formed LYVE-1-positive vascular tubes after implantation in vivo. Transcriptional profiling studies revealed increased expression of several extracellular matrix and adhesion molecules in BVECs, including versican, collagens, laminin, and N-cadherin, and of the growth factor receptors endoglin and vascular endothelial growth factor receptor-1/Flt-1. Differential immunostains of human skin confirmed the blood vessel-specific expression of these genes. During embryonic development, endoglin expression was gradually down-regulated on lymphatic endothelium whereas vascular endothelial growth factor receptor-1 was absent from lymphatics. We also identified several genes with specific expression in LECs. These results demonstrate that some lineage-specific genes are only expressed during distinct developmental stages and they identify new molecular markers for blood vascular and lymphatic endothelium with important implications for future studies of vascular development and function. In mammals, the lymphatic vascular system develops by budding of lymphatic progenitor endothelial cells from embryonic veins to form a distinct network of draining vessels with important functions in the immune response and in cancer metastasis. However, the lineage-specific molecular characteristics of blood vascular versus lymphatic endothelium have remained poorly defined. We isolated lymphatic endothelial cells (LECs) and blood vascular endothelial cells (BVECs) by immunomagnetic isolation directly from human skin. Cultured LECs but not BVECs expressed the lymphatic markers Prox1 and LYVE-1 and formed LYVE-1-positive vascular tubes after implantation in vivo. Transcriptional profiling studies revealed increased expression of several extracellular matrix and adhesion molecules in BVECs, including versican, collagens, laminin, and N-cadherin, and of the growth factor receptors endoglin and vascular endothelial growth factor receptor-1/Flt-1. Differential immunostains of human skin confirmed the blood vessel-specific expression of these genes. During embryonic development, endoglin expression was gradually down-regulated on lymphatic endothelium whereas vascular endothelial growth factor receptor-1 was absent from lymphatics. We also identified several genes with specific expression in LECs. These results demonstrate that some lineage-specific genes are only expressed during distinct developmental stages and they identify new molecular markers for blood vascular and lymphatic endothelium with important implications for future studies of vascular development and function. The lymphatic system consists of a vascular network of thin-walled capillaries that drain protein-rich lymph from the extracellular spaces within most organs and that play major roles in the immune response and in tumor metastasis.1Witte MH Bernas MJ Martin CP Witte CL Lymphangiogenesis and lymphangiodysplasia: from molecular to clinical lymphology.Microsc Res Tech. 2001; 55: 122-145Crossref PubMed Scopus (190) Google Scholar, 2Oliver G Detmar M The rediscovery of the lymphatic system. Old and new insights into the development and biological function of the lymphatic vascular system.Genes Dev. 2002; 16: 773-783Crossref PubMed Scopus (321) Google Scholar Lymphatic vessels provide the conduit for antigen-presenting cells from the organ exposed to antigens to the regional lymph nodes, involving active recruitment of antigen-presenting cells by chemokines and other mediators secreted by lymphatic endothelium.3Saeki H Moore AM Brown MJ Hwang ST Cutting edge: secondary lymphoid-tissue chemokine (SLC) and CC chemokine receptor 7 (CCR7) participate in the emigration pathway of mature dendritic cells from the skin to regional lymph nodes.J Immunol. 1999; 162: 2472-2475PubMed Google Scholar Moreover, the early dissemination of malignant tumors frequently occurs via lymphatic vessels to regional lymph nodes, and the recent discovery of active tumor lymphangiogenesis and its role in cancer metastasis has drawn considerable attention to the molecular mechanisms that control activation and proliferation of lymphatic endothelium.2Oliver G Detmar M The rediscovery of the lymphatic system. Old and new insights into the development and biological function of the lymphatic vascular system.Genes Dev. 2002; 16: 773-783Crossref PubMed Scopus (321) Google Scholar, 4Alitalo K Carmeliet P Molecular mechanisms of lymphangiogenesis in health and disease.Cancer Cell. 2002; 1: 219-227Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar In particular, overexpression of the lymphangiogenesis factors vascular endothelial growth factor (VEGF)-C and VEGF-D by tumor cells has been shown to promote tumor lymphangiogenesis by activation of VEGF receptor-3 (VEGFR-3) on tumor-associated lymphatic endothelium, resulting in enhanced rates of lymph node metastasis.5Skobe M Hawighorst T Jackson DG Prevo R Janes L Velasco P Riccardi L Alitalo K Claffey K Detmar M Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis.Nat Med. 2001; 7: 192-198Crossref PubMed Scopus (1524) Google Scholar, 6Stacker SA Caesar C Baldwin ME Thornton GE Williams RA Prevo R Jackson DG Nishikawa S Kubo H Achen MG VEGF-D promotes the metastatic spread of tumor cells via the lymphatics.Nat Med. 2001; 7: 186-191Crossref PubMed Scopus (1077) Google Scholar, 7Mandriota SJ Jussila L Jeltsch M Compagni A Baetens D Prevo R Banerji S Huarte J Montesano R Jackson DG Orci L Alitalo K Christofori G Pepper MS Vascular endothelial growth factor-C-mediated lymphangiogenesis promotes tumour metastasis.EMBO J. 2001; 20: 672-682Crossref PubMed Scopus (845) Google Scholar, 8Karpanen T Egeblad M Karkkainen MJ Kubo H Yla-Herttuala S Jaattela M Alitalo K Vascular endothelial growth factor C promotes tumor lymphangiogenesis and intralymphatic tumor growth.Cancer Res. 2001; 61: 1786-1790PubMed Google Scholar In contrast to the rapid progress made in elucidating the formation and molecular control of the blood vascular system,9Gale NW Yancopoulos GD Growth factors acting via endothelial cell-specific receptor tyrosine kinases: VEGFs, angiopoietins, and ephrins in vascular development.Genes Dev. 1999; 13: 1055-1066Crossref PubMed Scopus (707) Google Scholar, 10Carmeliet P Mechanisms of angiogenesis and arteriogenesis.Nat Med. 2000; 6: 389-395Crossref PubMed Scopus (3561) Google Scholar the mechanisms controlling the normal development of lymphatic vessels and the molecular regulation of their biological function have remained poorly understood, mainly because of the lack of molecular tools to specifically distinguish lymphatic vessels from blood vessels and to functionally characterize the lymphatic endothelium.11Detmar M Hirakawa S The formation of lymphatic vessels and its importance in the setting of malignancy.J Exp Med. 2002; 196: 713-718Crossref PubMed Scopus (64) Google Scholar Consequently, our understanding of the function of the lymphatic system and its role in disease is still rudimentary. Recently, several novel markers have been reported to be predominantly expressed by lymphatic endothelium. VEGFR-3, a receptor for the lymphangiogenesis factors VEGF-C and VEGF-D, is expressed by both blood vascular and lymphatic endothelium during embryonic development, whereas its expression becomes restricted to lymphatic vessels in adult life.12Lymboussaki A Partanen TA Olofsson B Thomas CJ Fletcher CD de Waal RM Kaipainen A Alitalo K Expression of the vascular endothelial growth factor C receptor VEGFR-3 in lymphatic endothelium of the skin and in vascular tumors.Am J Pathol. 1998; 153: 395-403Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar However, VEGFR-3 has been detected on blood vascular endothelium associated with tumors and healing wounds,13Valtola R Salven P Heikkila P Taipale J Joensuu H Rehn M Pihlajaniemi T Weich H deWaal R Alitalo K VEGFR-3 and its ligand VEGF-C are associated with angiogenesis in breast cancer.Am J Pathol. 1999; 154: 1381-1390Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar, 14Paavonen K Puolakkainen P Jussila L Jahkola T Alitalo K Vascular endothelial growth factor receptor-3 in lymphangiogenesis in wound healing.Am J Pathol. 2000; 156: 1499-1504Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar and blockade of VEGFR-3 also resulted in inhibition of tumor angiogenesis.15Kubo H Fujiwara T Jussila L Hashi H Ogawa M Shimizu K Awane M Sakai Y Takabayashi A Alitalo K Yamaoka Y Nishikawa SI Involvement of vascular endothelial growth factor receptor-3 in maintenance of integrity of endothelial cell lining during tumor angiogenesis.Blood. 2000; 96: 546-553Crossref PubMed Google Scholar Recently, podoplanin, a transmembrane mucoprotein, has been reported as a novel lymphatic-vessel marker,16Breiteneder-Geleff S Soleiman A Kowalski H Horvat R Amann G Kriehuber E Diem K Weninger W Tschachler E Alitalo K Kerjaschki D Angiosarcomas express mixed endothelial phenotypes of blood and lymphatic capillaries: podoplanin as a specific marker for lymphatic endothelium.Am J Pathol. 1999; 154: 385-394Abstract Full Text Full Text PDF PubMed Scopus (955) Google Scholar and both podoplanin and VEGFR-3 have been used for the isolation of lymphatic endothelial cells (LECs).17Kriehuber E Breiteneder GS Groeger M Soleiman A Schoppmann SF Stingl G Kerjaschki D Maurer D Isolation and characterization of dermal lymphatic and blood endothelial cells reveal stable and functionally specialized cell lineages.J Exp Med. 2001; 194: 797-808Crossref PubMed Scopus (446) Google Scholar, 18Maekinen T Veikkola T Mustjoki S Karpanen T Catimel B Nice EC Wise L Mercer A Kowalski H Kerjaschki D Stacker SA Achen MG Alitalo K Isolated lymphatic endothelial cells transduce growth, survival and migratory signals via the VEGF-C/D receptor VEGFR-3.EMBO J. 2001; 20: 4762-4773Crossref PubMed Scopus (695) Google Scholar The hyaluronan receptor LYVE-119Banerji S Ni J Wang SX Clasper S Su J Tammi R Jones M Jackson DG LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan.J Cell Biol. 1999; 144: 789-801Crossref PubMed Scopus (1335) Google Scholar is specifically expressed by LECs, but not by blood vessels in most organs including the skin,5Skobe M Hawighorst T Jackson DG Prevo R Janes L Velasco P Riccardi L Alitalo K Claffey K Detmar M Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis.Nat Med. 2001; 7: 192-198Crossref PubMed Scopus (1524) Google Scholar, 20Prevo R Banerji S Ferguson DJ Clasper S Jackson DG Mouse LYVE-1 is an endocytic receptor for hyaluronan in lymphatic endothelium.J Biol Chem. 2001; 276: 19420-19430Crossref PubMed Scopus (418) Google Scholar, 21Skobe M Detmar M Structure, function and molecular control of the skin lymphatic system.J Invest Dermatol Symp Proc. 2000; 5: 14-19Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 22Wigle JT Harvey N Detmar M Lagutina I Grosveld G Gunn MD Jackson DG Oliver G An essential role for Prox1 in the induction of the lymphatic endothelial cell phenotype.EMBO J. 2002; 21: 1505-1513Crossref PubMed Scopus (750) Google Scholar and recent studies have identified the transcription factor Prox1,2Oliver G Detmar M The rediscovery of the lymphatic system. Old and new insights into the development and biological function of the lymphatic vascular system.Genes Dev. 2002; 16: 773-783Crossref PubMed Scopus (321) Google Scholar a homeobox gene required for the development of the lymphatic system,23Wigle JT Oliver G Prox1 function is required for the development of the murine lymphatic system.Cell. 1999; 98: 769-778Abstract Full Text Full Text PDF PubMed Scopus (1282) Google Scholar as a master control gene in the program specifying LEC fate.22Wigle JT Harvey N Detmar M Lagutina I Grosveld G Gunn MD Jackson DG Oliver G An essential role for Prox1 in the induction of the lymphatic endothelial cell phenotype.EMBO J. 2002; 21: 1505-1513Crossref PubMed Scopus (750) Google Scholar, 24Hong Y-K Harvey N Noh Y-H Schacht V Hirakawa S Detmar M Oliver G Prox1 is a master control gene in the program specifying lymphatic endothelial cell fate.Dev Dyn. 2002; 225: 351-357Crossref PubMed Scopus (444) Google Scholar However, besides these few markers with specific expression in lymphatic endothelium, little is known about the distinct molecular characteristics of blood vascular endothelial cells (BVECs) versus LECs, and a comprehensive comparison of the lineage-specific differentiation of these cell types has been lacking. To identify novel lineage-specific molecules involved in the biological function of these distinct vascular cell types, we developed a new protocol for the selective isolation of LECs and BVECs directly from human skin. Here, we report that immunomagnetic selection of CD34−/CD31+ cells yields pure cultures of LECs that maintain expression of the specific lymphatic markers Prox1 and LYVE-1, whereas BVECs remain negative for these markers. Importantly, gene array profiling, combined with quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and double-immunofluorescence stains of normal skin, identified several novel genes with specific expression in lymphatic or blood vascular endothelium. Together, these results provide important new tools for molecular and functional studies of lymphatic and blood vascular endothelium in vascular development, tumor progression, and the immune response. Neonatal human foreskins were obtained after routine circumcisions. After enzymatic digestion, the epidermis was removed and dermal cells were mechanically released as previously described.25Richard L Velasco P Detmar M A simple immunomagnetic protocol for the selective isolation and long-term culture of human dermal microvascular endothelial cells.Exp Cell Res. 1998; 240: 1-6Crossref PubMed Scopus (116) Google Scholar CD34-positive BVECs were isolated by immunomagnetic purification25Richard L Velasco P Detmar M A simple immunomagnetic protocol for the selective isolation and long-term culture of human dermal microvascular endothelial cells.Exp Cell Res. 1998; 240: 1-6Crossref PubMed Scopus (116) Google Scholar with an anti-human CD34 antibody (BD Pharmingen, San Diego, CA) conjugated to immunomagnetic beads (Dynal, Lake Success, NY). Thereafter, the remaining CD34-negative cells were incubated with an immunomagnetic beads-conjugated anti-human CD31 antibody (Dynal) to isolate LECs. LECs were seeded onto fibronectin-coated (10 μg/ml; BD Biosciences, Bedford, MA) culture dishes and were propagated in endothelial cell basal medium (BioWhittaker, Walkersville, MD), supplemented with 10 μg/ml of hydrocortisone acetate, 2.5 × 10−2 mg/ml N-6,2′-O-dibutyryl-adenosine 3′,5′-cyclic monophosphate (Sigma, St. Louis, MO), 2 mmol/L l-glutamine, 20% fetal bovine serum (Life Technologies, Inc., Grand Island, NY), antibiotics, and 20 ng/ml of recombinant human VEGF165 (R&D Systems, Minneapolis, MN). VEGF was omitted after the first passage. LECs remained negative for CD34 expression for at least eight passages, as evaluated by immunocytochemistry. BVECs were cultured in the same medium without addition of VEGF. Confluent primary BVEC cultures were further purified by immunomagnetic E-selectin selection after 6 hours of stimulation with recombinant human tumor necrosis factor-α as described.25Richard L Velasco P Detmar M A simple immunomagnetic protocol for the selective isolation and long-term culture of human dermal microvascular endothelial cells.Exp Cell Res. 1998; 240: 1-6Crossref PubMed Scopus (116) Google Scholar Immunofluorescence stainings were performed on 6-μm cryostat sections of neonatal human foreskin or on 10-μm sections of mouse embryos as previously described,22Wigle JT Harvey N Detmar M Lagutina I Grosveld G Gunn MD Jackson DG Oliver G An essential role for Prox1 in the induction of the lymphatic endothelial cell phenotype.EMBO J. 2002; 21: 1505-1513Crossref PubMed Scopus (750) Google Scholar, 26Detmar M Brown LF Schön MP Elicker BM Velasco P Richard L Fukumura D Monsky W Claffey KP Jain RK Increased microvascular density and enhanced leukocyte rolling and adhesion in the skin of VEGF transgenic mice.J Invest Dermatol. 1998; 111: 1-6Crossref PubMed Scopus (467) Google Scholar using antibodies to Prox1,22Wigle JT Harvey N Detmar M Lagutina I Grosveld G Gunn MD Jackson DG Oliver G An essential role for Prox1 in the induction of the lymphatic endothelial cell phenotype.EMBO J. 2002; 21: 1505-1513Crossref PubMed Scopus (750) Google Scholar murine or human LYVE-1 (kindly provided by Dr. D. Jackson, John Radcliffe Hospital, Oxford, UK20Prevo R Banerji S Ferguson DJ Clasper S Jackson DG Mouse LYVE-1 is an endocytic receptor for hyaluronan in lymphatic endothelium.J Biol Chem. 2001; 276: 19420-19430Crossref PubMed Scopus (418) Google Scholar), human CD34 and CD31 (BD Pharmingen), mouse VEGFR-1 (MF1; kindly provided by Dr. DJ Hicklin, Imclone Systems, New York, NY) and human VEGFR-1/Flt-1 (Sigma), N-cadherin (Transduction Laboratories, San Diego, CA), versican (clone 12C5; Developmental Studies Hybridoma Bank, University of Iowa, Ames, IA), mouse endoglin (BD Pharmingen) and human endoglin (Neomarkers, Fremont, CA), macrophage mannose receptor (BD Biosciences), desmoplakin (Serotec, Raleigh, NC), MIP-3-α (Santa Cruz Biotechnology, Santa Cruz, CA), and corresponding secondary antibodies labeled with AlexaFluor488 or AlexaFluor594 (Molecular Probes, Eugene, Oregon). Nuclei were counterstained with 20 μg/ml of Hoechst bisbenzimide.27Skobe M Hamberg LM Hawighorst T Schirner M Wolf GL Alitalo K Detmar M Concurrent induction of lymphangiogenesis, angiogenesis, and macrophage recruitment by vascular endothelial growth factor-C in melanoma.Am J Pathol. 2001; 159: 893-903Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar Sections were examined by using a Nikon E-600 microscope (Nikon, Melville, NY) and images were captured with a SPOT digital camera (Diagnostic Instruments, Sterling Heights, MI). Antibody staining of cultured cells was observed using a Leica TCS NT4D confocal imaging system (Leica, Heidelberg, Germany). Total cellular RNA was isolated from confluent LEC and BVEC cultures at passages 4 and 5, using the TRIzol reagent (Invitrogen, Carlsbad, CA). Samples of RNA (10 μg each) were subjected to Northern blot analyses as described,26Detmar M Brown LF Schön MP Elicker BM Velasco P Richard L Fukumura D Monsky W Claffey KP Jain RK Increased microvascular density and enhanced leukocyte rolling and adhesion in the skin of VEGF transgenic mice.J Invest Dermatol. 1998; 111: 1-6Crossref PubMed Scopus (467) Google Scholar using a 2.0-kb human Prox1 cDNA probe and a 956-bp human type XVIII collagen probe (kindly provided by Dr. Y. Ninomiya). A 36B4 ribosomal-associated protein cDNA probe was used as a control for equal RNA loading.28Detmar M Brown LF Berse B Jackman RW Elicker BM Dvorak HF Claffey KP Hypoxia regulates the expression of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) and its receptors in human skin.J Invest Dermatol. 1997; 108: 263-268Crossref PubMed Scopus (237) Google Scholar For Western blot analyses, confluent LEC and BVEC cultures were homogenized in lysis buffer as described.5Skobe M Hawighorst T Jackson DG Prevo R Janes L Velasco P Riccardi L Alitalo K Claffey K Detmar M Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis.Nat Med. 2001; 7: 192-198Crossref PubMed Scopus (1524) Google Scholar Ten μg of protein per sample were analyzed by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were immunoblotted with polyclonal antibodies against human VEGFR-2/KDR or VEGFR-3/Flt4 (Santa Cruz Biotechnology) as described.5Skobe M Hawighorst T Jackson DG Prevo R Janes L Velasco P Riccardi L Alitalo K Claffey K Detmar M Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis.Nat Med. 2001; 7: 192-198Crossref PubMed Scopus (1524) Google Scholar The ABI Prism 7000 Sequence Detection System was used to perform either SYBR-Green based or dual-labeled probe based real-time RT-PCR reactions as described.29Hawighorst T Skobe M Streit M Hong YK Velasco P Brown LF Riccardi L Lange-Asschenfeldt B Detmar M Activation of the tie2 receptor by angiopoietin-1 enhances tumor vessel maturation and impairs squamous cell carcinoma growth.Am J Pathol. 2002; 160: 1381-1392Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar Sequences of the primers used for this study are provided in Table 3. For SYBR-Green based reactions, at least three sets of primers were used for each gene of interest. SYBR-Green PCR Master Mix was used for all SYBR-Green reactions with the addition of MultiScribe reverse transcriptase (Applied Biosystems, Foster City, CA). For dual-labeled probe-based real-time RT-PCR reactions, probes labeled with 6-FAM and TAMRA at their 5′ and 3′ end, respectively, were multiplexed with a GAPDH probe labeled with Joe and TAMRA at the 5′ and 3′ end as an internal control. TaqMan EZ RT-PCR Core Reagent was used for dual-labeled probe based reactions. Total RNAs were isolated as described above and were treated with RNase-free RQ-DNase (Promega, Madison, WI) before analyses. Twenty ng of total RNA were used for each reaction. The primers and probes were designed using Primer Express software (Perkin Elmer Life Sciences, Boston, MA) and were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA). Expression data were normalized based on the expression levels of GAPDH mRNA.Table 3Names of Genes and Primer Sequences for the SYBR-Green-Based Real-Time RT-PCRGene namesForward primersReverse primersCD44AAAGGAGCAGCACTTCAGGACTGTCTGTGCTGTCGGTGATCEA-CAMGTAGCAAAGCCCCAAATCAAAACGGATGGAGATTCCAGTGChondroitin sulfate proteoglycan 2 (versican)TTTGCCACCCAGTTACAACAGGGCCACAAGGACAGTAGTCCollagen type I, alpha 2 chainAGGAGTTGTTGGACCACAGGTCCCTTCAATCCATCCAGACG protein-coupled receptor 39AGCACAGAACAGAGGGGCTAGAGCAGGAGGGAGAGACAGAIntegrin alpha4GAGGAATTCCCACCACTTCAATTTCATGGGCACAAAACCIntegrin beta3 (GPIIIa, CD61)TGGTCCTGCTCTCAGTGATGGAATTCTTTTVCGGTCGTGGAIntestinal trefoil factorCCAGGCACTGTTCATCTCAGGAGCATGGGACCTTTATTCGMacrophage mannose receptor (MRC1)GTGGCCGGAGTAGTCATCATTCTTGAGGTAGGTGCACACGMembrane glycoprotein gp130TGAACGAGGGGAAGAAAATGTGTGTGTTGCCCATTCAGATN-cadherinTGGAGAACCCCATTGACATTTGATCCCTCAGGAACTGTCCReelinAGGACCGTTATGCTGGACACACATGTCAAAGGCGATCCTCVEGF receptor-1/Flt1GGCCTCTGATGGTGATTGTTGTGCTGCATCCTTGTTGAGANames of Genes and Primer/Probe Sequences for the Taqman-Based Real-Time RT-PCRGene namesForward primersReverse primersTaqman probesCCL21/SLCGGTTCTGGCCTTTGGCATCAGGCAACAGTCCTGAGCCCFAM-CCAGGACCCAAGGCAGTGATGGA-TAMRAGAPDHGATTCCACCCATGGCAATGAAGATGGTGATGGGATTTCJOE-CAAGCTTCCCGTTCTCAGCC-TAMRALYVE-1AGCTATGGCTGGGTTGGAGACCCCATTTTTCCCACACTTGFAM-TTCGTGGTCATCTCTAGGATTAGCCCAAACC-TAMRAPodoplaninAGGCGGCGTTGCCATGTCTTCGCTGGTTCCTGGAGFAM-CCAGGTGCCGAAGATGATGTGGTC-TAMRAProx1ACAAAATGGTGGCACGGACCTGATGTACTTCGGAGCCTGFAM-CCCAGTTTCCAAGCCAGCGGTCTCT-TAMRAVEGF-CCACCACCAAACATGCAGCTGTGAAAATCCTGGCTCACAAGCFAM-CGGCCATGTACGAACCGCCAG-TAMRAVEGFR3 (Flt4)TCTGCTACAGCTTCCAGGTGGGCAGCCAGGTCTCTGTGGATFAM-ATGGAGTTCCTGGCTTCCCGAAAGTG-TAMRA Open table in a new tab Confluent LEC and BVEC cultures at passage 5 were labeled with 5-chloromethylfluorescein diacetate (CMFDA, Cell Tracker Green; Molecular Probes) or with 5(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (CMTMR; Cell Tracker Orange) according to the manufacturer's instructions. LECs or BVECs (2.5 × 105) were seeded onto each well of Matrigel-coated (Becton Dickinson, Franklin Lakes, NJ) 24-well plates and were incubated for 24 hours at 37°C. Cells were analyzed by using a Nikon TE-300 microscope and images were captured with a SPOT digital camera. In vivo tube formation was investigated by mixing 1 × 106 LECs or BVECs with 500 μl of Matrigel, followed by subcutaneous injection into SCID CV17 mice (Charles River Laboratories, Wilmington, MA). All experiments were performed three times with comparable results. After 7 days, mice were sacrificed and tissue samples were fixed with 4% paraformaldehyde overnight and embedded in paraffin. Six-μm paraffin sections were either stained with hematoxylin and eosin or were double-stained with antibodies against human CD31 and LYVE-1 as described above. Total cellular RNA was extracted from confluent fifth passage LEC and BVEC cultures, maintained in complete endothelial growth medium without addition of VEGF. Oligonucleotide array analyses were performed using the human 95Av2 (12,625 genes) gene arrays (GeneChip; Affymetrix, Santa Clara, CA) according to the manufacturer's instructions. Arrays were scanned using an Affymetrix confocal scanner and analyzed by the Microarray Suite 5.0 software (Affymetrix). Intensity values were scaled so that the overall fluorescence intensity of each chip of the same type was equivalent. Genes with a P value of ≤0.002 were considered as significantly increased or decreased. BVECs or LECs (3.5 × 104) were seeded onto fibronectin-coated six-well plates. Triplicate dishes were treated without or with 100 ng/ml of recombinant human placental growth factor-1 (PlGF-1, R&D Systems), 100 ng/ml human VEGF-C (kindly provided by Dr. K. Alitalo), 100 ng/ml human VEGF-D (R&D Systems), or 10 ng/ml recombinant human VEGF-A (VEGF165, R&D Systems) in endothelial growth medium. After 48 hours, cells were trypsinized and cell numbers were determined using a hematocytometer. For thymidine incorporation assays, 2 × 104 cells were seeded into quadruplicate wells of 24-well plates in the presence or absence of the above growth factors. Thymidine incorporation was assessed after 36 hours as previously described.30Detmar M Mayer-da-Silva A Stadler R Orfanos CE Effects of azelaic acid on proliferation and ultrastructure of mouse keratinocytes in vitro.J Invest Dermatol. 1989; 93: 70-74Abstract Full Text PDF PubMed Google Scholar For apoptosis assays, 1 × 104 cells were seeded into triplicate wells of 96-well plates in endothelial cell basal medium containing 2% fetal calf serum, and were treated with the above growth factors for 72 hours. Apoptosis rates were determined by the Cellular DNA Fragmentation ELISA kit (Roche, Germany) according to the manufacturer's instructions. Statistical analyses were performed using the paired Student's t-test. Using the lymphatic-specific transcription factor Prox122Wigle JT Harvey N Detmar M Lagutina I Grosveld G Gunn MD Jackson DG Oliver G An essential role for Prox1 in the induction of the lymphatic endothelial cell phenotype.EMBO J. 2002; 21: 1505-1513Crossref PubMed Scopus (750) Google Scholar as a specific marker for lymphatic endothelium, we found that CD34 was selectively expressed by Prox1-negative blood vessels in human skin, whereas Prox1-positive lymphatic vessels did not express CD34 (Figure 1; A to C). These results were confirmed by immunofluorescence double stains with antibodies against the lymphatic-specific hyaluronan receptor LYVE-1 and against CD34, demonstrating mutually exclusive staining of lymphatic vessels with LYVE-1 and of blood vessels with CD34 (Figure 1; D to F). All Prox1-, LYVE-1-, or CD34-positive vessels also expressed the pan-vascular marker CD31 (data not shown). These results revealed that CD34 was specifically expressed by blood vascular, but not by lymphatic, endothelium. We next isolated CD34-positive BVECs and CD34-negative/CD31-positive LECs by immunomagnetic purification directly from enzymatically digested neonatal human foreskins. After seeding on fibronectin-coated culture dishes, both LECs and BVECs maintained a typical cobblestone-like endothelial morphology for at least eight passages (Figure 2, A and B).Figure 2Cultured human dermal LECs and BVECs maintain their lineage-specific differentiation in vitro. A and B: Cultured BVECs and LECs (passage 4) show comparable endothelial morphology with characteristic cobblestone appearance. Phase-contrast micrograph. C and D: All LECs express the lymphatic marker LYVE-1 in vitro (D), whereas BVECs are not labeled (C). E and F: Double immunostains for CD31 (green) and Prox1 (red) demonstrate CD31 membrane labeling in both cell types whereas Prox1 (nuclear stain, red) is selectively expressed in LECs. G: Northern blot analysis confirms selective Prox1 mRNA expression in cultured LECs (L), whereas no expression is found in BVECs (B). In contrast, the basement membrane component type XVIII collagen is expressed at higher levels in BVECs. Hybridization with a probe to ribosomal protein-associated RNA 36B4 demonstrates equal loading. Scale bars: 100 μm (D); 25 μm (F).View Large Image Figure ViewerDownload Hi-res image Download (PPT) All cultured LECs maintained expression of the hyaluronan receptor LYVE-1 during the first eight passages (Figure 2D), whereas LYVE-1 expression was absent from BVEC cultures (Figure 2C). Double-immunofluorescence stains with antibodies to the endothelial junction molecule CD31 (PECAM-131Dejana E Corada M Lampugnani MG Endothelial cell-to
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