Growth Hormone Promotes Lymphangiogenesis
2008; Elsevier BV; Volume: 173; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2008.080060
ISSN1525-2191
AutoresNadja E. Bänziger-Tobler, Cornelia Halin, Kentaro Kajiya, Michael Detmar,
Tópico(s)Lymphatic Disorders and Treatments
ResumoThe lymphatic system plays an important role in inflammation and cancer progression, although the molecular mechanisms involved are poorly understood. As determined using comparative transcriptional profiling studies of cultured lymphatic endothelial cells versus blood vascular endothelial cells, growth hormone receptor was expressed at much higher levels in lymphatic endothelial cells than in blood vascular endothelial cells. These findings were confirmed by quantitative real-time reverse transcriptase-polymerase chain reaction and Western blot analyses. Growth hormone induced in vitro proliferation, sprouting, tube formation, and migration of lymphatic endothelial cells, and the mitogenic effect was independent of vascular endothelial growth factor receptor-2 or -3 activation. Growth hormone also inhibited serum starvation-induced lymphatic endothelial cell apoptosis. No major alterations of lymphatic vessels were detected in the normal skin of bovine growth hormone-transgenic mice. However, transgenic delivery of growth hormone accelerated lymphatic vessel ingrowth into the granulation tissue of full-thickness skin wounds, and intradermal delivery of growth hormone resulted in enlargement and enhanced proliferation of cutaneous lymphatic vessels in wild-type mice. These results identify growth hormone as a novel lymphangiogenic factor. The lymphatic system plays an important role in inflammation and cancer progression, although the molecular mechanisms involved are poorly understood. As determined using comparative transcriptional profiling studies of cultured lymphatic endothelial cells versus blood vascular endothelial cells, growth hormone receptor was expressed at much higher levels in lymphatic endothelial cells than in blood vascular endothelial cells. These findings were confirmed by quantitative real-time reverse transcriptase-polymerase chain reaction and Western blot analyses. Growth hormone induced in vitro proliferation, sprouting, tube formation, and migration of lymphatic endothelial cells, and the mitogenic effect was independent of vascular endothelial growth factor receptor-2 or -3 activation. Growth hormone also inhibited serum starvation-induced lymphatic endothelial cell apoptosis. No major alterations of lymphatic vessels were detected in the normal skin of bovine growth hormone-transgenic mice. However, transgenic delivery of growth hormone accelerated lymphatic vessel ingrowth into the granulation tissue of full-thickness skin wounds, and intradermal delivery of growth hormone resulted in enlargement and enhanced proliferation of cutaneous lymphatic vessels in wild-type mice. These results identify growth hormone as a novel lymphangiogenic factor. Lymphatic vessels play an important role in tissue fluid homeostasis and lipid uptake as well as in pathological conditions such as inflammation and cancer dissemination.1Alitalo K Tammela T Petrova TV Lymphangiogenesis in development and human disease.Nature. 2005; 438: 946-953Crossref PubMed Scopus (961) Google Scholar, 2Cueni LN Detmar M New insights into the molecular control of the lymphatic vascular system and its role in disease.J Invest Dermatol. 2006; 126: 2167-2177Crossref PubMed Scopus (184) Google Scholar Recent studies have highlighted a direct correlation between the occurrence of lymph node metastases and lymphatic vessel density in several types of human cancer, including cutaneous malignant melanoma3Dadras SS Lange-Asschenfeldt B Velasco P Nguyen L Vora A Muzikansky A Jahnke K Hauschild A Hirakawa S Mihm MC Detmar M Tumor lymphangiogenesis predicts melanoma metastasis to sentinel lymph nodes.Mod Pathol. 2005; 18: 1232-1242Crossref PubMed Scopus (277) Google Scholar and head and neck cancer.4Beasley NJ Prevo R Banerji S Leek RD Moore J van Trappen P Cox G Harris AL Jackson DG Intratumoral lymphangiogenesis and lymph node metastasis in head and neck cancer.Cancer Res. 2002; 62: 1315-1320PubMed Google Scholar Moreover, in mouse models of cancer, tumor-induced lymphangiogenesis potently promotes tumor metastasis to lymph nodes and other sites.5Mattila MM Ruohola JK Karpanen T Jackson DG Alitalo K Harkonen PL VEGF-C induced lymphangiogenesis is associated with lymph node metastasis in orthotopic MCF-7 tumors.Int J Cancer. 2002; 98: 946-951Crossref PubMed Scopus (217) Google Scholar, 6Mandriota 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 (833) Google Scholar Lymphangiogenesis also occurs in tumor-draining lymph nodes, and lymph node lymphangiogenesis is associated with enhanced metastasis to distant organs in mouse tumor models and also in some types of human cancer.7Hirakawa S Kodama S Kunstfeld R Kajiya K Brown LF Detmar M VEGF-A induces tumor and sentinel lymph node lymphangiogenesis and promotes lymphatic metastasis.J Exp Med. 2005; 201: 1089-1099Crossref PubMed Scopus (571) Google Scholar, 8Hirakawa S Brown LF Kodama S Paavonen K Alitalo K Detmar M VEGF-C-induced lymphangiogenesis in sentinel lymph nodes promotes tumor metastasis to distant sites.Blood. 2007; 109: 1010-1017Crossref PubMed Scopus (414) Google Scholar, 9Van den Eynden GG Vandenberghe MK van Dam PJ Colpaert CG van Dam P Dirix LY Vermeulen PB Van Marck EA Increased sentinel lymph node lymphangiogenesis is associated with nonsentinel axillary lymph node involvement in breast cancer patients with a positive sentinel node.Clin Cancer Res. 2007; 13: 5391-5397Crossref PubMed Scopus (84) Google Scholar, 10Harrell MI Iritani BM Ruddell A Tumor-induced sentinel lymph node lymphangiogenesis and increased lymph flow precede melanoma metastasis.Am J Pathol. 2007; 170: 774-786Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar Thus, there has been a quest for identifying the molecular mediators of lymphangiogenesis. The first specific lymphangiogenic factors identified were vascular endothelial growth factor-C (VEGF-C) and VEGF-D, which activate VEGF-receptor-3 (VEGFR-3)11Jussila L Alitalo K Vascular growth factors and lymphangiogenesis.Physiol Rev. 2002; 82: 673-700Crossref PubMed Scopus (351) Google Scholar expressed on lymphatic endothelial cells (LECs) but not on blood vascular endothelial cells (BVECs) under normal conditions.12Partanen TA Paavonen K Lymphatic versus blood vascular endothelial growth factors and receptors in humans.Microsc Res Tech. 2001; 55: 108-121Crossref PubMed Scopus (44) Google Scholar During embryogenesis, VEGF-C is required for the development of the lymphatic vascular system,13Karkkainen MJ Haiko P Sainio K Partanen J Taipale J Petrova TV Jeltsch M Jackson DG Talikka M Rauvala H Betsholtz C Alitalo K Vascular endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic veins.Nat Immunol. 2004; 5: 74-80Crossref PubMed Scopus (1041) Google Scholar and overexpression of VEGF-C or -D in experimental tumors promotes tumor lymphangiogenesis and metastasis. Furthermore, VEGF-C and -D expression levels have been shown to correlate with the incidence of metastases in a large number of human tumor types.14Stacker SA Williams RA Achen MG Lymphangiogenic growth factors as markers of tumor metastasis.APMIS. 2004; 112: 539-549Crossref PubMed Scopus (67) Google Scholar More recently, additional lymphangiogenic factors have been identified, including VEGF-A,7Hirakawa S Kodama S Kunstfeld R Kajiya K Brown LF Detmar M VEGF-A induces tumor and sentinel lymph node lymphangiogenesis and promotes lymphatic metastasis.J Exp Med. 2005; 201: 1089-1099Crossref PubMed Scopus (571) Google Scholar, 15Hong YK Lange-Asschenfeldt B Velasco P Hirakawa S Kunstfeld R Brown LF Bohlen P Senger DR Detmar M VEGF-A promotes tissue repair-associated lymphatic vessel formation via VEGFR-2 and the alpha1beta1 and alpha2beta1 integrins.FASEB J. 2004; 18: 1111-1113Crossref PubMed Scopus (253) Google Scholar, 16Kunstfeld R Hirakawa S Hong YK Schacht V Lange-Asschenfeldt B Velasco P Lin C Fiebiger E Wei X Wu Y Hicklin D Bohlen P Detmar M Induction of cutaneous delayed-type hypersensitivity reactions in VEGF-A transgenic mice results in chronic skin inflammation associated with persistent lymphatic hyperplasia.Blood. 2004; 104: 1048-1057Crossref PubMed Scopus (268) Google Scholar, 17Nagy JA Vasile E Feng D Sundberg C Brown LF Detmar MJ Lawitts JA Benjamin L Tan X Manseau EJ Dvorak AM Dvorak HF Vascular permeability factor/vascular endothelial growth factor induces lymphangiogenesis as well as angiogenesis.J Exp Med. 2002; 196: 1497-1506Crossref PubMed Scopus (452) Google Scholar hepatocyte growth factor,18Kajiya K Hirakawa S Ma B Drinnenberg I Detmar M Hepatocyte growth factor promotes lymphatic vessel formation and function.EMBO J. 2005; 24: 2885-2895Crossref PubMed Scopus (254) Google Scholar angiopoietin-1,19Morisada T Oike Y Yamada Y Urano T Akao M Kubota Y Maekawa H Kimura Y Ohmura M Miyamoto T Nozawa S Koh GY Alitalo K Suda T Angiopoietin-1 promotes LYVE-1-positive lymphatic vessel formation.Blood. 2005; 105: 4649-4656Crossref PubMed Scopus (195) Google Scholar insulin-like growth factors I and II (IGF-I and -II),20Bjorndahl M Cao R Nissen LJ Clasper S Johnson LA Xue Y Zhou Z Jackson D Hansen AJ Cao Y Insulin-like growth factors 1 and 2 induce lymphangiogenesis in vivo.Proc Natl Acad Sci USA. 2005; 102: 15593-15598Crossref PubMed Scopus (195) Google Scholar and platelet-derived growth factor-BB.21Cao R Bjorndahl MA Religa P Clasper S Garvin S Galter D Meister B Ikomi F Tritsaris K Dissing S Ohhashi T Jackson DG Cao Y PDGF-BB induces intratumoral lymphangiogenesis and promotes lymphatic metastasis.Cancer Cell. 2004; 6: 333-345Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar However, the relative importance of these factors in physiological and pathological lymphangiogenesis is currently unclear, and additional factors are likely involved. Growth hormone receptor (GHR) belongs to the cytokine receptor superfamily and is expressed by a number of different cell types including fetal mesenchymal tissue22Edmondson SR Thumiger SP Werther GA Wraight CJ Epidermal homeostasis: the role of the growth hormone and insulin-like growth factor systems.Endocr Rev. 2003; 24: 737-764Crossref PubMed Scopus (167) Google Scholar from which vessels develop. On binding of its ligand, the pituitary gland-derived growth hormone (GH, also known as somatotropin), the GHR forms homodimers leading to receptor autophosphorylation and activation of insulin receptor substrate 1 and 2, janus kinase 2, and phosphatidylinositol-3 kinase.23Ridderstråle M Signaling mechanism for the insulin-like effects of growth hormone—another example of a classical hormonal negative feedback loop.Curr Drug Targets Immune Endocr Metabol Disord. 2005; 5: 79-92Crossref PubMed Scopus (19) Google Scholar In addition to production by the pituitary gland, ectopic GH expression has also been found in vivo in the developing rat lung,24Beyea JA Olson DM Harvey S Growth hormone expression in the perinatal and postnatal rat lung.Dev Dyn. 2005; 232: 1037-1046Crossref PubMed Scopus (15) Google Scholar normal and neoplastic human lymphoid tissues and endothelial cells,25Wu H Devi R Malarkey WB Localization of growth hormone messenger ribonucleic acid in the human immune system—a Clinical Research Center study.J Clin Endocrinol Metab. 1996; 81: 1278-1282PubMed Google Scholar in normal and cancerous breast tissue,26Stoll BA Breast cancer: further metabolic-endocrine risk markers?.Br J Cancer. 1997; 76: 1652-1654Crossref PubMed Scopus (25) Google Scholar as well as in pancreatic cancer.27Ezzat S Ezrin C Yamashita S Melmed S Recurrent acromegaly resulting from ectopic growth hormone gene expression by a metastatic pancreatic tumor.Cancer. 1993; 71: 66-70Crossref PubMed Scopus (28) Google ScholarIn vitro GH expression has been described in human dermal fibroblasts, in T-cell lymphoma and monocyte lymphoma cell lines,28Palmetshofer A Zechner D Luger TA Barta A Splicing variants of the human growth hormone mRNA: detection in pituitary, mononuclear cells and dermal fibroblasts.Mol Cell Endocrinol. 1995; 113: 225-234Crossref PubMed Scopus (54) Google Scholar in peripheral blood mononuclear cells,29Hattori N Shimatsu A Sugita M Kumagai S Imura H Immunoreactive growth hormone (GH) secretion by human lymphocytes: augmented release by exogenous GH.Biochem Biophys Res Commun. 1990; 168: 396-401Crossref PubMed Scopus (102) Google Scholar and in murine granulocytes and macrophages.30Kooijman R Malur A Van Buul-Offers SC Hooghe-Peters EL Growth hormone expression in murine bone marrow cells is independent of the pituitary transcription factor Pit-1.Endocrinology. 1997; 138: 3949-3955PubMed Google Scholar GH signaling has been implicated in promoting postnatal longitudinal growth, in carbohydrate metabolism, in adipocyte maturation, and in the maintenance and development of the immune system.31Kopchick JJ Parkinson C Stevens EC Trainer PJ Growth hormone receptor antagonists: discovery, development, and use in patients with acromegaly.Endocr Rev. 2002; 23: 623-646Crossref PubMed Scopus (293) Google Scholar GH also has been shown to enhance the proliferation of human retinal microvascular endothelial cells32Rymaszewski Z Cohen RM Chomczynski P Human growth hormone stimulates proliferation of human retinal microvascular endothelial cells in vitro.Proc Natl Acad Sci USA. 1991; 88: 617-621Crossref PubMed Scopus (66) Google Scholar and of bovine brain capillary endothelial cells,33Struman I Bentzien F Lee H Mainfroid V D'Angelo G Goffin V Weiner RI Martial JA Opposing actions of intact and N-terminal fragments of the human prolactin/growth hormone family members on angiogenesis: an efficient mechanism for the regulation of angiogenesis.Proc Natl Acad Sci USA. 1999; 96: 1246-1251Crossref PubMed Scopus (237) Google Scholar and to promote the formation of capillary-like structures by human umbilical cord vein cells in vitro.34Frerich B Kurtz-Hoffmann J Lindemann N Influence of growth hormone on maintenance of capillary-like structures in an in vitro model of stromal vascular tissue—results from morphometric analysis.Artif Organs. 2005; 29: 338-341Crossref PubMed Scopus (11) Google ScholarIn vivo, GH exerts an angiogenic effect in the late-stage chicken-chorioallantoic membrane assay,35Gould J Aramburo C Capdevielle M Scanes CG Angiogenic activity of anterior pituitary tissue and growth hormone on the chick embryo chorio-allantoic membrane: a novel action of GH.Life Sci. 1995; 56: 587-594Crossref PubMed Scopus (45) Google Scholar and transgenic mice expressing bovine GH under the rat phosphoenolpyruvate carboxykinase promoter (bGH tg) show enhanced vascularization of the wound granulation tissue.36Thorey IS Hinz B Hoeflich A Kaesler S Bugnon P Elmlinger M Wanke R Wolf E Werner S Transgenic mice reveal novel activities of growth hormone in wound repair, angiogenesis, and myofibroblast differentiation.J Biol Chem. 2004; 279: 26674-26684Crossref PubMed Scopus (38) Google Scholar However, the potential activity of GH in lymphatic vessel formation and function is not known. In previous gene expression profiling studies of cultured human LECs and BVECs, we detected that GHR expression levels were higher in LECs than in BVECs.37Hirakawa S Hong YK Harvey N Schacht V Matsuda K Libermann T Detmar M Identification of vascular lineage-specific genes by transcriptional profiling of isolated blood vascular and lymphatic endothelial cells.Am J Pathol. 2003; 162: 575-586Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar In the present study, we investigated the in vitro and in vivo expression and function of GHR and GH, respectively, with regard to potential activity on the lymphatic vascular system. We found that GHR was expressed at much higher levels in LECs than in BVECs, as evaluated by quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and by Western blot analyses. GH induced in vitro proliferation, sprouting, tube formation, and migration of LECs, and the mitogenic effect was independent of VEGFR-2 or -3 activation. GH also inhibited serum starvation-induced LEC apoptosis. No major alterations of lymphatic vessels were detected in the normal skin of bGH tg mice. However, transgenic delivery of GH accelerated lymphatic vessel ingrowth into the granulation tissue of full-thickness skin wounds, and intradermal delivery of GH resulted in enlargement and enhanced proliferation of cutaneous lymphatic vessels in wild-type mice. Together, these findings identify GH as a novel lymphangiogenic factor and suggest a potential role in pathological conditions associated with lymphatic vessel activation. Dermal BVECs and LECs were isolated from neonatal human foreskins by immunomagnetic purification as previously described.18Kajiya K Hirakawa S Ma B Drinnenberg I Detmar M Hepatocyte growth factor promotes lymphatic vessel formation and function.EMBO J. 2005; 24: 2885-2895Crossref PubMed Scopus (254) Google Scholar The lineage-specific differentiation was confirmed by real-time RT-PCR for the lymphatic vascular markers Prox1, LYVE-1, and podoplanin, and for the blood vascular endothelial markers VEGFR-1 and VEGF-C, as well as by immunostains for CD31, LYVE-1, and Prox1 as described.37Hirakawa S Hong YK Harvey N Schacht V Matsuda K Libermann T Detmar M Identification of vascular lineage-specific genes by transcriptional profiling of isolated blood vascular and lymphatic endothelial cells.Am J Pathol. 2003; 162: 575-586Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar Cells were cultured in endothelial basal medium (Cambrex, Verviers, Belgium) supplemented with 20% fetal bovine serum (Gibco, Paisley, UK), antibiotic antimycotic solution (1×; Fluka, Buchs, Switzerland), l-glutamine (2 mmol/L, Fluka), hydrocortisone (10 μg/ml, Fluka), and N6,2′-O-dibutyryladenosine-3′,5′-cyclic monophosphate sodium salt (25 μg/ml, Fluka) for up to 11 passages. Cells were grown in a humidified atmosphere at 37°C and 5% CO2. Total cellular RNA was extracted from confluent LECs or BVECs using the Trizol reagent (Invitrogen, Paisley, UK) and treated with RQ RNase-free DNase (Catalys AG, Wallisellen, Switzerland) in the presence of RNase inhibitor (Applied Biosystems, Rotkreuz, Switzerland). The expression levels of vascular lineage-specific genes and of GHR were examined by real-time PCR using the 7900HT fast real-time PCR system and TaqMan one-step RT-PCR master mix reagents (Applied Biosystems). Primer and probe sequences for detection of Prox-1, LYVE-1, and VEGFR-1 have been described previously.37Hirakawa S Hong YK Harvey N Schacht V Matsuda K Libermann T Detmar M Identification of vascular lineage-specific genes by transcriptional profiling of isolated blood vascular and lymphatic endothelial cells.Am J Pathol. 2003; 162: 575-586Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar For detection of GHR, the forward primer 5′-CATTGCCCTCAACTGGACTT-3′ and reverse primer 5′-GTGGTGCTTCCCATCTCACT-3′ were used in combination with SYBR Green PCR master mix (Applied Biosystems). IGF-I, VEGF-A, and VEGFR-2 mRNA expression levels were determined by TaqMan gene expression assays (Applied Biosystems) after treatment with 100 ng/ml of recombinant human GH (R&D Systems, Abingdon, UK) for 30 minutes, 4 hours, or 24 hours. All RT-PCR results were normalized by the expression levels of β-actin.18Kajiya K Hirakawa S Ma B Drinnenberg I Detmar M Hepatocyte growth factor promotes lymphatic vessel formation and function.EMBO J. 2005; 24: 2885-2895Crossref PubMed Scopus (254) Google Scholar LECs and BVECs were grown to 80% confluence. Total protein was extracted in lysis buffer (20 mmol/L Tris, 150 mmol/L sodium chloride, 5 mmol/L ethylenediaminetetraacetic acid, 1% Triton X, 25 mmol/L sodium fluoride, 1 mmol/L phenylmethyl sulfonyl fluoride, 1 mmol/L sodium metavanadate, 10% glycerol, one tablet of ethylenediaminetetraacetic acid-free protease inhibitor cocktail per 10 ml extraction buffer (Roche, Basel, Switzerland)) and the protein concentration was determined using the NanoOrange protein quantitation kit (Invitrogen). Eighty μg of total protein were immunoprecipitated with a mouse anti-GHR antibody (0.25 μg; Abcam, Cambridge, UK), separated in an 8% acrylamide separating gel and immunoblotted with the same GHR antibody (2 μg/ml) and an anti-mouse horseradish peroxidase antibody (1:2000; Amersham, Duebendorf, Switzerland). In an additional study, 2 ml of supernatant were obtained from LECs treated with 100 ng/ml of GH (R&D Systems) for 6 hours or 24 hours. After immunoprecipitation of IGF-I using a goat anti-IGF-I antibody (5 μg, R&D Systems), IGF-I was detected by incubation with a goat anti-IGF-I antibody (0.2 μg/ml, R&D Systems) and anti-goat horseradish peroxidase antibody (1:2000, Invitrogen). Recombinant human IGF-I (R&D Systems) was used as a positive control. Protein bands were visualized by the ECL plus Western blotting detection system (Amersham). For detection of IGF-I by enzyme-linked immunosorbent assay, 200 μl of immunoprecipitated tissue culture supernatant, and a dilution series of recombinant IGF-I were mixed with carbonate buffer (sodium hydrogen carbonate and sodium carbonate, 0.1 mol/L, pH 8.8) and incubated overnight in Nunc-Immuno 96-microwell plates (VWR, Dietikon, Switzerland) at 4°C. IGF-I was detected by incubation with 0.75 μg/ml goat anti-IGF I antibody (R&D Systems) and anti-goat horseradish peroxidase antibody (1:1000, Invitrogen). Detection was performed using BM Blue POD substrate (Roche) and stopped by applying sulfuric acid. Absorbance was measured by using a VersaMax microplate reader (Bucher Biotec AG, Basel, Switzerland). Immunofluorescence analyses were performed on 6-μm frozen sections of human neonatal foreskins and of mouse skin samples after implantation of Matrigel containing recombinant human GH or phosphate-buffered saline (PBS), as well as of 6-μm paraffin sections of skin samples obtained from bGH transgenic and age-matched wild-type mice (kindly provided by Dr. Sabine Werner, ETH Zurich, Switzerland).36Thorey IS Hinz B Hoeflich A Kaesler S Bugnon P Elmlinger M Wanke R Wolf E Werner S Transgenic mice reveal novel activities of growth hormone in wound repair, angiogenesis, and myofibroblast differentiation.J Biol Chem. 2004; 279: 26674-26684Crossref PubMed Scopus (38) Google Scholar The antibodies used were reactive against the lymphatic-specific markers podoplanin38Oliver G Detmar M The rediscovery of the lymphatic system: old and new insights into the development and biological function of the lymphatic vasculature.Genes Dev. 2002; 16: 773-783Crossref PubMed Scopus (309) Google Scholar (D2-40, 1:100; Signet, Dedham, USA) and LYVE-138Oliver G Detmar M The rediscovery of the lymphatic system: old and new insights into the development and biological function of the lymphatic vasculature.Genes Dev. 2002; 16: 773-783Crossref PubMed Scopus (309) Google Scholar (reactive against both human and mouse, 1:1000; Upstate, Dundee, UK), against the panendothelial marker CD3139Sauter B Foedinger D Sterniczky B Wolff K Rappersberger K Immunoelectron microscopic characterization of human dermal lymphatic microvascular endothelial cells. Differential expression of CD31, CD34, and type IV collagen with lymphatic endothelial cells vs blood capillary endothelial cells in normal human skin, lymphangioma, and hemangioma in situ.J Histochem Cytochem. 1998; 46: 165-176Crossref PubMed Scopus (140) Google Scholar (anti-human and anti-mouse, 1:50; Becton Dickinson, Allschwil, Switzerland) and against GH receptor (anti-human, 1:20; R&D Systems). Corresponding secondary antibodies were labeled with Alexa 488 or Alexa 594 (Invitrogen). Frozen sections were fixed in −20°C acetone for 2 minutes and 4°C 80% methanol for 5 minutes. Antigen retrieval was performed by boiling the sections for 10 minutes in citric acid buffer. For BrdU stains (Alexa 594-conjugated antibody, 1:60; Invitrogen), tissues were pretreated with 2 N HCl for 20 minutes at room temperature. Nuclei were counterstained with 20 μg/ml Hoechst trihydrochloride, trihydrate (nitrogen). Sections were examined by an Axioskop 2 plus microscope and pictures were taken using a AxioCam MRc (Carl Zeiss AG, Feldbach, Switzerland). The vessel density, vessel size, and average area occupied by vessels were determined in CD31/LYVE-1-stained sections obtained from wild-type and bGH transgenic mice as described,18Kajiya K Hirakawa S Ma B Drinnenberg I Detmar M Hepatocyte growth factor promotes lymphatic vessel formation and function.EMBO J. 2005; 24: 2885-2895Crossref PubMed Scopus (254) Google Scholar using the IPLab software (Scanalytics, Rockville, MD). Three pictures each were taken of hot-spot areas in normal tail skin (wild-type: n = 10 female and n = 6 male; transgenic: n = 9 female and n = 6 male), and four in areas surrounding or within the granulation tissue of 5-day-old skin wounds (wild-type: n = 5 female; transgenic: n = 6 female). For the Matrigel study, blood and lymphatic vessels as well as BrdU-positive LECs were analyzed in three areas in close proximity to the Matrigel. Morphometric analyses were performed by an investigator blinded to the identity of the samples. Statistical analyses were performed using the two-tailed unpaired Student's t-test (Graph Pad Prism 4; GraphPad Software Inc., San Diego, CA). Proliferation, migration, and tube formation assay were primarily performed as described.18Kajiya K Hirakawa S Ma B Drinnenberg I Detmar M Hepatocyte growth factor promotes lymphatic vessel formation and function.EMBO J. 2005; 24: 2885-2895Crossref PubMed Scopus (254) Google Scholar For proliferation assays, BVECs and LECs (1.25 to 1.5 × 103) were seeded into fibronectin- or collagen type I-coated 96-well plates and were treated with different concentrations of GH (0 to 1000 ng/ml, R&D Systems) or with an equal volume of PBS in endothelial basal medium containing 2% fetal bovine serum. LECs were also incubated with GH (100 ng/ml) together with goat anti-human GHR antibody (1 μg/ml, R&D Systems), human anti-human VEGFR-3 antibody (clone hF4-3C6, 1 μg/ml), human anti-human VEGFR-2 (clone 1121b, 10 μg/ml; kind gift of Dr. Bronek Pytowski, Imclone Systems Inc., New York, NY), goat anti-human IGF I antibody (10 μg/ml, R&D Systems), or control IgG (1 μg/ml or 10 μg/ml, respectively). After 72 hours, cells were incubated with 5-methylumbelliferylheptanoate as described.40Detmar M Tenorio S Hettmannsperger U Ruszczak Z Orfanos CE Cytokine regulation of proliferation and ICAM-1 expression of human dermal microvascular endothelial cells in vitro.J Invest Dermatol. 1992; 98: 147-153Abstract Full Text PDF PubMed Scopus (105) Google Scholar The intensity of fluorescence, proportional to the number of viable cells, was measured using a SpectraMax Gemini EM microplate reader (Bucher Biotec AG). Haptotactic cell migration was performed in the presence or absence of GH (10 to 1000 ng/ml) as described.18Kajiya K Hirakawa S Ma B Drinnenberg I Detmar M Hepatocyte growth factor promotes lymphatic vessel formation and function.EMBO J. 2005; 24: 2885-2895Crossref PubMed Scopus (254) Google Scholar In additional studies, cells were pre-incubated with a blocking anti-GHR antibody (1 μg/ml, R&D Systems), a blocking anti-integrin α9β1 antibody (1 μg/ml; Chemicon, Temecula, CA), or control IgG for 10 minutes, and were then seeded into the upper chambers of transwell migration chambers and incubated for 3 hours in the presence or absence of GH (100 ng/ml). Migrated cells were stained with Calcein AM (Invitrogen) and fixed in 4% paraformaldehyde. The fluorescence intensity, proportional to the number of transmigrated cells, was measured using a SpectraMax Gemini EM reader. For tube formation assays, confluent LEC monolayers were overlaid with collagen type I gels as described18Kajiya K Hirakawa S Ma B Drinnenberg I Detmar M Hepatocyte growth factor promotes lymphatic vessel formation and function.EMBO J. 2005; 24: 2885-2895Crossref PubMed Scopus (254) Google Scholar (1 mg/ml; Cohesion, Palo Alto, CA) containing GH (100 ng/ml) or an equal volume of PBS. Tubes were evaluated for up to 20 hours. Three pictures of hot-spots were taken per well and total tube length was analyzed using the IP Lab software as described.18Kajiya K Hirakawa S Ma B Drinnenberg I Detmar M Hepatocyte growth factor promotes lymphatic vessel formation and function.EMBO J. 2005; 24: 2885-2895Crossref PubMed Scopus (254) Google Scholar Spheroid sprouting assays were performed as described41Korff T Kimmina S Martiny-Baron G Augustin HG Blood vessel maturation in a 3-dimensional spheroidal coculture model: direct contact with smooth muscle cells regulates endothelial cell quiescence and abrogates VEGF responsiveness.FASEB J. 2001; 15: 447-457Crossref PubMed Scopus (312) Google Scholar with the following variation. Spheroids were generated in hanging drops without methylcellulose in Nunclone plates (1000 cells in 20 μl medium; Nunc GmbH, Wiesbaden, Germany). After 24 hours of incubation, spheroids were collected and mixed with collagen type I solution with or without GH (100 ng/ml). Spheroid sprouting was analyzed after 16 hours, using an Axiovert 200M microscope (Carl Zeiss AG). Quantitative sprout length analysis was performed using the IP Lab software. For apoptosis assays, LECs (one 60-mm plate each) were treated with 100 ng/ml GH or with PBS for 60 hours in endothelial basal medium containing 0.2% bovine serum albumin. As a positive control, 200 μmol/L hydrogen peroxide was applied for 24 hours. Cells were stained using the In Situ Cell Death Detection kit (Roche), and fluorescence was detected on a FACS DIVA flow cytometer (Becton Dickinson). E
Referência(s)