Chemotactic Properties of Angiopoietin-1 and -2, Ligands for the Endothelial-specific Receptor Tyrosine Kinase Tie2
1998; Elsevier BV; Volume: 273; Issue: 29 Linguagem: Inglês
10.1074/jbc.273.29.18514
ISSN1083-351X
AutoresBernhard Witzenbichler, Peter C. Maisonpierre, Pamela F. Jones, George D. Yancopoulos, Jeffrey M. Isner,
Tópico(s)Coronary Interventions and Diagnostics
ResumoAngiopoietin-1 and its putative natural antagonist, angiopoietin-2, were recently isolated, and the critical role of angiopoietin-1 in embryogenic angiogenesis was demonstrated by targeted gene disruption. Specific biological effects of angiopoietin-1, however, have yet to be defined. In this study we demonstrate that angiopoietin-1, but not angiopoietin-2, is chemotactic for endothelial cells. In contrast, angiopoietin-1 as well as angiopoietin-2 exhibit no proliferative effect on endothelial cells. Excess soluble Tie2, but not Tie1 receptor, abolish the chemotactic response of endothelial cells toward angiopoietin-1. Angiopoietin-2 dose-dependently blocks directed migration toward angiopoietin-1, consistent with the role of angiopoietin-2 as a naturally occurring inhibitor of angiopoietin-1. Fibroblasts stably transfected with Tie2 receptor exhibit chemotactic responses for both angiopoietin-1 and angiopoietin-2. Fibroblasts stably expressing a transfected chimeric receptor consisting of the ectodomain of TrkC fused to the cytoplasmic domain of Tie2 also exhibit a chemotactic response to neurotrophin 3 (NT-3), a specific ligand for TrkC. Endothelial cells are shown to express angiopoietin-2 mRNA and protein, indicating the potential for autocrine activation of angiopoietin/Tie2. Finally, the demonstration that Tie2 as well as angiopoietin-1 are expressed in normal human arteries and veins suggests that the role of angiopoietin/Tie2 may extend beyond embryonic angiogenesis to maintaining integrity of the adult vasculature. Angiopoietin-1 and its putative natural antagonist, angiopoietin-2, were recently isolated, and the critical role of angiopoietin-1 in embryogenic angiogenesis was demonstrated by targeted gene disruption. Specific biological effects of angiopoietin-1, however, have yet to be defined. In this study we demonstrate that angiopoietin-1, but not angiopoietin-2, is chemotactic for endothelial cells. In contrast, angiopoietin-1 as well as angiopoietin-2 exhibit no proliferative effect on endothelial cells. Excess soluble Tie2, but not Tie1 receptor, abolish the chemotactic response of endothelial cells toward angiopoietin-1. Angiopoietin-2 dose-dependently blocks directed migration toward angiopoietin-1, consistent with the role of angiopoietin-2 as a naturally occurring inhibitor of angiopoietin-1. Fibroblasts stably transfected with Tie2 receptor exhibit chemotactic responses for both angiopoietin-1 and angiopoietin-2. Fibroblasts stably expressing a transfected chimeric receptor consisting of the ectodomain of TrkC fused to the cytoplasmic domain of Tie2 also exhibit a chemotactic response to neurotrophin 3 (NT-3), a specific ligand for TrkC. Endothelial cells are shown to express angiopoietin-2 mRNA and protein, indicating the potential for autocrine activation of angiopoietin/Tie2. Finally, the demonstration that Tie2 as well as angiopoietin-1 are expressed in normal human arteries and veins suggests that the role of angiopoietin/Tie2 may extend beyond embryonic angiogenesis to maintaining integrity of the adult vasculature. The receptor tyrosine kinase family of cell surface proteins is known to play key roles in transducing intercellular signals to the cytoplasm (1Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4619) Google Scholar). A large diversity of receptor tyrosine kinases and receptor tyrosine kinase expression patterns, which are temporally modified during development and under pathologic conditions, determines cell fate and allows tissue-specific cell responses. Ligand binding to the large extracellular domain of receptor tyrosine kinases leads to receptor dimerization and autophosphorylation of tyrosine residues on the intracellular domain of the receptor (2Schlessinger J. Ullrich A. Neuron. 1992; 9: 383-391Abstract Full Text PDF PubMed Scopus (1295) Google Scholar). A variety of Src homology 2 domain-containing proteins, which are recruited to these phosphorylation sites, have been identified (3Marschall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4245) Google Scholar). These proteins are regarded as initiators of different signal cascades that finally lead to specific cellular responses including proliferation, migration, differentiation, and morphologic organization in the context of surrounding tissues (4Risau W. FASEB J. 1995; 9: 926-933Crossref PubMed Scopus (528) Google Scholar). On the basis of sequence similarity and structural characteristics, it is possible to classify receptor tyrosine kinases into subfamilies (1Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4619) Google Scholar). Two receptor tyrosine kinase subfamilies are characterized by their largely endothelium-specific expression. One of them consists of the three known vascular endothelial growth factor (VEGF) 1VEGF, vascular endothelial growth factor; EC, endothelial cell; FBS, fetal bovine serum; HUVEC, human umbilical vein endothelial cell; NT-3, neurotrophin 3; PDGF, platelet-derived growth factor; EGF, epidermal growth factor; SMC, smooth muscle cells; Ang, angiopoietin; HM, human microvascular; MTS, 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium; RT, reverse transcription; PCR, polymerase chain reaction; CM, conditioned media; rTie1-Fc, Tie1 receptor-Fc. receptors Flt-1/VEGF-R1, Flk-1/KDR/VEGF-R2, and Flt-4/VEGF-R3 (5Mustonen T. Alitalo K. J. Cell Biol. 1995; 129: 895-898Crossref PubMed Scopus (477) Google Scholar, 6Joukov V. Pajusola K. Kaipainen A. Chilov D. Lahtinen I. Kukk E. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1996; 15: 290-298Crossref PubMed Scopus (1160) Google Scholar). The critical role of these receptors in promoting vasculogenesis and angiogenesis during normal embryogenesis was demonstrated using mutant mice with targeted disruption of the flt-1 (7Fong G-H. Rossant J. Gerstenstein M. Breitman M.L. Nature. 1995; 376: 66-70Crossref PubMed Scopus (2224) Google Scholar) orflk-1 gene (8Shalaby F. Rossant J. Yamaguchi T.P. Gertsenstein M. Wu X-F. Breitman M.L. Schuh A.C. Nature. 1995; 376: 62-66Crossref PubMed Scopus (3371) Google Scholar). Moreover, expression of these receptors in the endothelial layer of adult blood vessels (9Couffinhal T. Kearney M. Witzenbichler B. Chen D. Murohara T. Losordo D.W. Symes J.F. Isner J.M. Am. J. Pathol. 1997; 150: 1673-1685PubMed Google Scholar) is thought to mediate VEGF-induced post-natal angiogenesis in pathologic (10Plate K.H. Breier G. Weich H.A. Risau W. Nature. 1992; 359: 845-848Crossref PubMed Scopus (2126) Google Scholar, 11Plate K.H. Breier G. Millauer B. Ullrich A. Risau W. Cancer Res. 1993; 53: 5822-5827PubMed Google Scholar, 12Folkman J. Nat. Med. 1995; 1: 27-30Crossref PubMed Scopus (7235) Google Scholar) or therapeutic settings (13Isner J.M. Pieczek A. Schainfeld R. Blair R. Haley L. Asahara T. Rosenfield K. Razvi S. Walsh K. Symes J. Lancet. 1996; 348: 370-374Abstract Full Text Full Text PDF PubMed Scopus (915) Google Scholar, 14Takeshita S. Zheng L.P. Brogi E. Kearney M. Pu L.Q. Bunting S. Ferrara N. Symes J.F. Isner J.M. J. Clin. Invest. 1994; 93: 662-670Crossref PubMed Scopus (1004) Google Scholar) but is also believed to be important in maintaining vessel integrity (15Tsurumi Y. Murohara T. Krasinski K. Dongfen C. Witzenbichler B. Kearney M. Couffinhal T. Isner J.M. Nat. Med. 1997; 3: 879-886Crossref PubMed Scopus (303) Google Scholar). The Tie (tyrosine kinase with immunoglobulin (Ig) and epidermal growth factor (EGF) homology domains) receptor family comprises a second endothelial-specific subfamily of receptor tyrosine kinases termed Tie1 (16Dumont D.J. Fong G-H. Fong M.C. Puri G. Gradwohl G. Alitalo K. Breitman M.L. Dev. Dyn. 1995; 203: 80-92Crossref PubMed Scopus (452) Google Scholar, 17Maisonpierre P.C. Goldfarb M. Yancopoulos G.D. Gao G. Oncogene. 1993; 8: 1631-1637PubMed Google Scholar, 18Sato T.N. Qin Y. Kozak C.A. Audus K.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9355-9358Crossref PubMed Scopus (405) Google Scholar, 19Ziegler S.F. Bird T.A. Schneringer K.A. Schooley K.A. Baum P.R. Oncogene. 1997; 8: 663-670Google Scholar) and Tie2/Tek (16Dumont D.J. Fong G-H. Fong M.C. Puri G. Gradwohl G. Alitalo K. Breitman M.L. Dev. Dyn. 1995; 203: 80-92Crossref PubMed Scopus (452) Google Scholar, 20Dumont D.J. Yamaguchi T.P. Conlon R.A. Rossant J. Breitman M.L. Oncogene. 1992; 7: 1471-1480PubMed Google Scholar, 21Schnurch H. Risau W. Development. 1993; 119: 957-968PubMed Google Scholar). Similar to the VEGF receptors, both receptors have been shown to be critically involved in the formation of embryonic vasculature. Tie1 null mice (22Sato T.N. Tozawa Y. Deutsch U. Wolburg-Buchholz K. Fujiwara Y. Gentron-Maguire M. Gridley T. Wolburg H. Risau W. Qin Y. Nature. 1995; 376: 70-74Crossref PubMed Scopus (1512) Google Scholar, 23Puri M.C. Rossant J. Alitalo K. Bernstein A. Partanen J. EMBO J. 1995; 14: 5884-5891Crossref PubMed Scopus (422) Google Scholar) as well as mice deficient in Tie2 (24Dumont D.J. Gradwohl G. Guo-Hua F. Puri M.C. Gertsenstein M. Auerbach A. Breitman M.L. Genes Dev. 1994; 8: 1897-1909Crossref PubMed Scopus (819) Google Scholar) display a lethal phenotype caused by severe defects in embryonic vasculature. Moreover, an activating mutation in the Tie2 receptor was shown to cause inherited venous malformations in humans (25Vikkula M. Boon L.M. Carraway III, K.L. Calvert J.T. Diamonti A.J. Goumnerov B. Pasyk K.A. Marchuk D.A. Warman M.L. Cantley L.C. Mulliken J.B. Olsen B.R. Cell. 1996; 87: 1181-1190Abstract Full Text Full Text PDF PubMed Scopus (615) Google Scholar); these malformed vessels exhibited a disproportionately large number of endothelial cells (ECs) versus smooth muscle cells (SMCs), underlining the importance of an intact Tie2 receptor system for EC-SMC communication in vascular morphogenesis. Although the ligands of the three VEGF receptors, their binding patterns, and their functions are relatively well characterized (5Mustonen T. Alitalo K. J. Cell Biol. 1995; 129: 895-898Crossref PubMed Scopus (477) Google Scholar, 6Joukov V. Pajusola K. Kaipainen A. Chilov D. Lahtinen I. Kukk E. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1996; 15: 290-298Crossref PubMed Scopus (1160) Google Scholar,26Olofsson B. Pajusola K. Kaipainen A. vonEuler G. Joukov V. Saksela O. Orpana A. Pettersson R.F. Alitalo K. Eriksson U. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2576-2581Crossref PubMed Scopus (629) Google Scholar), the ligand for the Tie2 receptor, named angiopoietin-1 (Ang1), was only recently isolated (27Davis S. Aldrich T.H. Jones P.F. Acheson A. Compton D.L. Jain V. Ryan T.E. Bruno J. Radziejewski C. Maisonpierre P.C. Yancopoulos G.D. Cell. 1996; 87: 1161-1169Abstract Full Text Full Text PDF PubMed Scopus (1698) Google Scholar), whereas the ligand(s) for the Tie1 receptors are still unknown. Targeted disruption of the Ang1 gene in mice resulted in an embryonic lethal phenotype by day 12.5 with generalized defects in vascular structure reminiscent of those previously observed in mice lacking Tie2 (28Suri C. Jones P.F. Patan S. Bartunkova S. Maisonpierre P.C. Davis S. Sato T. Yancopoulos G.D. Cell. 1996; 87: 1171-1180Abstract Full Text Full Text PDF PubMed Scopus (2406) Google Scholar). Ang1 protein phosphorylated Tie2 receptor in a variety of ECs but did not induce EC growth or tube formation (27Davis S. Aldrich T.H. Jones P.F. Acheson A. Compton D.L. Jain V. Ryan T.E. Bruno J. Radziejewski C. Maisonpierre P.C. Yancopoulos G.D. Cell. 1996; 87: 1161-1169Abstract Full Text Full Text PDF PubMed Scopus (1698) Google Scholar). In fact, no in vitro assay to date has demonstrated any biological function of Ang1 for ECs. Analysis of Ang1 mRNA expression in adult tissues revealed abundant expression in highly vascularized tissues such as skeletal muscle, prostate, ovary, uterus, and placenta (29Maisonpierre P. Suri C. Jones P. Bartunkova S. Wiegand S. Radziejewski C. Compton D. McClain J. Aldrich T. Papadopoulos N. Daly T. Davis S. Sato T. Yancopoulos G. Science. 1997; 277: 55-60Crossref PubMed Scopus (2975) Google Scholar), suggesting a role of Ang1 not only in embryonic angiogenesis but also in the adult vasculature. Given the paucity of in vitro data, however, the physiologic contribution of Ang1 to these processes remains enigmatic. The temporal appearance of embryonic Ang1 expression suggests that the Ang1/Tie2 system is not involved in the initial vasculogenic phase of vascular development (as shown for the VEGF system) but rather participates in angiogenic outgrowth (sprouts), vessel remodeling, and vascular maturation (22Sato T.N. Tozawa Y. Deutsch U. Wolburg-Buchholz K. Fujiwara Y. Gentron-Maguire M. Gridley T. Wolburg H. Risau W. Qin Y. Nature. 1995; 376: 70-74Crossref PubMed Scopus (1512) Google Scholar, 24Dumont D.J. Gradwohl G. Guo-Hua F. Puri M.C. Gertsenstein M. Auerbach A. Breitman M.L. Genes Dev. 1994; 8: 1897-1909Crossref PubMed Scopus (819) Google Scholar, 8Shalaby F. Rossant J. Yamaguchi T.P. Gertsenstein M. Wu X-F. Breitman M.L. Schuh A.C. Nature. 1995; 376: 62-66Crossref PubMed Scopus (3371) Google Scholar). The recent discovery of a natural antagonist for Tie2, named angiopoietin-2 (Ang2), that binds to Tie2 but does not transduce the receptor has added a further level of complexity to regulation by the Ang/Tie2 system (29Maisonpierre P. Suri C. Jones P. Bartunkova S. Wiegand S. Radziejewski C. Compton D. McClain J. Aldrich T. Papadopoulos N. Daly T. Davis S. Sato T. Yancopoulos G. Science. 1997; 277: 55-60Crossref PubMed Scopus (2975) Google Scholar). Findings described in the current study are the first to our knowledge to demonstrate an in vitro effect of Ang1 on ECs. We show that Ang1, but not Ang2, is a potent and specific chemotactic protein when administered to ECs. Neither Ang1 nor Ang2 was found to be mitogenic for ECs. In contrast to other angiogenic cytokines (30Haimovitz-Friedman A. Balaban N. McLoughlin M. Ehleiter D. Michaeli J. Vlodavsky I. Fuks Z. Cancer Res. 1994; 115: 245-247Google Scholar, 31Spyridopoulos I. Brogi E. Kearney M. Sullivan A.B. Cetrulo C. Isner J.M. Losordo D.W. J. Mol. Cell. Cardiol. 1997; 29: 1321-1330Abstract Full Text PDF PubMed Scopus (193) Google Scholar), both Ang1 and Ang2 were also devoid of anti- or pro-apoptotic effects. The chemotactic response of ECs toward Ang1 could be abolished by excess soluble Tie2, but not Tie1 receptor, as well as by increasing amounts of Ang2. In contrast to findings shown for Ang2 and ECs, fibroblasts stably transfected with Tie2 receptor exhibited strong chemotactic responses for Ang2 as well as Ang 1, consistent with previous observations that Ang2 can activate Tie2 receptors in non-ECs. Finally, the demonstration of Tie2 as well as Ang1 expression in adult normal arterial and venous specimens suggests a potential role for Tie2/Ang1 in the maintenance of vascular integrity. Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cord vein by collagenase treatment as described previously (32Jaffe E.A. Nachman R.L. Becker C.G. Minick C.R. J. Clin. Invest. 1973; 52: 2745-2756Crossref PubMed Scopus (6019) Google Scholar) and grown in medium 199 (M199) (Life Technologies, Inc.) supplemented with 20% fetal bovine serum (FBS) (Life Technologies), 100 μg/ml EC growth supplement, and 50 units/ml heparin (Sigma). HUVECs were used between passages 3 and 5. Human microvascular (HM) ECs of dermal origin were purchased from Clonetics, grown in EBM medium containing human epidermal growth factor (10 ng/ml), hydrocortisone (10 ng/ml), 5% FBS, and 0.4% bovine brain extract (Clonetics). Human microvascular endothelial cells were used between passages 4 and 6. Rat vascular smooth muscle cells were extracted from rats as described previously (33Mader S.L. J. Gerontol. Biol. Sci. 1992; 47: B32-B36Crossref Scopus (26) Google Scholar) and cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS. Human vascular smooth muscle cells were derived by explant outgrowth from unused portions of saphenous veins obtained at coronary bypass surgery as described previously (34Pickering J.G. Weir L. Rosenfield K. Stetz J. Jekanowski J. Isner J.M. J. Am. Coll. Cardiol. 1992; 20: 1430-1439Crossref PubMed Scopus (72) Google Scholar) and cultured in Dulbecco's modified Eagle's medium supplemented with 15% FBS. NIH 3T3 fibroblasts were purchased from the American Type Culture Collection and maintained in 10% FBS. Stable transfections of fibroblasts with an expression plasmid encoding full-length Tie2 receptor cDNA, a chimeric receptor consisting of the ectodomain of TrkC fused to the cytoplasmic domain of Tie2 or with empty vector (mock), were produced as before (29Maisonpierre P. Suri C. Jones P. Bartunkova S. Wiegand S. Radziejewski C. Compton D. McClain J. Aldrich T. Papadopoulos N. Daly T. Davis S. Sato T. Yancopoulos G. Science. 1997; 277: 55-60Crossref PubMed Scopus (2975) Google Scholar). Ang1* and Ang2 recombinant protein were produced using baculovirus vectors and purified using protein A-Sepharose as described previously (27Davis S. Aldrich T.H. Jones P.F. Acheson A. Compton D.L. Jain V. Ryan T.E. Bruno J. Radziejewski C. Maisonpierre P.C. Yancopoulos G.D. Cell. 1996; 87: 1161-1169Abstract Full Text Full Text PDF PubMed Scopus (1698) Google Scholar). (In the case of Ang1, the protein used for these in vitro studies was modified from the original and has been designated Ang1* (29Maisonpierre P. Suri C. Jones P. Bartunkova S. Wiegand S. Radziejewski C. Compton D. McClain J. Aldrich T. Papadopoulos N. Daly T. Davis S. Sato T. Yancopoulos G. Science. 1997; 277: 55-60Crossref PubMed Scopus (2975) Google Scholar)). The purity of proteins was greater than 95% as judged by reducing and nonreducing silver-stained SDS-polyacrylamide gel electrophoresis. Soluble Tie1 receptor-Fc (rTie1-Fc) and Tie2 receptor-Fc (rTie2-Fc) fusion proteins were constructed, produced, and purified as described recently (27Davis S. Aldrich T.H. Jones P.F. Acheson A. Compton D.L. Jain V. Ryan T.E. Bruno J. Radziejewski C. Maisonpierre P.C. Yancopoulos G.D. Cell. 1996; 87: 1161-1169Abstract Full Text Full Text PDF PubMed Scopus (1698) Google Scholar). Heterodimeric recombinant human VEGF165 protein purified from Escherichia coli was a generous gift of Drs. N. Ferrara, B. Keyt, and S. Bunting at Genentech Inc. Cell proliferation was assayed using the colorimetric MTS (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium) assay with the electron coupling reagent phenazine methosulfate (CellTiter 96 AQ, Promega). This assay was previously validated and demonstrated to positively correlate with cell number (35Buttke T.M. McCubrey J.A. Owen T.C. J. Immunol. Methods. 1993; 30: 233-240Crossref Scopus (290) Google Scholar). Cells were seeded in a fibronectin-coated 96-well plate (5 × 103cells/well) in 0.1 ml of serum-supplemented medium and allowed to attach overnight. Factors in 5% FBS were added to the wells for 48 h as indicated. MTS/phenazine methosulfate mixture (20 μl) was added to each well and allowed to incubate for 1 h at 37 °C before measuring absorbance at 490 nm in an enzyme-linked immunosorbent assay plate reader. Background absorbance from control wells was subtracted, and seven wells were performed in parallel for each condition. EC DNA synthesis was evaluated by [3H]thymidine incorporation assay. A total of 2 × 104 cells/well in M199 containing 5% FBS were seeded in 24-well plates, coated with 1.5% gelatin, and allowed to attach overnight. Specific conditions (see below) were employed for 48 h before the addition of 2 μl/ml [3H]thymidine (Amersham Pharmacia Biotech). After 4 h, cells were washed with phosphate-buffered saline, precipitated with 10% trichloroacetic acid, lysed in 0.25 n NaOH, and then transferred into glass vials filled with scintillation liquid. Radioactivity was measured in a Beckman counter and expressed as counts/min (cpm). The addition of phorbol 12-myristate 13-acetate (Sigma) was used as a positive control to induce [3H]thymidine incorporation. Each sample was done in triplicate. To quantify apoptosis, floating and adhesive cells were collected after treatment as indicated using trypsin and washed and resuspended in ice-cold 70% ethanol. For cell cycle analysis, only adhesive cells were considered. After fixing overnight at 4 °C, cells were stained with 50 μg/ml propidium iodide (Boehringer Mannheim) in phosphate-buffered saline, pH 7.4, containing 0.1% (w/v) Triton X-100 (Sigma), 0.5 mm EDTA, and 50 μg/ml RNase A (50 units/l, Sigma) at 4 °C for at least 1 h. DNA content was analyzed from 1 × 105 events/group using a fluorescent-activated cell sorter Scan cytometer (Becton Dickinson) with Lysys II software. Cells with less fluorescence intensity than the G1 peak were regarded as apoptotic and expressed as a percentage of total number of cells analyzed. Cell cycle statistics were calculated using CellFit Cell-Cycle software (Becton Dickinson). EC migration assays were performed using a 48-well microchemotaxis chamber (Neuroprobe) (36Falk W. Goodwin R.H. Leonard E.J. J. Immunol. Methods. 1980; 33: 239-247Crossref PubMed Scopus (251) Google Scholar). Polyvinylpyrrolidone-free polycarbonate filters with a pore size of 8 μm (Nuclepore) were coated with 50 μg/ml fibronectin and 0.1% gelatin in phosphate-buffered saline for at least 6 h at room temperature and dried under sterile air. Test substances were diluted to appropriate concentrations in M199 supplemented with 1% FBS, and 25 μl of the final dilution was placed in the lower chamber of a modified Boyden chamber. Confluent cell cultures were washed and trypsinized for the minimum time required to achieve cell detachment. After placing the filter between lower and upper chambers, 2.5 × 105 cells suspended in 50 μl of M199 containing 1% FBS were seeded in the upper compartment. The apparatus was then incubated for 5 h at 37 °C in a humidified chamber with 5% CO2 to allow cell migration. After the incubation period, the filter was removed, and the upper side of the filter containing the nonmigrated cells was scraped with a rubber policeman. The filters were fixed with methanol and stained with Giemsa solution (Diff-Quick, Baxter). Migration was quantified by counting cells in three random high power fields (100×) in each well. All groups were studied in quadruplicate. Unused segments of human internal mammary artery, radial artery, and saphenous vein were obtained during bypass surgery, as described previously (9Couffinhal T. Kearney M. Witzenbichler B. Chen D. Murohara T. Losordo D.W. Symes J.F. Isner J.M. Am. J. Pathol. 1997; 150: 1673-1685PubMed Google Scholar); these specimens were retrieved from the operating room at the time of excision and were promptly processed to avoid degradation of RNA. Care was taken to remove these segments in a nontraumatic manner, including limiting manipulation of the specimens to the ends of the vascular segments. Total RNA from these specimen or from cell cultures as indicated was immediately isolated by phenol/chloroform extraction (37Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar), and RNA concentration was calculated from absorbance at 260 nm. Reverse transcription (RT) (38Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York1989: 204-210Google Scholar) was performed by heating a 10 μl reaction mixture containing 1 μg of total RNA and 20 μg/ml oligodeoxythymidine (Life Technologies, Inc.) at 70 °C for 10 min. After cooling, 60 units of human placenta ribonuclease inhibitor (Promega) and 200 units of Moloney's murine leukemia virus RNase H reverse transcriptase (Life Technologies, Inc.) were added in a final 20-μl reaction mixture containing 1 mmol/l each dNTP (Amersham), 10 mmol/l dithiothreitol, 25 mmol/l Tris-HCl, pH 8.3, 75 mmol/l KCl, and 3mmol/l MgCl2, incubated for 1 h at 42 °C, heated 5 min at 95 °C, and diluted to 50 μl with double-distilled water. For the polymerase chain reaction (PCR) (39Saiki R.K. Gelfand D.H. Stoffel S. Scharf S.J. Higuchi R. Horn G.T. Mullis K.G. Ehrlich H.A. Science. 1988; 239: 487-491Crossref PubMed Scopus (13517) Google Scholar), a 50-μl reaction containing 5 μl of cDNA-RNA hybrids, 200 μmol/l dNTP (Amersham), 20 mmol/l Tris-HCl, pH 8.55, 2.5 mmol/l MgCl2,16 mmol/l (NH4)2SO4, 150 μg/ml BSA, 1 μmol/l each oligonucleotide primer, and 1.25 units ofTaq polymerase (Perkin-Elmer) was subjected to the following temperature cycles: 30 s of denaturing at 95 °C, 5 s of annealing at 56 °C, and 1 min of extension at 72 °C. At the end of the last cycle, a prolonged extension step was carried out for 10 min. PCR products were analyzed by electrophoreses of 10 μl of each PCR reaction mixture in a 1.5% agarose gel, and bands were visualized by ethidium bromide staining. Experiments were repeated ≥ triplicate. The primers chosen for sense (3′-5′) and antisense (5′-3′), respectively, were: for human Tie2 (GenBankTM accession number L06139) ATCCCATTTGCAAAGCTTCTGGCTGGC and TGTGAAGCGTCTCACAGGTCCAGGATG (512 base pairs of PCR product); for human Ang1 (GenBankTM accession number U83508) GGGGGAGGYYGGACTGTAAT and AGGGCACATTTGCACATACA (362 base pairs); for human Ang2 (GenBankTM accession number AF004227) GGATCTGGGGAGAGAGGAAC and CTCTGCACCGAGTCATCGTA (535 base pairs); and for human GAPDH (GenBankTM accession number X01677) TGAAGGTCGGAGTCAACGGATTTG and CATGTGGGCCATGAGGTCCACCAC (983 base pairs). The number of cycles was 40 for Tie2, Ang1, and Ang2 and 28 for glyceraldehyde-3-phosphate dehydrogenase. RT-PCR was performed in each case using exactly the same amount of RNA. PCR was always performed in parallel for each primer pair and the PCR products were separated on the same gel. The linear range of amplification was tested for all primer pairs by performing PCR with an increasing number of cycles. PCR for glyceraldehyde-3-phosphate dehydrogenase was performed as an internal control, demonstrating comparable starting amounts of cDNA for all extracts. Expression in human ECs of the angiopoietins, VEGF, Tie1, and Tie2 was examined on RNA blots. RNAs were isolated from low passage- number HUVECs, aorta (human aortic ECs), dermal microvasculature (all purchased from Clonetics, San Diego, CA), and cutaneous fat pad microvasculature (a gift from Dr. J. Springhorn, Alexion Pharmaceuticals) endothelial cells. The cells were grown on tissue culture plastic using recommended basal medium, 5% FBS, and supplements (Clonetics) at 37 °C in a humidified 5% CO2 atmosphere. To harvest RNAs, media was removed from confluent cultures, and the cell monolayer was briefly rinsed with phosphate-buffered saline then lysed with guanidinium thiocyanate and processed as described (37Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar). Total RNAs were resolved by electrophoresis on replicate formaldehyde-agarose gels, blotted to nylon membranes, and hybridized with radiolabeled cDNA probes. As a control, the blots also included poly(A) + RNAs from human placenta and lung (from CLONTECH). The Ang1, Ang2, Tie1, and Tie2 probes were radiolabeled inserts from human cDNAs; the VEGF probe was the entire insert of a mouse VEGF164cDNA. 2P. Maisonpierre, unpublished data. HUVEC expression of Ang2 RNAs led us to ask whether these cells secrete Ang2 protein into the culture medium. Conditioned media (CM) was collected from HUVEC cultures as follows. The medium from established HUVEC cultures was replaced with serum-free defined medium and grown for 24 h, whereupon the cells were refed with fresh-defined medium. After an additional 24 h of cell growth, this HUVEC-CM was harvested, concentrated 10-fold by filtration (Amicon, 10,000 MWCO), and then assayed for the presence of Tie2 binding activities on a Tie2-Fc-coupled BIAcore sensor chip (Amersham), as described previously (27Davis S. Aldrich T.H. Jones P.F. Acheson A. Compton D.L. Jain V. Ryan T.E. Bruno J. Radziejewski C. Maisonpierre P.C. Yancopoulos G.D. Cell. 1996; 87: 1161-1169Abstract Full Text Full Text PDF PubMed Scopus (1698) Google Scholar). HUVEC-CM contained 190 resonance units of Tie2-specific binding activity or roughly a 10-fold greater signal than that seen with CM from angiopoietin-negative cell media (data not shown). To distinguish whether this Tie2 binding activity was Ang2 protein, the HUVEC-CM was further concentrated to 50× (Centricon, 3000 MWCO), fractionated on nonreducing SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and then analyzed by immunodetection with antisera specific to Ang1 and Ang2. Results were expressed as mean ±S.E. of the mean. Statistical significance was evaluated using unpaired Student's t test for comparisons between two means and ANOVA followed by Scheffe's procedure for more than two means. A value of p < 0.05 was interpreted as statistically significant. To evaluate cellular proliferation in response to Ang1* or Ang2, we stimulated HUVECs with increasing amounts of Ang1*, Ang2, or VEGF, a known EC-specific mitogen, for 48 h and performed MTS analysis. This assay was previously shown to accurately reflect cell number (35Buttke T.M. McCubrey J.A. Owen T.C. J. Immunol. Methods. 1993; 30: 233-240Crossref Scopus (290) Google Scholar). VEGF treatment led to a significant dose-dependent increase in optical density, positively correlating with an increase in absolute cell number; a half-maximal response was between 1 and 10 ng/ml. In contrast, neither stimulation with Ang1* nor Ang2 over a wide dose range led to a significant increase in cell number (data not shown). In addition, the influence of Ang1* and -2 on DNA synthesis in HUVECs was evaluated by [3H]thymidine incorpo
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