Title: Tumor Necrosis Factor-α Regulates Expression of Vascular Endothelial Growth Factor Receptor-2 and of Its Co-receptor Neuropilin-1 in Human Vascular Endothelial Cells
Abstract: Tumor necrosis factor-α (TNF-α) modulates gene expression in endothelial cells and is angiogenic in vivo. TNF-α does not activate in vitro migration and proliferation of endothelium, and its angiogenic activity is elicited by synthesis of direct angiogenic inducers or of proteases. Here, we show that TNF-α up-regulates in a dose- and time-dependent manner the expression and the function of vascular endothelial growth factor receptor-2 (VEGFR-2) as well as the expression of its co-receptor neuropilin-1 in human endothelium. As inferred by nuclear run-on assay and transient expression of VEGFR-2 promoter-based reporter gene construct, the cytokine increased the transcription of the VEGFR-2 gene. Mithramycin, an inhibitor of binding of nuclear transcription factor Sp1 to the promoter consensus sequence, blocked activation of VEGFR-2, suggesting that the up-regulation of the receptor required Sp1 binding sites. TNF-α increased the cellular amounts of VEGFR-2 protein and tripled the high affinity125I-VEGF-A165 capacity without affecting theKd of ligand-receptor interaction. As a consequence, TNF-α enhanced the migration and the wound healing triggered by VEGF-A165. Since VEGFR-2 mediates angiogenic signals in endothelium, our data indicate that its up-regulation is another mechanism by which TNF-α is angiogenic and may provide insight into the mechanism of neovascularization as occurs in TNF-α-mediated pathological settings. Tumor necrosis factor-α (TNF-α) modulates gene expression in endothelial cells and is angiogenic in vivo. TNF-α does not activate in vitro migration and proliferation of endothelium, and its angiogenic activity is elicited by synthesis of direct angiogenic inducers or of proteases. Here, we show that TNF-α up-regulates in a dose- and time-dependent manner the expression and the function of vascular endothelial growth factor receptor-2 (VEGFR-2) as well as the expression of its co-receptor neuropilin-1 in human endothelium. As inferred by nuclear run-on assay and transient expression of VEGFR-2 promoter-based reporter gene construct, the cytokine increased the transcription of the VEGFR-2 gene. Mithramycin, an inhibitor of binding of nuclear transcription factor Sp1 to the promoter consensus sequence, blocked activation of VEGFR-2, suggesting that the up-regulation of the receptor required Sp1 binding sites. TNF-α increased the cellular amounts of VEGFR-2 protein and tripled the high affinity125I-VEGF-A165 capacity without affecting theKd of ligand-receptor interaction. As a consequence, TNF-α enhanced the migration and the wound healing triggered by VEGF-A165. Since VEGFR-2 mediates angiogenic signals in endothelium, our data indicate that its up-regulation is another mechanism by which TNF-α is angiogenic and may provide insight into the mechanism of neovascularization as occurs in TNF-α-mediated pathological settings. A well regulated angiogenesis is critical for embryonic growth, bone remodeling, menstrual cycle, corpus luteum formation, and tissue repair. The stable vascular bed occurring in these physiologic conditions results from a balance of signals that favor angiogenesis and those that promote vascular regression. In contrast, a deregulated angiogenesis is pivotal in tumor progression and inflammatory and viral diseases (1Bussolino F. Mantovani A. Persico G. Trends Biochem. Sci. 1997; 22: 251-256Abstract Full Text PDF PubMed Scopus (416) Google Scholar, 2Folkman J. Nat. Med. 1995; 1: 27-31Crossref PubMed Scopus (7209) Google Scholar, 3Risau W. Nature. 1997; 386: 671-674Crossref PubMed Scopus (4834) Google Scholar). A number of naturally occurring growth factors can directly induce angiogenesis by stimulating endothelial cell proliferation and migration or act indirectly by triggering endothelial cells themselves or accessory cells (monocyte/macrophage, mastocytes, T cells) to release direct angiogenic inducers (1Bussolino F. Mantovani A. Persico G. Trends Biochem. Sci. 1997; 22: 251-256Abstract Full Text PDF PubMed Scopus (416) Google Scholar, 2Folkman J. Nat. Med. 1995; 1: 27-31Crossref PubMed Scopus (7209) Google Scholar, 3Risau W. Nature. 1997; 386: 671-674Crossref PubMed Scopus (4834) Google Scholar). Tumor necrosis factor-α (TNF-α) 1The abbreviations used are: TNF-αtumor necrosis factor-αBSAbovine serum albuminFCSfetal calf serumFGFfibroblast growth factorPBSphosphate-buffered salineVEGFvascular endothelial growth factorVEGFRVEGF receptorkbkilobase pair(s)MOPS3-(N-morpholino)propanesulfonic acidTESN-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acidPIPESpiperazine-N,N′-bis(2-ethanesulfonic acid)CHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.1The abbreviations used are: TNF-αtumor necrosis factor-αBSAbovine serum albuminFCSfetal calf serumFGFfibroblast growth factorPBSphosphate-buffered salineVEGFvascular endothelial growth factorVEGFRVEGF receptorkbkilobase pair(s)MOPS3-(N-morpholino)propanesulfonic acidTESN-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acidPIPESpiperazine-N,N′-bis(2-ethanesulfonic acid)CHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. is a powerful activator of angiogenesis in vivo in several animal models when used at low doses (4Leibovich S.J. 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Blood. 1990; 75: 1991-1998Crossref PubMed Google Scholar), which is involved in the progression phase of angiogenesis characterized by a remodeling of extracellular matrix proteins by proteolytic enzymes (Ref. 16Bacharach E. Itin A. Keshet E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10686-10690Crossref PubMed Scopus (158) Google Scholar; for reviews, see Refs. 1Bussolino F. Mantovani A. Persico G. Trends Biochem. Sci. 1997; 22: 251-256Abstract Full Text PDF PubMed Scopus (416) Google Scholar, 2Folkman J. Nat. Med. 1995; 1: 27-31Crossref PubMed Scopus (7209) Google Scholar, 3Risau W. Nature. 1997; 386: 671-674Crossref PubMed Scopus (4834) Google Scholar). tumor necrosis factor-α bovine serum albumin fetal calf serum fibroblast growth factor phosphate-buffered saline vascular endothelial growth factor VEGF receptor kilobase pair(s) 3-(N-morpholino)propanesulfonic acid N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid piperazine-N,N′-bis(2-ethanesulfonic acid) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. tumor necrosis factor-α bovine serum albumin fetal calf serum fibroblast growth factor phosphate-buffered saline vascular endothelial growth factor VEGF receptor kilobase pair(s) 3-(N-morpholino)propanesulfonic acid N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid piperazine-N,N′-bis(2-ethanesulfonic acid) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. TNF-α cooperates with basic FGF, vascular endothelial growth factor-A (VEGF-A), and interleukin-8 to induce capillary-like tubular structure of human microvascular endothelial cell growth in a three-dimensional gel of extracellular matrix proteins (17Koolwijk P. van Erck M.G. de Vree W.J. Vermeer M.A. Weich H.A. Hanemaaijer R. van Hinsbergh V.W. J. Cell Biol. 1996; 132: 1177-1188Crossref PubMed Scopus (267) Google Scholar, 18Yoshida S. Ono M. Shono M. Izumi H. Ishibashi T. Suzuki H. Kuwano M. Mol. Cell. Biol. 1997; 17: 4015-4023Crossref PubMed Scopus (584) Google Scholar). In these systems, the type of extracellular matrix seems to address the features of the angiogenic model. TNF-α does not induce angiogenesis in vitro when the cells are plated on three-dimensional fibrin matrix, but it is permissive for the activity of basic FGF and VEGF-A. TNF-α up-regulates the activity of urokinase-type plasminogen activator, which is required for the formation of capillary structure in addition to the angiogenic molecules (17Koolwijk P. van Erck M.G. de Vree W.J. Vermeer M.A. Weich H.A. Hanemaaijer R. van Hinsbergh V.W. J. Cell Biol. 1996; 132: 1177-1188Crossref PubMed Scopus (267) Google Scholar). Otherwise, TNF-α induces in vitro angiogenesis of endothelium plated on collagen type I. This activity is mediated by the release of VEGF-A, basic FGF, and interleukin-8 (18Yoshida S. Ono M. Shono M. Izumi H. Ishibashi T. Suzuki H. Kuwano M. Mol. Cell. Biol. 1997; 17: 4015-4023Crossref PubMed Scopus (584) Google Scholar). Furthermore, TNF-α induces mesenchymal or tumor cells to release angiogenic molecules, including VEGF-A (7Ryuto M. Ono M. Izumi H. Yoshida S. Weich H.A. Kohno K. Kuwano M. J. Biol. Chem. 1996; 271: 28220-28228Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar, 19Gaudry M. Bregerie O. Andrieu V. Benna J. Pocidalo M.A. Hakim J. Blood. 1997; 90: 4153-4161Crossref PubMed Google Scholar). Finally, it has been reported that TNF-α regulates the expression of integrins involved in adhesion of endothelial cells to extracellular matrix and in angiogenesis (8Defilippi P. Truffa G. Stefanuto G. Altruda F. Silengo L. Tarone G. J. Biol. Chem. 1991; 266: 7638-7645Abstract Full Text PDF PubMed Google Scholar, 20Defilippi P. van Hinsbergh V. Bertolotto A. Rossino P. Silengo L. Tarone G. J. Cell Biol. 1991; 114: 855-863Crossref PubMed Scopus (151) Google Scholar). The puzzling effects of TNF-α on endothelial cells and new vessel growth suggest the presence of more than one angiogenic signaling pathway and that this cytokine may have different activities on endothelial cells depending on the microenvironment. In light of the relevance of the cooperation between TNF-α and VEGF-A (7Ryuto M. Ono M. Izumi H. Yoshida S. Weich H.A. Kohno K. Kuwano M. J. Biol. Chem. 1996; 271: 28220-28228Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar, 17Koolwijk P. van Erck M.G. de Vree W.J. Vermeer M.A. Weich H.A. Hanemaaijer R. van Hinsbergh V.W. J. Cell Biol. 1996; 132: 1177-1188Crossref PubMed Scopus (267) Google Scholar, 18Yoshida S. Ono M. Shono M. Izumi H. Ishibashi T. Suzuki H. Kuwano M. Mol. Cell. Biol. 1997; 17: 4015-4023Crossref PubMed Scopus (584) Google Scholar) in angiogenesis, we studied the effect of TNF-α on the expression and function of VEGF receptors. Adult endothelial cells express on their surface VEGF receptor (VEGFR)-1 encoded by Flt-1 (21De Vries C. Escobedo J.A. Ueno H. Houck K. Ferrara N. Williams L.T. Science. 1992; 255: 989-991Crossref PubMed Scopus (1892) Google Scholar) and VEGFR-2 by KDR/Flk-1 (22Millauer B. Wizigmann-Voos S. Schnurch H. Martinez R. Moller N.P. Risau W. Ullrich A. Cell. 1993; 72: 835-846Abstract Full Text PDF PubMed Scopus (1760) Google Scholar, 23Terman B.I. Carrion M.E. Kovacs E. Rasmussen B.A. Eddy R.L. Shows T.B. Oncogene. 1991; 6: 1677-1683PubMed Google Scholar), but recent findings suggest that the latter alone is able to mediate the mitogenic and chemotactic effect of VEGF-A in endothelial cells (22Millauer B. Wizigmann-Voos S. Schnurch H. Martinez R. Moller N.P. Risau W. Ullrich A. Cell. 1993; 72: 835-846Abstract Full Text PDF PubMed Scopus (1760) Google Scholar, 24Waltenberger J. Claesson-Welsh L. Siegbahn A. Shibuya M. Heldin C.H. J. Biol. Chem. 1994; 269: 26988-26995Abstract Full Text PDF PubMed Google Scholar, 25Thomas K.A. J. Biol. Chem. 1996; 271: 603-606Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar). More recently, it has been reported that neuropilin-1, a receptor that mediates neuronal cell guidance (26He Z. Tessier-Lavigne M. Cell. 1997; 90: 739-751Abstract Full Text Full Text PDF PubMed Scopus (968) Google Scholar), is expressed by endothelial cells and enhances the binding of VEGF-A165 isoform to VEGFR-2 (27Soker S. Takashima S. Miao H.Q. Neufeld G. Klagsbrun M. Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (2072) Google Scholar). Here, we demonstrate that the pretreatment of endothelial cells with TNF-α is followed by an increased migration and wound repair induced by VEGF-A165. An augmented expression of VEGFR-2 and neuropilin-1 genes causes this effect. Human umbilical vein endothelial cells, prepared and characterized as described previously (28Bussolino F. Di Renzo M.F. Ziche M. Bocchietto E. Olivero M. Naldini L. Gaudino G. Tamagnone L. Coffer A. Comoglio P.M. J. Cell Biol. 1992; 119: 629-641Crossref PubMed Scopus (1202) Google Scholar), were growth in medium 199 (Life Technologies, Inc.) supplemented with 20% fetal calf serum (FCS) (Irvine, Santa Ana, CA), endothelial cell growth supplement (100 μg/ml), porcine heparin (50 units/ml), 100 units/ml of penicillin, and 100 μg/ml of streptomycin (all from Sigma), in gelatin (Life Technologies, Inc.)-coated tissue culture plates (Falcon, Becton Dickinson, Plymouth, UK). They were used at early passages (I–III). Human fibrosarcoma 8378 cells, which respond to TNF-α (29Basile A. Sica A. d'Aniello E. Breviario F. Garrido G. Castellano M. Mantovani A. Introna M. J. Biol. Chem. 1997; 272: 8172-8178Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar), were maintained in Dulbecco's modified Eagle's medium containing 10% FCS. Porcine aortic endothelial cells transfected with human VEGFR-2 (24Waltenberger J. Claesson-Welsh L. Siegbahn A. Shibuya M. Heldin C.H. J. Biol. Chem. 1994; 269: 26988-26995Abstract Full Text PDF PubMed Google Scholar) were cultured in Ham's F-12 (Sigma) supplemented with 10% FCS. Human foreskin microvascular endothelial cells isolated as described previously (17Koolwijk P. van Erck M.G. de Vree W.J. Vermeer M.A. Weich H.A. Hanemaaijer R. van Hinsbergh V.W. J. Cell Biol. 1996; 132: 1177-1188Crossref PubMed Scopus (267) Google Scholar), were cultured on fibronectin-coated dishes in medium 199 buffered with 20 mm Hepes containing 10% human serum, 10% newborn calf serum, endothelial cell growth supplement (150 μg/ml), and porcine heparin (5 units/ml). To verify the effects of TNF-α on the expression of VEGF receptors and on the biological activities elicited by VEGF-A165, the following experimental conditions have been used: confluent endothelial cell growth at a CO2 level of 5% in atmospheric air was treated with TNF-α (1 × 107 units/mg of protein; Genentech, Inc., San Francisco, CA) in medium 199 supplemented with 20, 5, and 1% FCS or 1% bovine serum albumin (BSA) (lipopolysaccharide-free, Sigma), twice washed with medium 199, and then used to extract RNA. Alternatively, cells were stimulated with VEGF-A165 (a gift of Dr. H. A. Weich, GBF, Braunschweig, Germany) (30Barleon B. Sozzani S. Zhou D. Weich H.A. Mantovani A. Marme D. Blood. 1996; 87: 3336-3343Crossref PubMed Google Scholar) in medium 199 containing 1% FCS in chemotaxis or 3% BSA in wound healing experiments. In some experiments, endothelial cells were starved for 24 h in medium 199 containing 1% FCS and 1% BSA before adding TNF-α. The effect of mithramycin (Sigma), which inhibits gene expression by blocking Sp1 binding to the CG box (7Ryuto M. Ono M. Izumi H. Yoshida S. Weich H.A. Kohno K. Kuwano M. J. Biol. Chem. 1996; 271: 28220-28228Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar, 31Blume S.W. Snyder R.C. Ray R. Thomas S. Koller C.A. Miller D.M. J. Clin. Invest. 1991; 88: 1613-1621Crossref PubMed Scopus (297) Google Scholar), was studied by treating the cells for 12 h in medium 199 containing 5% FCS with or without TNF-α. Total cellular RNA was isolated by guanidinium isothiocyanate extraction and centrifugation through cesium chloride (32Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 7.3-7.87Google Scholar). Equal amounts of total RNA (15 μg/lane) were electrophoresed in 1% agarose gels containing 6.3% formaldehyde in MOPS buffer (Sigma) and blotted on a Nylon Duralon-UV membrane (Stratagene) by the traditional capillary system in 10× SSC (1.5m NaCl, 150 mm sodium citrate, pH 7) (32Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 7.3-7.87Google Scholar). Filters were cross-linked with UV light (0.5 J/cm2) and prehybridized for 4 h at 42 °C in 50% formamide deioinizate, 10% dextran sulfate, 1% SDS, 1 m NaCl, and 100 μg/ml denatured salmon sperm DNA. Hybridization was carried out overnight at 42 °C with [α-32P]dCTP-labeled (3000 Ci/mmol, Amersham, Buckinghamshire, United Kingdom, UK) human VEGFR-2 (a 0.729-kb HindIII–EcoRI of KDR cDNA) (23Terman B.I. Carrion M.E. Kovacs E. Rasmussen B.A. Eddy R.L. Shows T.B. Oncogene. 1991; 6: 1677-1683PubMed Google Scholar), VEGFR-1 (a 1.347-kb HindIII–BglII fragment of human FLT-1 cDNA) (21De Vries C. Escobedo J.A. Ueno H. Houck K. Ferrara N. Williams L.T. Science. 1992; 255: 989-991Crossref PubMed Scopus (1892) Google Scholar), neuropilin-1 (a 0.735-kbPstI–PstI fragment of human neuropilin-1 cDNA) (27Soker S. Takashima S. Miao H.Q. Neufeld G. Klagsbrun M. Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (2072) Google Scholar) and β-actin cDNAs (33Leavitt J. Gunning P. Porreca P. Sun-Yu N.G. Ching-Shwun L. Kedes L. Mol. Cell. Biol. 1984; 4: 1961-1969Crossref PubMed Scopus (77) Google Scholar). cDNAs were labeled using Rediprime random primer labeling kit (Amersham) according to manufacturer's instructions. Posthybridization washes were performed at high stringency (once in 2× SSC, 0.1% SDS for 30 min, once in 0.2× SSC, 0.1% SDS for 30 min and twice in 0.1× SSC, 0.1% SDS for 30 min) at 57 °C, and the membranes were exposed on autoradiography with Hyperfilm-MP (Amersham). Nuclei were isolated from cultured endothelial cells essentially according to Ref. 34Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acic Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9153) Google Scholar. Briefly, cells (2 × 107 cells/assay) were washed twice with ice-cold phosphate-buffered saline (PBS), scraped and collected in a 15-ml centrifuge tube by centrifugation at 500 × g for 5 min at 4 °C. Subsequent steps were performed at 4 °C. The cells were resuspended in 4 ml of lysis buffer (10 mm Tris-HCl, pH 7.4, 10 mm NaCl, 3 mm MgCl2, 0.5% Nonidet P-40) and allowed to stand on ice for 5 min. and then centrifuged at 500 × g at 4 °C for 5 min. Nuclei were resuspended in 200 μl of glycerol storage buffer (10 mm Tris-HCl, pH 8.3, 40% (v/v) glycerol, 5 mmMgCl2, 0.1 mm EDTA) and frozen in liquid N2. In vitro transcription and isolation of the resulting nuclear RNA were performed as described by Ikeda et al. (35Ikeda E. Achen M.G. Breier G. Risau W. J. Biol. Chem. 1995; 270: 19761-19766Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar). Two-hundred μl of frozen nuclei were thawed and mixed with 200 μl of 2× reaction buffer (10 mm Tris-HCl, pH 8.0, 5 mm MgCl2, 300 mm KCl 10 mm dithiothreitol, 400 units/ml placental ribonuclease inhibitor (Stratagene), 20 mm creatine phosphate (Sigma), 200 μg/ml creatine phosphokinase (Sigma), a 1 mmconcentration each of ATP, CTP, and GTP (Stratagene), and 100 μCi of [α-32P]UTP (3000 Ci/mmol, Amersham). Samples were incubated at 30 °C for 30 min with shaking and for 5 min in the presence of 20 units of DNase I (RNase-free, Life Technologies, Inc.). After the addition of proteinase K (150 μg/ml, Sigma) and SDS (0.5% final concentration), incubation was continued at 37 °C for 30 min. Extracted RNA was resuspended in TES buffer (10 mm TES, pH 7.4, 10 mm EDTA, 0.2% SDS) at 5 × 106cpm/ml. Linearized plasmids containing the target cDNAs (15 μg) were immobilized onto a nylon Duralon-UV membrane (Stratagene) using a Bio-Dot SF microfiltration apparatus (Bio-Rad). The filters were prehybridized overnight at 42 °C with hybridization buffer containing 20 mm PIPES (Sigma), pH 6.4, 50% formamide (Sigma), 2 mm EDTA, 0.8 m NaCl, 0.2% SDS, 1× Denhardt's solution (0.02% Ficoll, 0.02% BSA, 0.02% polyvinylpyrrolidone), 200 μg/ml E. coli tRNA (RNase-free, Stratagene). Hybridization was at 42 °C for 48 h in the same solution supplemented with 15 × 106 total cpm of labeled RNA. The filters were washed twice in 2× SSC, 0.5% SDS at 42 °C for 30 min, twice in 0.3× SSC, 0.5% SDS, at 42 °C for 30 min and then incubated with 10 μg/ml RNase A in 2× SSC at 37 °C for 30 min. Further washed were done in 2× SSC at 37 °C for 30 min and then in 0.3× SSC at 37 °C for 30 min. The filters were exposed on autoradiography with Hyperfilm-MP and intensifying screens at −80 °C. The amount of VEGFR-2 mRNA was standardized by comparison with the amount of β-actin mRNA. Densitometric analysis was performed with a GS250 Molecular Imager (Bio-Rad). Endothelial cells were washed twice with PBS and lysed on ice with 1 ml of 50 mm Tris buffer (pH 7.5) containing 150 mm NaCl, 0.1% Triton X-100, 10% glycerol, 5 mm EDTA, 50 μg/ml pepstatin, 100 μg/ml leupeptin, 10 μg/ml aprotinin, 2 mm phenylmethylsulfonyl fluoride, 500 μg/ml soybean trypsin inhibitor (all from Sigma). After centrifugation (20 min at 4 °C at 13,000 × g), protein were solubilized, separated by SDS-polyacrylamide gel electrophoresis (7%), transferred onto polyvinylidene difluoride membranes (Immobilon, Millipore Corp., Bedford, MA), probed with rabbit anti-VEGFR-2 antibody (C-1158, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and detected by ECL (Amersham). Recombinant VEGF-A165 (2 μg) was dissolved in 200 μl of sodium phosphate buffer 20 mm, pH 7.4, and transferred in IODO-GEN-coated tubes (50 μg/ml) (Pierce), where VEGF-A165 was iodinated (5 min, 4 °C) with 1 mCi of125I (Amersham). Twenty μl of phosphate buffer 20 mm, pH 7.2, containing 1% BSA, 0.4 m NaCl, 0.1% CHAPS (Pierce) was added, and the reaction products were separated on Sephadex-G10. The specific activity of the tracer was 90,000 cpm/ng. 125I-VEGF-A165 retained its biological activity as measured by migration of endothelial cells (28Bussolino F. Di Renzo M.F. Ziche M. Bocchietto E. Olivero M. Naldini L. Gaudino G. Tamagnone L. Coffer A. Comoglio P.M. J. Cell Biol. 1992; 119: 629-641Crossref PubMed Scopus (1202) Google Scholar). For specific binding studies confluent cells plated in 24-well plates were incubated an orbital shaker at 4 °C for 2 h in 200 μl/well of binding medium (medium 199 containing 20 mmHepes buffer, pH 7.4, 0.1% BSA, 100 μg/ml soybean trypsin inhibitor) with increasing concentrations of125I-VEGF-A165 in the presence of a 100-fold excess of unlabeled ligand. Endothelial cells were washed three times with ice-cold PBS containing 0.1% BSA and lysed in 200 μl/well of SDS 2% in PBS. Lysates were counted using a Beckman γ 5500B counter. Triplicate samples under each condition were obtained for each experiment. Specific binding, calculated subtracting from the total cpm bound after incubation with a 100-fold excess of unlabeled ligand, was approximately 80%. The Kd was estimated by Scatchard plot using the Ligand program (Elseviere-Biosoft, Cambridge, UK). The 2.0-kbXhoI/SacI fragment of Flk-1 promoter (36Gerber H.P. Condorelli F. Park J. Ferrara N. J. Biol. Chem. 1997; 272: 23659-23667Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar) was subcloned in pGL2basic plasmid (Promega, Madison, WI) to generate the luciferase reporter vector pGL2basicFlk-1. 2H. Gerber and J. Park, personal communication. Human fibrosarcoma 8387 cell line (2 × 105 cells/well) was transfected with 3 μg of pGL2basicFlk-1 or pGL2basic using Superfect Transfection Reagent according to the manufacturer's instructions (Qiagen, Inc., Valencia, CA).The generated plasmid-liposome complex in 0.6 ml of Dulbecco's modified Eagle medium containing 10% FCS was incubated with the cells for 3 h at 37 °C in 5% CO2. The medium was then replaced with fresh medium, and the cells were stimulated for 4 h with TNF-α (20 ng/ml) or left untreated. pSVgal construct (2.5 μg) was co-transfected to correct for the variability in transfection efficiency, and β-galactosidase activity was assayed with chlorophenol red β-d-galactopyranoside (Boehringer Mannheim GmbH, Mannheim, Germany) as a substrate. For final luciferase assay, cells were lysed in 0.2 ml of passive lysis buffer (dual luciferase assay, Promega) at 4 °C, and 20 μl of cleared (12,000 ×g for 2 min at 4 °C) cell extract containing 50 μg of protein were mixed with 0.1 ml of luciferase assay buffer. Light production was measured for 5 s in a luminometer (Magic Lite Analyzer, Ciba Corning, Milano, Italy), and results were normalized to the β-galactosidase activity. Migration assay was performed as described previously with Boyden's chamber technique (28Bussolino F. Di Renzo M.F. Ziche M. Bocchietto E. Olivero M. Naldini L. Gaudino G. Tamagnone L. Coffer A. Comoglio P.M. J. Cell Biol. 1992; 119: 629-641Crossref PubMed Scopus (1202) Google Scholar). Polycarbonate filters (5-μm pore size polyvinylpyrrolidone-free; Neuroprobe, Pleasanton, CA) were coated with 0.1% gelatin for 6 h at room temperature. VEGF-A165 in medium 199 supplemented with 1% FCS was seeded in the lower compartment of the chamber, and 1.25 × 105 resuspended cells in 50 μl of medium 199 containing 1% FCS were then seeded in the upper compartment. At the end of the incubation (37 °C in air with 5% CO2 for 6 h), filters were removed and stained with Diff-Quik (Baxter Spa, Rome, Italy), and 10 high power oil immersion fields were counted. The results obtained were analyzed by one-way analysis of variance and the Student-Newman-Keuls test (Statistic Software; Bio-Soft). Human endothelial cells were grown at confluence on 24 wells and monolayers were treated for 24 h with TNF-α (10 ng/ml) or vehicle alone in medium 199 containing 5% FCS. After washes, the monolayer was wounded with a razor blade (lesion surface: 20 mm2) as described (28Bussolino F. Di Renzo M.F. Ziche M. Bocchietto E. Olivero M. Naldini L. Gaudino G. Tamagnone L. Coffer A. Comoglio P.M. J. Cell Biol. 1992; 119: 629-641Crossref PubMed Scopus (1202) Google Scholar) and incubated in medium 199 containing 3% BSA with or without VEGF-A165. After 24 h, the cells were fixed and stained as described (28Bussolino F. Di Renzo M.F. Ziche M. Bocchietto E. Olivero M. Naldini L. Gaudino G. Tamagnone L. Coffer A. Comoglio P.M. J. Cell Biol. 1992; 119: 629-641Crossref PubMed Scopus (1202) Google Scholar). To quantify the repair process, phase-contrast microscopic pictures of wounded monolayer were recorded with a still video camera recorder (R5000H; Fuji Photo Film Co., Tokyo, Japan), and cell number was counted in 10 fields of 1 mm2 randomly selected, with a Cosmozone image analyzer (Nikon, Tokyo, Japan). Several studies have shown that 7.0-kb VEGFR-2 (23Terman B.I. 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