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• W1985403743 abstract "Semaphorin-3A (sema3A) is a neuropilin-1 (np1) agonist. It inhibits the binding of the 165-amino acid form of VEGF (VEGF165) to np1 and was reported to inhibit angiogenesis as a result. However, we find that sema3A concentrations that inhibit the mitogenic effects of VEGF165 do not inhibit VEGF165-induced phosphorylation of VEGF receptor-2 (VEGFR-2). Furthermore, sema3A inhibits the biological effects of VEGF121, a VEGF form that does not bind to neuropilins and basic fibroblast growth factor, a growth factor whose activity, unlike that of VEGF, is not inhibited by small interfering RNA directed against np1. Therefore, the mechanism by which sema3A inhibits VEGF165 activity does not depend on competition with VEGF165 for binding to np1. Sema3A induced rapid disappearance of focal contacts followed by collapse of the actin cytoskeleton in human umbilical vein-derived endothelial cells. HEK293 cells expressing sema3A repel human endothelial cells and at high concentrations induce their death by apoptosis. Furthermore, sema3A inhibited the formation of tubes from endothelial cells in an in vitro angiogenesis assay. Similar effects are induced by the neuropilin-2 (np2) agonist sema3F. These inhibitory effects are abrogated by small interfering RNAs directed against np1 or np2, respectively. The anti-proliferative effects of sema3A and sema3F are additive when the semaphorins are added as pure proteins. However, when sema3A and sema3F were co-expressed in HEK293 cells their pro-apoptotic and cell repellant activities appeared to be synergistic. These observations suggest that combinations of sema3A and sema3F may be able to inhibit tumor angiogenesis more effectively than single semaphorins. Semaphorin-3A (sema3A) is a neuropilin-1 (np1) agonist. It inhibits the binding of the 165-amino acid form of VEGF (VEGF165) to np1 and was reported to inhibit angiogenesis as a result. However, we find that sema3A concentrations that inhibit the mitogenic effects of VEGF165 do not inhibit VEGF165-induced phosphorylation of VEGF receptor-2 (VEGFR-2). Furthermore, sema3A inhibits the biological effects of VEGF121, a VEGF form that does not bind to neuropilins and basic fibroblast growth factor, a growth factor whose activity, unlike that of VEGF, is not inhibited by small interfering RNA directed against np1. Therefore, the mechanism by which sema3A inhibits VEGF165 activity does not depend on competition with VEGF165 for binding to np1. Sema3A induced rapid disappearance of focal contacts followed by collapse of the actin cytoskeleton in human umbilical vein-derived endothelial cells. HEK293 cells expressing sema3A repel human endothelial cells and at high concentrations induce their death by apoptosis. Furthermore, sema3A inhibited the formation of tubes from endothelial cells in an in vitro angiogenesis assay. Similar effects are induced by the neuropilin-2 (np2) agonist sema3F. These inhibitory effects are abrogated by small interfering RNAs directed against np1 or np2, respectively. The anti-proliferative effects of sema3A and sema3F are additive when the semaphorins are added as pure proteins. However, when sema3A and sema3F were co-expressed in HEK293 cells their pro-apoptotic and cell repellant activities appeared to be synergistic. These observations suggest that combinations of sema3A and sema3F may be able to inhibit tumor angiogenesis more effectively than single semaphorins. Semaphorins are axon guidance factors that induce localized collapse of neuronal growth cones (1Luo Y. Raible D. Raper J.A. Cell. 1993; 75: 217-227Abstract Full Text PDF PubMed Scopus (1019) Google Scholar). They are characterized by the presence of a sema domain located close to their N termini. The sema domain is essential for semaphorin signaling and determines the specificity of binding (2Gherardi E. Love C.A. Esnouf R.M. Jones E.Y. Curr. Opin. Struct. Biol. 2004; 14: 669-678Crossref PubMed Scopus (133) Google Scholar). Class 3 semaphorins are the only secreted vertebrate semaphorins and are distinguished in addition by the presence of a basic domain at their C termini. Most of the class 3 semaphorins bind to one or to both of the receptors belonging to the neuropilin family. The class 3 semaphorin sema3A 3The abbreviations used are: sema3Asemaphorin-3AHUVEChuman umbilical vein-derived endothelial cellsnp1neuropilin-1np2neuropilin-2sema3Fsemaphorin-3FsiRNAsmall inhibitory RNAVEGFvascular endothelial growth factorVEGF165165-amino acid long form of VEGF (other VEGF forms are labeled similarly)VEGFR-2VEGF receptor-2ERKextracellular signal-regulated kinasebFGFbasic fibroblast growth factorHEKhuman embryonic kidneyFCSfetal calf serumPBSphosphate-buffered salineFACSfluorescence-activated cell sorter. binds to np1 (3Kolodkin A.L. Levengood D.V. Rowe E.G. Tai Y.T. Giger R.J. Ginty D.D. Cell. 1997; 90: 753-762Abstract Full Text Full Text PDF PubMed Scopus (1002) Google Scholar, 4He Z. Tessier-Lavigne M. Cell. 1997; 90: 739-751Abstract Full Text Full Text PDF PubMed Scopus (970) Google Scholar), whereas the related sema3F binds to np2. The receptors of the plexin family play an important role in class 3 semaphorin signaling. Because the intracellular domain of the neuropilins is too short to enable signal transduction, neuropilins associate with plexins that serve as the signal transducing elements in neuropilin/plexin holoreceptors. Four type-A plexins as well as plexin-D1 have been found to associate with neuropilins (5Tamagnone L. Artigiani S. Chen H. He Z. Ming G.I. Song H. Chedotal A. Winberg M.L. Goodman C.S. Poo M. Tessier-Lavigne M. Comoglio P.M. Cell. 1999; 99: 71-80Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar, 6Takahashi T. Fournier A. Nakamura F. Wang L.H. Murakami Y. Kalb R.G. Fujisawa H. Strittmatter S.M. Cell. 1999; 99: 59-69Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar, 7Gitler A.D. Lu M.M. Epstein J.A. Dev. Cell. 2004; 7: 107-116Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). semaphorin-3A human umbilical vein-derived endothelial cells neuropilin-1 neuropilin-2 semaphorin-3F small inhibitory RNA vascular endothelial growth factor 165-amino acid long form of VEGF (other VEGF forms are labeled similarly) VEGF receptor-2 extracellular signal-regulated kinase basic fibroblast growth factor human embryonic kidney fetal calf serum phosphate-buffered saline fluorescence-activated cell sorter. In addition to their indispensable role in the shaping of the nervous system the neuropilins were also found to play important roles in developmental angiogenesis as revealed by gene targeting experiments that revealed major vascular defects in mice that lack either np1 or both np1 and np2 (8Kawasaki T. Kitsukawa T. Bekku Y. Matsuda Y. Sanbo M. Yagi T. Fujisawa H. Development. 1999; 126: 4895-4902Crossref PubMed Google Scholar, 9Takashima S. Kitakaze M. Asakura M. Asanuma H. Sanada S. Tashiro F. Niwa H. Miyazaki J.J. Hirota S. Kitamura Y. Kitsukawa T. Fujisawa H. Klagsbrun M. Hori M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3657-3662Crossref PubMed Scopus (323) Google Scholar). It was found that the neuropilins also function as receptors for heparin binding forms of the angiogenic factor VEGF and are expressed in endothelial cells (10Gitay-Goren H. Cohen T. Tessler S. Soker S. Gengrinovitch S. Rockwell P. Klagsbrun M. Levi B.-Z. Neufeld G. J. Biol. Chem. 1996; 271: 5519-5523Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 11Soker S. Takashima S. Miao H.Q. Neufeld G. Klagsbrun M. Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (2076) Google Scholar, 12Gluzman-Poltorak Z. Cohen T. Herzog Y. Neufeld G. J. Biol. Chem. 2000; 275: 18040-18045Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). Experiments in which the native np1 receptor of mice was replaced by a np1 variant that binds VEGF but not sema3A indicate that the cardiovascular abnormalities observed in mice lacking functional np1 receptors are probably caused by impaired VEGF signal transduction rather than impaired sema3A signaling (13Gu C. Limberg B.J. Whitaker G.B. Perman B. Leahy D.J. Rosenbaum J.S. Ginty D.D. Kolodkin A.L. J. Biol. Chem. 2002; 277: 18069-18076Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 14Gu C. Rodriguez E.R. Reimert D.V. Shu T. Fritzsch B. Richards L.J. Kolodkin A.L. Ginty D.D. Dev. Cell. 2003; 5: 45-57Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar). These observations do not mean that sema3A and sema3F cannot affect angiogenesis. Sema3A inhibits VEGF165-induced endothelial cell proliferation and migration. Sema3A inhibits the binding of VEGF165 to np1 and it is assumed that this is the cause of the inhibitory effects. Furthermore, sema3A inhibited sprouting in the rat aortic ring in vitro angiogenesis assay (15Miao H.Q. Soker S. Feiner L. Alonso J.L. Raper J.A. Klagsbrun M. J. Cell Biol. 1999; 146: 233-242Crossref PubMed Scopus (436) Google Scholar). Further studies revealed that VEGF165 can in turn inhibit sema3A-induced cell contraction (16Narazaki M. Tosato G. Blood. 2006; 107: 3892-3901Crossref PubMed Scopus (74) Google Scholar), that implantation of sema3A releasing beads can inhibit developmental angiogenesis in developing limbs of chick embryos (17Bates D. Taylor G.I. Minichiello J. Farlie P. Cichowitz A. Watson N. Klagsbrun M. Mamluk R. Newgreen D.F. Dev. Biol. 2003; 255: 77-98Crossref PubMed Scopus (148) Google Scholar), and that sema3A release from endothelial cells regulates branching of blood vessels in the developing chick embryo brain (18Serini G. Valdembri D. Zanivan S. Morterra G. Burkhardt C. Caccavari F. Zammataro L. Primo L. Tamagnone L. Logan M. Tessier-Lavigne M. Taniguchi M. Puschel A.W. Bussolino F. Nature. 2003; 424: 391-397Crossref PubMed Scopus (490) Google Scholar). However, it is not yet known whether sema3A can affect tumor development and tumor angiogenesis. In contrast, the gene encoding the np2 agonist sema3F was initially characterized as a tumor suppressor that is lost in small cells lung carcinoma (19Xiang R.H. Hensel C.H. Garcia D.K. Carlson H.C. Kok K. Daly M.C. Kerbacher K. van den Berg A. Veldhuis P. Buys C.H. Naylor S.L. Genomics. 1996; 32: 39-48Crossref PubMed Scopus (136) Google Scholar, 20Sekido Y. Bader S. Latif F. Chen J.Y. Duh F.M. Wei M.H. Albanesi J.P. Lee C.C. Lerman M.I. Minna J.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4120-4125Crossref PubMed Scopus (221) Google Scholar, 21Xiang R. Davalos A.R. Hensel C.H. Zhou X.J. Tse C. Naylor S.L. Cancer Res. 2002; 62: 2637-2643PubMed Google Scholar). There is evidence indicating that sema3F is an inhibitor of angiogenesis (22Kessler O. Shraga-Heled N. Lange T. Gutmann-Raviv N. Sabo E. Baruch L. Machluf M. Neufeld G. Cancer Res. 2004; 64: 1008-1015Crossref PubMed Scopus (190) Google Scholar, 23Bielenberg D.R. Hida Y. Shimizu A. Kaipainen A. Kreuter M. Kim C.C. Klagsbrun M. J. Clin. Investig. 2004; 114: 1260-1271Crossref PubMed Scopus (242) Google Scholar). The anti-angiogenic effects of sema3F do not seem to depend upon competition with VEGF165 for binding to np2 because sema3F seems to initiate np2-dependent signaling that results in inhibition of VEGF-induced ERK1/2 phosphorylation and cell proliferation (22Kessler O. Shraga-Heled N. Lange T. Gutmann-Raviv N. Sabo E. Baruch L. Machluf M. Neufeld G. Cancer Res. 2004; 64: 1008-1015Crossref PubMed Scopus (190) Google Scholar). Additional evidence indicates that sema3F also affects the behavior of some tumor cells directly, by inhibiting their migration and attachment (23Bielenberg D.R. Hida Y. Shimizu A. Kaipainen A. Kreuter M. Kim C.C. Klagsbrun M. J. Clin. Investig. 2004; 114: 1260-1271Crossref PubMed Scopus (242) Google Scholar, 24Nasarre P. Kusy S. Constantin B. Castellani V. Drabkin H.A. Bagnard D. Roche J. Neoplasia. 2005; 7: 180-189Crossref PubMed Scopus (87) Google Scholar, 25Nasarre P. Constantin B. Rouhaud L. Harnois T. Raymond G. Drabkin H.A. Bourmeyster N. Roche J. Neoplasia. 2003; 5: 83-92Crossref PubMed Google Scholar). We report that sema3A inhibits the proliferation and induces apoptosis of endothelial cells. It does that using a mechanism that does not require competition with VEGF165 for a shared receptor because it is also able to inhibit the survival promoting effects of VEGF121, a VEGF form that does not bind to neuropilins. We show that the effects of sema3A and sema3F are synergistic at low concentrations. We also show that sema3A inhibits the spontaneous organization of endothelial cells into tube-like structures in an in vitro angiogenesis assay. Materials—Chemicals were from Sigma unless otherwise indicated. The silver stain kit was from ICN. Mediums and sera for cell culture were from Biological Industries Inc. (Kibbutz Beth-Haemek, Israel). Lipofectamine and Oligofectamine were from Invitrogen. Antibodies directed against c-myc, VEGFR-2, phosphorylated Y-951 of VEGFR-2, phosphorylated ERK1/2, and ERK2 (total ERK) were purchased from Santa Cruz Biotechnology Inc. (San Diego, CA). Antibodies directed against phosphorylated Y-1175 of VEGFR-2 were purchased from Cell Signaling. Antibodies against β-actin, FLAG, anti-FLAG M2 affinity resin, and AP-conjugated agarose beads were purchased from Sigma. Antibodies against active caspase-3 were purchased from MBL (Woburn, MA). Antibodies directed against human placental alkaline phosphatase were purchased from DAKO (Glostrup, Denmark). Antibodies directed against human semaphorin-3A were purchased from R&D Systems (Minneapolis, MN). The AP-sema3A encoding cDNA was kindly given by Dr. Tessiere-Lavinge (Genentech, South San Francisco, CA). The fluorescent vital dye DiI was purchased from Molecular Probes (Eugene, OR). Cy2-conjugated donkey anti-mouse antibodies were from Jackson ImmunoResearch. Antibodies directed against vinculin were from Sigma. Alexa-conjugated phalloidin was from Molecular Probes. bFGF was produced as previously described (26Tessler S. Neufeld G. J. Cell. Physiol. 1990; 145: 310-317Crossref PubMed Scopus (88) Google Scholar). Cells—HUVEC and HEK293 cells expressing sema3A, sema3F, or empty vector were cultured as previously described (22Kessler O. Shraga-Heled N. Lange T. Gutmann-Raviv N. Sabo E. Baruch L. Machluf M. Neufeld G. Cancer Res. 2004; 64: 1008-1015Crossref PubMed Scopus (190) Google Scholar). Radial artery and saphenous vein-derived endothelial cells were a kind gift from Dr. Flugelman (Lady Davis Carmel Medical Center, Haifa). Human radial artery and sapheneous vein-derived endothelial cells were cultured as previously described for HUVEC cells (10Gitay-Goren H. Cohen T. Tessler S. Soker S. Gengrinovitch S. Rockwell P. Klagsbrun M. Levi B.-Z. Neufeld G. J. Biol. Chem. 1996; 271: 5519-5523Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Endothelial cells were not used beyond passage 8. HEK293, which express sema3A, were generated by transfecting cells with the sema3A-FLAG/pcDNA3.1/Hygro plasmid. To simultaneously express sema3A and sema3F, sema3A expressing HEK293 cells were transfected with the sema3F-myc/pcDNA3.1/neo plasmid (22Kessler O. Shraga-Heled N. Lange T. Gutmann-Raviv N. Sabo E. Baruch L. Machluf M. Neufeld G. Cancer Res. 2004; 64: 1008-1015Crossref PubMed Scopus (190) Google Scholar). Clones expressing sema3F at similar levels to those found in the parental sema3F expressing HEK293 cells were selected using G418 (0.5 mg/ml), followed by isolation of clones and Western blot screens for characterization of sema3A and sema3F expression levels. Production and Purification of Sema3A—The sema3A cDNA was ligated in-frame to a FLAG epitope tag inserted in-frame before the stop codon. The cDNA was ligated into the pcDNA3.1/hygro plasmid to generate the sema3A-FLAG/pcDNA3.1/hygro expression plasmid. HEK293 cells were transfected using Lipofectamine and stable sema3A-FLAG expressing clones were isolated using hygromycin (0.3 mg/ml). HEK293 cells transfected with the sema3A expression vector or with empty expression vector were cultured to 90% confluence. The medium was changed to serum-free Dulbecco's modified Eagle's medium. Sodium butyrate (5 mm) was added after 24 h to the control and to the sema3A expressing cells, and the medium was collected after an additional 24 h. HEPES buffer (10 mm, pH 7.3) and protease inhibitors (phenylmethylsulfonyl fluoride, 0.2 mg/ml; leupeptin, 5 μg/ml; aprotinin, 2 μg/ml, and 1 mm EDTA) were added. The medium was incubated overnight at 4 °C with anti-FLAG M2 affinity gel (0.5 ml beads/0.5 liter of medium). The column was washed thoroughly with TBST (10 mm Tris-HCl, pH 8.0, 150 mm NaCl, 0.02% Tween 20) and bound sema3A eluted using 0.1 m glycine (pH 3.0) into a neutralizing volume of 1 m Tris/HCl (pH 8.0) to obtain a final concentration of 150 mm Tris/HCl (pH 7.3). Sema3A containing fractions and the corresponding fractions from the control were frozen in liquid nitrogen. Endothelial Proliferation and Repulsion Assays—HUVEC (passages 4–8) were seeded in gelatin-coated 48- or 24-well dishes at a concentration of 104 or 2 × 104 cells/well, respectively, in M199 medium supplemented with 10% FCS. Angiogenic growth factors and either sema3A (0.3 μg/ml) or a corresponding volume from a control fraction were added after the cells adhered. On day 2, the factors were re-added. Adherent cells were counted on day 4 using a Coulter counter. In proliferation or repulsion experiments using semaphorin-secreting HEK293 cells, endothelial cells were seeded in gelatinized 48-(2 × 104 cells/well) or 24-(5 × 104 cells/well) well dishes in M199 containing 20% FCS and 5 ng/ml bFGF. The following day up to 5% HEK293 cells expressing various semaphorins were seeded on top of the endothelial cells in M199 containing 10% FCS with or without 0.5 ng/ml bFGF. In some of the experiments the cells were incubated prior to seeding with 5 μg/ml of the fluorescent vital dye DiI for 30 min to enable easy detection of the cells in mixed cell cultures. Cells were photographed using an inverted phase-contrast/fluorescence microscope after 24–48 h and counted in a Coulter counter. ERK1/2 and VEGFR-2 Phosphorylation—HUVEC cells were seeded in 6-well gelatinized dishes at a concentration of 4 × 105 cells/well in growth medium containing 10% FCS. Cells were allowed to attach and were incubated 16 h at 37 °C. The cells were transferred to room temperature and incubated 15 min with sema3A (0.5 μg/ml) or a corresponding volume of a control fraction purified similarly from cells transfected with empty expression vector. Subsequently, VEGF121 (10 ng/ml) or VEGF165 (3 ng/ml) were added or not and the cells were incubated for 10 more minutes. The cells were then washed with ice-cold PBS and lysed with 0.03 ml of lysis buffer containing HEPES (50 mm, pH 7.4), 4 mm EDTA, 1% Triton X-100, 0.5 mg/ml Na3VO4, 4.5 mg/ml Na4P2O7, and fresh protease inhibitors (phenylmethylsulfonyl fluoride, 0.2 mg/ml; leupeptin, 5 μg/ml; and aprotinin, 2 μg/ml). The cells were scraped off, nonsoluble debris was removed by low speed centrifugation at 4 °C, and aliquots of cell lysate containing 20–60 μg of protein separated on an SDS-PAGE gel. Proteins were blotted onto a nitrocellulose filter and probed with an antibody directed against phosphorylated ERK1/2 or phosphorylated Y-951 of VEGFR-2. The blot was then stripped and re-probed with an antibody directed against ERK2 (total ERK) or VEGFR-2 (Total VEGFR-2). Quantification of band intensity was performed using a Fuji Film image reader LAS-3000 machine and the ratio between phosphorylated protein and the total amount of a target protein determined using the Multi-Gauge program. Down-regulation of Neuropilin Expression in HUVEC Using siRNA—The np1-specific siRNA r(AAGGAAACCUUGGUGGGAU)d(TT) and the np2-specific siRNA r(CCAGAAGAUUGUCCUC AAC)d(TT) or a control siRNA r(UUCUCCGAACGUGUCACGU)dTdT were transfected into HUVEC using Oligofectamine at a final concentration of 120 nm. The cells were trypsinized 1 day following transfection, and seeded at desired concentrations. To verify down-regulation of neuropilins, cells were lysed 72 h following transfection, 40 μg of protein from the cell lysates were separated on an SDS-PAGE gel, blotted onto nitrocellulose, and probed with antibodies directed against np1 or np2. To verify that the amount of protein in each lane is similar the membrane was stripped and re-probed with antibodies directed against β-actin. Silver Staining—Silver staining was performed according to the instructions of the vendor. Apoptosis Assays—For fluorescence-activated cell sorter (FACS) analysis, HUVEC cells were seeded in gelatinized 6-cm dishes (6 × 105 cells/dish). The following day HEK293 cells (2.5 × 104 cells/dish) co-expressing sema3A and sema3F or HEK293 cells expressing sema3A, sema3F, or empty vector-transfected cells (5 × 104 cells/dish) were seeded on top of HUVEC cells in M199 medium containing 10% FCS. After 28 h the supernatant and the trypsinized cells from each plate were centrifuged, washed once in cold PBS, and cells fixed in ice-cold 70% EtOH overnight. The next day the cells were incubated with 0.2 mg/ml RNase A and 20 μg/ml propidium iodide for 30 min at 37 °C. FACS analysis was performed using a Callibur flow cytometer (BD Biosciences). The proportion of cells with hypodiploid DNA content was quantified using the CellQuest program. To quantify apoptosis by active caspase-3 content HUVEC cells were seeded in gelatinized 6-well dishes (4 × 105 cells/well) in medium containing 20% FCS and 5 ng/ml bFGF. The following day, the medium was aspirated, replaced with fresh medium lacking bFGF, and 1 μg/ml of sema3A or sema3F or corresponding volumes of control fractions were added. Serum-starved cells were used as a positive control and cells treated with 5 ng/ml bFGF were used as a negative control. The supernatant and the trypsinized cells from each well were lysed 24 h later and 100 μg of protein were loaded on a 14% SDS-PAGE gel, blotted onto nitrocellulose, and probed with an antibody directed against active caspase-3. Blots were then stripped and the total amount of protein assessed using antibodies directed against β-actin. Microscopic determination of DNA fragmentation by the TUNEL method was performed using a commercial kit according to the manufacturer's instructions (DeadEnd™ Colorimetric TUNEL System, Promega). Briefly, HUVEC were seeded in gelatinized 24-well dishes (5 × 104/well) in M199 medium containing 20% FCS and various factors were added. After 36 h the cells were fixed with 4% paraformaldehyde in PBS for 30 min and permeabilized by incubation with 0.2% Triton® X-100 in PBS. DNA strand breaks were labeled with biotinylated nucleotide mixture using terminal deoxynucleotidyl transferase. Horseradish peroxidase-conjugated streptavidin was bound to the biotinylated nucleotides incorporated into the 3′-OH end of damaged DNA and detected by diaminobenzidine staining. Positive cells were counted blindly and the results expressed as the percentage of TUNEL positive cells. The total number of cells counted in each group was at least 100. Tube Formation Assay—Fibrin gels were prepared by dissolving bovine fibrinogen immediately before use in M199 medium to a final protein concentration of 2.5 mg/ml and filtered through a 0.22-μm filter. Fibrinogen was added to 24-well dishes (0.3 ml/well). Bovine thrombin was added to a final concentration of 0.2 units/ml and the dishes were then incubated at 37 °C for 30 min. HUVEC cells (2 × 105 cells/well) were then seeded on top of the fibrin gel. After spreading, the cells were covered with a similar coat of fibrin gel. M199 medium supplemented with 20% FCS and 1 μg/ml sema3A or a corresponding control fraction was added after polymerization of the covering gel. Developing capillaries were photographed using a phase-contrast microscope. HEK293 Proliferation Experiments—HEK293 cells transfected with empty expression vector or HEK293 cells expressing recombinant sema3A or sema3F were seeded in 48-well gelatin-coated dishes (5 × 103 cells/well). Cells were trypsinized and counted in a Coulter counter every day. Immunofluoresence Experiments—HUVEC were seeded in 8-well gelatin-coated chamber slides (4 × 104 cells/chamber). Following a 7-min incubation with sema3A at 37 °C, cells were washed with PBS and fixed in 4% paraformaldehyde 15 min at room temperature. The cells were washed with PBS and permeabilized using 0.5% Triton for 1 min. Following 3 more washes in PBS and blocking with 10% goat serum in PBS (1 h at room temperature), the cells were then incubated with anti-vinculin antibody followed by washes with PBS. Bound antibody was visualized using a Cy2-conjugated anti-mouse antibody and photographed using a fluorescent microscope. A similar procedure was used to stain actin fibers with Alexa-conjugated phalloidin except that the cells were stimulated with sema3A for 30 min prior to fixation. Sema3A Inhibits VEGF121 as Well as VEGF165 and bFGF-induced Proliferation of Endothelial Cells—Sema3A was previously found to inhibit VEGF165-induced proliferation, to compete with VEGF165 for binding to np1, and to inhibit VEGF-induced angiogenesis in in vitro angiogenesis assays (15Miao H.Q. Soker S. Feiner L. Alonso J.L. Raper J.A. Klagsbrun M. J. Cell Biol. 1999; 146: 233-242Crossref PubMed Scopus (436) Google Scholar). We have previously observed that sema3F inhibits proliferation of endothelial cells and angiogenesis by a mechanism that does not require competition with VEGF for binding to shared receptors (22Kessler O. Shraga-Heled N. Lange T. Gutmann-Raviv N. Sabo E. Baruch L. Machluf M. Neufeld G. Cancer Res. 2004; 64: 1008-1015Crossref PubMed Scopus (190) Google Scholar). Here we have used sema3A tagged at the C-terminal with a FLAG epitope tag, which we have purified on an anti-FLAG affinity resin (Fig. 1A, lanes 1 and 4). A fraction purified similarly from the conditioned medium of cells transfected with empty expression vector was used as a control in all the experiments in which purified sema3A was used (Fig. 1A, lanes 2, 5, and 6). VEGF165 promotes the proliferation and survival of endothelial cells. These activities were inhibited by Sema3A (Fig. 1B). It was reported previously that sema3A and VEGF165 compete for a common binding site on np1 (15Miao H.Q. Soker S. Feiner L. Alonso J.L. Raper J.A. Klagsbrun M. J. Cell Biol. 1999; 146: 233-242Crossref PubMed Scopus (436) Google Scholar). However, we observed that sema3A was also able to inhibit the activity of VEGF121, a VEGF form that does not bind to neuropilins (Fig. 1C) (12Gluzman-Poltorak Z. Cohen T. Herzog Y. Neufeld G. J. Biol. Chem. 2000; 275: 18040-18045Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). Sema3A was also able to inhibit bFGF-induced proliferation of HUVEC (Fig. 3F) even though the mitogenic response of HUVEC to bFGF is not affected when the expression of np1 is inhibited (data not shown). Furthermore, sema3A compromised the survival of endothelial cells even in the absence of VEGF, indicating that sema3A transduces signals that compromise endothelial cell survival regardless of the presence or absence of VEGF (Fig. 1D). To make sure that the inhibition was due to sema3A and not due to nonspecific factors such as endotoxin we have heated the sema3A. Endotoxin is heat resistant (27Sharma S.K. Biotechnol. Appl. Biochem. 1986; 8: 5-22PubMed Google Scholar) but the inhibitory activity was completely abolished by heating (data not shown). We have also tested the concentration of endotoxin in our assays and it was less than 10 pg/ml, which is a non-toxic concentration (28Ferro D. Quintarelli C. Lattuada A. Leo R. Alessandroni M. Mannucci P.M. Violi F. Hepatology. 1996; 23: 1377-1383Crossref PubMed Google Scholar).FIGURE 3Sema3A inhibits the activity of bFGF even though neuropilin-1 is not required for bFGF-induced signaling.A, HUVEC cells were seeded in gelatinized 6-well dishes (4.5 × 105 cells/well) in growth medium containing 10% FCS. After 16 h the medium was aspirated and replaced with conditioned medium of empty vector-transfected HEK293 cells (pcDNA)(lanes 1, 3, and 5) or HEK293 cells expressing sema3A (s3a)(lanes 2, 4, and 6). For conditioning, HEK293 cells expressing or not expressing sema3A (80% confluent) were incubated 24 h in M199 containing 10% FCS. Following a 15-min preincubation at room temperature, 0.5 ng/ml bFGF (lanes 3 and 4) or 1 ng/ml (lanes 5 and 6) were added or not added (lanes 1 and 2) to the cells. The experiment was terminated after 10 more minutes. Phospho-ERK1/2 and total ERK1/2 were visualized as described. B, the ratio between phosphorylated ERK1/2 and the total amount of ERK1/2 shown in A was quantified as described under “Experimental Procedures.” C, HUVEC were transfected with nonspecific siRNA (SiC) or with siRNA directed against np1 (Sinp1). ERK1/2 activation in response to the indicated bFGF concentrations was then assayed as described under A. D, the ratio between phosphorylated ERK1/2 and the total amount of ERK1/2 shown in C was quantified as described under “Experimental Procedures.” E, HUVEC were transfected with nonspecific siRNA or siRNA directed against np1 and the expression of the neuropilins determined by Western blot analysis as described. F, HUVEC were seeded in gelatinized 24-well dished (2 × 104 cells/well) in conditioned medium from HEK293 cells expressing Sema3A (s3a) or in conditioned medium from empty vector-transfected HEK293 cells (C) in the presence or absence of bFGF (0.5 ng/ml) (bF). Adherent cells were counted after 3 days using a Coulter counter.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To make sure that the inhibition takes place" @default.
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• W1985403743 date "2007-09-01" @default.
• W1985403743 modified "2023-09-27" @default.
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