Placenta Growth Factor - Semantic Scholar

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Acknowledgments-We thank William J . Henzel for protein microse- quencing .... Quinn, T. P., Peters, K. G., De Vries, C., Ferrara, N., and Williams, L. T. (1993).
THEJOURNALOF BIOL~CICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 41, Issue of October 14, pp. 2564625654, 1994 Printed in U.S.A.

Placenta Growth Factor POTENTIATION O F VASCULAR ENDOTHELIAL GROWTH FACTOR BIOACTIVITY, IN VITRO AND IN VIVO,

AND HIGH AFFINITY BINDING TO Flt-1 BUT NOT TO Flk-l/KDR* (Received forpublication, April 12, 1994, and in revised form, July 18, 1994)

John E. Park, Helen H.Chen, Jane Winer, Keith A.. HouckS, and Napoleone FerraraO From Genentech, Inc., South San Francisco, California 94080

The recently identified placenta growth factor (PlGF) factor a, and angiogenin (5-14). The recently described vascuis a member of the vascular endothelial growth factor lar endothelial growth factor (VEGF)' has several attractive (VEGF) family of growth factors. PlGF displays a 53% features as a regulator of normal andpathological angiogenesis identity with the platelet-derived growth factor-like re- (15).VEGF is a n endothelial cell-specific mitogen in vitroand an gion of VEGF. By alternative splicing of RNA, two PlGF angiogenic inducer in uiuo. In addition,VEGF is able to induce isoforms are generated PlGF,,,(PlGF-1) and PlGF,,, vascular leakage in theMiles assay (7,8). On the basis of this (PlGF-2). Relative to PIGF,,,, PlGF,,, has a 21-amino acid activity, VEGF has been also implicated as a mediator of the insertion enriched in basic amino acids. Little is known abnormal permeability propertiesof tumor blood vessels (7, 8). at the present time about the significance and function By alternative splicing of mRNA, VEGF may exist in four of these proteins. To assess their potential role, we different homodimeric molecular species, each monomer havcloned the cDNAs coding for both isoforms, expressed them in mammalian cells, and purified to apparentho- ing, respectively, 121, 165, 189, or 206 amino acids (VEGF,,,, mogeneity the recombinant proteins. Like VEGF, the VEGF,,,,VEGF,,,, VEGF,,,). VEGF,,, and VEGF,,, are diffusible proteins, whereasVEGF,,, and VEGF,,, are mostly bound PlGF isoforms are homodimeric glycoproteins. PlGF,,, is a non-heparin binding protein, whereas PIGFIG, to heparin-containingproteoglycans in the extracellular matrix strongly binds to heparin. We examined the ability of (16, 17). Recent studies have suggested that VEGF is an imPlGF to bind to soluble VEGF receptors, Flt-1 and portant regulatorof both developmental and ovarianangiogenFlk-l/KDR,and characterized the binding of PlGF to en- esis (18-21). Furthermore, inhibitionof VEGF action results in dothelial cells. Whilethe PlGF proteins bound with high marked suppressionof the growthof several human tumorcell affinity to Flt-1, they failedto bind to Flk-l/KDR. Bind- lines in nude mice (22). identified as putative VEGF ing of l2%P1GFto human endothelial cells revealed two Two tyrosine kinases have been classes of sites, having high and low affinity. The high receptors (23,241. The fms-like tyrosine kinase (Flt-1) (25) and affinity site is consistent with Flt-1; the identity of the the kinasedomain region(KDR) (26) proteins have been shown low affinity site remains to be determined. Purified to bind VEGF with high affinity. The murine homologue of PlGF isoforms had little or no direct mitogenic or per- KDR, known as fetal liver kinase-1(Flk-1) (271, has been also meability-enhancing activity. However, they were able shown to bind VEGF (28, 29). Flt-1 and Flk-l/KDR receptors to significantly potentiate the action of low concentra- have a single signal sequence,one transmembrane domain, tions of VEGF in vitro and, more strikingly,in vivo. seven immunoglobulin-like domains in their extracellular domain, and a consensus tyrosine kinase sequence domain. In addition, a cDNAencoding a truncated form of Flt-1 lacking the The vascular endothelium is one of the most versatile sys- seventh immunoglobulin-like domain, thecytoplasmic domain, tems in the body, serving a variety of essential exchange and and transmembrane sequence has been identified in human regulatory functions. A fundamental property of vascular en- umbilical vein endothelial cells (30). Recently, a cDNA encoding a protein having a 53% identity form a network of dothelial cells is the ability to proliferate and of VEGF has capillaries. This process, known as angiogenesis, is prominent with theplatelet-derived growth factor-like region during embryonic development (1,2). Ina normal adult,angio- been isolated from a human placental cDNA library (31).The genesis occurs only following injury or, in a cyclical fashion, in encoded protein, named placenta growth factor (PlGF), was acids. Subsequently, a longer PlGF the endometrium andin the ovary (3). Angiogenesis, however, expected to have 149 amino is known to play a critical role in the pathogenesisof a variety cDNA was identified (32,331. Compared to theoriginally idenexpected to have of disorders, most notablygrowthandmetastasis of solid tified PlGF species, the long PlGF protein was a 21-amino acid insertion highly enriched in basic residues. tumors (4). Several potential positive regulators of angiogenesis have The two isoforms, resulting from alternative splicing of RNA, been described including acidic and basic fibroblast growth were named, respectively, PlGF-1 and PlGF-2 (32, 33). In confactors, transforming growth factors a and p, tumor necrosis trast to the widespread distributionof the VEGF mRNA (151, expression of PlGF mRNA appears to be restricted t o placenta, trophoblastic tumors, and cultured human endothelial cells * The costsof publication of this article were defrayed in part by the (31-33). Based on the homology with VEGF, PlGF was propayment of page charges. This article must therefore be hereby marked "a'aduertisement"in accordance with 18 U.S.C.Section 1734 solely to The abbreviationsused are: VEGF, vascular endothelialgrowth facindicate this fact. $ Presentaddress:SphinxPharmaceuticalsCorp.,Durham, NC tor; Flt-1, fms-like tyrosine kinase; Flk-1, fetal liver kinase 1; KDR, kinase domain region; PlGF, placenta growth factor; PAGE, polyacryl27717. 8 To whom correspondence andreprint requests should be addressed: amide gel electrophoresis; FGF, fibroblast growth factor; rh, recombiDept. of Cardiovascular Research, Genentech Inc., 460 Point SanBruno nant human; b, basic; PBS, phosphate-buffered saline; ACCE, adrenal Blvd., South San Francisco, CA 94080. Tel.: 415-225-2968; Fax: 415cortex-derivedcapillary endothelial; HUVE, human umbilical vein endothelial. 225-6327.

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of each receptor and the IgG. Below each receptor-IgG, sequence of the original receptor is shown for comparison. Underlined amino acids or nucleotides indicate modifications of the original sequences. IgG residues are enclosed in boxes. Construction of VEGF Receptor-IgG Chimeras-The extracellular doposed to be an angiogenic factor, and initialevidence suggested mains of Flt-l andKDR were cloned by polymerase chain reaction using that conditioned media of transfected cells expressing PlGF cDNA served as the template. Primwere weakly mitogenic to endothelialcells derived from bovine Pfu polymerase. Human placental ers encompassed the entire extracellular domains, including signal peppulmonary arteryor aorta (31,331. To date, PlGF hasnot been tides (25, 26). The cDNAs for each receptor extracellular domain were reported to have been purified to homogeneity. Two sets of primers (shown cloned in twopieces to facilitate sequencing. In the present study, we purified t o apparent homogeneity below) were used, and the resulting bands (-1 kilobase) were digested the two PlGF isoforms and tested them in vitro for mitogenic with the appropriate enzymes and subcloned into pBluescript I1 or of Flt-1 cells and in pSL301. The cDNAs produced encoded the first 758 amino acids activity on bovine and human vascular endothelial a n in vivo system, the Miles vascular permeability assay. Fur- and the first762 amino acidsof KDR. Since the 5’ end of the published thermore, we examined their ability t o bind to soluble VEGF KDR sequence is incomplete (26), the initiator methionine and glutamate were added to enable secretion of the fusion protein, resulting in receptors, Flt-1 and Flk-l/KDR, and characterized the binding a 764-amino acid extracellular domain. TheKDR primers add a silent of lZ5I-PlGFto endothelial cells. PlGF was able to bind with internal SpeI site for cloning purposes (Fig. 1).Full-length KDR extraa receptor thought cellular domaincDNA was createdby ligating the two parts high affinity to Flt-1 but not to Flk-liKDR, at thenovel to be critically involved in mediatingW G F biological actions. SpeI site, while full-length Flt-1 extracellular domain cDNA was crePlGF had littleor no direct mitogenic or permeability-enhanc- ated by ligating the two Flt-1 polymerase chain reaction clones at a ing activity. Intriguingly, however, it was able to significantly unique natural MunI site. potentiate the effects of low concentrations of W G F , both in Flt-1 Set 1: 5‘ TCTAGAGAATTCCATGGTCAGCTACTGGGACACC 3’ vitro and in vivo. 5’ CCAGGTCATTTGAACTCTCGTGTTC 3’ EXPERIMENTAL PROCEDURES

Flt-1 Set 2

5‘ TACTTAGAGGCCATACTCTTGTCCT 3’ 5‘ GGATCCTTCGAAATTAGACTTGTCCGAGGTTC 3’ 5’ GAATTCATCGATGGAGAGCAAGGTGCTGCTGGCCGTC 3’ 5’ ACACAACTAGTGAGACCACATGGCTCTGCTTCTC 3‘ 5: GGTCTCACTAGTTGTGTATGTCCCACCCCAGATT 3 ‘ 5‘ GAATTCGGATCCAAGTTCGTCTTTTCCGGGCA 3‘

Materials-Tissue culture reagents and media were obtained from Life Technologies, Inc. through the Genentech media facility. Restric- KDR Set 1 tion enzymes were from New England Biolabs except for Pfu polymerKDR Set 2 ase which was from Stratagene. Q-Sepharose Resource fast protein liquid chromatographycolumn (6 ml), protein A-Sepharose, wheat germ agglutinin agarose, heparin-Sepharose, and S-Sepharose fastflow resThese primers changed amino acid 757 t o phenylalanine and introduced ins were from Pharmacia Biotech. The C4 reversed phase high performa BstBI siteat the 3’ end of the Flt-1 extracellular domain (Fig.1).The ance liquid chromatography column (4.6 x 100 mm) was purchased from 3’ end of the KDR extracellular SynChrom (Lafayette, IN). The preparative TSK 3000 GS column x (21KDR primers addeda BamHI site to the domain. A BstBI mutation which eliminated any linker sequences was 300mm)was from Hewlett-Packard. Trifluoroacetic acid wasfrom Pierce. Acetonitrile was purchased from Fisher Scientific. Tissue cul- introduced at the 5’ end of CH,CH,, an IgGylheavy chain cDNA clone (37). Flt-1 extracellular domain sequences were fused to the coding ture plates werefrom Costar except for large scale Nunc plates (24.5x 24.5 cm), which werefrom Applied Scientific. Molecular weight stand- sequences for amino acids 2 1 6 4 4 3 of this IgGyl heavy chainclone via ards for gelchromatographyandprestained low molecularweight the unique BstBI siteat the 3’ end of the extracellular domain coding markers for SDSPAGE gel were purchased from Bio-Rad. Recombinant region while KDR extracellular domain sequences were fused t o the Fig. 1). Both constructs were then subcloned human (rh)basic FGF (bFGF) was from R& D Systems (Minneapolis, upstream BamHI site (see MN). rhVEGF,,, was purified from transfected Chinese hamster ovary into pHEB023for expression in CEN4cells (17). The authenticity of all cells as described (34). Iodination of VEGFle, and PlGF proteins was clones was verifiedby DNA sequencing. The strategies for construction and cloning of Flk-1 IgG were essentially similar to those applied to performed by the indirect IODOGEN method (35). Specific activities KDR IgG. ranged from18,000 to 32,000 c p d n g for lZ5I-P1GFand 48,000 to 97,000 c p d n g for lZ5I-VEGF.Rabbit polyclonal antibodies to Flk-l/KDR and Purification of VEGF Receptor-ZgG Chimeras-Conditioned media of Flt-1 were produced by immunizing New ZealandWhiterabbits transfected CEN4 cells expressing the three chimeric receptors were monthly with purified Flk-1 IgG or Flt-1 IgG. concentrated 5- to 10-fold by ultrafiltration and then applied ontoproCloning of PLGF-Oligonucleotide primers (5’GAATTCTCTAGAT- tein A-Sepharose columns (10 ml) that had been pre-equilibrated in GCCGGTCA TGAGGCTGT 3’ and 5’ GAATTCTCTAGAGGTTACCTC- PBS. The flow rate was 2 mumin. Absorbance was monitored at 280 nm. CGGGGAACAGCATC 3’) were designed to amplifya full-length PlGF The columns were washed with PBS until the absorbance at 280 nm cDNA clone (31).The polymerase chain reaction was performed using became negligible and then eluted with 100 mM citric acid, pH 3.0. human placenta cDNA as the template. The polymerase chain reactionSufficient 2 M Tris base was added to tubes to immediately neutralize products were subjected t o polyacrylamide gel electrophoresis. the acid. A silver-stained SDSPAGE gel revealed the presence of a Ethidium bromide stainingof the gel revealed two reaction products at single major bandat >300 kDa in nonreducing and -180 kDa in reduc475 and 538 base pairs,respectively. The lower molecular weight prod- ing conditions. The authenticity of the proteins was verified by NH,uctisconsistentwithPlGF-1,thehigherwiththelonger isoform, terminal amino acid sequence. PlGF-2 (31-33). The bands were cut, electroeluted, and subcloned into Purification of PZGF Isoforms-Serum-free conditioned media from the XbaI siteof pHEB023 for expression in CEN4cells. Such cells are transfected CEN4 cells expressing each PlGF molecular species(-2.5 a derivative of the human embryonic kidney 293 cell line that stably liters) were concentrated 5- to 10-fold by ultrafiltration using Amicon expresses the Epstein-Barr virus nuclear antigen-1, requiredepisofor stir cells CYM 10 membrane). For purification of PlGF,,JPlGF-2 (premal replication of pHEB023 vector (36). Transfectionsand selection of dictably a basic protein), the concentrated conditioned medium was transformants were performed as described previously (16,17). The extensively dialyzed against 25 mM sodium phosphate, pH 6.0, and then authenticity of all clones was verified by DNA sequencing. applied onto a cation exchange S-Sepharose fastflow resin packed in a

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PlGFPotentiates Flt-1 and Binds to

glass column (Omni, 10 x 100 mm) pre-equilibrated with the same buffer. The flow rate was 2 mumin. After loading, the column was washed with 22 ml of 25 m~ sodium phosphate, pH 6.0, containing 0.4 M NaC1. This wash resulted in a major peak of absorbance at 280 nm that contained less than 10% of the activity able to compete with lZ5IVEGF,,, for binding to Flt-IgG. Such radioreceptor assay was used at all steps to monitor the purification. Elution of bound PlGF was with a linear gradient (0.4-1.0 M) of NaCl over 30 min. Fractions containing the highest competing activity (0.5-0.7 M NaCl) were pooled, diluted 6-fold in water containing 0.1% trifluoroacetic acid, and applied by multiple injections into a C4 reversed phase column that had been pre-equilibrated with 20% acetonitrile/ 0.1% trifluoroacetic acid. The flow rate was 0.6 mumin. The column was washed with 5 ml of equilibration buffer and was then eluted with a lineargradient of acetonitrile (2045%)in 95 min. The activity eluted as a broad and asymmetric peak of absorbance a t 210 nm between 30 and 32% acetonitrile. A silverstained SDSffAGE gel of the most active fractions revealed the presence of a single band at -32 kDa in reducing conditions. Such fractions were pooled and subjected to microsequencing. The yield in purified protein was approximately 0.5 mgfliter, as estimated by amino acid analysis. The recovery, as assessed by radioreceptor assay, was -40%. For purification of PlGF,,,/PlGF-l, concentrated conditioned medium wasdialyzed against 20 m~ Tris, pH 8.0, and then applied onto a Q-Sepharose Resource anion exchange column. The flow rate was 2 mumin. The column waseluted with a lineargradient of NaCl(0-0.5 M) in 30 min. The activity capable of competing with lZ5I-VEGF for binding to Flt-IgG was eluted in the presence of -0.2 M NaC1. The most active fractions were pooled and then applied onto a TSK 3000 GS column equilibrated with 100 mM KH,PO,, pH 6.8. The flow rate was 3 mumin. The activity eluted with an apparent molecular weight of -60,000. Fractions containing such activity were further purified by reverse phase chromatography, exactly as described for PlGF-2. Purified protein was quantified by amino acid analysis. Protein yield and recovery were very similar to those described for PlGF-2. Binding Assays with Soluble Receptors-Ninety-six-well breakaway enzyme-linked immunosorbent assay plates (Nunc, Kampstrup, Denmark) were coatedovernight a t 4 "C with 2 pg/ml affinity-purified goat anti-human Fc IgG(Organon-Teknika)in 50 mM Na,CO,, pH 9.6. Plates were blocked for 1 h with 10% fetal bovine serum in PBS (buffer B). Blocking bufferwas then removed. To each well, IgGfusion protein (-1 ng), "'1-VEGF or 1251-P1GF, and cold competitor were added to a final volume of 100 1.11 in buffer B. Unless noted otherwise, 12,000 cpm of radioligand were added to the assay. When noted, binding experiments contained 1-10 pg/ml heparin. Binding was carried out at room temperature for 3.5 h followed by 6 washes with buffer B. Binding was determined by counting individual wells in a y counter. Data were analyzed by a 4-parameter nonlinear curve-fitting program developed a t Genentech. Endothelial Cell Culture and Mitogenic Assays-Bovine adrenal cortex-derived capillary endothelial (ACCE) cellsor human umbilical vein endothelial (HUVE) cells wereused for binding and mitogenic assays. Stock plates ofACCE cells were maintained in the presence oflow glucose Dulbecco'smodified Eagle's medium supplemented with 10% calf serum, 2 mM glutamine (growth medium) plus bFGF at a final concentration of 2 ng/ml (6, 17). For proliferation assays, ACCE cells wereseeded at 7,00O/well in 6-multiwell plates in the presence of growth medium. HUVE cells were maintained in endothelial growth medium (Clonetics)supplemented with 5% fetal bovine serum and bovine pituitary extract, according tothe instruction of the manufacturer. For proliferation assays, HUVE cells were seeded at 12,00O/well in 6-multiwell plates in the presence of endothelial growth medium containing 2% serum, without pituitary extract. Heparin (1 or 10 pg/ml) was added when noted. After5-8 days, cells were dissociatedby trypsin, and cell numbers were determined with a Coulter counter. Binding to Endothelial Cells-ACCE or HUVE cells were grown to confluence in 6-well plates in the appropriate growth media. The media were removed, and binding was carried out in low glucose Dulbecco's modified Eagle's mediumcontaining 10%fetal bovine serum, 0.2% gelatin, 10 &ml heparin, and 10 mM HEPES, pH 7.4 (binding medium) on ice. One mlof binding medium containing 50,000 cpm of lZ5I-VEGF(or 'zsI-PIGF) and various competitive agents were added to eachwell. When noted, amounts of radioligand were varied. Monolayers were washed three times with binding medium and thenextracted with 1ml of 1 N NaOH to recover bound lZ5I-VEGF (or '261-P1GF).The entire NaOH extract was counted in a y counter. Data were analyzed by the method of Scatchard (38). Cross-linking ofLigands-HUVE cells were grown to confluence in 10-cm dishes. All subsequent steps were performed at 4 "C or on ice.

VEGF Activity Media were replaced with the binding medium (detailed above) containing 1261-VEGF (110PM) or '251-P1GF(230 PM) ? 2 pgiml cold VEGF, as indicated, for 2 h. Cells werewashed twice with cold PBS and incubated for 20 min at 4 "C with 0.5 mM BS3cross-linkingagent (Pierce)dissolved in PBS. The reaction was quenched with one-tenth volume of 200 mM glycine, 10 mM Tris-HC1, pH 8. The dishes were washed twicewith cold PBS and extracted with 1.25 ml of lysis buffer containing 1%Triton X-100, 137 mM NaCl, 10%glycerol, 2 m~ EDTA, 1 mM vanadate, 1 m~ phenylmethylsulfonylfluoride, 0.15 unit/ml aprotinin, 20 p~ leupeptin, 20 mM Tris HC1, pH 8.0 (39). The extracts were spun to remove debris, and the supernatants were saved. The supernatants were split into equal volumes and then immunoprecipitated with rabbit antisera to Flt-1, Flk-l/KDR, or with control antisera at the final dilution of 150 for 1 h. Protein A-agarose was added and mixed for an additional hour at 4 "C. The beads were pelletedand washed twicewith lysis buffer before addition of double-strength Laemmli sample buffer to elute proteins from the beads. The eluates were boiled for 1min and electrophoresed on 6 2 0 % SDS-polyacrylamide gels.The gels weredried and exposed t o a phosphor-imaging plate for -36 h. The plate was read on a BAS-2000 image analyzer (Fuji, Japan). nrosine Phosphorylation Assay-ACCE cells were grown to confluence in 60-mm dishes without added growth factors. Confluent cultures were preincubated for 24 h in the presence of low glucose Dulbecco's modified Eagle's medium supplemented with 0.5% calf serum (deprived medium). Media werethen removed and cells wereincubated for 5 min at 37"C in the presence of deprived medium containing 50ng/ml VEGF,,, or varying amounts of PlGF-2 (PIGF,,,). At the end of the incubation, media were removed and cells were washedwith cold PBS containing 1mM sodium vanadate and then extracted with 0.25 ml of lysis buffer (see above). This and all subsequent steps were performed at 4 "C. The extracts were centrifuged briefly in a Microfuge to pellet debris. The supernatants were mixed for 1h with 50 1.11of wheat germ agglutinin agarose beads to collect membrane proteins. The beads were pelleted and washed once with lysis buffer. Double-strength Laemmli sample buffer was added to elute proteins, boiled for1min, and subjected to electrophoresis on4-20% SDS-polyacrylamide gels. The gels were electroblotted to polyvinylidene difluoridemembranes. Following incubation with 4G10 anti-phosphotyrosinemonoclonal antibody (UBI, Lake Placid, N Y ) , immunoreactive bands were visualized with an ABC kit, according t o the instructions of the manufacturer (Vector Laboratories). Miles VascularPermeabilityAssay-Induction of vascular permeability was determined by the Miles assay essentially as described previously (8, 40). Oneml of 0.5% (w/v)Evans blue was injected intracardially into anesthetized guinea pigs. One hour later, PBS alone, different concentrations of VEGF or PlGF, individually or in combination, were injected intradermally in 200-1.11 aliquots in the dorsal area. Leakage of protein-bound dye wasdetected by a blue spot surrounding the injection site. RESULTS

Biochemical Characterization of PlGF-The two PlGF proteins were purified to apparenthomogeneity. Anion and cation exchange chromatographies were used as initial purification steps for PIGF,,l/PIGF-l and PlGF,,@GF-2, respectively. The final purification step for both isoforms was reversed phase chromatography on a C4 column. Analysis of the purified materials by silver-stained SDS-PAGE gel in reducing conditions revealed major bands (Fig. 2)at -32 kDa (PlGF,,,, lane 4 ) and -28 kDa (PlGF,,,, lane 61, respectively. In nonreducing conditions, bands of approximately twice that size were evidenced (PIGF,,, (lune 1 ) and PlGF,,, (lane 3 ) ) .This is consistent with a homodimeric structure. Microsequencing of the purified material revealed in both cases a single NH,-terminal amino acid sequence: LPAW. Therefore, the PlGF proteins are generated following cleavage of a n 18-amino acid signal sequence (31,321. The mature PlGF monomers are expected t o have, respectively, 131 and 152 amino acids. By analogy with theW G F polypeptides, they may be called PlGF,,, and PlGF,,,. Both PlGF isoforms reveal higher apparent molecular weight than WGFlG5 (Fig. 2), in spite of the fact that their predicted sequence is shorter. A higher level of glycosylation probably accounts for such differences. This is consistent with the presence of two putative glycosylation sites ineach PlGF monomer (31-33) uersus only one in VEGF (6). Furthermore, the finding that both

PlGFPotentiates Flt-1 and Binds to VEGF Activity PlGF isoforms are eluted as broad and asymmetric peaks by reversed phase HPLC (data not shown)is consistent withheavily glycosylated proteins. The PlGF proteins were then compared for their ability to bind to heparin-Sepharoseor S-Sepharose. As shown in Fig. 3, P1GFl3, did not bind appreciably to S-Sepharose or heparin-Sepharose. In contrast, PIGF,,, bound strongly to both matrices and was eluted in the presence of 20.5 M and 0.9 M NaCl, respectively. The differential chromatographic behavior of PlGF,,, and PlGF,,, is clearly reminiscent of VEGF,,, and VEGF,,, (16) and is consistent with the primary amino acid sequence of the PlGFproteins (31-33). PlGF Bindsto Flt-1 IgG but Not to KDR ZgG-Flt-1 IgG or KDR IgG were first tested incompetition binding assays using I2,I-VEGF as the radioligand (Fig. 4, A and B). The IC,, for VEGF,,, binding to Flt-1 IgG was 22 -c 3 PM, while that for VEGF,,, binding to KDR IgG was 125 2 20 PM. In both cases,

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25649 *251-VEGF,,,bound to thesoluble receptor with anaffinity very similar to that of the full-length receptor expressed in transfected cells (23, 24, 28). Therefore, these soluble receptor-IgGs provide suitable in vitro model systems to study each VEGF receptor independently of the other. In preliminary experiments, we determined that conditioned medium derived from transfected CEN4 cells expressing either PlGF,,, or PIGF,,, was able to compete for 1251-VEGF,,,binding to Flt-1 IgG. Such activity provided a convenient assay for the purification of both PlGF species. Like the conditioned medium, highly purified PlGF was able to compete with lZ5IVEGF,,, for binding to Flt-1 IgG (Fig. 5A). In contrast, PlGF (up to48 nM) was unable todisplace 12,1-VEGF bound to KDR IgG (Fig. 5A) or Flk-1 IgG (not shown). Both '251-P1GF,,2 and 1251-PIGF13, bound to Flt-1IgG with high affinity (Fig. 5, B-D). The ED,, values were250 ? 35 PM and 186 2 40 PM,respectively. However, lZ5I-P1GF failed to bind to KDR IgG (Fig. 5E). In panels D and E, binding experiments were performed in the presence of 1 pg/ml heparin, although experiments without heparin or using 1251-P1GF,31, which fails to bind heparin, gave similar results. Similar results were also obtained when we tested the binding of lZ5I-P1GFon transfected COS cells expressing full-length Flt-1 or KDR receptors (data not shown). Binding of VEGF or PlGF tosoluble receptors, endothelialcells, or transfected cells wasnot affected bybFGF, hepatocyte growth factor, or insulin (data not shown), indicating that the binding was specific. Thus, Flt-1, but not Flk-l/KDR, is a potential high affinity receptor for PlGF. PlGF Binds to Endothelial Cells-Ligand binding to endothelial cells was performed to determine whether binding sites consistent with those observed in vitro were duplicated on endothelial cells. As shown in Fig. 6 A , 1251-VEGF,6,bound to ACCE and could be only partially displaced by PlGF,indicating the presence of both PlGF-sensitive and -insensitive VEGF receptors on ACCE cells. Similar resultswere observed in HUVE cells (data not shown). Since HUVE cells consistently displayed

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60 FIG.3. Chromatographic behavior of PlGF,,, (A and B ) or PlGF,,, (Cand D ) on S-Sepharose(A and C ) or heparin-Sepharose( B andD) columns. In A and C , concentrated conditioned medium from transfected cells expressing either PlGF isoform was equilibrated in 25 mM sodium phosphate, pH 6.0. The material was loaded onto S-Sepharose columns (1 ml), washed with starting buffer, and eluted stepwise with increasing concentrations of NaCI, as indicated. In B and D, concentrated conditioned medium was equilibrated in 10 mM Tris HCI, pH 7.2, containing 50 mM NaCI. The material was loaded onto heparin-Sepharose columns (1 ml), washed with starting buffer, and eluted with increasing concentrations of NaCI. PlGF was detected by inhibition of '2sI-VEGFbinding to Flt-1 IgG.

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25650 Potentiates Flt-1 and PlGF Binds to VEGF Activity higher expression of PlGF-sensitive VEGF binding sites than ACCE cells, we chose to characterize PlGF binding to HUVE cells ingreaterdetail (Fig.6B).Binding of1251-PlGFl,, to

HUVE cells demonstrated two classes of sites, havingaffinities of 230 2 16 PM and >2 nM. H W E cells express approximately 15,000 high affinity and >30,000low affinity PlGF receptors per cell. These results demonstrate that endothelial cells express displaceable cell surface PlGF receptors. The high affin100 ity PlGF binding siteconsistent is with Flt-1,while the identity A of the low af'finity site remains tobe determined. Cross-linking of 1251-VEGF,,Sor '251-P1GFl,, to HUVE cells, followed by immunoprecipitation of these complexes with antibodies to Flt-1or Flk-l/KDR, was performed in the attempt to identify the receptors involved in the binding events. Immunoprecipitation of '251-VEGF,,,-cross-linkedcomplexes with antiserum directed against Flt-1 yielded a relatively faint band at -190,000 and lower molecular weight bands consistent with un-cross-linked VEGF (Fig. 7, lane 1 ) . The anti-Flt-1 antiserum also immunoprecipitated 12,1-P1GF (Fig. 7, lane 3 ) . However, 1 1000 10 100 cross-linked 1251-P1GFl,2~Flt-1 complexes at - 190,000 could not be clearly visualized. Lower molecular weight complexes con100 B sistent with possible oligomers of PlGF were observed (Fig. 7, lane 3 ) . Immunoprecipitation of cross-linked 1251-VEGF1,,with anti-Flk-1lKDR antiserum yielded a strong receptor-ligand complex band at -210 kDa. Un-cross-linked VEGF was also observed (Fig. 7, lane 5).In contrast, theanti-Flk-1/KDR antiserum failed to precipitatesignificant lZ5I-P1GF(Fig. 7, lane 7). Allof the observed labeling events could be inhibited by an excess of cold VEGF (Fig. 7, lanes 2,4,and 6). Preimmune sera failed to precipitate lZ5I-VEGFor lZ5I-P1GF(data not shown). PlGF Fails to Induce Tyrosine Phosphorylation in Endothe0 2n - . play a direct role lial Cells-To determine whether PlGF might 1 10 100 1000 10000 in intracellular signal transduction pathways, we tested VEGF or PlGF for their ability t o trigger tyrosine phosphorylation of VEGF165 (pM) ACCE cells. In agreement withprevious FIG.4.Binding of VEGF to Flt-1 IgG or to KDR IgG. Microtiter membrane proteins in plates were coatedwith goat anti-human Fc IgGovernight and blocked studies (41), VEGF,,, stimulated tyrosine phosphorylation of with 10% fetal bovine serum in PBS. Wells were incubated with lZ5I- an -200-kDa membrane protein. In contrast, P1GFl,2, at all VEGF,,, (-12,000 cpdwell) and either Flt-1 IgG or KDR IgG and concentrations tested, failed to stimulate tyrosine phosphorylvarious concentrations of cold VEGF as described under "Experimental ation (Fig. 8). Procedures." Wells were washed and individually counted in ay counter PlGF Potentiates VEGF Mitogenic Activity in Cultured Ento determine binding. Nonspecific binding (determined in the absence of dothelial Cells-We next examined the effects of PlGF alone, or receptor-IgG) was subtracted.

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FIG.5. PlGF binds to Flt-1 IgG but not to KDR IgG. In A X , wells were incubated with radioligand, as indicated (-12,000 cpdwell), receptor-IgG, and various concentrations of cold VEGFor PlGF, as described under "Experimental Procedures." Panel A shows that PlGF (548m) can displace '261-VEGF,,, bound to Flt-1 IgG but not to KDR IgG. In B , '251-P1GFl,,binding to Flt-1 IgG is competed by native PlGF,,,. In C , '251-P1GF,,, binding to Flt-1 IgG is competed by cold PlGF,,, or by VEGF,,,. Flt-1 IgG showsconcentration-dependentbinding of both 1251-VEGF,,, and '251-P1GF,,, (panel D).In contrast, KDR IgG binds only 1251-VEGF,6,(panel E ) .

PlGF Binds Flt-1 and to

Potentiates VEGF Activity

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FIG.7. Cross-linking and immunoprecipitation of 1261-VEGF,, or llbI-PIGF,,, bound toHUVE cells. HUVE cells were incubated in medium containing 1""IVEGF,6, (lanes 1, 2, 5, and 6 ) or 12sII-PIGF,,, (lanes 3 , 4 , 7, and 8).Lanes 2,4,6, and 8 reflect the addition of 2 pg/ml cold VEGF,,,. Cells were washed and incubated with 0.5 mMBE?' crosslinking agent. After cross-linking, the reaction was quenched, and then the dishes were washed and extracted. The extracts were equally divided and immunoprecipitated with rabbit antisera to Flt-1 (lanes 1 4 ) or Flk-l/KDR (lanes 5-8). The immunoprecipitation productswere subjected to electrophoresis on 4-20% SDS-polyacrylamide gels. The gels were dried andexposed to a phosphor-imaging plate and readon BAS2000 image analyzer.

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125l-PLGF152 (pM) FIG.6.Identificationof 1261-VEGF,,binding sites displaceable by PlGF on ACCE cells and concentration dependenceof lz5IPlGF,,, binding to HUVE cells. In A, confluent ACCE cells were incubated with12sI-VEGF,65 and varying concentrationsof cold PlGF,5p. Bindingconditionswere a s describedunder"Experimental Procedures." In B , confluent HUVE cells were incubated with varying concentrations of 1251-P1GF15p. Data plotted by the method of Scatchard (38) are shown in the inset. Nonspecific binding was determinedin each case with 5 pg of VEGF,, and was always515% of the total binding.

in combination with VEGF, on cultured endothelial cells. Conditioned media of transfected CEN4 cells or partially purified PlGF isoforms had little or no mitogenic activity for ACCE or HUVE cells (datanot shown). Likewise, highly purified PlGF,,,, tested up to 530 ng/ml, did not promote the growth of ACCE cells (Fig. 9A). PlGF,,, also failed to stimulateACCE cell growth, in thepresence or in theabsence of heparin (Fig. 9B). HUVE cells showed a "20% growth stimulation at concentrations of PlGF > 100 ng/ml. In contrast, rhVEGF,,,, at the concentrations of 2.5 or 10 ng/ml, induced a 3- to 4-fold increase in finalcell count (Fig. 9, A-D). Purified PlGF did not affect the maximal mitogenic response (efficacy) to VEGF16, in ACCE cells (Fig. 9C). However, when PlGF wasadded in thepresence of low, marginally efficacious concentrations of VEGF,,,, this resulted in a dose-dependent potentiation of the mitogenic activity of VEGF. In ACCE cells, addition of 190 ng/ml PlGF,,2 to 0.03 ng/ml VEGF resulted ina 4 0 4 0 % increase in cell number over that observed with VEGF alone. However, PIGF,,, failed to potentiate low doses of bFGF, demonstrating that the observed potentiation is specific for VEGF. Fig. 9D illustrates a representative experiment. A similar potentiation of VEGF bioactivity by PlGF was observed in HUVE cells (data not shown). PlGF Does Not Directly Induce Vascular Permeability but It Dramatically Potentiates VEGF Action-We then tested PlGF

200

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FIG.8.Effect of VEGF or PlGF ontyrosine phosphorylationof ACCE cellsurfaceglycoproteins. ACCE cells culturedin 6-cm disheswerestimulatedwith VEGF,, orvariousconcentrations of PlGF,,, for 5 min a t 37 "C, as indicated. Cell extracts were prepared and then precipitated with wheat germ agglutinin agarose, followed by elution with Laemmli sample buffer. The eluates were subjected to electrophoresis, transferred to polyvinylidene difluoride membranes, and immunoblotted with an anti-phosphotyrosine monoclonal antibody.

in an in vivo system, the Miles vascular permeability assay (Fig. 10). This assay provides a rapid andreproducible measure of one of the known activities of VEGF and is suitablefor the screening of multiple samples ina single animal. Tested alone up to 500 ng/site, PlGF,,, showed no activity (Fig. 10, 2 4 , while VEGF16, was able to induce Evans blue extravasation in a dose-dependent manner (Fig. 10, 8-12), in agreement with previous studies (7,8, 40). Once again, PlGF was able to potentiate the activity of minimally effective doses of VEGF. In the presence of 500 ng of PIGF,,,, a dose of VEGF,,, (10 ng) that alone gave a barely detectable response induced a near-maximal increase in dye extravasation, comparable to thatinduced by 250 ng of VEGF,,, (Fig. 10, 14-18). This potentiation was dose-dependent. However, PlGF did not increase the maximal

PlGF Binds Flt-1 to

25652 100

and Potentiates VEGF Activity

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FIG.9.Effects of PlGF on endothelial cell growth. ACCE cells were seeded into 6-well plates at 7,000 cells per well in thepresence of VEGF, PlGF, or bFGF, as indicated. After 5-7 days, cells were trypsinized and cell counts were determined using a Coulter counter. In A, cells were cultured in the presence of varying amounts of PIGFIB,.In B , varying amounts of PlGF,,, were added, in the presence or absence of 10 pg/ml heparin. In C , each well received2.5 ng/ml VEGF,,, in thepresence or in the absence of varying amounts of PlGF,,,. In D , each well received either 0.03 ng/ml VEGF,,, or 0.015 ng/ml bFGF and varying amounts of PIGF,,,. VEGF,,, at 10 ng/ml and bFGF at 2 ng/ml served as positive controls.

response (efficacy) of VEGF nor could bFGF potentiate the activity of VEGF in this assay (data not shown).

lular domain contains all the information required for high affinity binding. PlGF bound with high affinity to Flt-1 IgG. That Flt-1 is DISCUSSION truly expressed in HUVE cells and available to bind PlGF is Angiogenesis is a tightly regulated process, integral to nor- demonstrated by the identification of a cross-linked receptormal and pathologic conditions, including wound healing, cycli- lz5I-VEGFband of the predicted size (-190 kDa) following imcal growth of the corpus luteum, rheumatoid arthritis, and munoprecipitationwith an anti-Flt-1 antiserum. Such antigrowth and metastasis of solid tumors. Recent evidence impli- serum also immunoprecipitatedFlt-1-bound '251-P1GF. Our cates VEGF as a major regulator of these events (18-22, 42,inability to directlyvisualizePlGF-Flt-1 cross-linked com43).In addition, VEGF has been proposed to play a role in the plexes is likely to reflect both the lower affinity of PlGF comregulation of microvascular permeability (7,8). Yet, the signals pared toVEGF as well as a low cross-linking efficiency of both which regulate or modulate the actions of VEGF on the vascu- ligands to Flt-1. That such a receptor may also be present in lar endothelium are largely unknown. The recently identified ACCE cells is suggested by the finding that thesecells express PlGF may share some of the functions of the VEGF polypep- high affinity VEGF binding sites consistent with Flt-1 (44). tides since: (i) it belongs to the same gene family, (ii) displays Also, PlGF is able tocompete about half of the lz5I-VEGFbindsignificant sequencehomology to WGF, and(iii) the PlGFgene ing to such cells. Furthermore, recent i n situhybridization undergoes alternative exon splicing events reminiscent of the studies have demonstrated the ubiquitous expression of Flt-1 mRNA in endothelial cells (45).However, a recent study (44) VEGF gene. Therefore, it is possible that VEGF and PlGF interact with similarpathways. To better understand this po- failed to detect expression of the Flt-1 gene inACCE cells by tential interaction, we chose to purify to homogeneity the two Northern blot analysis of total RNA using a heterologous cDNA PlGF isoforms and test whether they are able to bind to the probe. The RNA blotting methods employed in that studymay known W G F receptors. The construction of soluble chimeric be less sensitive or specific than the radioligand binding or i n Flt-1, KDR, and Flk-1 receptors represented a very useful tool situ hybridization studies. Alternatively, it is possible that as it allowed us to individually characterize the properties of ACCE cells express a novel Flt-1-like VEGFPIGF receptor. each receptor both i n vitro and, potentially, i n vivo, without Remarkably, the PlGF proteins did not demonstrate high interference from the otherreceptor or other factors. The bind- affinity bindingto Flk-1IgG or to KDR IgG. In agreementwith ing characteristicsof such soluble receptors are very similar to these findings, an anti-Flk-l/KDR antiserum failed to precipithose of the full-length receptors, indicating that theextracel- tate '251-P1GFbound to HUVE cells. InFontrast, the antiserum

PlGF Binds Potentiates Flt-1 and to

VEGF Activity

25653

to preferentially occupy its higher affinity receptors (i.e. Flt-1). However, PlGF did not affect the mitogenic response to VEGF a t concentrations where the latterwould be expected to occupy both Flt-1 and Flk-lKDRreceptors. Potentiation of VEGF action by PlGF was replicated i n vivo in the Miles vascular permeability assay. In fact, the potentiation of such activity of VEGF by PlGF was much more striking than thepotentiation of the mitogenic action. Such an effect required approximately a 10-20-fold molar excess of PlGF over VEGF. Interestingly, this is similar to the difference in the relative affinities of VEGF and PlGF for Flt-1. As mentioned above, recent in situ hybridization studies havedocumented the widespread expression of the Flt-1 mRNA in endothelial cells in normal adult tissues, including the skin (45). This suggests’that interaction of PlGF with Flt-1 may take place in vivo. It will be of significant interest to determine whether PlGF is also able to enhance major in vivo actions of VEGF such as the ability to promote angiogenesis in tumors (22) or in ischemic limbs (47). One possible explanation for the effects that we report here is that Flt-1 (or other unidentified high affinity VEGFPIGF receptor) behaves as a “decoy,” having little or no transducing activity alone. Therefore, binding to this receptor would limit the bioactivity of VEGF by preventing its binding to a signal transducing receptor. According to thishypothesis, PlGFwould act t o release VEGF from Flt-1 andincrease its availability to the more relevant Flk-VKDR. Such a mechanism would be similar to thatrecently described for IL-1 receptors type I and type I1 (48). Type I receptor is responsible for mediating IL-1 biological activities, whereas type I1 receptor has been shown to play an inhibitory role by acting as a “decoy” target for IL-1. Regulation of Flt-1 and/or PlGF expression would provide a sensitivity of the endothelium to VEGF. In FIG.10. Potentiation of VEGF action by PlGF in the Miles vas- means to adjust the cular permeability assay. Evans Blue was injected intracardially intothis context, recent studies have shown that thePlGF mRNAis guinea pigs. After 1 h, various doses of VEGF1,,, PlGF,,, or a combi- expressed in HUVE cells (33). Therefore, PlGF may modulate nation of both, were administered intradermally in 0.2 ml of PBS. PBS (sites1, 7, and 13)was used a s control. Sites 2-6 reflect the response to VEGF action by an autocrine mechanism. Alternatively, the formation of heterodimers between KDR 500-,250-,125-, 50-,and 25-ng doses of PIGF,,,. Sites 8-12 show the response to 250-, 125-,50-, 20-, and 10-ng doses of VEGF,6,. Sites 14-18 and Flt-1 might confer new properties or ligand specificities show the effect of the administrationof a constant dose of VEGF,,, (10 upon these receptors. Both possibilities are currently being ng) in the presenceof 500-, 250-, 125-, 50-,and 25-ng doses of PIGF,,,. examined. In addition, we attempted toascertain whether KDR (or other receptor) could acquire high affinity binding for PlGF efficiently immunoprecipitated 1251-VEGF-receptorcomplexes. following stimulation with VEGF. So far, we have found no Although we cannot rule out the possibility that PlGF has a evidence for such a mechanism in HUVE or ACCE cells. Furvery low affinity for Flk-l/KDR (Kd > 100 nM), such a low thermore, low concentrations of VEGF spiked into binding asaffinity binding would unlikely be physiologically relevant. A says did not reveal cryptic, VEGF-dependent, binding sites for recent report suggests that heparin enhancescross-linking of 1251-P1GF,,2in KDR IgG (data not shown). VEGF to Flk-1 receptors (46). We observed only a modest inThe lack of pronounced effects of PlGF on endothelial cell crease inbinding of VEGF or PlGF to soluble receptors in the growth and vascular permeability suggest that this protein presence of heparin (data not shown). However, since all bind- may not be responsible for the direct induction of angiogenesis/ ing assays described here were performed in the presence of permeability and thus differs from VEGF. In this respect, it is serum (which may contain heparin sulfate proteoglycans), the interesting to point out that high PlGF expression has been need for exogenous heparin may have been obviated. reported in trophoblastic hydatiform mole and choriocarcinoPurified PlGF showed little or no mitogenic activity for mas (321, conditions characterized by paucity or absence of blood ACCE and HUVE cells. Likewise, PlGF failed to induce extrav- vessels (49). Rather, PlGFmay function to enhance the activity asation in the Miles vascular permeability assay. These find- ofVEGF in situationswhere the concentrations of the latter are ings suggest that binding to Flt-1 (and/or other unidentified limiting. Interestingly,the PlGFmRNAis expressed in theplareceptor) is not sufficient to trigger an effective mitogenic re- centa a t substantially higher levels than the VEGF mRNA.’ sponse or to induce vascular permeability. Unlike the Flk-1 Therefore, the molar excess of PlGF versus VEGF required to receptor (28, 291, Flt-1 fails to demonstrate VEGF-dependent elicit the effects that we describe here may occur i n vivo. tyrosine phosphorylation (23). PIGF, tested over a wide dose The PlGF isoforms share several biochemical properties with range, did not induce tyrosine phosphorylation in ACCE cells. the VEGF polypeptides. PIGF,,, lacks the highly basic domain Interaction with Flk-l/KDR may be a critical requirement to characteristic of PIGF,,,. Accordingly, PlGF,,, fails to bind to induce the full spectrum of VEGF actions. In this context, a heparin or to cation-exchange resins and isa diffusible protein. negative-dominant Flk-1 mutant has been recently shown to This behavior is very similar to that displayed by VEGF,,, (16). suppress tumor angiogenesis in vivo (43). Although the 21-amino acid insertion confers strong heparinIntriguingly, however, PlGF was able to potentiate action the of low, marginally efficacious, concentrations of VEGF on en’J. E. Park, H. H. Chen, J. Winer, K. A. Houck, and N. Ferrara, dothelial cell growth. At such concentrations, VEGF is expected unpublished observations.

25654

PlGFPotentiates Flt-1 and Binds to VEGF Activity

binding properties on PIGF,,,, thisprotein differs from 19. Phillips, H. S., Hains, J., Leung, D. W., and Ferrara, N. (1990) Endocrinology 127,965-967 VEGF18, or VEGF,,, since it is substantially diffusible. In this 20. Jakeman, L. B., Armanini, M., Phillips, H. S., and Ferrara, N. (1993) Endorespect, the behavior of PIGF,,, is reminiscent of that of crinology 133, 848459 VEGF,,, (16), a diffusible protein that nevertheless displays 21. Breier, G., Albrecht, U., Sterrer, S., and Risau, W. (1992) Development 114, 521-532 significant binding t o heparin-containing proteoglycans. Fur- 22. Kim, K. J., Li, B., Winer, J., Armanini, M., Gillett, N., Phillips, H. S., and ther studies are required to assess whether PIGF,,, binds to the Ferrara, N.(1993) Nature 362,8414344 extracellular matrix or whether proteolysis may play a role in 23. deVries, C., Escobedo, J. A,, Ueno, H., Houck, K., Ferrara, E, andWilliams, L. T. (1992) Science 256,989-991 regulating PlGF bioavailability, by analogy with the VEGF 24. Terman, B. I., Dougher-Vermazen, M., Carrion, M. E., Dimitrov, D., Armellino, D. C., Gospodarowicz, D., and Bohlen, P. (1992) Biochem. Biophys. Res. polypeptides (16, 17). Commun. 187,1579-1586 It hasbeen observed that PlGFsequences are present in the 25. Shibuya, M., Yamaguchi, S., Yamane, A,, Ikeda, T., Tojo, A,, Matsushime, H., genome of a variety of species including Drosophila melanoand Sato, M. (1990) Oncogene 5,519-524 gaster, suggesting important or even essential functions (32). 26. Terman, B. I., Carrion, M. E., Kovacs, E., Rasmussen, B. A., Eddy, R. L., and Shows, T. B. (1991) Oncogene 6,1677-1683 The availability of highly purified recombinant PlGF should 27. Matthews, W., Jordan, C. T., Gavin, M., Jenkins, N. A,, Copeland, N. G., and facilitate addressing such questions and may also permit the Lemischka, I. R. (1991)Proc. Natl. Acad. Sci. U. S. A. 88,902G9030 identification of novel receptors which could in turn shed fur- 28. Quinn, T. P., Peters, K. G., De Vries, C., Ferrara, N., and Williams, L. T. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7533-7537 ther light on the significance of this protein. Furthermore, by 29. Millauer, B., Wizigmann-Voos, S., Schnurch, H., Martinez,R., Moller, N. P. H., virtue of its selective interaction with VEGF receptors, PlGF Risau, W., and Ullrich, A. (1993) Cell 72, 8354346 may constitute an importantmolecular tool t o analyze thedif- 30. Kendell, R.L., and Thomas, K. A. (1993) Proc. Natl. Acad. Sei. U. S. A. 90, 10705-10709 ferential role of such receptors in mediating the various biolog- 31. Maglione, D., Guerriero, V., Vigliette, G., Delli-Bovi, P., and Persico, M.G. ical actions of this family of proteins and to dissect critical (1991)Proc. Natl. Acad. Sci. U. S. A. 88,9267-9271 32. Maglione, D., Guerriero, V., Viglietto, G., Ferrara, M. G., Aprelikova, O., Alistructural domains involved in receptor binding. Acknowledgments-We thank William J . Henzel for protein microsequencing, Allan Padua for amino acid analysis, Brian Fendlyfor antibodies, Eileen Soriano-Szatkowski for performing the Miles vascular permeability assay, and Louis Tamayo for graphics. We are grateful t o Joffre Baker for helpful discussions and advice.

33. 34. 35. 36.

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