Proliferation of Neointimal Smooth Muscle Cells after Arterial Injury (2004)

The growth factor signaling mechanisms responsible for neointimal smooth muscle cell (SMC) proliferation and accumulation, a characteristic feature of many vascular pathologies that can lead to restenosis after angioplasty, remain to be identified. Here, we examined the contribution of fibroblast growth factor receptors (FGFRs) 2 and 3 as well as novel fibroblast growth factors (FGFs) to such proliferation. Balloon catheter injury to the rat carotid artery stimulated the expression of two distinctly spliced FGFR-2 isoforms, differing only by the presence or absence of the acidic box, and two distinctly spliced FGFR-3 isoforms containing the acidic box and differing only by the presence of either the IIIb or IIIc exon. Post-injury arterial administration of recombinant adenoviruses expressing dominant negative mutant forms of these FGFRs were used to assess the roles of the endogenous FGFR isoforms in neointimal SMC proliferation. Dominant negative FGFR-2 containing the acidic box inhibited such proliferation by 40%, whereas the dominant negative FGFR-3 forms had little effect. Expression of FGF-9, known to be capable of binding to all four neointimal FGFR-2/-3 isoforms, was abundant within the neointima. FGF-9 markedly stimulated both the proliferation of neointimal SMCs and the activation of extracellular signal-related kinases 1/2, effects which were abrogated by the administration of antisense FGF-9 oligonucleotides to injured arteries and the expression of the dominant negative FGFR-2 adenovirus in cultured neointimal SMCs. These studies demonstrate that, although multiple FGFRs are induced in neointimal SMCs following arterial injury, specific interactions between distinctly spliced FGFR-2 isoforms and FGF-9 contribute to the proliferation of these SMCs. The growth factor signaling mechanisms responsible for neointimal smooth muscle cell (SMC) proliferation and accumulation, a characteristic feature of many vascular pathologies that can lead to restenosis after angioplasty, remain to be identified. Here, we examined the contribution of fibroblast growth factor receptors (FGFRs) 2 and 3 as well as novel fibroblast growth factors (FGFs) to such proliferation. Balloon catheter injury to the rat carotid artery stimulated the expression of two distinctly spliced FGFR-2 isoforms, differing only by the presence or absence of the acidic box, and two distinctly spliced FGFR-3 isoforms containing the acidic box and differing only by the presence of either the IIIb or IIIc exon. Post-injury arterial administration of recombinant adenoviruses expressing dominant negative mutant forms of these FGFRs were used to assess the roles of the endogenous FGFR isoforms in neointimal SMC proliferation. Dominant negative FGFR-2 containing the acidic box inhibited such proliferation by 40%, whereas the dominant negative FGFR-3 forms had little effect. Expression of FGF-9, known to be capable of binding to all four neointimal FGFR-2/-3 isoforms, was abundant within the neointima. FGF-9 markedly stimulated both the proliferation of neointimal SMCs and the activation of extracellular signal-related kinases 1/2, effects which were abrogated by the administration of antisense FGF-9 oligonucleotides to injured arteries and the expression of the dominant negative FGFR-2 adenovirus in cultured neointimal SMCs. These studies demonstrate that, although multiple FGFRs are induced in neointimal SMCs following arterial injury, specific interactions between distinctly spliced FGFR-2 isoforms and FGF-9 contribute to the proliferation of these SMCs. Atherosclerosis, vasculopathies, post-transplant arteriosclerosis, and restenosis are characterized by expansion of the arterial intima as a result of the infiltration of mononuclear leukocytes, the accumulation of the extracellular matrix, and the proliferation of vascular smooth muscle cells (SMCs) 1The abbreviations used are: SMC, smooth muscle cell; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; BrdUrd, bromodeoxyuridine; RT, reverse transcription; DN, dominant negative; GFP, green fluorescent protein; ERK, extracellular signal-related kinase. (1Ross R. N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19647) Google Scholar, 2Ventura H.O. Am. Heart J. 1995; 129: 791-799Crossref PubMed Scopus (108) Google Scholar, 3Miller M.J. Kuntz R.E. Friedrich S.P. Leidig G.A. Fishman R.F. Schnitt S.J. Baim D.S. Safian R.D. Am. J. Cardiol. 1993; 71: 652-658Abstract Full Text PDF PubMed Scopus (50) Google Scholar). To date, the agents responsible for the proliferation of SMCs within the intima have eluded identification, and strategies aimed at inhibiting intimal SMC proliferation after arterial injury by either preventing platelet-derived growth factor expression or neutralizing fibroblast growth factor-2 (FGF-2) have been unsuccessful (4Wong J. Rauhoft C. Dilley R.J. Agrotis A. Jennings G.L. Bobik A. Circulation. 1997; 96: 1631-1640Crossref PubMed Scopus (47) Google Scholar, 5Olson N.E. Kozlowski J. Reidy M.A. J. Biol. Chem. 2000; 275: 11270-11277Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). This has led to postulates stating that phenotypically unique SMCs that can exhibit autonomous growth in the intima and secrete unknown heparin binding growth factors may be responsible for expansion of the SMC population in the intima (6Bochaton-Piallat M.L. Clowes A.W. Clowes M.M. Fischer J.W. Redard M. Gabbiani F. Gabbiani G. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 949-954Crossref PubMed Scopus (25) Google Scholar, 7Frid M.G. Aldashev A.A. Nemenoff R.A. Higashito R. Westcott J.Y. Stenmark K.R. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 2884-2893Crossref PubMed Scopus (35) Google Scholar). Findings that fibroblast growth factor receptors (FGFRs), (8Lee S.H. Schloss D.J. Swain J.L. J. Biol. Chem. 2000; 275: 33679-33687Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) but not FGF-1 or FGF-2, are critical for vascular development implicate other members of the FGF superfamily in vessel development (9Zhou M. Sutliff R.L. Paul R.J. Lorenz J.N. Hoying J.B. Haudenschild C.C. Yin M. Coffin J.D. Kong L. Kranias E.G. Luo W. Boivin G.P. Duffy J.J. Pawlowski S.A. Doetschman T. Nat. Med. 1998; 4: 201-207Crossref PubMed Scopus (304) Google Scholar, 10Miller D.L. Ortega S. Bashayan O. Basch R. Basilico C. Mol. Cell. Biol. 2000; 20: 2260-2268Crossref PubMed Scopus (251) Google Scholar). Such FGFs are most likely also responsible for SMC proliferation within the intima of healing arteries following arterial injury during angioplasty and during the development of arteriosclerosis. Immunohistochemical studies have identified the presence of multiple FGFR types in healing and atherosclerotic arteries, including FGFR-1, FGFR-2, and FGFR-3 (11Koyama H. Reidy M.A. Circ. Res. 1997; 80: 408-417Crossref PubMed Scopus (42) Google Scholar, 12Hughes S.E. Cardiovasc. Res. 1996; 32: 557-569Crossref PubMed Google Scholar, 13Brogi E. Winkles J.A. Underwood R. Clinton S.K. Alberts G.F. Libby P. J. Clin. Investig. 1993; 92: 2408-2418Crossref PubMed Scopus (194) Google Scholar). Although FGFR-1 has been shown to be involved in the medial SMC proliferation within healing arteries, it does not play a major role in neointimal SMC proliferation (11Koyama H. Reidy M.A. Circ. Res. 1997; 80: 408-417Crossref PubMed Scopus (42) Google Scholar). Furthermore, the contribution of FGFR-2 and FGFR-3 to neointimal SMC proliferation has not been elucidated. The structural features of FGFRs include an extracellular ligand binding domain, a single transmembrane domain, and an intracellular tyrosine kinase domain. The extracellular region of FGFRs prototypically consists of three immunoglobulin-like domains, a short motif of acidic amino acids (the "acidic box") located just distal to Ig-like domain I, and a heparin binding domain also interposed between the first and second Ig-like domains; however, alternative RNA splicing can result in several isoforms of an FGFR from the one respective gene that differ in their extracellular sequence and possess unique ligand binding properties (14Powers C.J. Mcleskey S.W. Wellstein A. Endocr. Relat. Cancer. 2000; 7: 165-197Crossref PubMed Scopus (1140) Google Scholar). One such splicing event results in a deletion of the exon encoding the amino-terminal Ig-like domain (domain I), resulting in a shorter, two-Ig-like domain isoform of the receptor (15Wang F. Kan M. Yan G. Xu J. McKeehan W.L. J. Biol. Chem. 1995; 270: 10231-10235Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar); such shorter FGFRs ("β-forms") frequently have higher affinities for some FGFs than do the corresponding longer three-Ig-like domain FGFRs ("α-forms") and, in some instances, have been associated with tumor progression (16Yamaguchi F. Saya H. Bruner J.M. Morrison R.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 484-488Crossref PubMed Scopus (225) Google Scholar). Another splicing event that can dramatically alter the FGF binding ability of FGFR-1, -2, and -3 involves the alternate usage of either the "IIIb" or "IIIc" exon to encode the carboxyl-terminal portion of Ig-like domain III (14Powers C.J. Mcleskey S.W. Wellstein A. Endocr. Relat. Cancer. 2000; 7: 165-197Crossref PubMed Scopus (1140) Google Scholar). Expression of these FGFR isoforms appears to be regulated in a tissue-specific manner, with IIIb exon-containing isoforms apparently restricted in expression to epithelial cell lineages and IIIc exon-containing isoforms restricted in expression to mesenchymal cell lineages (17Ornitz D.M. Xu J. Colvin J.S. McEwen D.G. MacArthur C.A. Coulier F. Gao G. Goldfarb M. J. Biol. Chem. 1996; 271: 15292-15297Abstract Full Text Full Text PDF PubMed Scopus (1449) Google Scholar). As a consequence of such splicing events, FGF ligands can have either a diverse or an extremely restricted FGFR binding profile. For example, FGF-1 binds to all FGFR isoforms with high affinity, FGF-2 exhibits a high affinity for binding to FGFR-1IIIb, FGFR-1IIIc, and FGFR-3IIIc, and FGF-9 exhibits a high affinity for binding to FGFR-2IIIc, FGFR-3IIIb, and FGFR-3IIIc (17Ornitz D.M. Xu J. Colvin J.S. McEwen D.G. MacArthur C.A. Coulier F. Gao G. Goldfarb M. J. Biol. Chem. 1996; 271: 15292-15297Abstract Full Text Full Text PDF PubMed Scopus (1449) Google Scholar); conversely, FGF-7 and FGF-10 are restricted in binding, with high affinity only to FGFR-2IIIb (14Powers C.J. Mcleskey S.W. Wellstein A. Endocr. Relat. Cancer. 2000; 7: 165-197Crossref PubMed Scopus (1140) Google Scholar, 17Ornitz D.M. Xu J. Colvin J.S. McEwen D.G. MacArthur C.A. Coulier F. Gao G. Goldfarb M. J. Biol. Chem. 1996; 271: 15292-15297Abstract Full Text Full Text PDF PubMed Scopus (1449) Google Scholar, 18Yeh B.K. Igarashi M. Eliseenkova A.V. Plotnikov A.N. Sher I. Ron D. Aaronson S.A. Mohammadi M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2266-2271Crossref PubMed Scopus (154) Google Scholar). Finally, it has become increasingly apparent that the cellular potency of FGFR signaling is also influenced by post-translational receptor modification, in particular the enhanced mitogenic capacity of FGFR-2β isoforms arising from glycosaminoglycan modification at the acidic box motif (19Sakaguchi K. Lorenzi M.V. Bottaro D.P. Miki T. Mol. Cell. Biol. 1999; 19: 6754-6764Crossref PubMed Scopus (14) Google Scholar). Here, we demonstrate that arterial injury induces the predominant expression by neointimal SMCs of two distinctly spliced FGFR-2IIIc β-isoforms, differing from each other only by the presence/absence of the acidic box motif, and two distinctly spliced FGFR-3 α-isoforms, both containing the acidic box motif and differing from each other only by the alternate IIIb/IIIc composition of Ig-like domain III. Using adenoviruses expressing dominant negative mutants of specific receptor types truncated at their transmembrane domain and devoid of tyrosine kinase activity, we demonstrate that signaling through FGFR-2IIIc is a major contributor to neointimal SMC proliferation. We further demonstrate that a specific FGF ligand, FGF-9, known to have high affinity for the neointimal FGFR-2/-3 isoforms, shows an elevated neointimal expression after arterial injury and contributes to neointimal SMC proliferation through an interaction with FGFR-2IIIc. These findings, for the first time, demonstrate a major regulatory role for an FGFR2-IIIc/FGF-9 interaction in the stimulation of neointimal SMC proliferation in the injured arterial wall. Carotid Artery Injury—Balloon catheter injury to the rat carotid artery was carried out as described previously (4Wong J. Rauhoft C. Dilley R.J. Agrotis A. Jennings G.L. Bobik A. Circulation. 1997; 96: 1631-1640Crossref PubMed Scopus (47) Google Scholar, 20Ward M.R. Tsao P.S. Agrotis A. Dilley R.J. Jennings G.L. Bobik A. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 208-213Crossref PubMed Scopus (52) Google Scholar). Briefly, after anesthetizing the rats with a mixture of pentobarbitone (30 mg/kg), methohexitone (40 mg/kg), and atropine sulfate (3 mg/kg) administered by intraperitoneal injection, a 2F Fogarty arterial embolectomy catheter (Baxter) was passed through an arteriotomy in the left common femoral artery and into the left common carotid artery to its bifurcation. The balloon was inflated with 25 μl of saline and withdrawn with a rotating action to the aortic arch and then reintroduced and withdrawn a further two times. The femoral artery was then ligated, the incision was closed, and the animals were allowed to recover in a humidified, warmed chamber for 1–2 h. Subsequently, the animals either underwent a second surgical procedure (see below) or were sacrificed 2 to 14 days later with an overdose of pentobarbitone (100 mg/kg intraperitoneal), and the carotid arteries were collected for mRNA analyses, Western blotting, or immunohistochemistry. Delivery of Adenoviruses/Antisense Oligonucleotides to Injured Arteries and Determination of SMC Proliferation—After anesthetizing the rats (see above), a midline neck incision was made, and blunt end dissection to the carotid bifurcation was performed. A fine catheter was then inserted through an arteriotomy in the external carotid artery and placed into the common carotid artery. Temporary ligatures were then placed around the catheter in the common carotid artery before flushing the vessel with a solution of adenovirus or oligonucleotide and then placing a second ligature on the vessel toward the aortic arch. Solutions of either adenovirus (5 × 109 plaque-forming units in 100 μl saline) or oligonucleotides (20 μm in 100 μl of LipofectAMINE/Opti-MEM) were then infused into the ligated segment of the common carotid artery for 30 min at a pressure of 80–100 mm Hg. The ligatures and catheter were then removed, the external carotid ligated, the incision closed, and the animals allowed to recover (see above). To measure cell proliferation in the healing vessels, 50 mg/kg bromodeoxyuridine (BrdUrd) was administered by intraperitoneal injection 24 and 8 h before sacrifice. Tissue Collection and Immunohistochemistry—Carotid arteries were removed from the sacrificed animals and cleaned of non-vascular tissue. Then, arteries intended for mRNA analyses or Western blotting underwent careful removal of their neointima under a dissecting microscope, and the neointimal tissue was then rapidly frozen in liquid nitrogen. Carotid arteries for immunohistochemical analyses were mounted in OCT compound (Tissue Tek; Miles Scientific), frozen in pentane over liquid nitrogen, and stored at -70 °C. Immunohistochemical detection of FGFR and FGF peptide expression and BrdUrd incorporation was carried out using 6 μm of frozen cross-sections and the avidin-biotinperoxidase complex kit (Vector Laboratories) with 3,3′-diaminobenzidine tetrachloride as the chromogenic substrate, essentially as described previously (21Ward M.R. Agrotis A. Kanellakis P. Dilley R.J. Jennings G. Bobik A. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 2461-2470Crossref PubMed Scopus (56) Google Scholar). Primary antibodies to FGFR-2 (1:500, rabbit anti-human polyclonal; catalog number sc-122), FGFR-3 (1:500, rabbit anti-human polyclonal; catalog number sc-123), and FGF-9 (1:500, goat anti-human polyclonal; catalog number sc-1368) were from Santa Cruz Biotechnology Inc., the anti-FGF-1 antibody (1:500, rabbit antibovine polyclonal; catalog number G5081) was from Promega, the anti-FGF-2 antibody (1:500, mouse anti-human monoclonal; catalog number DE6) was from du Pont du Nemours and Company, and the anti-BrdUrd antibody (1:100, mouse monoclonal; catalog number 03-3900) was from Zymed Laboratories Inc.. The appropriate peroxidase-labeled secondary antibodies (Vector Laboratories) were used at a concentration of 1:200. After completing the immunohistochemical procedures, the sections were lightly counterstained with hematoxylin. Reverse Transcription PCR—RNA was isolated from 2–3 neointimas and treated with DNase (Promega) essentially as described previously (21Ward M.R. Agrotis A. Kanellakis P. Dilley R.J. Jennings G. Bobik A. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 2461-2470Crossref PubMed Scopus (56) Google Scholar). Approximately 300 ng of RNA was reverse transcribed using a GeneAmp RNA-PCR kit (PerkinElmer Life Sciences), and specific FGFR-2, FGFR-3, and FGF-9 fragments were amplified using 35 PCR cycles (see next paragraph) as described previously (20Ward M.R. Tsao P.S. Agrotis A. Dilley R.J. Jennings G.L. Bobik A. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 208-213Crossref PubMed Scopus (52) Google Scholar). For determination of FGFR cDNA structures and verification of FGF-9 amplification specificity, nucleotide sequencing (TaqTrack, Promega) was carried out on gel-purified fragments cloned into pGEM-T vectors (Promega) (22Agrotis A. Condron M. Bobik A. FEBS Lett. 2000; 467: 128-132Crossref PubMed Scopus (15) Google Scholar). Oligonucleotide Primers for RT-PCR and Antisense FGF-9 Oligonucleotides—FGFR-2 and FGFR-3 primers were designed to provide definitive structural information on distinct regions of the receptors encoded by each amplified cDNA (seeFig. 2A). Specifically, to detect the presence/absence of Ig-like domain I and the acidic box motif in the extracellular region of each receptor, primer sets designated as "Region A" were utilized in which the sense and antisense primers were chosen such that they were sufficiently distal from the 5′ and 3′ splicing boundaries of both consecutive exons that code for Ig-like domain I and the acidic box; for detection of alternatively spliced IIIb or IIIc exons in the carboxyl terminus of Ig-like domain III and in the presence of the transmembrane-spanning region, primer sets designated as "Region B-TM" in the case of FGFR-2 and "Region A-IIIb", "Region A-IIIc", or Region B-TM in the case of FGFR-3 were utilized. The precise location of each of the primer pairs within each cDNA and the predicted size of their amplification products were as follows. For FGFR-2 cDNA (GenBank™ accession numbers Z35138, L19104, and D83495), in Region A the sense primer comprised nucleotides 19–42 and the antisense primer comprised nucleotides 585–614 (596 bp); in Region B-TM the sense primer comprised nucleotides 884–913 and the antisense primer comprised nucleotides 1426–1445 (562 bp). To detect the presence in this cDNA fragment of the IIIb or IIIc exon, the restriction endonuclease HpaI, a site for which is present in the IIIc exon but not the IIIb exon, was used to digest the amplified product. For FGFR-3 cDNA (GenBank™ accession numbers M81342 and L26492), in Region A the sense primer comprised nucleotides 6–33 and the antisense primer comprised nucleotides 433–452 (447 bp). To detect either the IIIb exon or IIIc exon in the carboxyl-terminal half of Ig-like domain III, the sense primer of Region A was used with a IIIb exon-specific antisense primer (Region A-IIIb, nucleotides 914–939; 934 bp) or a IIIc exon-specific antisense primer (Region A-IIIc, nucleotides 956–981; 976 bp). For the transmembrane-spanning domain (Region B-TM), the sense primer comprised nucleotides 913–942 and the antisense primer comprised nucleotides 1676–1696 (784 bp). To detect FGF-9 cDNA (GenBank™ accession number D14839), the sense primer comprised nucleotides 222–247 and the antisense primer comprised nucleotides 508–537 (316 bp). Phosphorothioate-modified FGF-9 antisense and sense oligonucleotides were made, corresponding to nucleotides 170–184 of the rat cDNA. In Situ RT-PCR—To detect FGF-9 mRNA in the neointima, 6-μm cross-sections of carotid arteries fixed onto gelatinized slides with formalin were subjected to in situ RT-PCR as described previously (23Kalinina N. Agrotis A. Tararak E. Antropova Y. Kanellakis P. Ilyinskaya O. Quinn M.T. Smirnov V. Bobik A. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 2037-2043Crossref PubMed Scopus (104) Google Scholar). Briefly, FGF-9 mRNA was detected by a "one-step" RT-PCR method (35 cycles) utilizing EZ rTth polymerase (PerkinElmer Life Sciences) according to the manufacturer's protocol and digoxigenin-11-dUTP. Messenger RNA expression/localization was then determined with an anti-digoxigenin antibody coupled to alkaline phosphatase; color detection was carried out using the substrate nitro blue tetrazolium. Controls included tissue sections not exposed to PCR primers and reaction mixtures not subjected to reverse transcription. Dominant FGFR-2 and FGFR-3 and were from R. Y. D.J. T. H. R.D. Circ. Res. 2002; PubMed Scopus Google Scholar). expressing dominant negative of Ig-like domain I and containing the acidic and containing all three Ig-like and the acidic box and differing from each other only in the alternate presence of either the IIIb or IIIc exon in the carboxyl terminus of Ig-like domain were by their from rat SMC RNA and these at the transmembrane domain with the of a distal to the amino of the transmembrane domain. These were cloned into using the following PCR primers and restriction nucleotides of site is and and the is and and nucleotides of site is and is and and the is in and or nucleotides of site is and and the is and and nucleotides of the site is and the is and and the is in The of each was by and the between either the or the was by the of restriction specific for either the IIIb or IIIc exon. adenoviruses were by between containing and amplification and using cells was then carried out as described previously S. J. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). SMC and SMCs were isolated by of 2–3 neointimas from healing carotid arteries and cultured as described previously (21Ward M.R. Agrotis A. Kanellakis P. Dilley R.J. Jennings G. Bobik A. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 2461-2470Crossref PubMed Scopus (56) Google Scholar). These SMCs the described previously for such SMCs M.L. P. Gabbiani F. Gabbiani G. Arterioscler. Thromb. Vasc. Biol. 1996; PubMed Scopus Google Scholar). stimulated by FGF-9 was determined on neointimal SMC that had been for h to the of the growth factor and then for a further h with the of cellular essentially as described previously A. J. Bobik A. 1994; PubMed Scopus Google Scholar). For such SMCs at were Western was from the neointima of injured arteries and subjected to Western using either the antibody (see above), an antibody catalog number or an antibody to green fluorescent mouse catalog number and the enhanced Western blotting essentially as described previously (4Wong J. Rauhoft C. Dilley R.J. Agrotis A. Jennings G.L. Bobik A. Circulation. 1997; 96: 1631-1640Crossref PubMed Scopus (47) Google Scholar). To measure adenovirus expression in neointimal SMCs h the cells were in saline and using a solution of mm mm mm 2 mm 100 mm and 100 μm The solution was by at × for and the was using the kit using as was using by to and of peptide expression was carried out by using the The ability of FGF-9 to the of kinases in the neointimal either with or was with Western using a mouse anti-human kinase antibody catalog number in these SMCs were using a rabbit anti-human antibody to Cruz Biotechnology catalog number and of were using the are as S.E. The of the adenoviruses and the oligonucleotide on neointimal SMC proliferation in injured arteries was using was by a of FGFR Expression in SMCs of the FGFRs FGF members with and alternative splicing to receptor isoforms that possess unique FGF binding properties (14Powers C.J. Mcleskey S.W. Wellstein A. Endocr. Relat. Cancer. 2000; 7: 165-197Crossref PubMed Scopus (1140) Google Scholar, 17Ornitz D.M. Xu J. Colvin J.S. McEwen D.G. MacArthur C.A. Coulier F. Gao G. Goldfarb M. J. Biol. Chem. 1996; 271: 15292-15297Abstract Full Text Full Text PDF PubMed Scopus (1449) Google we the expression of two FGFR-2 and FGFR-3, in the rapidly SMCs within the neointima of healing rat carotid arteries injured with an inflated balloon days after injury, high of FGFR-2 and FGFR-3 were in the neointima and the of these arteries A and days FGFR-2 were but FGFR-3 to be FGFR-2 and FGFR-3 were also in the of these as as 2 days after injury but not in carotid arteries and To an into the role of these receptors in neointimal SMC proliferation and the FGF ligands with which they we determined the expression of distinctly spliced FGFR-2 and FGFR-3 isoforms by RT-PCR of their isolated from the neointima, by nucleotide sequencing of specific amplified of FGFR-2 mRNA was using oligonucleotide primers in the extracellular region that Ig-like I and (Region nucleotides of the rat and the region that Ig-like domain III and the transmembrane-spanning domain (Region nucleotides of the rat using Region A primers that only of FGFR-2 containing all three Ig-like was as shown by the of the PCR (596 an two FGFR-2 cDNA the fragment were determined by restriction endonuclease not to from splicing within the Ig-like domain I exon. The two predominant amplification products were in the size and were