Genomic Organization of Human and Mouse Genes for Vascular Endothelial Growth Factor C
1997; Elsevier BV; Volume: 272; Issue: 40 Linguagem: Inglês
10.1074/jbc.272.40.25176
ISSN1083-351X
AutoresDmitri Chilov, Eola Kukk, Suvi Taira, Michael Jeltsch, Jaakko Kaukonen, Aarno Palotie, Vladimir Joukov, Kari Alitalo,
Tópico(s)Atherosclerosis and Cardiovascular Diseases
ResumoWe report here the cloning and characterization of human and mouse genes for vascular endothelial growth factor C (VEGF-C), a newly isolated member of the vascular endothelial growth factor/platelet-derived growth factor (VEGF/PDGF) family. Both VEGF-C genes comprise over 40 kilobase pairs of genomic DNA and consist of seven exons, all containing coding sequences. The VEGF homology domain of VEGF-C is encoded by exons 3 and 4. Exons 5 and 7 encode cysteine-rich motifs of the type C6C10CRC, and exon 6 encodes additional C10CXCXC motifs typical of a silk protein. A putative alternatively spliced rare RNA form lacking exon 4 was identified in human fibrosarcoma cells, and a major transcription start site was located in the human VEGF-C gene 523 base pairs upstream of the translation initiation codon. The upstream promoter sequences contain conserved putative binding sites for Sp-1, AP-2, and NF-κB transcription factors but no TATA box, and they show promoter activity when transfected into cells. The VEGF-C gene structure is thus assembled from exons encoding propeptides and distinct cysteine-rich domains in addition to the VEGF homology domain, and it shows both similarities and distinct differences in comparison with other members of the VEGF/PDGF gene family. We report here the cloning and characterization of human and mouse genes for vascular endothelial growth factor C (VEGF-C), a newly isolated member of the vascular endothelial growth factor/platelet-derived growth factor (VEGF/PDGF) family. Both VEGF-C genes comprise over 40 kilobase pairs of genomic DNA and consist of seven exons, all containing coding sequences. The VEGF homology domain of VEGF-C is encoded by exons 3 and 4. Exons 5 and 7 encode cysteine-rich motifs of the type C6C10CRC, and exon 6 encodes additional C10CXCXC motifs typical of a silk protein. A putative alternatively spliced rare RNA form lacking exon 4 was identified in human fibrosarcoma cells, and a major transcription start site was located in the human VEGF-C gene 523 base pairs upstream of the translation initiation codon. The upstream promoter sequences contain conserved putative binding sites for Sp-1, AP-2, and NF-κB transcription factors but no TATA box, and they show promoter activity when transfected into cells. The VEGF-C gene structure is thus assembled from exons encoding propeptides and distinct cysteine-rich domains in addition to the VEGF homology domain, and it shows both similarities and distinct differences in comparison with other members of the VEGF/PDGF gene family. The process of growth and development of new blood vessels from preexisting ones is termed angiogenesis. Angiogenesis plays a critical role in providing growing tissues with oxygen and nutrients and also occurs in some pathological conditions, including tumor growth and metastasis, inflammatory lesions, wound healing, and endocrine diseases (1Folkman J. Nature Medicine. 1995; 1: 27-31Crossref PubMed Scopus (7176) Google Scholar). Specific growth factors for the vascular endothelium, namely vascular endothelial growth factor (VEGF), 1The abbreviations used are: VEGF, vascular endothelial growth factor; PlGF, placenta growth factor; PDGF, platelet-derived growth factor; VEGFR, vascular endothelial growth factor receptor; bp, base pair(s); kb, kilobase pair(s); PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-polymerase chain reaction.1The abbreviations used are: VEGF, vascular endothelial growth factor; PlGF, placenta growth factor; PDGF, platelet-derived growth factor; VEGFR, vascular endothelial growth factor receptor; bp, base pair(s); kb, kilobase pair(s); PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-polymerase chain reaction. VEGF-B, VEGF-C, and placenta growth factor (PlGF) form a subfamily of the platelet-derived growth factor family (2Ferrara N. Davis-Smyth T. Endocr. Rev. 1997; 18: 4-25Crossref PubMed Scopus (3668) Google Scholar, 3Neufeld G. Tessler S. Gitay-Goren H. Cohen T. Levi B.-Z. Prog. Growth Factor Res. 1994; 5: 89-97Abstract Full Text PDF PubMed Scopus (210) Google Scholar, 4Dvorak H.F. Brown L.F. Detmar M. Dvorak A.M. Am. J. Pathol. 1995; 146: 1029-1039PubMed Google Scholar, 5Klagsburn M. D'Amore P.A. Cytokine Growth Factor Rev. 1996; 7: 259-270Crossref PubMed Scopus (401) Google Scholar). Certain other angiogenic growth factors may also act indirectly on endothelial cells via the induction of VEGF secretion in adjacent cells (4Dvorak H.F. Brown L.F. Detmar M. Dvorak A.M. Am. J. Pathol. 1995; 146: 1029-1039PubMed Google Scholar, 6Pertovaara L. Kaipainen A. Mustonen T. Orpana A. Ferrara N. Saksela O. Alitalo K. J. Biol. Chem. 1994; 269: 6271-6274Abstract Full Text PDF PubMed Google Scholar). Besides angiogenesis, VEGF is implicated in vasculogenesis or formation of blood vessels de novo by in situ differentiation from mesodermal precursor cells (7Risau W. Sariola H. Zerwes H.-G. Sasse J. Ekblom P. Kemler R. Doetschman T. Development. 1988; 102: 471-478PubMed Google Scholar). Both vasculogenesis and angiogenesis appear to be further regulated by inhibitory signals from cells and pericellular matrices (8Folkman J. Shing Y. J. Biol. Chem. 1992; 267: 10931-10934Abstract Full Text PDF PubMed Google Scholar, 9Hanahan D. Folkman J. Cell. 1996; 86: 353-364Abstract Full Text Full Text PDF PubMed Scopus (6016) Google Scholar).In situ hybridization studies have revealed that the three VEGF receptors VEGFR-1 (Flt-1), VEGFR-2 (Flk-1/KDR), and VEGFR-3 (Flt4) have specific, although partly overlapping, expression patterns in developing fetal tissues (10Kaipainen A. Korhonen J. Pajusola K. Aprelikova O. Persico M.G. Terman B.I. Alitalo K. J. Exp. Med. 1993; 178: 2077-2088Crossref PubMed Scopus (215) Google Scholar). VEGFR-3 expression has been localized in early embryonic blood vessels, especially in regions where the sprouting of developing lymphatic vessels takes place during the embryonic period (11Kaipainen A. Korhonen J. Mustonen T. van Hinsbergh V.W.M. Fang G.-H. Dumont D. Breitman M. Alitalo K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3566-3570Crossref PubMed Scopus (1174) Google Scholar). Its continued expression primarily in the lymphatic endothelium of adult tissues suggests that it functions as a regulator of these vessels. Notably, the expression of VEGF-C is often found in mesenchymal tissues surrounding the developing lymphatic vessels (12Kukk E. Lymboussaki A. Taira S. Kaipainen A. Jeltsch M. Joukov V. Alitalo K. Development. 1996; 122: 3829-3837PubMed Google Scholar), and overexpression of VEGF-C induces lymphatic hyperplasia in transgenic mice (13Jeltsch M. Kaipainen A. Joukov V. Meng X. Lakso M. Rauvala H. Swartz M.D.F. Jain R. Alitalo K. Science. 1997; 276: 1423-1425Crossref PubMed Scopus (1103) Google Scholar).Amino acid sequences of the PDGF/VEGF growth factor family are distinguished by eight conserved cysteine residues. In the case of PDGF, the cysteine residues have been shown to be essential for the correct folding and dimerization of the protein (14Andersson M. Östman A. Bäckström G. Hellman U. George-Nascimento C. Westermark B. Heldin C.-H. J. Biol. Chem. 1992; 267: 11260-11266Abstract Full Text PDF PubMed Google Scholar, 15Oefner C. DArcy A. Winkler F.K. Eggimann B. Hosang M. EMBO J. 1992; 11: 3921-3926Crossref PubMed Scopus (185) Google Scholar), although activity was preserved when the interchain disulfide bonds were mutated (16Kenney W.C. Haniu M. Herman A.C. Arakawa T. Costigan V.J. Lary J. Yphantis D.A. Thomason A.R. J. Biol. Chem. 1994; 269: 12351-12359Abstract Full Text PDF PubMed Google Scholar). The VEGF-C dimers consist of polypeptides of 29- and 31-kDa, generated by proteolytic cleavage of the precursor between residues 227 and 228, and 21 kDa, produced via further N-terminal cleavages (17Hu J.-S. Hastings G. Cherry S. Gentz R. Ruben S. Coleman T. FASEB J. 1997; 11: 498-504Crossref PubMed Scopus (19) Google Scholar, 18Joukov V. Pajusola K. Kaipainen A. Chilov D. Lahtien I. Kukk E. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1996; 15: 290-298Crossref PubMed Scopus (1143) Google Scholar, 19Joukov V. Sorsa T. Kumar V. Jeltsch M. Claesson-Welsh L. Cao Y. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1997; 16: 3898-3911Crossref PubMed Scopus (638) Google Scholar). In its C terminus, the VEGF-C precursor contains a unique domain of cysteine rich sequences resembling those of a silk protein. Other VEGF/PDGF family members typically have short C-terminal stretches of basic amino acid residues, which are encoded by alternatively spliced mRNAs and mediate VEGF binding to heparan sulfate proteoglycans (20Soker S. Fidder H. Neufeld G. Klagsbrun M. J. Biol. Chem. 1996; 271: 5761-5767Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar).The isolation and characterization of the VEGF gene promoter has been reported from human, rat, and mouse species (21Levy A.P. Levy N.S. Wegner S. Goldberg M.A. J. Biol. Chem. 1995; 270: 13333-13340Abstract Full Text Full Text PDF PubMed Scopus (876) Google Scholar, 22Tischer E. Mitchell R. Hartman T. Silva M. Gospodarowicz D. Fiddes J.C. Abraham J.A. J. Biol. Chem. 1991; 266: 11947-11954Abstract Full Text PDF PubMed Google Scholar, 23Shima D.T. Kuroki M. Deutsch U. Ng Y.-S. Adamis A.P. D'Amore P.A. J. Biol. Chem. 1996; 271: 3877-3883Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). The human promoter was shown to contain multiple binding sites for Sp-1, AP-1, and AP-2 transcription factors and hypoxia-regulated elements (21Levy A.P. Levy N.S. Wegner S. Goldberg M.A. J. Biol. Chem. 1995; 270: 13333-13340Abstract Full Text Full Text PDF PubMed Scopus (876) Google Scholar, 22Tischer E. Mitchell R. Hartman T. Silva M. Gospodarowicz D. Fiddes J.C. Abraham J.A. J. Biol. Chem. 1991; 266: 11947-11954Abstract Full Text PDF PubMed Google Scholar, 23Shima D.T. Kuroki M. Deutsch U. Ng Y.-S. Adamis A.P. D'Amore P.A. J. Biol. Chem. 1996; 271: 3877-3883Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). This promoter is TATA-less and responds to various effectors, including hypoxia, tumor necrosis factor-α, and basic fibroblast growth factor (24Ryuto M. Ono M. Izumi H. Yoshida S. Weich H.A. Kohno K. Kuwano M. J. Biol. Chem. 1996; 271: 28220-28228Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar). A 1.2-kb 5′-flanking region of the mouse VEGF gene contains basal promoter activity (21Levy A.P. Levy N.S. Wegner S. Goldberg M.A. J. Biol. Chem. 1995; 270: 13333-13340Abstract Full Text Full Text PDF PubMed Scopus (876) Google Scholar, 23Shima D.T. Kuroki M. Deutsch U. Ng Y.-S. Adamis A.P. D'Amore P.A. J. Biol. Chem. 1996; 271: 3877-3883Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). However, although the promoter also has a hypoxia-regulated element, VEGF gene induction by hypoxia is also effected via mRNA stabilization (25Levy A.P. Levy N.S. Goldberg M.A. J. Biol. Chem. 1996; 271: 25492-25497Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar).We report here the cloning and characterization of the human and mouse VEGF-C genes. The genomic structure of the VEGF-C gene has been analyzed, and one alternatively spliced human mRNA isoform is described. We show that human genomic fragments containing the transcription initiation site are sufficient for basal promoter activity in transfected cells.DISCUSSIONVEGF-C is the fourth member of the VEGF subfamily of PDGF-related growth factors and the largest VEGF gene described so far. The genomic structures of the previously described members, VEGF, VEGF-B, and PlGF show a high degree of similarity between each other in exon-intron junctions and some similarities of alternative splicing. In the case of VEGF-C, the exon structure resembles that of the other VEGF family members, particularly in exons 3 and 4 encoding the core growth factor domain, which contains the eight cysteine residues and the signature sequence PXCVXXXRCXGCC conserved in all PDGF/VEGF family members. The most variable parts of these genes are the regions encoded by the sixth and seventh exons, which are alternatively spliced in all other members except VEGF-C. A short fifth exon is present in all splicing variants of the other genes, but VEGF-C appears to lack such an exon. Instead, there is a C-terminal extension in the sequences encoded by exon 4. Also, sequence corresponding to exon 6, which encodes part of a heparin binding domain present in VEGF145, VEGF165, VEGF189, and PlGF152 is missing in VEGF-C (22Tischer E. Mitchell R. Hartman T. Silva M. Gospodarowicz D. Fiddes J.C. Abraham J.A. J. Biol. Chem. 1991; 266: 11947-11954Abstract Full Text PDF PubMed Google Scholar, 33Poltorak Z. Cohen T. Sivan R. Kandelis Y. Spira G. Vlodavsky I. Keshet E. Neufeld G. J. Biol. Chem. 1997; 272: 7151-7158Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar).All VEGFs have a cysteine-rich C terminus. In the case of VEGF-C, the cysteine-rich domain is extended in a repeated motif and divided into three exons. Exons 5–7 show no significant homology with VEGF, although the C6C10CRC cysteine motifs, which are encoded by exons 5 and 7 of VEGF-C may be related to the CXCXC6C11CRC pattern distributed in exons 7 and 8 of VEGF (22Tischer E. Mitchell R. Hartman T. Silva M. Gospodarowicz D. Fiddes J.C. Abraham J.A. J. Biol. Chem. 1991; 266: 11947-11954Abstract Full Text PDF PubMed Google Scholar) and 6A and 7 of VEGF-B (34Lagercrantz J. Larsson C. Grimmond S. Fredriksson M. Weber G. Piehl F. Biochem. Biophys. Res. Commun. 1996; 220: 147-152Crossref PubMed Scopus (21) Google Scholar,35Olofsson B. Pajusola K. Kaipainen A. Euler G. Joukov V. Saksela O. Orpana A. Pettersson R. Alitalo K. Eriksson U. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2576-2581Crossref PubMed Scopus (619) Google Scholar). The distribution of functional domains in the VEGF-C gene resembles to some extent that of the PDGF-B gene, where the N- and C-terminal propeptides are also cleaved off after polypeptide biosynthesis (36Heldin C.-H. Westermark B. Trends Genet. 1989; 5: 108-111Abstract Full Text PDF PubMed Scopus (130) Google Scholar).The most striking differences between VEGF-C and the other VEGFs concern the potential alternative splicing variants. We have previously shown hybridization of mRNA species of 2.4 and 2.0–2.2 kb with the VEGF-C probe. Lee et al. (37Lee J. Gray A. Yuan J. Luoh S.-M. Avraham H. Wood W.I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1988-1992Crossref PubMed Scopus (329) Google Scholar) reported two VEGF-C cDNA clones containing deletions of 152 or 557 nucleotides corresponding to exon 4 or exons 2–4, respectively. These variant cDNAs were considered to be the result of alternative splicing. We report here a similar, alternatively spliced version of VEGF-C lacking exon 4. Deletion of exon 4 would result in a premature stop codon caused by a frameshift after the alternative splice acceptor of exon 5. This protein would not contain the C-terminal cleavage site (amino acids 227 and 228) of the precursor and would lack 2 of the 8 conserved Cys residues of the VEGF homology domain (18Joukov V. Pajusola K. Kaipainen A. Chilov D. Lahtien I. Kukk E. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1996; 15: 290-298Crossref PubMed Scopus (1143) Google Scholar). Indeed, a recombinant protein of the expected size was produced from a baculovirus vector containing the variant cDNA, but in preliminary experiments, we have been unable to show that this protein is capable of stimulating the autophosphorylation of VEGFR-3. 2D. Chilov, E. Kukk, S. Taira, M. Jeltsch, J. Kaukonen, A. Palotie, V. Joukov, and Kari Alitalo, unpublished data. Studies of VEGF crystal structure have shown that the corresponding region contains the receptor binding sites and essential cysteine residues involved in inter- and intrachain disulfide bonding (38Muller Y. Li B. Christinger H. Wels J. Cunningham B. Devos A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7192-7197Crossref PubMed Scopus (363) Google Scholar). All isoforms of the VEGFs described earlier contain this part of the protein. Furthermore, RT-PCR analysis suggested that the PC-3 and HT-1080 cells as well as several tissues express only one major form of VEGF-C. Therefore, the alternatively spliced mRNA detected in the HT-1080 cell library may represent a rare transcript that is unlikely to have significant biological relevance in the cells and tissues analyzed. A similar conclusion was made from RT-PCR analysis of the mouse VEGF-C coding region.The transcription of the human VEGF-C gene starts 523 bp before the first coding base; thus, the mRNA has a long untranslated 5′-leader. As for the VEGF gene (22Tischer E. Mitchell R. Hartman T. Silva M. Gospodarowicz D. Fiddes J.C. Abraham J.A. J. Biol. Chem. 1991; 266: 11947-11954Abstract Full Text PDF PubMed Google Scholar), the VEGF-C gene promoter lacks consensus TATA and CCAAT sequences. Instead, it has putative binding sites for Sp-1, a ubiquitous nuclear protein that can initiate transcription from TATA-less genes (39Pugh B.F. Tjian R. Cell. 1990; 61: 1187-1197Abstract Full Text PDF PubMed Scopus (733) Google Scholar) and a long GC-rich 5′-untranslated region, which is typical for several growth factor genes and is postulated to have a role in translational regulation (22Tischer E. Mitchell R. Hartman T. Silva M. Gospodarowicz D. Fiddes J.C. Abraham J.A. J. Biol. Chem. 1991; 266: 11947-11954Abstract Full Text PDF PubMed Google Scholar,23Shima D.T. Kuroki M. Deutsch U. Ng Y.-S. Adamis A.P. D'Amore P.A. J. Biol. Chem. 1996; 271: 3877-3883Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, 28Bonthron D. Orkin S.H. Eur J. Biochem. 1988; 171: 51-57Crossref PubMed Scopus (22) Google Scholar, 40Rao C.D. Igarashi H. Chiu I.-M. Robbins K. Aaronson S.A. Proc. Natl. Acad. Sci. 1986; 83: 2392-2396Crossref PubMed Scopus (79) Google Scholar). In addition, sequences upstream of the VEGF-C translation start site were found to contain consensus binding sites for the AP-2 transcription factor. This suggests that the cAMP-dependent protein kinase and TGF-α as activators of the AP-2 transcription factor can regulate VEGF-C transcription (41Imagawa M. Chiu R. Karin M. Cell. 1987; 51: 251-260Abstract Full Text PDF PubMed Scopus (1029) Google Scholar, 42Gille J. Swerlick R. Caughman S. EMBO J. 1997; 16: 750-759Crossref PubMed Scopus (250) Google Scholar, 43Mitchell P.J. Wang C. Tjian R. Cell. 1987; 50: 847-861Abstract Full Text PDF PubMed Scopus (627) Google Scholar). Besides, we have shown here that the VEGF-C promoter activity is approximately 2-fold induced by serum stimulation. The corresponding mRNA increase was 2–5-fold in different cells, although some of this increase could be attributed to mRNA stabilization (32Enholm B. Paavonen K. Ristimaki A. Kumar V. Gunji Y. Klefstrom J. Kivinen L. Laiho M. Olofsson B. Joukov V. Eriksson U. Alitalo K. Oncogene. 1997; 14: 2475-2483Crossref PubMed Scopus (385) Google Scholar). Thus, important serum-responsive elements may be located outside of the promoter fragments used in the present work, for example in the first intron of this large gene.Potential binding site for the NF-κB transcription factor important for the lymphoid and immune systems was also found. NF-κB is associated with rapid response activation mechanisms and is known to be induced by a variety of inflammatory stimuli, being a mediator of tissue-specific gene expression (44Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2917) Google Scholar, 45Lenardo M.J. Baltimore D. Cell. 1989; 58: 227-229Abstract Full Text PDF PubMed Scopus (1253) Google Scholar). The presence of NF-κB sites suggests that NF-κB may be implicated in the induction of the VEGF-C mRNA by interleukin-1 and tumor necrosis factor-α. 3A. Ristimaki, D. Chilov, E. Kukk, S. Taira, M. Jeltsch, J. Kaukonen, A. Palotie, V. Joukov, and Kari Alitalo, unpublished data.Unlike the VEGF gene, the VEGF-C gene promoter does not contain putative binding sites for hypoxia-regulated factors. Numerous studies have shown that another major control point for the hypoxic induction of the VEGF gene is the regulation of the steady-state level of mRNA (21Levy A.P. Levy N.S. Wegner S. Goldberg M.A. J. Biol. Chem. 1995; 270: 13333-13340Abstract Full Text Full Text PDF PubMed Scopus (876) Google Scholar, 46Shweiki D. Itin A. Soffer D. Keshet E. Nature. 1992; 359: 843-845Crossref PubMed Scopus (4124) Google Scholar, 47Levy A.P. Levy N.S. Goldberg M.A. J. Biol. Chem. 1996; 271: 2746-2753Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar). The VEGF mRNA stability is considered to be determined by the presence of protein-binding sequence motifs in its 3′-untranslated region (5Klagsburn M. D'Amore P.A. Cytokine Growth Factor Rev. 1996; 7: 259-270Crossref PubMed Scopus (401) Google Scholar, 25Levy A.P. Levy N.S. Goldberg M.A. J. Biol. Chem. 1996; 271: 25492-25497Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). The 3′-untranslated region of the VEGF-C gene also contains a motif of this type, but at least our preliminary analyses indicate that the mRNA steady-state levels are not increased by hypoxia. Thus, besides the significant differences in its exon structure and encoded protein domains, the VEGF-C gene also has interesting differences in its regulation and function in comparison with the VEGF prototype of this gene family (13Jeltsch M. Kaipainen A. Joukov V. Meng X. Lakso M. Rauvala H. Swartz M.D.F. Jain R. Alitalo K. Science. 1997; 276: 1423-1425Crossref PubMed Scopus (1103) Google Scholar, 32Enholm B. Paavonen K. Ristimaki A. Kumar V. Gunji Y. Klefstrom J. Kivinen L. Laiho M. Olofsson B. Joukov V. Eriksson U. Alitalo K. Oncogene. 1997; 14: 2475-2483Crossref PubMed Scopus (385) Google Scholar). Interestingly, if gaps introduced into the amino acid sequences for the purposes of alignment are ignored for calculations, the newly cloned FIGF/VEGF-D is about 48% identical with human VEGF-C and about 31% identical with human VEGF (48Orlandini M. Marconcini L. Ferruzzi R. Oliviero S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11675-11680Crossref PubMed Scopus (262) Google Scholar, 52Maglione D. Guerriore V. Viglietto G. Ferraro M.G. Aprelikova O. Alitalo K. Vecchio S.D. Lie K.-J. Chou J.Y. Persico G. Oncogene. 1993; 8: 925-931PubMed Google Scholar). On the basis of their multiple amino acid sequence alignments, VEGF-C and VEGF-D are likely to have similar structural and functional properties and gene structures. Perhaps the PDGF-A/PDGF-B and VEGF-C/VEGF-D genes have diverged from VEGF/VEGF-B and from PlGF earlier than from each other during evolution. The process of growth and development of new blood vessels from preexisting ones is termed angiogenesis. Angiogenesis plays a critical role in providing growing tissues with oxygen and nutrients and also occurs in some pathological conditions, including tumor growth and metastasis, inflammatory lesions, wound healing, and endocrine diseases (1Folkman J. Nature Medicine. 1995; 1: 27-31Crossref PubMed Scopus (7176) Google Scholar). Specific growth factors for the vascular endothelium, namely vascular endothelial growth factor (VEGF), 1The abbreviations used are: VEGF, vascular endothelial growth factor; PlGF, placenta growth factor; PDGF, platelet-derived growth factor; VEGFR, vascular endothelial growth factor receptor; bp, base pair(s); kb, kilobase pair(s); PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-polymerase chain reaction.1The abbreviations used are: VEGF, vascular endothelial growth factor; PlGF, placenta growth factor; PDGF, platelet-derived growth factor; VEGFR, vascular endothelial growth factor receptor; bp, base pair(s); kb, kilobase pair(s); PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-polymerase chain reaction. VEGF-B, VEGF-C, and placenta growth factor (PlGF) form a subfamily of the platelet-derived growth factor family (2Ferrara N. Davis-Smyth T. Endocr. Rev. 1997; 18: 4-25Crossref PubMed Scopus (3668) Google Scholar, 3Neufeld G. Tessler S. Gitay-Goren H. Cohen T. Levi B.-Z. Prog. Growth Factor Res. 1994; 5: 89-97Abstract Full Text PDF PubMed Scopus (210) Google Scholar, 4Dvorak H.F. Brown L.F. Detmar M. Dvorak A.M. Am. J. Pathol. 1995; 146: 1029-1039PubMed Google Scholar, 5Klagsburn M. D'Amore P.A. Cytokine Growth Factor Rev. 1996; 7: 259-270Crossref PubMed Scopus (401) Google Scholar). Certain other angiogenic growth factors may also act indirectly on endothelial cells via the induction of VEGF secretion in adjacent cells (4Dvorak H.F. Brown L.F. Detmar M. Dvorak A.M. Am. J. Pathol. 1995; 146: 1029-1039PubMed Google Scholar, 6Pertovaara L. Kaipainen A. Mustonen T. Orpana A. Ferrara N. Saksela O. Alitalo K. J. Biol. Chem. 1994; 269: 6271-6274Abstract Full Text PDF PubMed Google Scholar). Besides angiogenesis, VEGF is implicated in vasculogenesis or formation of blood vessels de novo by in situ differentiation from mesodermal precursor cells (7Risau W. Sariola H. Zerwes H.-G. Sasse J. Ekblom P. Kemler R. Doetschman T. Development. 1988; 102: 471-478PubMed Google Scholar). Both vasculogenesis and angiogenesis appear to be further regulated by inhibitory signals from cells and pericellular matrices (8Folkman J. Shing Y. J. Biol. Chem. 1992; 267: 10931-10934Abstract Full Text PDF PubMed Google Scholar, 9Hanahan D. Folkman J. Cell. 1996; 86: 353-364Abstract Full Text Full Text PDF PubMed Scopus (6016) Google Scholar). In situ hybridization studies have revealed that the three VEGF receptors VEGFR-1 (Flt-1), VEGFR-2 (Flk-1/KDR), and VEGFR-3 (Flt4) have specific, although partly overlapping, expression patterns in developing fetal tissues (10Kaipainen A. Korhonen J. Pajusola K. Aprelikova O. Persico M.G. Terman B.I. Alitalo K. J. Exp. Med. 1993; 178: 2077-2088Crossref PubMed Scopus (215) Google Scholar). VEGFR-3 expression has been localized in early embryonic blood vessels, especially in regions where the sprouting of developing lymphatic vessels takes place during the embryonic period (11Kaipainen A. Korhonen J. Mustonen T. van Hinsbergh V.W.M. Fang G.-H. Dumont D. Breitman M. Alitalo K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3566-3570Crossref PubMed Scopus (1174) Google Scholar). Its continued expression primarily in the lymphatic endothelium of adult tissues suggests that it functions as a regulator of these vessels. Notably, the expression of VEGF-C is often found in mesenchymal tissues surrounding the developing lymphatic vessels (12Kukk E. Lymboussaki A. Taira S. Kaipainen A. Jeltsch M. Joukov V. Alitalo K. Development. 1996; 122: 3829-3837PubMed Google Scholar), and overexpression of VEGF-C induces lymphatic hyperplasia in transgenic mice (13Jeltsch M. Kaipainen A. Joukov V. Meng X. Lakso M. Rauvala H. Swartz M.D.F. Jain R. Alitalo K. Science. 1997; 276: 1423-1425Crossref PubMed Scopus (1103) Google Scholar). Amino acid sequences of the PDGF/VEGF growth factor family are distinguished by eight conserved cysteine residues. In the case of PDGF, the cysteine residues have been shown to be essential for the correct folding and dimerization of the protein (14Andersson M. Östman A. Bäckström G. Hellman U. George-Nascimento C. Westermark B. Heldin C.-H. J. Biol. Chem. 1992; 267: 11260-11266Abstract Full Text PDF PubMed Google Scholar, 15Oefner C. DArcy A. Winkler F.K. Eggimann B. Hosang M. EMBO J. 1992; 11: 3921-3926Crossref PubMed Scopus (185) Google Scholar), although activity was preserved when the interchain disulfide bonds were mutated (16Kenney W.C. Haniu M. Herman A.C. Arakawa T. Costigan V.J. Lary J. Yphantis D.A. Thomason A.R. J. Biol. Chem. 1994; 269: 12351-12359Abstract Full Text PDF PubMed Google Scholar). The VEGF-C dimers consist of polypeptides of 29- and 31-kDa, generated by proteolytic cleavage of the precursor between residues 227 and 228, and 21 kDa, produced via further N-terminal cleavages (17Hu J.-S. Hastings G. Cherry S. Gentz R. Ruben S. Coleman T. FASEB J. 1997; 11: 498-504Crossref PubMed Scopus (19) Google Scholar, 18Joukov V. Pajusola K. Kaipainen A. Chilov D. Lahtien I. Kukk E. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1996; 15: 290-298Crossref PubMed Scopus (1143) Google Scholar, 19Joukov V. Sorsa T. Kumar V. Jeltsch M. Claesson-Welsh L. Cao Y. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1997; 16: 3898-3911Crossref PubMed Scopus (638) Google Scholar). In its C terminus, the VEGF-C precursor contains a unique domain of cysteine rich sequences resembling those of a silk protein. Other VEGF/PDGF family members typically have short C-terminal stretches of basic amino acid residues, which are encoded by alternatively spliced mRNAs and mediate VEGF binding to heparan sulfate proteoglycans (20Soker S. Fidder H. Neufeld G. Klagsbrun M. J. Biol. Chem. 1996; 271: 5761-5767Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). 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Chem. 1991; 266: 11947-11954Abstract
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