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Thromb Haemost 2003; 90(03): 501-510
DOI: 10.1160/TH03-02-0106
Vascular Development and Vessel Remodeling
Schattauer GmbH

Potent inhibition of angiogenesis by D,L-peptides derived from vascular endothelial growth factor receptor 2

Christine Piossek
1   Cytos Biotechnology AG, Zurich, Switzerland
,
Karl-Heinz Thierauch
2   Experimental Oncology, Research Laboratories, Schering AG, Berlin, Germany
,
Jens Schneider-Mergener
3   Institut für Medizinische Immunologie, Universitätsklinikum Charité, Humboldt-Universität zu Berlin, Berlin, Germany
4   Jerini AG, Berlin, Germany
,
Rudolf Volkmer-Engert
3   Institut für Medizinische Immunologie, Universitätsklinikum Charité, Humboldt-Universität zu Berlin, Berlin, Germany
,
Martin F. Bachmann
1   Cytos Biotechnology AG, Zurich, Switzerland
,
Thomas Korff
5   Department of Vascular Biology & Angiogenesis Research, Institute of Molecular Oncology, Tumor Biology Center, Freiburg, Germany
,
Hellmut G. Augustin
5   Department of Vascular Biology & Angiogenesis Research, Institute of Molecular Oncology, Tumor Biology Center, Freiburg, Germany
,
Lothar Germeroth
6   IBA GmbH, Göttingen, Germany
› InstitutsangabenFinancial support: Supported by a grant from the Deutsche Forschungsgemeinschaft (SPP1069, Au83/3-2).
Weitere Informationen

Publikationsverlauf

Received 19. Februar 2003

Accepted after resubmission 02. Juni 2003

Publikationsdatum:
05. Dezember 2017 (online)

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Summary

Vascular endothelial growth factor (VEGF) is a potent mitogen for endothelial cells and plays a central role in angiogenesis and vasculogenesis. Therefore, VEGF and its receptors VEGFR-1 and VEGFR-2 are prime targets for anti-angiogenic intervention which is thought to be one of the most promising approaches in cancer therapy. Recently, we have discovered a VEGFR-2-derived peptide (247RTELNVGIDFNWEYP261) representing a potential binding site to VEGF. Using the spot synthesis technique, systematic D-amino acid substitutional analyses of this peptide were conducted and the resulting D, L-peptides inhibit VEGF binding to VEGFR-2 at half maximal concentration of 30 nM.The serum-stable D, L-peptides further inhibited auto-phosphorylation of the VEGFR-2 at nanomolar concentrations. Testing of the peptides in a spheroid-based angiogenesis assay demonstrated a potent anti-angiogenic effect in vitro. The rational design of potent and stable anti-angiogenic peptide inhibitors from their parent receptors provides a feasible route to develop novel leads for anti-angiogenic medicines.

Keywords

VEGFR-2 - angiogenesis - endothelial cells - spheroid sprouting assay - D - L-peptides
 

  • References

  • 1 Risau W. Mechanisms of angiogenesis. Nature 1997; 386 6626 671-4.
    CrossrefPubMedGoogle Scholar
  • 2 Aiello LP, Pierce EA, Foley ED, Takagi H, Chen H, Riddle L. et al. Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins. Proc Natl Acad Sci U S A 1995; 92 (23) 10457-61.
    CrossrefPubMedGoogle Scholar
  • 3 Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1995; 1 (01) 27-31.
    CrossrefPubMedGoogle Scholar
  • 4 Ferrara N, Alitalo K. Clinical applications of angiogenic growth factors and their inhibitors. Nat Med 1999; 5 (12) 1359-64.
    CrossrefPubMedGoogle Scholar
  • 5 Ferrara N, Houck K, Jakeman L, Leung DW. Molecular and biological properties of the vascular endothelial growth factor family of proteins. Endocr Rev 1992; 13 (01) 18-32.
    CrossrefPubMedGoogle Scholar
  • 6 Gospodarowicz D, Abraham JA, Schilling J. Isolation and characterization of a vascular endothelial cell mitogen produced by pituitary-derived folliculo stellate cells. Proc Natl Acad Sci U S A 1989; 86 (19) 7311-5.
    CrossrefPubMedGoogle Scholar
  • 7 Neufeld G, Tessler S, Gitay-Goren H, Cohen T, Levi BZ. Vascular endothelial growth factor and its receptors. Prog Growth Factor Res 1994; 5 (01) 89-97.
    CrossrefPubMedGoogle Scholar
  • 8 Dvorak HF, Nagy JA, Feng D, Brown LF, Dvorak AM. Vascular permeability factor/vascular endothelial growth factor and the significance of microvascular hyperpermeability in angiogenesis. Curr Top Microbiol Immunol 1999; 237: 97-132.
    PubMedGoogle Scholar
  • 9 Plouet J, Schilling J, Gospodarowicz D. Isolation and characterization of a newly identified endothelial cell mitogen produced by AtT-20 cells. Embo J 1989; 8 (12) 3801-6.
    PubMedGoogle Scholar
  • 10 Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 1989; 246 4935 1306-9.
    CrossrefPubMedGoogle Scholar
  • 11 Ferrara N, Carver-Moore K, Chen H, Dowd M, Lu L, O’Shea KS. et al. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature 1996; 380 6573 439-42.
    CrossrefPubMedGoogle Scholar
  • 12 Carmeliet P, Ferreira V, Breier G, Pollefeyt S, Kieckens L, Gertsenstein M. et al. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 1996; 380 6573 435-9.
    CrossrefPubMedGoogle Scholar
  • 13 Schuh AC, Faloon P, Hu QL, Bhimani M, Choi K. In vitro hematopoietic and endothelial potential of flk-1(-/-) embryonic stem cells and embryos. Proc Natl Acad Sci USA 1999; 96 (05) 2159-64.
    CrossrefPubMedGoogle Scholar
  • 14 Dumont DJ, Jussila L, Taipale J, Lymboussaki A, Mustonen T, Pajusola K. et al. Cardiovascular failure in mouse embryos deficient in VEGF receptor-3. Science 1998; 282 5390 946-9.
    CrossrefPubMedGoogle Scholar
  • 15 Fong GH, Rossant J, Gertsenstein M, Breitman ML. Role of the Flt-1 receptor tyro-sine kinase in regulating the assembly of vascular endothelium. Nature 1995; 376 6535 66-70.
    CrossrefPubMedGoogle Scholar
  • 16 Shalaby F, Rossant J, Yamaguchi TP, Gertsenstein M, Wu XF, Breitman ML. et al. Failure of blood-island formation and vasculo-genesis in Flk-1-deficient mice. Nature 1995; 376 6535 62-6.
    CrossrefPubMedGoogle Scholar
  • 17 Jakeman LB, Winer J, Bennett GL, Altar CA, Ferrara N. Binding sites for vascular endothelial growth factor are localized on endothelial cells in adult rat tissues. J Clin Invest 1992; 89 (01) 244-53.
    CrossrefPubMedGoogle Scholar
  • 18 Makinen T, Jussila L, Veikkola T, Karpanen T, Kettunen MI, Pulkkanen KJ. et al. Inhibition of lymphangiogenesis with resulting lymph-edema in transgenic mice expressing soluble VEGF receptor-3. Nat Med 2001; 7 (02) 199-205.
    CrossrefPubMedGoogle Scholar
  • 19 Brekken RA, Overholser JP, Stastny VA, Waltenberger J, Minna JD, Thorpe PE. Selective inhibition of vascular endothelial growth factor (VEGF) receptor 2 (KDR/Flk-1) activity by a monoclonal anti-VEGF antibody blocks tumor growth in mice. Cancer Res 2000; 60 (18) 5117-24.
    PubMedGoogle Scholar
  • 20 Borgstrom P, Hillan KJ, Sriramarao P, Ferrara N. Complete inhibition of angiogenesis and growth of microtumors by anti-vascular endothelial growth factor neutralizing antibody: novel concepts of angiostatic therapy from intravital videomicroscopy. Cancer Res 1996; 56 (17) 4032-9.
    PubMedGoogle Scholar
  • 21 Kim KJ, Li B, Winer J, Armanini M, Gillett N, Phillips HS. et al. Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo. Nature 1993; 362 6423 841-4.
    CrossrefPubMedGoogle Scholar
  • 22 Prewett M, Huber J, Li Y, Santiago A, O’Connor W, King K. et al. Antivascular endothelial growth factor receptor (fetal liver kinase 1) monoclonal antibody inhibits tumor angiogenesis and growth of several mouse and human tumors. Cancer Res 1999; 59 (20) 5209-18.
    PubMedGoogle Scholar
  • 23 Skobe M, Rockwell P, Goldstein N, Vosseler S, Fusenig NE. Halting angiogenesis suppresses carcinoma cell invasion. Nat Med 1997; 3 (11) 1222-7.
    CrossrefPubMedGoogle Scholar
  • 24 Witte L, Hicklin DJ, Zhu Z, Pytowski B, Kotanides H, Rockwell P. et al. Monoclonal antibodies targeting the VEGF receptor-2 (Flk1/KDR) as an anti-angiogenic therapeutic strategy. Cancer Metastasis Rev 1998; 17 (02) 155-61.
    CrossrefPubMedGoogle Scholar
  • 25 Lin P, Sankar S, Shan S, Dewhirst MW, Polverini PJ, Quinn TQ. et al. Inhibition of tumor growth by targeting tumor endothelium using a soluble vascular endothelial growth factor receptor. Cell Growth Differ 1998; 9 (01) 49-58.
    PubMedGoogle Scholar
  • 26 Goldman CK, Kendall RL, Cabrera G, Soroceanu L, Heike Y, Gillespie GY. et al. Paracrine expression of a native soluble vascular endothelial growth factor receptor inhibits tumor growth, metastasis, and mortality rate. Proc Natl Acad Sci USA 1998; 95 (15) 8795-800.
    CrossrefPubMedGoogle Scholar
  • 27 Pan B, Li B, Russell SJ, Tom JY, Cochran AG, Fairbrother WJ. Solution structure of a phage-derived peptide antagonist in complex with vascular endothelial growth factor. J Mol Biol 2002; 316 (03) 769-87.
    CrossrefPubMedGoogle Scholar
  • 28 Binetruy-Tournaire R, Demangel C, Malavaud B, Vassy R, Rouyre S, Kraemer M. et al. Identification of a peptide blocking vascular endothelial growth factor (VEGF)-mediated angiogenesis. Embo J 2000; 19 (07) 1525-33.
    CrossrefPubMedGoogle Scholar
  • 29 Fong TA, Shawver LK, Sun L, Tang C, App H, Powell TJ. et al. SU5416 is a potent and selective inhibitor of the vascular endothelial growth factor receptor (Flk-1/KDR) that inhibits tyrosine kinase catalysis, tumor vascularization, and growth of multiple tumor types. Cancer Res 1999; 59 (01) 99-106.
    PubMedGoogle Scholar
  • 30 Wood J. Pharmacological profile of a potent and orally active inhibitor of VEGF receptor tyrosine kinases. Proc. Am. Assoc. Cander Res 1998; 39: 96.
    PubMedGoogle Scholar
  • 31 Hamby JM, Showalter HD. Small molecule inhibitors of tumor-promoted angiogenesis, including protein tyrosine kinase inhibitors. Pharmacol Ther 1999; 82 (2-3) 169-93.
    CrossrefPubMedGoogle Scholar
  • 32 Hennequin LF, Thomas AP, Johnstone C, Stokes ES, Pl inverted question marke PA, Lohmann JJ. et al. Design and structure-activity relationship of a new class of potent VEGF receptor tyrosine kinase inhibitors. J Med Chem 1999; 42 (26) 5369-89.
    CrossrefPubMedGoogle Scholar
  • 33 Piossek C, Schneider-Mergener J, Schirner M, Vakalopoulou E, Germeroth L, Thierauch KH. Vascular endothelial growth factor (VEGF) receptor II-derived peptides inhibit VEGF. J Biol Chem 1999; 274 (09) 5612-9.
    CrossrefPubMedGoogle Scholar
  • 34 Frank RW. Tetrahedron. 1992; 48: 9217-9232.
    PubMedGoogle Scholar
  • 35 Kramer A. Comp. Methods Enzymol 1994; 6: 388-395.
    CrossrefPubMedGoogle Scholar
  • 36 Gausepohl H, Boulin C, Kraft M, Frank RW. Automated multiple peptide synthesis. Pept Res 1992; 5 (06) 315-20.
    PubMedGoogle Scholar
  • 37 Korff T, Augustin HG. Tensional forces in fibrillar extracellular matrices control directional capillary sprouting. J Cell Sci 1999; 112 (Pt 19) 3249-58.
    PubMedGoogle Scholar
  • 38 Korff T, Kimmina S, Martiny-Baron G, Augustin HG. Blood vessel maturation in a 3-dimensional spheroidal coculture model: direct contact with smooth muscle cells regulates endothelial cell quiescence and abrogates VEGF responsiveness. Faseb J 2001; 15 (02) 447-57.
    CrossrefPubMedGoogle Scholar
  • 39 Goodman M, Chorev M. On the concept of linear modified retro-peptide structures. Accounts Chem Res 1979; 12: 1-7.
    CrossrefPubMedGoogle Scholar
  • 40 Chorev M, Goodman M. Recent developments in retro peptides and proteins - an ongoing topochemical exploration. Trends Biotechnol 1995 1995; 13: 438-445.
    PubMedGoogle Scholar
  • 41 Schumacher TN, Mayr LM, Minor Jr. DL, Milhollen MA, Burgess MW, Kim PS. Identification of D-peptide ligands through mirror-image phage display. Science 1996; 271 5257 1854-7.
    CrossrefPubMedGoogle Scholar
  • 42 Eckert DM, Malashkevich VN, Hong LH, Carr PA, Kim PS. Inhibiting HIV-1 entry: discovery of D-peptide inhibitors that target the gp41 coiled-coil pocket. Cell 1999; 99 (01) 103-15.
    CrossrefPubMedGoogle Scholar
  • 43 Kramer A, Stigler RD, Knaute T, Hoffmann B, Schneider-Mergener J. Stepwise transformation of a cholera toxin and a p24 (HIV-1) epitope into D-peptide analogs. Protein Eng 1998; 11 (10) 941-8.
    CrossrefPubMedGoogle Scholar
  • 44 Kramer A, Reineke U, Dong L, Hoffmann B, Hoffmuller U, Winkler D. et al. Spot synthesis: observations and optimizations. J Pept Res 1999; 54 (04) 319-27.
    CrossrefPubMedGoogle Scholar
  • 45 Hagedorn M, Bikfalvi A. Target molecules for anti-angiogenic therapy: from basic research to clinical trials. Crit Rev Oncol Hematol 2000; 34 (02) 89-110.
    CrossrefPubMedGoogle Scholar