Download PDF
Thromb Haemost 2000; 84(03): 460-467
DOI: 10.1055/s-0037-1614045
Commentary
Schattauer GmbH

Adenoviral Gene Transfer of a u-PA Receptor-binding Plasmin Inhibitor and Green Fluorescent Protein: Inhibition of Migration and Visualization of Expression

M. L. M. Lamfers
1   From the Gaubius Laboratory TNO-PG, Leiden, The Netherlands
,
M. J. Wijnberg
1   From the Gaubius Laboratory TNO-PG, Leiden, The Netherlands
,
J. M. Grimbergen
1   From the Gaubius Laboratory TNO-PG, Leiden, The Netherlands
,
L. G. M. Huisman
1   From the Gaubius Laboratory TNO-PG, Leiden, The Netherlands
,
M. C. Aalders
1   From the Gaubius Laboratory TNO-PG, Leiden, The Netherlands
,
F. N. B. Cohen
1   From the Gaubius Laboratory TNO-PG, Leiden, The Netherlands
,
J. H. Verheijen
1   From the Gaubius Laboratory TNO-PG, Leiden, The Netherlands
,
V. W. M. van Hinsbergh
1   From the Gaubius Laboratory TNO-PG, Leiden, The Netherlands
,
P. H. A. Quax
1   From the Gaubius Laboratory TNO-PG, Leiden, The Netherlands
› Author AffiliationsThis study is supported by the Netherlands Heart foundation, grants 95-126 and M93-001.
Further Information

Publication History

Received 17 December 1999

Accepted after revision 21 February 2000

Publication Date:
14 December 2017 (online)

    Permissions and Reprints

Summary

Smooth muscle cell migration plays a role in the development of intimal hyperplasia. Given the established role of the plasminogen activation system in cell migration, an approach to therapy is to overexpress an inhibitor of plasmin. Therefore, an adenoviral vector was constructed encoding the hybrid protein ATF.BPTI, which contains the active domain of bovine pancreas trypsin inhibitor (BPTI), fused to ATF, the amino terminal fragment or receptor-binding domain of u-PA. Adenoviral vectors expressing ATF and BPTI individually were also constructed, and a fourth vector was constructed encoding ATF.BPTI linked by an internal ribosomal entry site to Green Fluorescent Protein (ABIG). Both the expression and functionality of the recombinant proteins were established in human vascular smooth muscle cells. Adenoviral gene transfer of ATF.BPTI inhibited SMC migration more efficiently than the expression of ATF or BPTI individually. Expression of ABIG resulted in the co-expression of ATF.BPTI and Green Fluorescent Protein, thereby providing a tool to monitor transfection efficiency and the behavior of the transfected cells.

Key words

SMC migration - plasminogen activators - plasmin - gene therapy - green fluorescent protein
 

  • References

  • 1 Plow EF, Freaney DE, Plescia J, Miles LA. The plasminogen system and cell surfaces: evidence for plasminogen and urokinase receptors on the same cell type. J Cell Biol 1986; 103: 2411-20.
    CrossrefPubMedGoogle Scholar
  • 2 Reuning U, Magdolen V, Wilhelm O, Fischer K, Lutz V, Graeff H, Schmitt M. Multifunctional potential of the plasminogen activation system in tumor invasion and metastasis. Int J Oncol 1998; 13: 893-906.
    PubMedGoogle Scholar
  • 3 Blasi F. The urokinase receptor and cell migration. Semin Thromb Hemost 1996; 22: 513-6.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 4 Pepper MS, Montesano R, Mandriota SJ, Orci L, Vassalli JD. Angiogenesis: a paradigm for balanced extracellular proteolysis during cell migration and morphogenesis. Enzyme Protein 1996; 49: 138-62.
    CrossrefPubMedGoogle Scholar
  • 5 Tkachuk V, Stepanova V, Little PJ, Bobik A. Regulation and role of urokinase plasminogen activator in vascular remodelling. Clin Exp Pharmacol Physiol 1996; 23: 759-65.
    CrossrefPubMedGoogle Scholar
  • 6 Wijnberg MJ, Quax PHA, Nieuwenbroek NM, Verheijen JH. The migration of human smooth muscle cells in vitro is mediated by plasminogen activation and can be inhibited by alpha2-macroglobulin receptor associated protein. Thromb Haemost 1997; 78: 880-6.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 7 Okada SS, Grobmyer SR, Barnathan ES. Contrasting effects of plasminogen activators, urokinase receptor, and LDL receptor-related protein on smooth muscle cell migration and invasion. Arterioscler Thromb Vasc Biol 1996; 16: 1269-76.
    CrossrefPubMedGoogle Scholar
  • 8 Herbert JM, Lamarche I, Carmeliet P. Urokinase and tissue-type plasminogen activator are required for the mitogenic and chemotactic effects of bovine fibroblast growth factor and platelet-derived growth factor-BB for vascular smooth muscle cells. J Biol Chem 1997; 272: 23585-91.
    CrossrefPubMedGoogle Scholar
  • 9 Jackson CL, Reidy MA. The role of plasminogen activation in smooth muscle cell migration after arterial injury. Ann N Y Acad Sci 1992; 667: 141-50.
    CrossrefPubMedGoogle Scholar
  • 10 Reidy MA, Irvin C, Lindner V. Migration of arterial wall cells. Expression of plasminogen activators and inhibitors in injured rat arteries. Circ Res 1996; 78: 405-14.
    CrossrefPubMedGoogle Scholar
  • 11 Carmeliet P, Moons L, Ploplis V, Plow E, Collen D. Impaired arterial neointima formation in mice with disruption of the plasminogen gene. J Clin Inves 1997; 99: 200-8.
    CrossrefPubMedGoogle Scholar
  • 12 Carmeliet P, Moons L, Herbert JM, Crawley J, Lupu F, Lijnen R, Collen D. Urokinase but not tissue plasminogen activator mediates arterial neointima formation in mice. Circ Res 1997; 81: 829-39.
    CrossrefPubMedGoogle Scholar
  • 13 Appella E, Blasi F. The growth factor module of urokinase is the binding sequence for its receptor. Ann N Y Acad Sci 1987; 511: 192-5.
    CrossrefPubMedGoogle Scholar
  • 14 Appella E, Robinson EA, Ullrich SJ, Stoppelli MP, Corti A, Cassani G, Blasi F. The receptor-binding sequence of urokinase. A biological function for the growth-factor module of proteases. J Biol Chem 1987; 262: 4437-40.
    PubMedGoogle Scholar
  • 15 Behrendt N, Ronne E, Ploug M, Petri T, Lober D, Nielsen LS, Schleuning WD, Blasi F, Appella E, Dano K. The human receptor for urokinase plasminogen activator. NH2-terminal amino acid sequence and glycosylation variants. J Biol Chem 1990; 265: 6453-60.
    PubMedGoogle Scholar
  • 16 Kanse SM, Benzakour O, Kanthou C, Kost C, Lijnen HR, Preissner KT. Induction of vascular SMC proliferation by urokinase indicates a novel mechanism of action in vasoproliferative disorders. Arterioscler Thromb Vasc Biol 1997; 17: 2848-54.
    CrossrefPubMedGoogle Scholar
  • 17 Rabbani SA, Gladu J, Mazar AP, Henkin J, Goltzman D. Induction in human osteoblastic cells (SaOS2) of the early response genes fos, jun, and myc by the amino terminal fragment (ATF) of urokinase. J Cell Physiol 1997; 172: 137-45.
    CrossrefPubMedGoogle Scholar
  • 18 Kirchheimer JC, Christ G, Binder BR. Growth stimulation of human epidermal cells by urokinase is restricted to the intact active enzyme. Eur J Biochem 1989; 181: 103-7.
    CrossrefPubMedGoogle Scholar
  • 19 Hall SW, Humphries JE, Gonias SL. Inhibition of cell surface receptorbound plasmin by alpha 2-antiplasmin and alpha 2-macroglobulin. J Biol Chem 1991; 266: 12329-36.
    PubMedGoogle Scholar
  • 20 Reuning U, Dixon EP, Little SP, Bang NU. Mitogen crosstalk accompanying urokinase receptor expression in stimulated vascular smooth muscle cells. FEBS Letters 1996; 392: 125-8.
    CrossrefPubMedGoogle Scholar
  • 21 Quax PHA, Lamfers MLM, Grimbergen JM, Verheijen JH, Vanhinsbergh VWM. Inhibition of neointima formation in cultured human saphenous vein segments by an adenovirus expressing a urokinase receptor-binding plasmin inhibitor. Circulation 1997; 96: I-669 (Abstract).
    PubMedGoogle Scholar
  • 22 Gerdes HH, Kaether C. Green fluorescent protein: applications in cell biology. FEBS Lett 1996; 389: 44-7.
    CrossrefPubMedGoogle Scholar
  • 23 Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC. Green fluorescent protein as a marker for gene expression. Science 1994; 263: 802-5.
    CrossrefPubMedGoogle Scholar
  • 24 Kocan RM, Moss NS, Benditt EP. Human arterial wall cells and tissues in culture. Methods Cell Biol 1980; 21A: 153-66.
    PubMedGoogle Scholar
  • 25 Fallaux FJ, Kranenburg O, Cramer SJ, Houweling A, van Ormondt H, Hoeben RC, van der Eb AJ. Characterization of 911: a new helper cell line for the titration and propagation of early region 1-deleted adenoviral vectors. Hum Gene Ther 1996; 07: 215-22.
    CrossrefPubMedGoogle Scholar
  • 26 Quax PHA, Grimbergen JM, Lansink M, Bakker AHF, Blatter MC, Belin D, Vanhinsbergh VWM, Verheijen JH. Binding of human urokinase-type plasminogen activator to its receptor residues involved in species specificity and binding. Arterioscler Thromb Vasc Biol 1998; 18: 693-701.
    CrossrefPubMedGoogle Scholar
  • 27 Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate- phenol-chloroform extraction. Anal Biochem 1987; 162: 156-9.
    PubMedGoogle Scholar
  • 28 Binnema DJ, van Iersel JJ, Dooijewaard G. Quantitation of urokinase antigen in plasma and culture media by use of an ELISA. Thromb Res 1986; 43: 569-77.
    CrossrefPubMedGoogle Scholar
  • 29 Vanboheemen P, Vandenhoogen C, Koolwijk P. Comparison of the inhibition of urokinase-type plasminogen activator (u-PA) activity by monoclonal antibodies specific for u-PA as assessed by different assays. Fibrinolysis 1995; 09: 343-9.
    PubMedGoogle Scholar
  • 30 Lefkovits J, Topol EJ. Pharmacological approaches for the prevention of restenosis after percutaneous coronary intervention. Prog Cardiovasc Dis 1997; 40: 141-58.
    CrossrefPubMedGoogle Scholar
  • 31 Herrman JP, Hermans WR, Vos J, Serruys PW. Pharmacological approaches to the prevention of restenosis following angioplasty. The search for the Holy Grail? (Part II). Drugs 1993; 46: 249-62.
    PubMedGoogle Scholar
  • 32 Mignatti P. Extracellular matrix remodeling by metalloproteinases and plasminogen activators. Kidney Int 1995; 49: S12-4.
    PubMedGoogle Scholar
  • 33 Nielsen LS, Kellerman GM, Behrendt N, Picone R, Dano K, Blasi F. A 55,000-60,000 Mr receptor protein for urokinase-type plasminogen activator. Identification in human tumor cell lines and partial purification. J Biol Chem 1988; 263: 2358-63.
    PubMedGoogle Scholar
  • 34 Ellis V, Behrendt N, Dano K. Plasminogen activation by receptor-bound urokinase. A kinetic study with both cell-associated and isolated receptor. J Biol Chem 1991; 266: 12752-8.
    PubMedGoogle Scholar
  • 35 Stepanova V, Bobik A, Bibilashvily R, Belogurov A, Rybalkin I, Domogatsky S, Little PJ, Goncharova E, Tkachuk V. Urokinase plasminogen activator induces smooth muscle cell migration: key role of growth factorlike domain. FEBS Lett 1997; 414: 471-4.
    CrossrefPubMedGoogle Scholar
  • 36 Fazioli F, Resnati M, Sidenius N, Higashimoto Y, Appella E, Blasi F. A urokinase-sensitive region of the human urokinase receptor is responsible for its chemotactic activity. EMBO J 1997; 16: 7279-86.
    CrossrefPubMedGoogle Scholar
  • 37 Stahl A, Mueller BM. Melanoma cell migration on vitronectin: regulation by components of the plasminogen activation system. Int J Cancer 1997; 71: 116-22.
    CrossrefPubMedGoogle Scholar
  • 38 Li H, Lu H, Griscelli F, Opolon P, Sun LQ, Ragot T, Legrand Y, Belin D, Soria J, Soria C, Perricaudet M, Yeh P. Adenovirus-mediated delivery of a uPA/uPAR antagonist suppresses angiogenesis-dependent tumor growth and dissemination in mice. Gene Therapy 1998; 05: 1105-13.
    CrossrefPubMedGoogle Scholar
  • 39 Kroon ME, Koolwijk P, van Goor H, Weidle UH, Collen A, van der Pluijm G, van Hinsbergh VWM. Role and localization of urokinase receptor in the formation of new microvascular structures in fibrin matrices. Am J Pathol 1999; 154: 1731-42.
    CrossrefPubMedGoogle Scholar
  • 40 De Petro G, Copeta A, Barlati S. Urokinase-type and tissue-type plasminogen activators as growth factors of human fibroblasts. Exp Cell Res 1994; 213: 286-94.
    CrossrefPubMedGoogle Scholar
  • 41 Koopman JL, Slomp J, de Bart AC, Quax PH, Verheijen JH. Mitogenic effects of urokinase on melanoma cells are independent of high affinity binding to the urokinase receptor. J Biol Chem 1998; 273: 33267-72.
    CrossrefPubMedGoogle Scholar
  • 42 Baker AH, Zaltsman AB, George SJ, Newby AC. Divergent effects of tissue inhibitor of metalloproteinase- 1, -2, or -3 overexpression on rat vascular smooth muscle cell invasion, proliferation, and death in vitro – TIMP-3 promotes apoptosis. J Clin Invest 1998; 101: 1478-87.
    CrossrefPubMedGoogle Scholar
  • 43 Baker AH, George SJ, Zaltsman AB, Murphy G, Newby AC. Inhibition of invasion and induction of apoptotic cell death of cancer cell lines by overexpression of TIMP-3. Br J Cancer 1999; 79: 1347-55.
    CrossrefPubMedGoogle Scholar