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Thromb Haemost 2002; 88(01): 131-136
DOI: 10.1055/s-0037-1613165
Review Article
Schattauer GmbH

Identification of Potentially Effective Antisense Oligodeoxyribonucleotide (ODN) Sequences for Inhibiting Plasminogen Activator Inhibitor-2 (PAI-2) Production by Monocytes

Julia Humphries
1   Academic Dept of Surgery, GKT Medical School, Kings College, St Thomas´ Hospital, London
,
Kevin G. Burnand
1   Academic Dept of Surgery, GKT Medical School, Kings College, St Thomas´ Hospital, London
,
Philip Cunningham
2   Dept of Bioinformatics, Kings College, London
,
Adrian Brock
3   Dimbleby Cancer Research Unit, ICRF Laboratories, The Rayne Institute, Kings College, St Thomas´ Hospital, London
,
Nigel Westwood
4   Leukemia Science Laboratories, The Rayne Institute, Kings College, London
,
Alberto Smith
1   Academic Dept of Surgery, GKT Medical School, Kings College, St Thomas´ Hospital, London
› Author Affiliations
Further Information

Publication History

Received 28 September 2001

Accepted after resubmission 25 February 2002

Publication Date:
09 December 2017 (online)

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Summary

Objective

Monocyte fibrinolytic activity may influence thrombus resolution. The balance between uPA and PAI-2 could determine the fibrinolytic activity of the monocyte. Inhibiting PAI-2 production using specific antisense sequences might alter this balance. Selecting effective sequences is a problem as prediction of the secondary structure of target mRNA is difficult. This study reports the modification of a cell free system for rapid antisense screening.

Methods

Five 18-19 mer oligodeoxynucleotides (ODN), sequences A, B, K, T and Q, and their matched scrambled controls were designed and screened using a modified rabbit reticulocyte lysate transcription and translation system (RRL). Intracellular uptake of ODNs was confirmed by fluorescence microscopy, scanning laser confocal microscopy and fluorimetery. Monocytes were transfected with a liposome/ODN complex using sequences A, B, A + B combined, or T and PAI-2 levels measured by ELISA. Inhibition of PAI-2 production was calculated as a percentage of control levels (baseline and scrambled).

Results

(i) RRL System – Sequence A was the most effective inhibitor of PAI-2 production in this system (median 63%) compared with sequences, B median 9%, K median 14%, T median 11% and Q median –8% respectively (n = 3). Sequence A was the only sequence, which always inhibited PAI-2. This was confirmed using fluorescently labelled protein (n = 2). (ii) Monocyte transfection Fluorescence microscopy and fluorimetry showed that intracellular delivery of labelled antisense was only achieved when a liposome was used. Transfection of monocytes extracted from 5 subjects showed that sequence A significantly reduced PAI-2 production (mean % 41.4, sem 9.1) compared with sequences B (mean% 3.4, sem 8.9, p = 0.04), A + B (mean % 0.4, sem 7.8, p = 0.04), and T (mean % 5.4, sem 4.9, p = 0.01). Futher studies using sequence A on cells from 10 subjects showed a significant reduction in monocyte PAI-2 production (27.6 ng/ml, sem 3.9) compared with matched scrambled controls (mean 38.3 ng/ml, sem 4.5, p = 0.0112) and baseline (mean 51.4 ng/ml, sem 6.7, p = 0.0009).

Conclusion

Use of the RRL screening system allowed the selection of a novel antisense sequence, which significantly reduced PAI-2 production in monocytes.

Keywords

Plasminogen activator inhibitor–2 (PAI-2) - antisense oligodeoxynucleotides - monocyte
 

  • References

  • 1 Saksela O. Plasminogen activation and regulation of pericellular proteolysis. Biochim Biophys Acta 1985; 823: 35-65.
    PubMedSearch in Google Scholar
  • 2 Sappino AP, Huarte J, Belin D. et al. Plasminogen activators in tissue remodeling and invasion: mRNA localization in mouse ovaries and implanting embryos. J Cell Biol 1989; 109: 2471-9.
    CrossrefPubMedSearch in Google Scholar
  • 3 Hart PH, Burgess DR, Vitti GF. et al. Interleukin-4 stimulates human monocytes to produce tissue-type plasminogen activator. Blood 1989; 74: 1222-5.
    PubMedSearch in Google Scholar
  • 4 Manchanda N, Schwartz BS. Lipopolysaccharide-induced modulation of human monocyte urokinase production and activity. J Immunol 1990; 145: 4174-80.
    PubMedSearch in Google Scholar
  • 5 Castellote JC, Grau E, Linde MA. et al. Detection of both type 1 and type 2 plasminogen activator inhibitors in human monocytes. Thromb Haemost 1990; 63: 67-71.
    Thieme ConnectPubMedSearch in Google Scholar
  • 6 Wohlwend A, Belin D, Vassalli JD. Plasminogen activator-specific inhibitors produced by human monocytes/macrophages. J Exp Med 1987; 165: 320-39.
    CrossrefPubMedSearch in Google Scholar
  • 7 Vassalli JD, Baccino D, Belin D. A cellular binding site for the Mr 55,000 form of the human plasminogen activator, urokinase. J Cell Biol 1985; 100: 86-92.
    CrossrefPubMedSearch in Google Scholar
  • 8 Chapman Jr HA, Stone OL. A fibrinolytic inhibitor of human alveolar macrophages. Induction with endotoxin. Am Rev Respir Dis 1985; 132: 569-75.
    PubMedSearch in Google Scholar
  • 9 Dickinson JL, Bates EJ, Ferrante A. et al. Plasminogen activator inhibitor type 2 inhibits tumor necrosis factor alpha-induced apoptosis. Evidence for an alternate biological function. J Biol Chem 1995; 270: 27894-904.
    CrossrefPubMedSearch in Google Scholar
  • 10 Kruithof EK, Baker MS, Bunn CL. Biological and clinical aspects of plasminogen activator inhibitor type 2. Blood 1995; 86: 4007-24.
    PubMedSearch in Google Scholar
  • 11 Estelles A, Gilabert J, Aznar J. et al. Changes in the plasma levels of type 1 and type 2 plasminogen activator inhibitors in normal pregnancy and in patients with severe preeclampsia. Blood 1989; 74: 1332-8.
    PubMedSearch in Google Scholar
  • 12 Dougherty KM, Pearson JM, Yang AY. et al. The plasminogen activator inhibitor-2 gene is not required for normal murine development or survival. Proc Natl Acad Sci USA 1999; 96: 686-91.
    CrossrefPubMedSearch in Google Scholar
  • 13 Northeast AD, Soo KS, Bobrow LG. et al. The tissue plasminogen activator and urokinase response in vivo during natural resolution of venous thrombus. J Vasc Surg 1995; 22: 573-9.
    CrossrefPubMedSearch in Google Scholar
  • 14 Soo KS, Northeast AD, Happerfield LC. et al. Tissue plasminogen activator production by monocytes in venous thrombolysis. J Pathol 1996; 178: 190-4.
    CrossrefPubMedSearch in Google Scholar
  • 15 Goodchild J, Carroll III E, Greenberg JR. Inhibition of rabbit beta-globin synthesis by complementary oligonucleotides: identification of mRNA sites sensitive to inhibition. Arch Biochem Biophys 1988; 263: 401-9.
    CrossrefPubMedSearch in Google Scholar
  • 16 Loke SL, Stein C, Zhang X. et al. Delivery of c-myc antisense phosphorothioate oligodeoxynucleotides to hematopoietic cells in culture by liposome fusion: specific reduction in c-myc protein expression correlates with inhibition of cell growth and DNA synthesis. Curr Top Microbiol Immunol 1988; 141: 282-9.
    PubMedSearch in Google Scholar
  • 17 Zhang H, Hanecak R, Brown-Driver V. et al. Antisense oligonucleotide inhibition of hepatitis C virus (HCV) gene expression in livers of mice infected with an HCV-vaccinia virus recombinant. Antimicrob Agents Chemother 1999; 43: 347-53.
    CrossrefPubMedSearch in Google Scholar
  • 18 Chen TZ, Lin SB, Wu JC. et al. A method for screening antisense oligodeoxyribonucleotides effective for mRNA translation-arrest. J Biochem (Tokyo) 1996; 119: 252-5.
    CrossrefPubMedSearch in Google Scholar
  • 19 Chen TZ, Wu JC, Au LC. et al. Specific inhibition of delta antigen by in vitro system by antisense oligodeoxynucleotide: implications for translation mechanism and treatment. J Virol Methods 1997; 65: 183-9.
    CrossrefPubMedSearch in Google Scholar
  • 20 Ritchie H, Fragoyannis A. Thrombin inhibits apoptosis of monocytes and plasminogen activator inhibitor 2 (PAI-2) is not responsible for this inhibition. Exp Cell Res 2000; 260: 20-9.
    CrossrefPubMedSearch in Google Scholar
  • 21 Graziani-Bowering GM, Graham JM, Filion LG. A quick, easy and inexpensive method for the isolation of human peripheral blood monocytes. J Immunol Methods 1997; 207: 157-68.
    CrossrefPubMedSearch in Google Scholar
  • 22 Taylor MF, Weller DD, Kobzik L. Effect of TNF-alpha antisense oligomers on cytokine production by primary murine alveolar macrophages. Antisense Nucleic Acid Drug Dev 1998; 08: 199-205.
    CrossrefPubMedSearch in Google Scholar
  • 23 Rojanasakul Y, Weissman DN, Shi X. et al. Antisense inhibition of silicainduced tumor necrosis factor in alveolar macrophages. J Biol Chem 1997; 272: 3910-4.
    CrossrefPubMedSearch in Google Scholar
  • 24 Ho SP, Bao Y, Lesher T. et al. Mapping of RNA accessible sites for antisense experiments with oligonucleotide libraries. Nat Biotechnol 1998; 16: 59-63.
    PubMedSearch in Google Scholar
  • 25 Sohail M, Akhtar S, Southern EM. The folding of large RNAs studied by hybridization to arrays of complementary oligonucleotides. RNA 1999; 05: 646-55.
    CrossrefPubMedSearch in Google Scholar
  • 26 Storey A, Oates D, Banks L. et al. Anti-sense phosphorothioate oligonucleotides have both specific and non-specific effects on cells containing human papillomavirus type 16. Nucleic Acids Res 1991; 19: 4109-14.
    CrossrefPubMedSearch in Google Scholar
  • 27 Milner N, Mir KU, Southern EM. Selecting effective antisense reagents on combinatorial oligonucleotide arrays. Nat Biotechnol 1997; 15: 537-41.
    CrossrefPubMedSearch in Google Scholar
  • 28 Boiziau C, Thuong NT, Toulme JJ. Mechanisms of the inhibition of reverse transcription by antisense oligonucleotides. Proc Natl Acad Sci USA 1992; 89: 768-72.
    CrossrefPubMedSearch in Google Scholar
  • 29 Kozak M. Interpreting cDNA sequences: some insights from studies on translation. Mamm Genome 1996; 07: 563-74.
    CrossrefPubMedSearch in Google Scholar
  • 30 Schwartz BS, Monroe MC, Levin EG. Increased release of plasminogen activator inhibitor type 2 accompanies the human mononuclear cell tissue factor response to lipopolysaccharide. Blood 1988; 71: 734-41.
    PubMedSearch in Google Scholar
  • 31 Hartmann G, Krug A, Waller-Fontaine K. et al. Oligodeoxynucleotides enhance lipopolysaccharide-stimulated synthesis of tumor necrosis factor: dependence on phosphorothioate modification and reversal by heparin. Mol Med 1996; 02: 429-38.
    PubMedSearch in Google Scholar
  • 32 Akhtar S, Hughes MD, Khan A. et al. The delivery of antisense therapeutics. Adv Drug Deliv Rev 2000; 44: 3-21.
    CrossrefPubMedSearch in Google Scholar
  • 33 Erbacher P, Roche AC, Monsigny M. et al. Putative role of chloroquine in gene transfer into a human hepatoma cell line by DNA/lactosylated polylysine complexes. Exp Cell Res 1996; 225: 186-94.
    CrossrefPubMedSearch in Google Scholar
  • 34 Hasan MT, Subbaroyan R, Chang TY. High-efficiency stable gene transfection using chloroquine-treated Chinese hamster ovary cells. Somat Cell Mol Genet 1991; 17: 513-7.
    CrossrefPubMedSearch in Google Scholar
  • 35 Luthman H, Magnusson G. High efficiency polyoma DNA transfection of chloroquine treated cells. Nucleic Acids Res 1983; 11: 1295-308.
    CrossrefPubMedSearch in Google Scholar
  • 36 Kronenwett R, Steidl U, Kirsch M. et al. Oligodeoxyribonucleotide uptake in primary human hematopoietic cells is enhanced by cationic lipids and depends on the hematopoietic cell subset. Blood 1998; 91: 852-62.
    PubMedSearch in Google Scholar
  • 37 Steidl U, Haas R, Kronenwett R. Intercellular adhesion molecular 1 on monocytes mediates adhesion as well as trans-endothelial migration and can be downregulated using antisense oligonucleotides. Ann Hematol 2000; 79: 414-23.
    CrossrefPubMedSearch in Google Scholar
  • 38 Williams SA, Chang L, Buzby JS. et al. Cationic lipids reduce time and dose of c-myc antisense oligodeoxynucleotides required to specifically inhibit Burkitt’s lymphoma cell growth. Leukemia 1996; 10: 1980-9.
    PubMedSearch in Google Scholar
  • 39 Mayne M, Ni W, McKenna R. et al. Antisense oligodeoxynucleotides targeting internal exon sequences efficiently regulate TNF-alpha expression. Antisense Nucleic Acid Drug Dev 1999; 09: 135-44.
    CrossrefPubMedSearch in Google Scholar
  • 40 van Leeuwen EB, van der Veen AY, Hoekstra D. et al. Transfection of small numbers of human endothelial cells by electroporation and synthetic amphiphiles. Eur J Vasc Endovasc Surg 1999; 17: 9-14.
    CrossrefPubMedSearch in Google Scholar
  • 41 Bielinska A, Kukowska-Latallo JF, Johnson J. et al. Regulation of in vitro gene expression using antisense oligonucleotides or antisense expression plasmids transfected using starburst PAMAM dendrimers. Nucleic Acids Res 1996; 24: 2176-82.
    CrossrefPubMedSearch in Google Scholar
  • 42 Kukowska-Latallo JF, Bielinska AU, Johnson J. et al. Efficient transfer of genetic material into mammalian cells using Starburst polyamidoamine dendrimers. Proc Natl Acad Sci USA 1996; 93: 4897-902.
    CrossrefPubMedSearch in Google Scholar
  • 43 Derossi D, Chassaing G, Prochiantz A. Trojan peptides: the penetratin system for intracellular delivery. Trends Cell Biol 1998; 08: 84-7.
    CrossrefPubMedSearch in Google Scholar
  • 44 Morris MC, Vidal P, Chaloin L. et al. A new peptide vector for efficient delivery of oligonucleotides into mammalian cells. Nucleic Acids Res 1997; 25: 2730-6.
    CrossrefPubMedSearch in Google Scholar
  • 45 Putney SD, Brown J, Cucco C. et al. Enhanced anti-tumor effects with microencapsulated c-myc antisense oligonucleotide. Antisense Nucleic Acid Drug Dev 1999; 09: 451-8.
    CrossrefPubMedSearch in Google Scholar
  • 46 Lewis KJ, Irwin WJ, Akhtar S. Development of a sustained-release biodegradable polymer delivery system for site-specific delivery of oligonucleotides: characterization of P(LA-GA) copolymer microspheres in vitro. J Drug Target 1998; 05: 291-302.
    CrossrefPubMedSearch in Google Scholar
  • 47 Ferkol T, Perales JC, Mularo F. et al. Receptor-mediated gene transfer into macrophages. Proc Natl Acad Sci USA 1996; 93: 101-5.
    CrossrefPubMedSearch in Google Scholar
  • 48 Ferkol T, Mularo F, Hilliard J. et al. Transfer of the human Alpha1-antitrypsin gene into pulmonary macrophages in vivo. Am J Respir Cell Mol Biol 1998; 18: 591-601.
    CrossrefPubMedSearch in Google Scholar
  • 49 Liang WW, Shi X, Deshpande D. et al. Oligonucleotide targeting to alveolar macrophages by mannose receptor-mediated endocytosis. Biochim Biophys Acta 1996; 1279: 227-34.
    CrossrefPubMedSearch in Google Scholar