Pharmaceutical Research

, Volume 21, Issue 5, pp 736–741

Sp1-Dependent Regulation of the RTP801 Promoter and Its Application to Hypoxia-Inducible VEGF Plasmid for Ischemic Disease

  • Minhyung Lee
  • Malavosklish Bikram
  • Seungjoon Oh
  • David A. Bull
  • Sung Wan Kim


Purpose. Gene therapy using vascular endothelial growth factor (VEGF) is a new potential treatment of ischemic disease. To be safe and effective, VEGF expression should be enhanced locally in ischemic tissue. In this study, we identified the cis-regulatory element for the hypoxia induction of the RTP801 promoter. In addition, pRTP801-VEGF was evaluated as a therapeutic plasmid in vitro.

Methods. The cis-regulatory element for hypoxia induction was identified by deletion and mutation analyses. Antisense oligonucleotide co-transfection assay was performed to evaluate the role of Sp1. pRTP801-VEGF was constructed by the insertion of the RTP801 promoter into the VEGF plasmid. The hypoxia-inducible expression of VEGF was evaluated by in vitro transfection assay.

Results. In luciferase assay, the region between -495 and -446 was responsible for the hypoxia-induced transcription. The mutation of the Sp1 site in this region reduced hypoxia-induced transcription. In addition, co-transfection with antisense Sp1 oligonucleotide suggests that hypoxia induction of the RTP801 promoter is mediated by Sp1. In vitro transfection showed that pRTP801-VEGF had higher VEGF expression than pEpo-SV-VEGF. In addition, VEGF expression by pRTP801-VEGF was induced under hypoxia.

Conclusions. With strong basal promoter activity and induction under hypoxia, pRTP801-VEGF may be useful for gene therapy for ischemic disease.

hypoxia RTP801 Sp1 transcriptional regulation vascular endothelial growth factor 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    M. Azrin. Angiogenesis, protein and gene delivery. Br. Med. Bull. 59:211-215 (2001).Google Scholar
  2. 2.
    J. Kastrup, E. Jorgensen, and V. Drvota. Vascular growth factor and gene therapy to induce new vessels in the ischemic myocardium. Therapeutic angiogenesis. Scand. Cardiovasc. J. 35:291-296 (2001).Google Scholar
  3. 3.
    J. M. Isner. Myocardial gene therapy. Nature 415:234-239 (2002).Google Scholar
  4. 4.
    J. F. Symes, D. W. Losordo, P. R. Vale, K. G. Lathi, D. D. Esakof, M. Mayskiy, and J. M. Isner. Gene therapy with vascular endothelial growth factor for inoperable coronary artery disease. Ann. Thorac. Surg. 68:836-837 (1999).Google Scholar
  5. 5.
    P. R. Vale, D. W. Losordo, C. E. Milliken, M. Maysky, D. D. Esakof, J. F. Symes, and J. M. Isner. Left ventricular electromechanical mapping to assess efficacy of phVEGF(165) gene transfer for therapeutic angiogenesis in chronic myocardial ischemia. Circulation 102:965-974 (2000).Google Scholar
  6. 6.
    C. Sylven, N. Sarkar, A. Ruck, V. Drvota, S. Y. Hassan, B. Lind, A. Nygren, Q Kallner, P. Blomberg, J. van der Lindern, D. Lindblom, L. A. Brodin, and K. B. Islam. Myocardial Doppler tissue velocity improves following myocardial gene therapy with VEGF-A165 plasmid in patients with inoperable angina pectoris. Coron. Artery Dis. 12:239-243 (2001).Google Scholar
  7. 7.
    D. G. Affleck, L. Yu, D. A. Bull, S. H. Bailey, and S. W. Kim. Augmentation of myocardial transfection using TerplexDNA: a novel gene delivery system. Gene Ther. 8:349-353 (2001).Google Scholar
  8. 8.
    M. Lee, J. Rentz, S. Han, D. A. Bull, and S. W. Kim. Water soluble lipopolymer as an efficient carrier for gene delivery to myocardium. Gene Ther. 10:585-593 (2003).Google Scholar
  9. 9.
    E. Brogi, G. Schatteman, T. Wu, E. A. Kim, L. Varticovski, B. Keyt, and J. M. Isner. Hypoxia-induced paracrine regulation of vascular endothelial growth factor receptor expression. J. Clin. Invest. 97:469-476 (1996).Google Scholar
  10. 10.
    J. S. Lee and A. M. Feldman. Gene therapy for therapeutic myocardial angiogenesis: a promising synthesis of two emerging technologies. Nature Med. 4:739-742 (1998).Google Scholar
  11. 11.
    M. L. Springer, A. S. Chen, P. E. Kraft, M. Bednarski, and H. M. Blau. VEGF gene delivery to muscle: potential role for vasculogenesis in adults. Mol. Cell 2:549-558 (1998).Google Scholar
  12. 12.
    R. J. Lee, M. L. Springer, W. E. Blanco-Bose, R. Shaw, P. C. Ursell, and H. M. Blau. VEGF gene delivery to myocardium: deleterious effects of unregulated expression. Circulation 102:898-901 (2000).Google Scholar
  13. 13.
    M. Lee, J. Rentz, M. Bikram, S. Han, D. A. Bull, and S. W. Kim. Hypoxia inducible VEGF gene delivery to ischemic myocardium using water-soluble lipopolymer. Gene Ther. 10:1535-1542 (2003).Google Scholar
  14. 14.
    H. Su, J. Arakawa-Hoyt, and Y. W. Kan. Adeno-associated viral vector-mediated hypoxia response element-regulated gene expression in mouse ischemic heart model. Proc. Natl. Acad. Sci. USA 99:9480-9485 (2002).Google Scholar
  15. 15.
    T. Shoshani, A. Faerman, I. Mett, E. Zelin, T. Tenne, S. Corodin, Y. Moshel, S. Elbaz, A. Budanov, A. Chajut, H. Kalinski, I. Kamer, A. Rozen, O. Mor, E. Keshet, D. Leshkowitz, P. Einat, R. Skaliter, and E. Feinstein. Identification of a novel hypoxia-inducible factor 1-responsive gene, RTP801, involved in apoptosis. Mol. Cell. Biol. 22:2283-2293 (2002).Google Scholar
  16. 16.
    G. L. Wang, B.-H. Jiang, E. Rue, and G. L. Semenza. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc. Natl. Acad. Sci. USA 92:5510-5514 (1995).Google Scholar
  17. 17.
    R. H. Wenger and M. Gassmann. Oxygen(es) and the hypoxia-inducible factor-1. Biol. Chem. 378:609-616 (1997).Google Scholar
  18. 18.
    B.-H. Jiang, E. Rue, G. L. Wang, R. Roe, and G. L. Semenza. Dimerization, DNA binding, and transactivation properties of hypoxia-inducible factor 1. J. Biol. Chem. 271:17771-17778 (1996).Google Scholar
  19. 19.
    G. L. Semenza, B. H. Jiang, S. W. Leung, R. Passantino, J. P. Concordet, P. Maire, and A. Giallongo. Hypoxia response elements in the aldolase A, enolase 1, and lactate dehydrogenase A gene promoters contain essential binding sites for hypoxia-inducible factor 1. J. Biol. Chem. 271:32529-32537 (1996).Google Scholar
  20. 20.
    G. L. Wang and G. L. Semenza. General involvement of hypoxia-inducible factor 1 in transcriptional response to hypoxia. Proc. Natl. Acad. Sci. USA 90:4304-4308 (1993).Google Scholar
  21. 21.
    Q. Xu, Y.-S. Ji, and J. F. Schmedtje Jr. Sp1 increases expression of cyclooxygenase-2 in hypoxic vascular endothelium. Implications for the mechanisms of aortic aneurysm and heart failure. J. Biol. Chem. 275:24583-24589 (2000).Google Scholar
  22. 22.
    L. E. Huang, J. Gu, M. Schau, and H. F. Bunn. Regulation of hypoxia-inducible factor 1α is mediated by an O2-dependent degradation domain via the ubiquitin-proteasome pathway. Proc. Natl. Acad. Sci. USA 95:7987-7992 (1998).Google Scholar
  23. 23.
    T. Sanchez-Elsner, L. M. Botella, B. Velasco, C. Langa, and C. Bernabeu. Endoglin expression is regulated by transcriptional cooperation between the hypoxia and transforming growth pathways. J. Biol. Chem. 277:43799-43808 (2002).Google Scholar

Copyright information

© Plenum Publishing Corporation 2004

Authors and Affiliations

  • Minhyung Lee
    • 1
  • Malavosklish Bikram
    • 2
  • Seungjoon Oh
    • 3
  • David A. Bull
    • 4
  • Sung Wan Kim
    • 2
  1. 1.Clinical Research Center, College of MedicineInha UniversityInchonKorea
  2. 2.Center for Controlled Chemical Delivery, Department of Pharmaceutics and Pharmaceutical ChemistryUniversity of UtahSalt Lake CityUSA
  3. 3.Department of Internal Medicine, College of MedicineKyung Hee UniversitySeoulKorea
  4. 4.Department of Surgery, Division of Cardiothoracic SurgeryUniversity of Utah Health Sciences CenterSalt Lake CityUSA

Personalised recommendations