Pharmaceutical Research

, Volume 33, Issue 9, pp 2250–2258 | Cite as

Delivery of Hypoxia-Inducible Heme Oxygenase-1 Gene for Site-Specific Gene Therapy in the Ischemic Stroke Animal Model

  • Manbok Choi
  • Jungju Oh
  • Taiyoun RhimEmail author
  • Minhyung LeeEmail author
Research Paper



To reduce side effects due to non-specific expression, the heme oxygenase-1 (HO-1) gene under control of a hypoxia-inducible erythropoietin (Epo) enhancer (pEpo-SV-HO-1) was developed for site-specific gene therapy of ischemic stroke.


pEpo-SV-HO-1 was constructed by insertion of the Epo enhancer into pSV-HO-1. Dexamethasone-conjugated polyamidoamine (PAMAM-Dexa) was used as a gene carrier. In vitro transfection assays were performed in the Neuro2A cells. In vivo efficacy of pEpo-SV-HO-1 was evaluated in the transient middle cerebral artery occlusion (MCAO) model.


In vitro transfection assay with the PAMAM-Dexa/pEpo-SV-HO-1 complex showed that pEpo-SV-HO-1 had higher HO-1 gene expression than pSV-HO-1 under hypoxia. In addition, pEpo-SV-HO-1 reduced the level of apoptosis more efficiently than pSV-HO-1 in Neuro2A cells under hypoxia. For in vivo evaluation, the PAMAM-Dexa/pEpo-SV-HO-1 complex was injected into the ischemic brain of the transient MCAO model. pEpo-SV-HO-1 increased HO-1 expression and reduced the number of apoptotic cells in the ischemic brain, compared with the pSV-HO-1 injection group. As a result, the infarct volume was more efficiently decreased by pEpo-SV-HO-1 than by pSV-HO-1.


pEpo-SV-HO-1 induced HO-1 gene expression and therapeutic effect in the ischemic brain. Therefore, pEpo-SV-HO-1 may be useful for site-specific gene therapy of ischemic stroke.


gene delivery heme oxygenase-1 hypoxia-inducible gene ischemic stroke site-specific gene therapy 







Hypoxia-inducible factor-1


Heme oxygenase-1


Hypoxia response elements


Lactate dehydrogenase


Middle cerebral artery occlusion


N-methyl-D-aspartic acid


Polyamidoamine generation 2


Plasmid DNA


Reactive oxygen species


Sprague Dawley


Simian virus 40


2, 3, 5-triphenyl tetrazolium chloride


Vascular endothelial growth factor



This work was supported by a grant from the National Research Foundation of Korea, funded by the Ministry of Science, ICT and Future Planning (NRF-2013R1A1A2059236).


  1. 1.
    Hinkle JL, Guanci MM. Acute ischemic stroke review. J Neurosci Nurs. 2007;39(5):285–93. 310.CrossRefPubMedGoogle Scholar
  2. 2.
    Fagan SC, Hess DC, Hohnadel EJ, Pollock DM, Ergul A. Targets for vascular protection after acute ischemic stroke. Stroke. 2004;35(9):2220–5.CrossRefPubMedGoogle Scholar
  3. 3.
    Rhim T, Lee DY, Lee M. Drug delivery systems for the treatment of ischemic stroke. Pharm Res. 2013;30(10):2429–44.CrossRefPubMedGoogle Scholar
  4. 4.
    Kim JB, Sig Choi J, Yu YM, Nam K, Piao CS, Kim SW, et al. HMGB1, a novel cytokine-like mediator linking acute neuronal death and delayed neuroinflammation in the postischemic brain. J Neurosci. 2006;26(24):6413–21.CrossRefPubMedGoogle Scholar
  5. 5.
    Kim JB, Lim CM, Yu YM, Lee JK. Induction and subcellular localization of high-mobility group box-1 (HMGB1) in the postischemic rat brain. J Neurosci Res. 2008;86(5):1125–31.CrossRefPubMedGoogle Scholar
  6. 6.
    Qiu J, Nishimura M, Wang Y, Sims JR, Qiu S, Savitz SI, et al. Early release of HMGB-1 from neurons after the onset of brain ischemia. J Cereb Blood Flow Metab. 2008;28(5):927–38.CrossRefPubMedGoogle Scholar
  7. 7.
    Hoshida S, Nishida M, Yamashita N, Igarashi J, Aoki K, Hori M, et al. Heme oxygenase-1 expression and its relation to oxidative stress during primary culture of cardiomyocytes. J Mol Cell Cardiol. 1996;28(9):1845–55.CrossRefPubMedGoogle Scholar
  8. 8.
    Panahian N, Yoshiura M, Maines MD. Overexpression of heme oxygenase-1 is neuroprotective in a model of permanent middle cerebral artery occlusion in transgenic mice. J Neurochem. 1999;72(3):1187–203.CrossRefPubMedGoogle Scholar
  9. 9.
    Hyun H, Won YW, Kim KM, Lee J, Lee M, Kim YH. Therapeutic effects of a reducible poly (oligo-D-arginine) carrier with the heme oxygenase-1 gene in the treatment of hypoxic-ischemic brain injury. Biomaterials. 2010;31(34):9128–34.CrossRefPubMedGoogle Scholar
  10. 10.
    Hyun H, Lee J, Hwang do W, Kim S, Hyun DK, Choi JS, et al. Combinational therapy of ischemic brain stroke by delivery of heme oxygenase-1 gene and dexamethasone. Biomaterials. 2011;32(1):306–15.CrossRefPubMedGoogle Scholar
  11. 11.
    Lee J, Hyun H, Kim J, Ryu JH, Kim HA, Park JH, et al. Dexamethasone-loaded peptide micelles for delivery of the heme oxygenase-1 gene to ischemic brain. J Control Release. 2012;158:131–8.CrossRefPubMedGoogle Scholar
  12. 12.
    Kang HC, Lee M, Bae YH. Polymeric gene carriers. Crit Rev Eukaryot Gene Expr. 2005;15(4):317–42.CrossRefPubMedGoogle Scholar
  13. 13.
    Bae YM, Choi H, Lee S, Kang SH, Kim YT, Nam K, et al. Dexamethasone-conjugated low molecular weight polyethylenimine as a nucleus-targeting lipopolymer gene carrier. Bioconjug Chem. 2007;18:2029–36.CrossRefGoogle Scholar
  14. 14.
    Kim H, Kim HA, Bae YM, Choi JS, Lee M. Dexamethasone-conjugated polyethylenimine as an efficient gene carrier with an anti-apoptotic effect to cardiomyocytes. J Gene Med. 2009;11(6):515–22.CrossRefPubMedGoogle Scholar
  15. 15.
    Kim HA, Park JH, Lee S, Choi JS, Rhim T, Lee M. Combined delivery of dexamethasone and plasmid DNA in an animal model of LPS-induced acute lung injury. J Control Release. 2011;156(1):60–9.CrossRefPubMedGoogle Scholar
  16. 16.
    Choi JS, Ko KS, Park JS, Kim YH, Kim SW, Lee M. Dexamethasone conjugated poly(amidoamine) dendrimer as a gene carrier for efficient nuclear translocation. Int J Pharm. 2006;320(1–2):171–8.CrossRefPubMedGoogle Scholar
  17. 17.
    Jeon P, Choi M, Oh J, Lee M. Dexamethasone-conjugated polyamidoamine dendrimer for delivery of the heme oxygenase-1 gene into the Ischemic brain. Macromol Biosci. 2015;15(7):1021–8.CrossRefPubMedGoogle Scholar
  18. 18.
    Chauhan AS, Diwan PV, Jain NK, Tomalia DA. Unexpected in vivo anti-inflammatory activity observed for simple, surface functionalized poly(amidoamine) dendrimers. Biomacromolecules. 2009;10(5):1195–202.CrossRefPubMedGoogle Scholar
  19. 19.
    Tang YL, Tang Y, Zhang YC, Qian K, Shen L, Phillips MI. Protection from ischemic heart injury by a vigilant heme oxygenase-1 plasmid system. Hypertension. 2004;43(4):746–51.CrossRefPubMedGoogle Scholar
  20. 20.
    Platt JL, Nath KA. Heme oxygenase: protective gene or Trojan horse. Nat Med. 1998;4(12):1364–5.CrossRefPubMedGoogle Scholar
  21. 21.
    Jiang BH, Semenza GL, Bauer C, Marti HH. Hypoxia-inducible factor 1 levels vary exponentially over a physiologically relevant range of O2 tension. Am J Physiol. 1996;271(4 Pt 1):C1172–80.PubMedGoogle Scholar
  22. 22.
    Lee M, Rentz J, Bikram M, Han S, Bull DA, Kim SW. Hypoxia-inducible VEGF gene delivery to ischemic myocardium using water-soluble lipopolymer. Gene Ther. 2003;10(18):1535–42.CrossRefPubMedGoogle Scholar
  23. 23.
    Lee M, Lee ES, Kim YS, Choi BH, Park SR, Park HS, et al. Ischemic injury-specific gene expression in the rat spinal cord injury model using hypoxia-inducible system. Spine. 2005;30(24):2729–34.CrossRefPubMedGoogle Scholar
  24. 24.
    Lee M, Choi D, Choi MJ, Jeong JH, Kim WJ, Oh S, et al. Hypoxia-inducible gene expression system using the erythropoietin enhancer and 3’-untranslated region for the VEGF gene therapy. J Control Release. 2006;115(1):113–9.CrossRefPubMedGoogle Scholar
  25. 25.
    Kim H, Kim HA, Choi JS, Lee M. Delivery of hypoxia inducible heme oxygenase-1 gene using dexamethasone cojugated polyethylenimine for protection of cardiomyocytes under hypoxia. Bull Kor Chem Soc. 2009;30(4):897–901.CrossRefGoogle Scholar
  26. 26.
    Kim JY, Ryu JH, Hyun H, Kim HA, Choi JS, Yun Lee D, et al. Dexamethasone conjugation to polyamidoamine dendrimers G1 and G2 for enhanced transfection efficiency with an anti-inflammatory effect. J Drug Target. 2012;20(8):667–77.CrossRefPubMedGoogle Scholar
  27. 27.
    Maxwell PH, Pugh CW, Ratcliffe PJ. Inducible operation of the erythropoietin 3’ enhancer in multiple cell lines: evidence for a widespread oxygen-sensing mechanism. Proc Natl Acad Sci U S A. 1993;90(6):2423–7.CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Huang LE, Gu J, Schau M, Bunn HF. Regulation of hypoxia-inducible factor 1alpha is mediated by an O2-dependent degradation domain via the ubiquitin-proteasome pathway. Proc Natl Acad Sci U S A. 1998;95(14):7987–92.CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Koike S, Ogasawara Y, Shibuya N, Kimura H, Ishii K. Polysulfide exerts a protective effect against cytotoxicity caused by t-buthylhydroperoxide through Nrf2 signaling in neuroblastoma cells. FEBS Lett. 2013;587(21):3548–55.CrossRefPubMedGoogle Scholar
  30. 30.
    Nakamichi N, Taguchi T, Hosotani H, Wakayama T, Shimizu T, Sugiura T, et al. Functional expression of carnitine/organic cation transporter OCTN1 in mouse brain neurons: possible involvement in neuronal differentiation. Neurochem Int. 2012;61(7):1121–32.CrossRefPubMedGoogle Scholar
  31. 31.
    Godbey WT, Wu KK, Mikos AG. Size matters: molecular weight affects the efficiency of poly(ethylenimine) as a gene delivery vehicle. J Biomed Mater Res. 1999;45(3):268–75.CrossRefPubMedGoogle Scholar
  32. 32.
    Rondon IJ, MacMillan LA, Beckman BS, Goldberg MA, Schneider T, Bunn HF, et al. Hypoxia up-regulates the activity of a novel erythropoietin mRNA binding protein. J Biol Chem. 1991;266(25):16594–8.PubMedGoogle Scholar
  33. 33.
    Choi BH, Ha Y, Ahn CH, Huang X, Kim JM, Park SR, et al. A hypoxia-inducible gene expression system using erythropoietin 3’ untranslated region for the gene therapy of rat spinal cord injury. Neurosci Lett. 2007;412(2):118–22.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Bioengineering, College of EngineeringHanyang UniversitySeongdong-guSouth Korea

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