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Effective treatment of vascular endothelial growth factor refractory hindlimb ischemia by a mutant endothelial nitric oxide synthase gene

Gene Therapy volume 13, pages 1342–1350 (2006)Cite this article

Abstract

Gene delivery of angiogenic growth factors is a promising approach for the treatment of ischemic cardiovascular diseases. However, success of this new therapeutic principle is hindered by the lack of critical understanding as to how disease pathology affects the efficiency of gene delivery and/or the downstream signaling pathways of angiogenesis. Critical limb ischemia occurs in patients with advanced atherosclerosis often exhibiting deficiency in endothelial nitric oxide production. Similar to these patients, segmental femoral artery resection progresses into severe ischemic necrosis in mice deficient in endothelial nitric oxide synthase (ecNOS-KO) as well as in balb/c mice. We used these models to evaluate the influence of severe ischemia on transfection efficiency and duration of transgene expression in the skeletal muscle following plasmid injection in combination with electroporation. Subsequently, we also explored the potential therapeutic effect of the phosphomimetic mutant of ecNOS gene (NOS1177D) using optimized delivery parameters, and found significant benefit both in ecNOS-KO and balb/c mice. Our results indicate that NOS1177D gene delivery to the ischemic skeletal muscle can be efficient to reverse critical limb ischemia in pathological settings, which are refractory to treatments with a single growth factor, such as vascular endothelial growth factor.

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References

  1. Rissanen TT, Vajanto I, Yla-Herttuala S . Gene therapy for therapeutic angiogenesis in critically ischaemic lower limb – on the way to the clinic. Eur J Clin Invest 2001; 31: 651–666.

    Article  CAS  PubMed  Google Scholar 

  2. Khan TA, Sellke FW, Laham RJ . Gene therapy progress and prospects: therapeutic angiogenesis for limb and myocardial ischemia. Gene Therapy 2003; 10: 285–291.

    Article  CAS  PubMed  Google Scholar 

  3. Li S, Huang L . Nonviral gene therapy: promises and challenges. Gene Therapy 2000; 7: 31–34.

    Article  CAS  PubMed  Google Scholar 

  4. Mir LM, Bureau MF, Gehl J, Rangara R, Rouy D, Caillaud JM et al. High efficiency gene transfer into skeletal muscle mediated by electric pulses. Proc Natl Acad Sci USA 1999; 96: 4262–4267.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Kodama K, Kusuoka H, Sakai A, Adachi T, Hasegawa S, Ueda Y et al. Collateral channels that develop after an acute myocardial infarction prevent subsequent left ventricular dilation. J Am Coll Cardiol 1996; 27: 1133–1139.

    Article  CAS  PubMed  Google Scholar 

  6. Anversa P, Li P, Sonnenblick EH, Olivetti G . Effects of aging on quantitative structural properties of coronary vasculature and microvasculature in rats. Am J Physiol 1994; 267: H1062–H1073.

    CAS  PubMed  Google Scholar 

  7. Gerhard M, Roddy MA, Creager SJ, Creager MA . Aging progressively impairs endothelium-dependent vasodilation in forearm resistance vessels of humans. Hypertension 1996; 27: 849–853.

    Article  CAS  PubMed  Google Scholar 

  8. Rivard A, Fabre JE, Silver M, Chen D, Murohara T, Kearney M et al. Age-dependent impairment of angiogenesis. Circulation 1999; 99: 111–120.

    Article  CAS  PubMed  Google Scholar 

  9. Couffinhal T, Silver M, Kearney M, Sullivan A, Witzenbichler B, Magner M et al. Impaired collateral vessel development associated with reduced expression of vascular endothelial growth factor in ApoE−/− mice. Circulation 1999; 99: 3188–3198.

    Article  CAS  PubMed  Google Scholar 

  10. Panchal VR, Rehman J, Nguyen AT, Brown JW, Turrentine MW, Mahomed Y et al. Reduced pericardial levels of endostatin correlate with collateral development in patients with ischemic heart disease. J Am Coll Cardiol 2004; 43: 1383–1387.

    Article  CAS  PubMed  Google Scholar 

  11. Pola R, Ling LE, Aprahamian TR, Barban E, Bosch-Marce M, Curry C et al. Postnatal recapitulation of embryonic hedgehog pathway in response to skeletal muscle ischemia. Circulation 2003; 108: 479–485.

    Article  PubMed  Google Scholar 

  12. Masaki I, Yonemitsu Y, Yamashita A, Sata S, Tanii M, Komori K et al. Angiogenic gene therapy for experimental critical limb ischemia: acceleration of limb loss by overexpression of vascular endothelial growth factor 165 but not of fibroblast growth factor-2. Circ Res 2002; 90: 966–973.

    Article  CAS  PubMed  Google Scholar 

  13. Fukino K, Sata M, Seko Y, Hirata Y, Nagai R . Genetic background influences therapeutic effectiveness of VEGF. Biochem Biophys Res Commun 2003; 310: 143–147.

    Article  CAS  PubMed  Google Scholar 

  14. Murohara T, Asahara T, Silver M, Bauters C, Masuda H, Kalka C et al. Nitric oxide synthase modulates angiogenesis in response to tissue ischemia. J Clin Invest 1998; 101: 2567–2578.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Zeiher A M, Drexler H, Wollschläger H, Just H . Endothelial dysfunction of coronary microvasculature is associated with impaired coronary blood flow regulation in patients with early atherosclerosis. Circulation 1991; 84: 1984–1992.

    Article  PubMed  Google Scholar 

  16. Kauser K, Rubanyi GM . ‘Nitric oxide deficiency’ in cardiovascular diseases. Cardiovascular protection by restoration of endothelial nitric oxide production. In: Rubanyi GM (ed). Mechanisms of Vasculoprotection. Springer-Verlag: NY, 2002, pp 1–31.

    Google Scholar 

  17. Ziche M, Morbidelli L . Nitric oxide and angiogenesis. J Neurooncol 2000; 50: 139–148.

    Article  CAS  PubMed  Google Scholar 

  18. Katusic ZS . Therapeutic angiogenesis. Arterioscler Thromb Vasc Biol 2002; 22: 1254–1255.

    Article  CAS  PubMed  Google Scholar 

  19. Qian HS, Liu P, Kauser K, Rubanyi GM . Nitric oxide deficiency leads to impaired angiogenesis and severe dysfunction of microcirculation in a mouse hind limb ischemia model. In: Proceedings of the 7th World Congress of Microcirculation. Monduzzi Editore: Sydney, Australia, 2001, pp 525–529.

    Google Scholar 

  20. Smith RS, Lin K-F, Agata J, Chao L, Chao J . Human endothelial nitric oxide synthase gene delivery promotes angiogenesis in a rat model of hindlimb ischemia. Arterioscler Thromb Vasc Biol 2002; 22: 1279–1285.

    Article  CAS  PubMed  Google Scholar 

  21. Gregg AR, Schauer A, Shi O, Liu Z, Lee CG, O'Brien WE . Limb reduction defects in endothelial nitric oxide synthase-deficient mice. Am J Physiol 1998; 275: H2319–H2324.

    CAS  PubMed  Google Scholar 

  22. Fulton D, Gratton JP, McCabe TJ, Fontana J, Fujio Y, Walsh K et al. Regulation of endothelium derived nitric oxide production by the protein kinase Akt. Nature 1999; 399: 597–601.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Dimmeler S, Dernbach E, Zeiher AM . Phosphorylation of the endothelial nitric oxide synthase at ser-1177 is required for VEGF-induced endothelial cell migration. FEBS Lett 2000; 477: 258–262.

    Article  CAS  PubMed  Google Scholar 

  24. Rivard A, Silver M, Chen D, Kearney M, Magner M, Annex B et al. Rescue of diabetes-related impairment of angiogenesis by intramuscular gene therapy with adeno-VEGF. Am J Pathol 1999; 154: 355–363.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Couffinhal T, Silver M, Zheng LP, Kearney M, Witzenbichler B, Isner JM . Mouse model of angiogenesis. Am J Pathol 1998; 152: 1667–1679.

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Takeshita S, Isshiki T, Sato T . Increased expression of direct gene transfer into skeletal muscles observed after acute ischemic injury in rats. Lab Invest 1996; 74: 1061–1065.

    CAS  PubMed  Google Scholar 

  27. Hoffmann J, Haendeler J, Aicher A, Rossig L, Vasa M, Zeiher AM et al. Aging enhances the sensitivity of endothelial cells toward apoptotic stimuli: Important role of nitric oxide. Circ Res 2001; 89: 709–715.

    Article  CAS  PubMed  Google Scholar 

  28. Endo A, Fukuhara S, Masuda M, Ohmori T, Mochizuki N . Selective inhibition of vascular endothelial growth factor receptor-2 (VEGFR-2) identifies a central role for VEGFR-2 in human aortic endothelial cell responses to VEGF. J Recept Signal Transduct Res 2003; 23: 239–254.

    Article  CAS  PubMed  Google Scholar 

  29. Sato Y, Kanno S, Oda N, Abe M, Ito M, Shitara K et al. Properties of two VEGF receptors, Flt-1 and KDR, in signal transduction. Ann NY Acad Sci 2000; 902: 201–205.

    Article  CAS  PubMed  Google Scholar 

  30. Obeso J, Weber J, Auerbach R . A hemangioendothelioma-derived cell line: its use as a model for the study of endothelial cell biology. Lab Invest 1990; 63: 259–269.

    CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Department of Pharmacology, Berlex Biosciences, Richmond, CA, USA

    H S Qian, P Liu, L-Y Huw, M Halks-Miller, S M Hill & R Vergona

  2. Department of Gene Therapy, Berlex Biosciences, Richmond, CA, USA

    A Orme, F Jin, P Kretschmer, L Cashion, P Szymanski, R Harkins, G M Rubanyi & K Kauser

  3. Department of Immunology, Berlex Biosciences, Richmond, CA, USA

    E Blasko

  4. Yale University, New Haven, CT, USA

    J Yu & W C Sessa

  5. Cardiovascular Research, Berlex Biosciences, Richmond, CA, USA

    W P Dole

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Correspondence to K Kauser.

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Qian, H., Liu, P., Huw, LY. et al. Effective treatment of vascular endothelial growth factor refractory hindlimb ischemia by a mutant endothelial nitric oxide synthase gene. Gene Ther 13, 1342–1350 (2006). https://doi.org/10.1038/sj.gt.3302781

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  • DOI: https://doi.org/10.1038/sj.gt.3302781

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