Molecular Biotechnology

, Volume 37, Issue 1, pp 38–47 | Cite as

The Rational Design of β Cell Cytoprotective Gene Transfer Strategies: Targeting Deleterious iNOS Expression

Original Paper

Abstract

Islet transplantation represents a promising therapeutic strategy for the treatment of type 1 diabetes mellitus (T1DM) [Hakim and Papalois (Ann Ital Chir 75:1–7, 2004); Jaeckel et al. (Internist (Berl) 45:1268–1280, 2004); Sutherland et al. (Transplant Proc 36:1697–1699, 2004)]. The insulin-secreting pancreatic β cells of the islet allograft are, however, subject to recurrent immune-mediated damage. Principal among the molecular culprits involved in this destructive process is the proinflammatory cytokine IL-1β. IL-1β-induced β cell destruction may be mediated by the generation of NO and/or ROS, although the relative importance of NO and ROS in this process remains unclear. This study broadly encompassed three arms of investigation: the first of these was geared toward the establishment of a robust in vitro cell system for the study of IL-1β-induced pathophysiology; the second arm aimed to provide a comparative analysis of the gene transfer profiles of the three most commonly used gene transfer vehicles, namely plasmid vectors, adenoviral vectors, and lentiviral vectors, in the aforementioned cell system; the final arm aimed to screen an array of potentially cytoprotective gene transfer strategies incorporating the optimal gene transfer vectors. Briefly, we established an in vitro β cell system that accurately reflected primary β cell cytokine-induced pathophysiology. That is, IL-1β exposure (100 U/ml) induced a time-dependent decrease in rat insulinoma (RIN) cell viability, which coincided with an induction in iNOS expression and nitrite accumulation. Gene transfer studies using plasmid, adenoviral, or lentiviral vectors underscored the superiority of viral vector-based gene transfer strategies for the manipulation of this β cell line. Using these vectors, we provide evidence that NF-κB-based iNOS inhibition confers significant protection against IL-1β-induced damage whereas antioxidant overexpression fails to provide protection. Conferred cytoprotection was associated with a suppression of iNOS expression and nitrite accumulation. From a therapeutic standpoint, gene transfer strategies employing efficient viral vectors to target iNOS activation may harbour therapeutic potential in preserving β cell survival against proinflammatory cytokine exposure.

Keywords

β Cells Cytokines iNOS ROS Antioxidants NFκB Gene transfer Cytoprotection Adenovirus shRNA Lentivirus Rat insulinoma cells Cytokines IL-1β 

References

  1. 1.
    Hakim, N., & Papalois, V. (2004). Pancreas and islet transplantation. Annali Italiani di Chirurgia, 75, 1–7.PubMedGoogle Scholar
  2. 2.
    Jaeckel, E., Becker, T., & Manns, M. P. (2004). Organ transplantation in endocrinology. Islet cells and pancreas. Internist (Berl), 45, 1268–1280.CrossRefGoogle Scholar
  3. 3.
    Sutherland, D. E., Gruessner, R., Kandswamy, R., Humar, A., Hering, B., & Gruessner, A. (2004). Beta-cell replacement therapy (pancreas and islet transplantation) for treatment of diabetes mellitus: an integrated approach. Transplantation Proceedings, 36, 1697–1699.PubMedCrossRefGoogle Scholar
  4. 4.
    Rabinovitch, A., Suarez-Pinzon, W. L., Strynadka, K., Lakey, J. R., & Rajotte, R. V. (1996). Human pancreatic islet beta-cell destruction by cytokines involves oxygen free radicals and aldehyde production. Journal of Clinical Endocrinology and Metabolism, 81, 3197–3202.PubMedCrossRefGoogle Scholar
  5. 5.
    Corbett, J. A., Sweetland, M. A., Wang, J. L., Lancaster, J. R. Jr., & McDaniel, M. L. (1993). Nitric oxide mediates cytokine-induced inhibition of insulin secretion by human islets of Langerhans. Proceedings of National Academy Sciences USA, 90, 1731–1735.CrossRefGoogle Scholar
  6. 6.
    Delaney, C. A., & Eizirik, D. L. (1996). Intracellular targets for nitric oxide toxicity to pancreatic beta-cells. Brazilian Journal of Medical and Biological Research, 29, 569–579.PubMedGoogle Scholar
  7. 7.
    Southern, C., Schulster, D., & Green, I. C. (1990). Inhibition of insulin secretion by interleukin-1 beta and tumour necrosis factor-alpha via an l-arginine-dependent nitric oxide generating mechanism. FEBS Letter, 276, 42–44.CrossRefGoogle Scholar
  8. 8.
    McCabe, C., & O’Brien, T. (2007). Beta cell cytoprotection using lentiviral vector-based iNOS-specific shRNA delivery. Biochemical and Biophysical Research Communication, 357, 75–80.CrossRefGoogle Scholar
  9. 9.
    McCabe, C., Samali, A., & O’Brien, T. (2006). Beta cell cytoprotective strategies: Establishing the relative roles for iNOS and ROS. Biochemical and Biophysical Research Communication, 342, 1240–1248.CrossRefGoogle Scholar
  10. 10.
    Lenzen, S., Drinkgern, J., & Tiedge, M. (1996). Low antioxidant enzyme gene expression in pancreatic islets compared with various other mouse tissues. Free Radical Biology and Medicine, 20, 463–466.PubMedCrossRefGoogle Scholar
  11. 11.
    McCabe, C., Samali, A., & O’Brien, T. (2006). Cytoprotection of beta cells: Rational gene transfer strategies. Diabetes–Metabolism Research and Reviews, 22, 241–252.PubMedCrossRefGoogle Scholar
  12. 12.
    Lortz, S., Tiedge, M., Nachtwey, T., Karlsen, A. E., Nerup, J., & Lenzen, S. (2000). Protection of insulin-producing RINm5F cells against cytokine-mediated toxicity through overexpression of antioxidant enzymes. Diabetes, 49, 1123–1130.PubMedCrossRefGoogle Scholar
  13. 13.
    Chen, G., Hohmeier, H. E., Gasa, R., Tran, V. V., & Newgard, C. B. (2000). Selection of insulinoma cell lines with resistance to interleukin-1beta- and gamma-interferon-induced cytotoxicity. Diabetes, 49, 562–570.PubMedCrossRefGoogle Scholar
  14. 14.
    Moriscot, C., Pattou, F., Kerr-Conte, J., Richard, M. J., Lemarchand, P., & Benhamou, P. Y. (2000). Contribution of adenoviral-mediated superoxide dismutase gene transfer to the reduction in nitric oxide-induced cytotoxicity on human islets and INS-1 insulin-secreting cells. Diabetologia, 43, 625–631.PubMedCrossRefGoogle Scholar
  15. 15.
    Azevedo-Martins, A. K., Lortz, S., Lenzen, S., Curi, R., Eizirik, D. L., & Tiedge, M. (2003). Improvement of the mitochondrial antioxidant defense status prevents cytokine-induced nuclear factor-kappaB activation in insulin-producing cells. Diabetes, 52, 93–101.PubMedCrossRefGoogle Scholar
  16. 16.
    Tiedge, M., Lortz, S., Munday, R., & Lenzen, S. (1999). Protection against the co-operative toxicity of nitric oxide and oxygen free radicals by overexpression of antioxidant enzymes in bioengineered insulin-producing RINm5F cells. Diabetologia, 42, 849–855.PubMedCrossRefGoogle Scholar
  17. 17.
    Tiedge, M., Lortz, S., Munday, R., & Lenzen, S. (1998). Complementary action of antioxidant enzymes in the protection of bioengineered insulin-producing RINm5F cells against the toxicity of reactive oxygen species. Diabetes, 47, 1578–1585.PubMedCrossRefGoogle Scholar
  18. 18.
    Bertera, S., Crawford, M. L., Alexander, A. M., Papworth, G. D., Watkins, S. C., Robbins, P. D., & Trucco, M. (2003). Gene transfer of manganese superoxide dismutase extends islet graft function in a mouse model of autoimmune diabetes. Diabetes, 52, 387–393.PubMedCrossRefGoogle Scholar
  19. 19.
    Karsten, V., Sigrist, S., Moriscot, C., Benhamou, P. Y., Lemarchand, P., Belcourt, A., Poindron, P., Pinget, M., & Kessler, L. (2002). Reduction of macrophage activation after antioxidant enzymes gene transfer to rat insulinoma INS-1 cells. Immunobiology, 205, 193–203.PubMedCrossRefGoogle Scholar
  20. 20.
    Moriscot, C., Richard, M. J., Favrot, M. C., & Benhamou, P. Y. (2003). Protection of insulin-secreting INS-1 cells against oxidative stress through adenoviral-mediated glutathione peroxidase overexpression. Diabetes Metabolism, 29, 145–151.PubMedCrossRefGoogle Scholar
  21. 21.
    Li, X., Chen, H., & Epstein, P. N. (2004). Metallothionein protects islets from hypoxia and extends islet graft survival by scavenging most kinds of reactive oxygen species. Journal of Biological Chemistry, 279, 765–771.PubMedCrossRefGoogle Scholar
  22. 22.
    Schroppel, B., Zhang, N., Chen, P., Chen, D., Bromberg, J. S., & Murphy, B. (2005). Role of donor-derived monocyte chemoattractant protein-1 in murine islet transplantation. Journal of American Society Nephrology, 16, 444–451.CrossRefGoogle Scholar
  23. 23.
    Giannoukakis, N., Rudert, W. A., Trucco, M., & Robbins, P. D. (2000). Protection of human islets from the effects of interleukin-1beta by adenoviral gene transfer of an Ikappa B repressor. Journal of Biological Chemistry, 275, 36509–36513.PubMedCrossRefGoogle Scholar
  24. 24.
    Heimberg, H., Heremans, Y., Jobin, C., Leemans, R., Cardozo, A. K., Darville, M., & Eizirik, D. L. (2001). Inhibition of cytokine-induced NF-kappaB activation by adenovirus-mediated expression of a NF-kappaB super-repressor prevents beta-cell apoptosis. Diabetes, 50, 2219–2224.PubMedCrossRefGoogle Scholar
  25. 25.
    Kwon, G., Corbett, J. A., Rodi, C. P., Sullivan, P., & McDaniel, M. L. (1995). Interleukin-1 beta-induced nitric oxide synthase expression by rat pancreatic beta-cells: evidence for the involvement of nuclear factor kappa B in the signaling mechanism. Endocrinology, 136, 4790–4795.PubMedCrossRefGoogle Scholar
  26. 26.
    Zanetti, M., Sato, J., Katusic, Z. S., & O’Brien, T. (2000). Gene transfer of endothelial nitric oxide synthase alters endothelium-dependent relaxations in aortas from diabetic rabbits. Diabetologia, 43, 340–347.PubMedCrossRefGoogle Scholar
  27. 27.
    Oberholzer, J., Shapiro, A. M., Lakey, J. R., Ryan, E. A., Rajotte, R. V., Korbutt, G. S., Morel, P., & Kneteman, N. M. (2003). Current status of islet cell transplantation. Advances in Surgery, 37, 253–282.PubMedGoogle Scholar
  28. 28.
    Ryan, E. A., Paty, B. W., Senior, P. A., & Shapiro, A. M. (2004). Risks and side effects of islet transplantation. Current Diabetes Report, 4, 304–309.CrossRefGoogle Scholar
  29. 29.
    Abdelli, S., Ansite, J., Roduit, R., Borsello, T., Matsumoto, I., Sawada, T., Allaman-Pillet, N., Henry, H., Beckmann, J. S., Hering, B. J., & Bonny, C. (2004). Intracellular stress signaling pathways activated during human islet preparation and following acute cytokine exposure. Diabetes, 53, 2815–2823.PubMedCrossRefGoogle Scholar
  30. 30.
    Chen, H., Li, X., & Epstein, P. N. (2005). MnSOD and catalase transgenes demonstrate that protection of islets from oxidative stress does not alter cytokine toxicity. Diabetes, 54, 1437–1446.PubMedCrossRefGoogle Scholar
  31. 31.
    Delaney, C. A., Tyrberg, B., Bouwens, L., Vaghef, H., Hellman, B., & Eizirik, D. L. (1996). Sensitivity of human pancreatic islets to peroxynitrite-induced cell dysfunction and death. FEBS Letter, 394, 300–306.CrossRefGoogle Scholar
  32. 32.
    Beeharry, N., Chambers, J. A., Faragher, R. G., Garnett, K. E., & Green, I. C. (2004). Analysis of cytokine-induced NO-dependent apoptosis using RNA interference or inhibition by 1400W. Nitric Oxide, 10, 112–118.PubMedCrossRefGoogle Scholar
  33. 33.
    Mahato, R. I., Henry, J., Narang, A. S., Sabek, O., Fraga, D., Kotb, M., & Gaber, A. O. (2003). Cationic lipid and polymer-based gene delivery to human pancreatic islets. Molecular Therapy, 7, 89–100.PubMedCrossRefGoogle Scholar
  34. 34.
    Benhamou, P. Y., Moriscot, C., Prevost, P., Rolland, E., Halimi, S., & Chroboczek, J. (1997). Standardization of procedure for efficient ex vivo gene transfer into porcine pancreatic islets with cationic liposomes. Transplantation, 63, 1798–1803.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2007

Authors and Affiliations

  1. 1.Regenerative Medicine Institute, National Centre for Biomedical Engineering SciencesNational University of IrelandGalwayIreland
  2. 2.Department of MedicineNational University of Ireland, GalwayGalwayIreland

Personalised recommendations