The Rational Design of β Cell Cytoprotective Gene Transfer Strategies: Targeting Deleterious iNOS Expression
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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.
Keywordsβ Cells Cytokines iNOS ROS Antioxidants NFκB Gene transfer Cytoprotection Adenovirus shRNA Lentivirus Rat insulinoma cells Cytokines IL-1β
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