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

Abstract

It has been established from numerous studies that oxygen and nitrogen free radicals can contribute to the islet beta-cell destruction in Type I (insulin-dependent) diabetes mellitus models [1±2]. The deleterious actions of activated mononuclear cells could be mediated by cytokines through an increase in the intracellular generation of superoxide anions, nitric oxide (NO) and peroxynitrite [3±5]. Islet beta cells appear to have an exquisite sensitivity to these radicals because of constitutively low expression of antioxidant enzymes [6±7]. Several reports explained the rationale of increasing antioxidant enzyme expression Diabetologia (2000) AbstractAims/hypothesis. Vulnerability of pancreatic islets to oxygen free radicals and nitric oxide contributes to islet transplantation obstacles. This susceptibility can be linked to the low expression levels of antioxidant enzymes in islets. Our aim was to investigate the effect of overexpressing Cu/Zn superoxide dismutase in human islets through a simple procedure on the cytotoxic effects of two nitric oxide donors: 3-morpholinosydnonimine (SIN-1) and S-Nitroso-N-acetyl-d,lpenicillamine (SNAP). Methods. Cultured human islets and INS-1 rat-derived insulin-secreting cells were transfected by an E1-deleted adenovirus carrying Cu/Zn SOD cDNA under the control of a cytomegalovirus (CMV) promoter (AdSOD). The viability of the cells was tested by the WST-1 assay (Roche, Indianapolis, Ind., USA). Results. The AdSOD procedure allowed SOD activity to increase by twofold to threefold for 2 to 8 days following transfection. Adenovirus-driven SOD overexpression was associated with a significant reduction of SIN-1-induced cytotoxicity on human islets (69.9 ± 10.5 % protection at 200 mmol/l and 40.5 ± 8.9 % protection at 400 mmol/l) and INS-1 cells (82.2 ± 8.8 % protection at 200 mmol/l and 31.1 ± 5.8 % protection at 400 mmol/l). Protection against increasing doses of SNAP was AdSOD dose-dependent. Transfected islets released significantly more insulin than control islets in glucose-theophyllinstimulated conditions, without or following exposure to SNAP. Conclusions/interpretation. We thus established that adenoviral-induced overexpression of Cu/Zn SOD can be beneficial to human islet endocrine function and resistance to nitric oxide cytotoxicity. These data could be relevant for the development of new strategies aimed at preventing NO-induced beta-cell damage in an islet transplantation setting. [Diabetologia (2000) 43: 625±631] Keywords Islet transplantation, nitric oxide, oxidant stress, superoxide dismutase, reactive oxygen species, adenovirus, gene transfer. Corresponding author: Dr P. Y. Benhamou, Department of Endocrinology, CHU, BP 217X, 38 043 Grenoble, France Abbreviations: AdSOD, Adenovirus-driven superoxide dismutase; IEQ, equivalent number of islets; MOI, multiplicity of infection; NO, nitric oxide; pfu, plaque-forming units; ROS, reactive oxygen species; SIN-1, 3-Morpholino sydnonimine.HCl; SNAP, S-Nitroso-N-acetyl-d,l-penicillamine; SOD, superoxide dismutase.

The protective effect of cardiac gene transfer of CuZn–sod in comparison with the cardioprotector monohydroxyethylrutoside against doxorubicin-induced cardiotoxicity in cultured cells

“…Others, however, studied the protection of CuZn-sod against hypoxia-reoxygenation damage, which is also free radical mediated. 14,18,32,33 In the present experiment, significant levels of CuZnsod were produced in NeRCaMs at the third day of infection with AdCuZn-sod compared to noninfected cells. However, the occurrence of cellular cytotoxicity limited the MOI of AdCuZn-sod to 25 PFU/cell.…”

“…CuZn-sod activity increased two and eight folds in A549 cells infected with an MOI of 20 and 50 of AdCuZn-sod at the third day postinfection, respectively. 18 Others showed a 2-3-fold increase in the activity of CuZn-sod in islets 33 and aortic endothelial cells 34 3 days postinfection.…”

Heme Oxygenase-1 Induction in Islet Cells Results in Protection From Apoptosis and Improved In Vivo Function After Transplantation

“…RNA extraction was performed using the RNA NOW-LM kit (Biogentex, Seabrook, TX). Synthesis of first-strand cDNA was performed from DNase-treated RNA (DNase I; Gibco-BRL), using the SuperScript II RT kit (Gibco-BRL), with 25 ng/l oligo(dT) [12][13][14][15][16][17][18] as primer. The RT product was amplified by PCR in the LightCycler, which allows real-time quantification of the PCR product, based on the incorporation of a fluorescent dye in the neosynthesized DNA and its measurement at the end of each PCR cycle.…”

“…Inflammation can not only functionally impair or reduce the mass of implanted islets but can also result in the amplification of the subsequent immune response in a transplantation setting. In the transplanted islets, the induction of cytoprotective genes that are capable of scavenging oxygen radicals and preventing the toxic effects of proinflammatory cytokines might therefore promote early function (12)(13)(14)(15)(16).…”

Protective effects of Lagerstroemia speciosa on 3-morpholinosydnonimine (SIN-1)-induced oxidative stress in HIT-T15 pancreatic β cells

“…However, LWE treatment caused an increase in the activity of these antioxidant enzymes in HIT-T15 cells, indicating that LWE reduced SIN-1-induced oxidative stress. A number of studies have reported that the overexpression of CuZnSOD had a protective effect in NO-induced human islets, INS-1 insulin-secreting cells (28) and alloxan-and streptozotocin-induced diabetes (29,30). CAT has also exhibited a protective effect against H 2 O 2 and streptozotocin-induced oxidative stress in vivo (31).…”