iNOS gene silencing prevents inflammatory cytokine-induced beta-cell apoptosis

Human islet transplantation has great potential as an effective means of treating insulin-dependent diabetes mellitus. Upregulation of inducible nitric oxide synthase (iNOS) and subsequent product of radical nitric oxide (NO) impair islet beta-cell function. Therefore, we hypothesize that iNOS gene silencing will prevent beta-cell death and improve the survival and function of islets. Small interfering RNA duplex (siRNA) inhibited rat iNOS gene expression and NO production in rat beta-cell lines (INS-1E) in a dose- and sequence-dependent manner. iNOS gene silencing also protected these beta-cells from inflammatory cytokine-induced apoptosis and increased their capacity to secret insulin. Three siRNA sequences against human iNOS were then designed and transfected into human islets. Although there was dose- and sequence-dependent iNOS gene silencing and NO production in human islets, the effect of iNOS gene silencing on apoptosis of islets was only moderate, as evidenced by 25-30% reduction in caspase 3 activity and in the percentage of apoptotic cells. Since an islet is a cluster of 200-1,000 cells, the transfection efficiency of lipid/siRNA complexes into human islets was only 21-28%, compared to effective transfection efficiency (>90%) in beta-cell lines. Nevertheless, these results suggest that siRNA may penetrate beyond the periphery into a larger percentage of an islet mass than previously thought.