Abstract 1916: Manipulation of Intracellular Redox State using siRNA-Mediated Knockdown of Thiol-Metabolizing Pathways Leads to Differential Modulation of Endothelial Nitric Oxide Pathways
Cellular redox state is stringently maintained by thiol-based antioxidants to prevent the adverse consequences of generating excessive quantities of reactive oxygen species. The relative contributions of the thioredoxin (Trx) and glutathione/glutaredoxin systems to intracellular redox balance are incompletely understood, as are the consequences of altered thiol metabolism on eNOS and NO-dependent pathways in the endothelium. We designed duplex siRNA constructs to specifically “knock down” the expression of key thiol metabolizing enzymes in cultured aortic endothelial cells (EC). Transfection of siRNA constructs targeting glutathione reductase (GR), cytosolic Trx reductase (TrxR1), or mitochondrial Trx reductase (TrxR2) decreased levels of these proteins by 83 ± 4%, 70 ± 4%, or 88 ± 4% (n = 10 for each construct, p < 0.01), and decreased the intracellular reduced glutathione/oxidized glutathione ratio by 53 ± 10%, 56 ± 9%, or 54 ± 8% (each n = 4, p <0.01), respectively. siRNA-mediated knockdown of either GR, TrxR1 or TrxR2 markedly suppressed VEGF-induced NO production (measured by an electrochemical NO sensor) by 83 ± 2%, 92 ± 2%, or 96 ± 2% respectively (n = 4, p < 0.01), and also blocked eNOS enzyme activity (using the [3H]-arginine/[3H]-citrulline assay) by 97 ± 2%, 85 ± 2%, or 101 ± 1% (n = 4, p < 0.01). Pretreatment of EC with BCNU, an inhibitor of GR and TrxR, significantly decreased VEGF-induced NO production by 75 ± 5% (n = 4, p < 0.01). TrxR2 knockdown led to a marked increase in hydrogen peroxide (H2O2) production in EC (89 ± 5% increase; n = 4, p < 0.01); this TrxR2-mediated increase in H2O2 production was entirely unaffected by siRNA-mediated knockdown of eNOS. In contrast, knockdown of GR or TrxR1 slightly but significantly increased H2O2 production by 38 ± 4% or 32± 5% (n = 4, p < 0.01); these effects were abrogated by simultaneous eNOS knockdown. These studies show that the differential regulation of thiol-metabolizing proteins has pleiotropic effects on endothelial function, leading to critical changes in oxidative and nitrosative stress pathways. Greater understanding of the differential regulation of thiol-metabolizing proteins may lead to the development of new pharmacological targets for diseases associated with oxidative stress in the vascular wall.
This research has received full or partial funding support from the American Heart Association, AHA Founders Affiliate (Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, New York, Rhode Island, Vermont).