Thiol-metabolizing proteins and endothelial redox state: differential modulation of eNOS and biopterin pathways

The intracellular redox state is stringently maintained by thiol-based antioxidants to establish a balance for the physiological and pathophysiological roles 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 endothelial nitric oxide (NO) synthase (eNOS) and NO-dependent pathways in the endothelium. We designed duplex small interfering RNA (siRNA) constructs to specifically "knock down" the expression of three key thiol-metabolizing enzymes in cultured aortic endothelial cells. Transfection of siRNA constructs targeting glutathione reductase (GR), cytosolic Trx reductase (TrxR1), or mitochondrial Trx reductase (TrxR2) significantly decreased the intracellular reduced glutathione-to-oxidized glutathione ratio. siRNA-mediated knockdown of either GR, TrxR1, or TrxR2 markedly suppressed VEGF-induced NO production (measured by an electrochemical NO sensor) and also blocked eNOS enzyme activity (using the [(3)H]arginine/[(3)H]citrulline assay). Pretreatment of endothelial cells with N,N'-bis(2-chloroethyl)-N-nitrosourea, an inhibitor of GR and TrxR, significantly decreased VEGF-induced NO production. siRNA-mediated TrxR2 knockdown led to a marked increase in hydrogen peroxide (H(2)O(2)) production in endothelial cells. In contrast, knockdown of GR or TrxR1 only slightly increased H(2)O(2) production. Supplementation of endothelial cells with tetrahydrobiopterin prevented the increase in H(2)O(2) generation seen with siRNA-mediated knockdown of GR. These studies show that the differential regulation of thiol-metabolizing proteins leads 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.