Details

Autor(en) / Beteiligte

• Sugiyama, Toru
• Michel, Thomas

Titel

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

Ist Teil von

• American journal of physiology. Heart and circulatory physiology, 2010-01, Vol.298 (1), p.H194-H201

Ort / Verlag

United States: American Physiological Society

Erscheinungsjahr

2010

Link zum Volltext

Quelle

EZB Free E-Journals

Beschreibungen/Notizen

• Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts Submitted August 17, 2009 ; accepted in final form October 30, 2009 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. oxidative stress; endothelial function; thioredoxin; glutathione; nitric oxide; tetrahydrobiopterin Address for reprint requests and other correspondence: T. Michel, Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, 75 Francis St., Thorn Bldg., Rm. 1210A, Boston, MA 02115 (e-mail: thomas_michel{at}harvard.edu ).