Abstract-Reactive

Objective-Human endothelial cells use the multidrug resistance protein-1 (MRP1) to export glutathione disulfide (GSSG).This can promotes thiol loss during states of increased glutathione oxidation. We investigated how MRP1 modulates blood pressure and vascular function during angiotensin II-induced hypertension. Methods and Results-Angiotensin II-induced hypertension altered vascular glutathione flux by increasing GSSG export and decreasing vascular levels of glutathione in wild-type (FVB) but not in MRP1 Ϫ/Ϫ mice. Aortic endotheliumdependent vasodilatation was reduced in FVB after angiotensin II infusion, but unchanged in MRP1 Ϫ/Ϫ mice. Aortic superoxide (O 2 ⅐Ϫ ) production and expression of several NADPH oxidase subunits were increased by angiotensin II in FVB. These effects were markedly blunted in MRP1 Ϫ/Ϫ vessels. The increase in O 2 ⅐Ϫ production in FVB vessels caused by angiotensin II was largely inhibited by L-NAME, suggesting eNOS uncoupling. Accordingly, aortic tetrahydrobiopterin and levels of NO were decreased by angiotensin II in FVB but were unchanged in MRP1 Ϫ/Ϫ . Finally, the hypertension caused by angiotensin II was markedly blunted in MRP1 Ϫ/Ϫ mice (137Ϯ4 versus 158Ϯ6 mm Hg). Key Words: endothelial function Ⅲ glutathione Ⅲ hypertension Ⅲ MRP1 Ⅲ oxidative stress H ypertension increases vascular production of reactive oxygen species (ROS). 1 There is accumulating evidence that this contributes to the pathophysiologic consequences of hypertension. 2 A major intracellular defense against ROS is the tripeptide glutathione (GSH). 3 GSH serves as a cosubstrate for the glutathione peroxidases, which scavenge hydrogen peroxide and lipid peroxides. GSH also directly reacts with strong oxidants such as peroxynitrite. Depletion of the glutathione pool or inhibition of glutathione peroxidase alters vascular tone and leads to hypertension. 4,5 The multidrug resistance proteins (MRPs) are members of the ATP-binding cassette (ABC) superfamily which use ATP for active transport of a variety of endogenously produced and exogenously administered molecules. 6 MRP1 is a transporter of chemotherapeutic agents complexed to glutathione, leukotriene C4, glutathione disulfide (GSSG), and estrogen. Other MRPs have a myriad of cell functions including export of xenobiotics, cyclic nucleotides, lipids, and billirubin. Recently, we found that MRP1 is the major transporter of GSSG in human endothelial cells. 7 In this prior study, oxidative stress caused by oscillatory shear markedly enhanced the release of GSSG from cultured human aortic endothelial cells in an MRP1 dependent fashion. This led to a depletion of intracellular GSH and ultimately apoptosis. Blockade of MRP1 prevented these untoward effects of oxidative stress. We also showed that endothelial GSSG export was increased in an in vivo model of oxidative stress caused by desoxycorticosterone acetate-salt (DOCA-salt) hypertension in normal but not MRP1 Ϫ/Ϫ mice. We selected DOCA-salt hypertension as a model of in vivo oxidative stress in this prior study because th...

Abstract-Glutathione (GSH) is the major source of intracellular sulfhydryl groups. Oxidized GSH (GSSG) can be recycled to GSH by the GSH reductase or exported from the cell. The mechanism by which GSSG is exported and the consequence of its export from endothelial cells has not been defined previously. We found that human endothelial cells express the multidrug resistance protein-1 (MRP1) and use this as their major exporter of GSSG. Oscillatory shear stress, which is known to stimulate endothelial cell production of reactive oxygen species, decreased intracellular GSH. In contrast, laminar shear significantly increased intracellular GSH. Oscillatory shear also caused a robust export of GSSG that was prevented by the MRP1 inhibitor MK571 and by MRP1 small interfering RNA. MRP1 inhibition prevented the decline in intracellular GSH, preserved the intracellular GSH Nernst potential, and reduced apoptosis caused by oscillatory shear. In aortas of hypertensive mice, endothelial disulfide export was doubled, and this was prevented by MK571 and was not observed in aortas of hypertensive MRP1 Ϫ/Ϫ mice. Further, the altered endothelium-dependent vasodilatation caused by hypertension was ameliorated in MRP1 Ϫ/Ϫ mice. GSSG export by MRP1 leads to a perturbation of endothelial redox state and ultimately endothelial cell apoptosis. Endothelial MRP1 may provide a novel therapeutic target for prevention of vascular disease. (Circ Res. 2005;97:637-644.) Key Words: endothelial cells Ⅲ glutathione Ⅲ MRP1 Ⅲ oscillatory shear stress Ⅲ oxidative stress A major cellular defense against oxidative stress is reduced glutathione (GSH), a tripeptide consisting of cysteine, glutamate, and glycine. 1,2 The antioxidant properties of GSH relate to its ability to directly reduce strong oxidants such as peroxynitrite and to its role as a cosubstrate for the enzyme GSH peroxidase. 3 Of the total GSH pool, oxidized GSH (GSSG) normally represents Ͻ2%. Perturbations of the GSH/GSSG ratio can affect the ratio of other redox couples, such as NADH/NAD ϩ and NADPH/NADP ϩ , and can induce cellular apoptosis. 4,5 This ratio can be quantified as a Nernst potential for the GSH/GSSG couple, and this is generally regulated tightly to maintain cellular homeostasis. 6 To maintain the GSH/GSSG Nernst potential in the setting of oxidative stress, cells may either synthesize GSH de novo or modulate GSSG levels. Cells use two competing mechanisms to maintain GSSG at low levels. The first is reduction by the enzyme GSH reductase, and the second involves GSSG export from the cell. For example, organs perfused with peroxide release GSSG. 7 To date, it is unclear whether these competing mechanisms have similar effects on cellular function.Within the endothelium, oxidative stress has been implicated in the pathogenesis of atherosclerosis, hypertension, altered vasomotion, and apoptosis. 8 Endothelial production of reactive oxygen species (ROS) is increased by a variety of pathophysiological stimuli, including cytokines, hypertension, and altered mechanical forces. In p...

IntroductionAtherosclerosis is a chronic inflammatory disease characterized by endothelial cell damage, infiltration, proliferation and accumulation of macrophages, lymphocytes and transformed vascular smooth muscle cells within the vascular wall and procoagulation processes involving activation of plasmatic coagulation events and platelets. Numerous studies suggested a close interaction between thrombin action and atherogenesis, but possibly underlying mechanisms are multiple and specific treatment options were missing until now.Material and methodsAtherosclerosis prone 12 weeks old ApoE–/– mice were fed a cholesterol rich diet for 4 weeks and were concomitantly treated orally with placebo or the thrombin inhibitor dabigatran (1.2 g/kg/day).ResultsThe thrombin time (HEMOCLOT®) was significant extended in dabigatran treated animals. Vascular oxidative stress was significantly reduced during thrombin inhibition, as assessed by L012 chemiluminescence in aortic segments (212 ±84 vs. 69 ±21 RLU/s/mg dry weight, p = 0.048). Organ chamber experiments of isolated aortic rings showed that dabigatran treatment significantly improved endothelium-derived vasorelaxation (p < 0.001). Dabigatran treated mice developed less atherosclerotic lesions (6.2 ±0.2% vs. 9 ±1.1%, p = 0.037) and showed less infiltration of atherosclerotic lesions with macrophages (2.59 ±0.3% vs. 5.14 ±0.7%, p = 0.0046), as determined by systematic histological and immunohistological analyses of the aortic root. Blood pressure, body weight and food intake were not altered by the treatment.ConclusionsThe thrombin inhibitor dabigatran reduces vascular oxidative stress and inflammation, improves endothelial function and decreases atherosclerosis in mice.

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