Sandrine Delbosc

Overproduction of reactive oxygen species in end-stage renal disease patients: A potential component of hemodialysis-associated inflammation

During the past decade, hemodialysis (HD)‐induced inflammation has been linked to the development of long‐term morbidity in end‐stage renal disease (ESRD) patients on regular renal replacement therapy. Because interleukins and anaphylatoxins produced during HD sessions are potent activators for nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, an example of an enzyme that is responsible for overproduction of reactive oxygen species (ROS), this may constitute a link between leukocyte activation and cell or organ toxicity. Oxidative stress, which results from an imbalance between oxidant production and antioxidant defense mechanisms, has been documented in ESRD patients using lipid and/or protein oxidative markers.

Simvastatin Prevents Angiotensin II–Induced Cardiac Alteration and Oxidative Stress

The influence of the HMG-CoA reductase inhibitor simvastatin was assessed on the cardiovascular alterations and production of free radicals associated with chronic angiotensin II (Ang II) infusion. Simvastatin (60 mg/kg per day PO) or placebo were given concomitantly for 10 days in Sprague-Dawley rats infused with Ang II (200 ng/kg per minute SC, osmotic pump). In addition, simvastatin or placebo was also given in vehicle-infused rats. Tail-cuff pressure and albuminuria were measured before and at the end of the treatment period. Cardiac weight, carotid structure, production of reactive oxygen species (ROS, by chemiluminescence) by polymorphonuclear leukocytes and aortic wall as well as protein and lipid oxidation products were determined at the end of the study. Ang II increased tail-cuff pressure by 56±12 mm Hg and simvastatin blunted the development of hypertension by ≈70% (19±5 mm Hg). Increases in heart weight index and carotid cross-sectional area induced by Ang II were obliterated by simvastatin (3.18±0.09 versus 3.46±0.11 mg/g body wt and 0.125±0.010 versus 0.177±0.010 mm2, respectively). The Ang II–induced increases in leukocyte and aortic production of ROS as well as protein and lipid oxidation products were prevented by simvastatin. No effect of simvastatin was detected in non–Ang II–infused rats. These results indicate that simvastatin prevented the development of hypertension and cardiovascular hypertrophy together with inhibition of the induced angiotensin II production of ROS. Therefore, inhibition of HMG CoA reductase by statins may have a beneficial effect on cardiovascular alterations through its antioxidant action in experimental Ang II–dependent hypertension.

Statins, 3-hydroxy-3-methylglutaryl Coenzyme A Reductase Inhibitors, Are Able to Reduce Superoxide Anion Production by Nadph Oxidase in Thp-1-derived Monocytes

Reactive oxygen species formation by phagocytes and subsequent modifications of vascular wall are involved in the early step of human atherogenesis. This study looked for the effect of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors on NADPH oxidase–dependent superoxide anion production in THP-1 cells, a monocyte-derived cell line, and on the translocation of p21 Rac 2 and p67phox. A 30-min incubation with simvastatin (50 &mgr;M) inhibited phorbol 12-myristate 13-acetate–induced superoxide anion production by monocytes (32%) and a maximum inhibition was obtained at 3 h of incubation (69.5%). In addition, after 3 h of incubation a dose-dependent inhibition was obtained in the range 10–50 &mgr;M of simvastatin with a median inhibitory concentration of 36 ± 2.3 &mgr;M. Mevalonic acid (100 and 300 &mgr;M) and geranylgeraniol (100 &mgr;M) totally prevented the simvastatin-induced inhibitory effect of superoxide production by monocytes whereas farnesyl PP (100 &mgr;M) partially prevented (50%) this effect. In addition, simvastatin inhibited the translocation of p21 rac 2 and p67phox, suggesting that geranylgeranylation is required for NADPH oxidase activation. In another set of experiments, the rank order of potency of different statins on NADPH oxidase was determined (pravastatin < cerivastatin < lovastatin < fluvastatin < simvastatin). In conclusion, inhibition of superoxide formation by HMG CoA reductase inhibitors is highly suitable to prevent or limit the oxidative stress involved in the atherosclerosis process.

Inhibitors of NADPH oxidase reduce the organ injury in hemorrhagic shock.

Reactive oxygen species contribute to the multiple organ dysfunction syndrome in hemorrhagic shock. Here, we investigate the effects of two chemically distinct inhibitors of NADPH oxidase on the circulatory failure and the organ dysfunction and injury associated with hemorrhagic shock in the anesthetized rat. Hemorrhage (sufficient to lower mean arterial blood pressure of 45 mmHg for 90 min) and subsequent resuscitation with shed blood resulted (within 4 h after resuscitation) in a delayed fall in blood pressure and in renal dysfunction and liver injury. Treatment of rats upon resuscitation with the NADPH oxidase inhibitors diphenylene iodonium (DPI, 1 mg/kg i.v.) reduced renal dysfunction and liver injury, whereas apocynin (3 mg/kg i.p.) did reduce the liver injury, but not the renal dysfunction caused by hemorrhagic shock. DPI and apocynin also attenuated the lung and intestinal injury (determined by histology) caused by hemorrhage and resuscitation. In the liver, DPI and apocynin abolished the increase in the formation of superoxide anions associated with hemorrhagic shock. However, neither DPI nor apocynin had a significant effect on the delayed circulatory failure caused by hemorrhage and resuscitation. In addition, DPI and apocynin did not reduce the increase in nitric oxide synthesis caused by hemorrhagic shock. Moreover, DPI reduced the activation of the transcription factor activator protein-1 caused by severe hemorrhage and resuscitation in the liver. Thus, we propose that an enhanced formation of superoxide anions by NADPH oxidase contributes to the liver injury caused by hemorrhagic shock, and that inhibitors of NADPH oxidase may represent a novel therapeutic approach for the therapy of hemorrhagic shock.

Superoxide anion overproduction in sepsis: Effects of vitamin E and simvastatin

Oxidative stress during sepsis induces tissue damage, leading to organ dysfunction and high mortality. The antioxidant effects of vitamin E have been reported in several diseases, but not in sepsis. Statins have cholesterol-independent anti-inflammatory effects that are related to a decrease of isoprenoid proteins and oxidative stress. Therefore, we evaluated superoxide anion (O2°−) production and ex vivo effects of vitamin E and simvastatin in sepsis. Fourteen healthy volunteers, 14 intensive care unit (ICU) nonseptic, and 14 ICU patients with sepsis were included in this prospective study. Plasma cholesterol, triglyceride, and vitamin E levels were determined by routine laboratory tests. Superoxide anion production was measured in the venous blood by chemiluminescence technique after phorbol myristate acetate stimulation. Effects of vitamin E and simvastatin on O2°− production was investigated ex vivo. Luminescence was indexed to the leukocyte count. We also investigated the in vitro effect of simvastatin on translocation of NADPH oxidase p21 Rac2 subunit in THP-1 monocytic cell line. The ratio of vitamin E/cholesterol + triglycerides was significantly decreased in septic as compared with nonseptic patients and volunteers. The O2°− production was significantly higher in the group of septic patients than in the others, regardless of the polymorphonuclear leukocyte count. Vitamin E and simvastatin induced ex vivo an inhibition of O2°− production of 20% and 40% respectively. In vitro, simvastatin inhibited phorbol myristate acetate-induced- O2°− production by monocytes through NADPH oxidase inactivation. We conclude that sepsis is associated with a significant decrease in vitamin E and an overproduction of O2°−. Vitamin E and simvastatin lessen this phenomenon through NADPH oxidase inactivation.

Simvastatin reverses target organ damage and oxidative stress in Angiotensin II hypertension: comparison with apocynin, tempol, and hydralazine.

The ability of the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor simvastatin to reverse established cardiovascular and renal alterations and oxidative stress was assessed in angiotensin II (AngII) hypertension. Sprague-Dawley rats infused with AngII (200 ng/kg per minute for 17 days) were concomitantly treated or not for the last 7 days with simvastatin, apocynin, tempol, and hydralazine (60, 60, 30, and 15 mg/kg per day, respectively). Only hydralazine lowered AngII hypertension. Simvastatin and apocynin lowered cardiac hypertrophy by 52% and 54% and reversed the marked rise in albuminuria by 25% and 70%. Neither tempol nor hydralazine affected cardiac mass or albuminuria. None of the treatments modified the AngII-induced increase in carotid media thickness. The rise in cardiac superoxide anion production (lucigenin-enhanced chemiluminescence method) induced by AngII was reversed by all treatments. Enhanced plasma concentration of advanced oxidation protein products (spectrophotometry using chloramine T) was unaffected by simvastatin and tempol, but it was reversed by apocynin and hydralazine. Our results indicate that simvastatin reverse established cardiac and renal alterations in AngII hypertension independently of arterial pressure. It is suggested that oxidative stress participates in the maintenance of target organ damage and that antioxidant properties are involved in the beneficial influence of the statin.

Thrombus Neutrophil Extracellular Traps Content Impair tPA-Induced Thrombolysis in Acute Ischemic Stroke

Background and Purpose— Neutrophil Extracellular Traps (NETs) are DNA extracellular networks decorated with histones and granular proteins produced by activated neutrophils. NETs have been identified as major triggers and structural factors of thrombosis. A recent study designated extracellular DNA threads from NETs as a potential therapeutic target for improving tissue-type plasminogen activator (tPA)-induced thrombolysis in acute coronary syndrome. The aim of this study was to assess the presence of NETs in thrombi retrieved during endovascular therapy in patients with acute ischemic stroke (AIS) and their impact on tPA-induced thrombolysis. Methods— We analyzed thrombi from 108 AIS patients treated with endovascular therapy. Thrombi were characterized by hematoxylin/eosin staining, immunostaining, and ex vivo enzymatic assay. Additionally, we assessed ex vivo the impact of deoxyribonuclease 1 (DNAse 1) on thrombolysis of AIS thrombi. Results— Histological analysis revealed that NETs contributed to the composition of all AIS thrombi especially in their outer layers. Quantitative measurement of thrombus NETs content was not associated with clinical outcome or AIS pathogenesis but correlated significantly with endovascular therapy procedure length and device number of passes. Ex vivo, recombinant DNAse 1 accelerated tPA-induced thrombolysis, whereas DNAse 1 alone was ineffective. Conclusions— This study suggests that thrombus NETs content may be responsible for reperfusion resistance, including mechanical or pharmacological approaches with intravenous tPA, irrespectively of their etiology. The efficacy of a strategy involving an administration of DNAse 1 in addition to tPA should be explored in the setting of AIS. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT02907736.