Significance: Enhanced levels of reactive oxygen species (ROS) have been associated with different disease states. Most attempts to validate and exploit these associations by chronic antioxidant therapies have provided disappointing results. Hence, the clinical relevance of ROS is still largely unclear. Recent Advances: We are now beginning to understand the reasons for these failures, which reside in the many important physiological roles of ROS in cell signaling. To exploit ROS therapeutically, it would be essential to define and treat the disease-relevant ROS at the right moment and leave physiological ROS formation intact. This breakthrough seems now within reach. Critical Issues: Rather than antioxidants, a new generation of protein targets for classical pharmacological agents includes ROS-forming or toxifying enzymes or proteins that are oxidatively damaged and can be functionally repaired. Future Directions: Linking these target proteins in future to specific disease states and providing in each case proof of principle will be essential for translating the oxidative stress concept into the clinic. Antioxid. Redox Signal. 23, 1171–1185.

Significance: Oxidative stress is suggested to be a disease mechanism common to a wide range of disorders affecting human health. However, so far, the pharmacotherapeutic exploitation of this, for example, based on chemical scavenging of pro-oxidant molecules, has been unsuccessful. Recent Advances: An alternative emerging approach is to target the enzymatic sources of disease-relevant oxidative stress. Several such enzymes and isoforms have been identified and linked to different pathologies. For some targets, the respective pharmacology is quite advanced, that is, up to late-stage clinical development or even on the market; for others, drugs are already in clinical use, although not for indications based on oxidative stress, and repurposing seems to be a viable option. Critical Issues: For all other targets, reliable preclinical validation and drug ability are key factors for any translation into the clinic. In this study, specific pharmacological agents with optimal pharmacokinetic profiles are still lacking. Moreover, these enzymes also serve largely unknown physiological functions and their inhibition may lead to unwanted side effects. Future Directions: The current promising data based on new targets, drugs, and drug repurposing are mainly a result of academic efforts. With the availability of optimized compounds and coordinated efforts from academia and industry scientists, unambiguous validation and translation into proof-of-principle studies seem achievable in the very near future, possibly leading towards a new era of redox medicine. Antioxid. Redox Signal. 23, 1113–1129.

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16Unphysiological reactive oxygen species (ROS) formation is considered an important 17 pathomechanism for several disease phenotypes with high unmet medical need. Therapeutically, 18 antioxidants have failed multiple times. Instead, focusing on only disease-relevant, enzymatic 19sources of ROS appears to be a more promising and highly validated approach. Here the family of 20 five NADPH oxidases (NOX) stands out as drug targets. Validation has been restricted, however, 21 mainly to genetically modified rodents and is lacking in other species including human. It is thus 22 unclear whether the different NOX isoforms are sufficiently distinct to allow selective 23 pharmacological modulation. Here we show for five of the most advanced NOX inhibitors that 24 indeed isoform selectivity can be achieved. NOX1 was most potently (IC 50 ) targeted by ML171 (0.1 25 µM); NOX2, by VAS2870 (0.7 µM); NOX4, by M13 (0.01 µM) and NOX5, by ML090 (0.01 µM). 26Conditions need to be carefully controlled though as previously unrecognized non-specific 27 antioxidant and assay artefacts may limit the interpretation of data and this included, surprisingly, 28 one of the most advanced NOX inhibitors, GKT136901. As proof-of-principle that now also 29 pharmacological and non-rodent target validation of different NOX isoforms is possible, we used a 30 human blood-brain barrier model and NOX inhibitor panel at IC 50 concentrations. The protective 31 efficacy pattern of this panel confirmed the predominant role of NOX4 in stroke from previous 32 genetic models. Our findings strongly encourage further lead optimization efforts for isoform-33 selective NOX inhibitors and clinical development and provide an experimental alternative when 34 genetic validation of a NOX isoform is not an option. 35 [2][3][4]. This paradox was initially explained by these compounds being underdosed, thereby not 41 reaching efficacy. It is now understood, however, that ROS are not only harmful metabolic by-42 products, but also serve important protective, metabolic and signaling functions, such as the 43 regulation of cell proliferation, differentiation, migration and survival, innate immune response, 44 vascular tone, neuronal signaling as well as inflammation [5][6][7][8]. Anti-oxidants are likely to 45 simultaneously interfere with both qualities of ROS, the physiological and pathophysiological ones 46 with overall neutral or even deleterious outcomes. Thus, ROS should not be modulated in a 47

Pentaerythritol tetranitrate (PETN) treatment reduces progression of atherosclerosis and endothelial dysfunction and decreases oxidation of low-density lipoprotein (LDL) in rabbits. These effects are associated with decreased vascular superoxide production, but the underlying molecular mechanisms remain unknown. Previous studies demonstrated that endogenous nitric oxide could regulate the expression of extracellular superoxide dismutase (ecSOD) in conductance vessels in vivo. We investigated the effect of PETN and overexpression of endothelial nitric oxide synthase (eNOS++) on the expression and activity of ecSOD. C57BL/6 mice were randomized to receive placebo or increasing doses of PETN for 4 weeks and eNOS++ mice with a several fold higher endothelial-specific eNOS expression were generated. The expression of ecSOD was determined in the lung and aortic tissue by real-time PCR and Western blot. The ecSOD activity was measured using inhibition of cytochrome C reduction. There was no effect of PETN treatment or eNOS overexpression on ecSOD mRNA in the lung tissue, whereas ecSOD protein expression increased from 2.5-fold to 3.6-fold (P < 0.05) by 6 mg PETN/kg body weight (BW)/day and 60 mg PETN/kg BW/day, respectively. A similar increase was found in aortic homogenates. eNOS++ lung cytosols showed an increase of ecSOD protein level of 142 ± 10.5% as compared with transgene-negative littermates (P < 0.05), which was abolished by Nω-nitro-L-arginine treatment. In each animal group, the increase of ecSOD expression was paralleled by an increase of ecSOD activity. Increased expression and activity of microvascular ecSOD are likely induced by increased bioavailability of vascular nitric oxide. Up-regulation of vascular ecSOD may contribute to the reported antioxidative and anti-atherosclerotic effects of PETN.

KeywordsACE I/D polymorphism, angio-oedema, bradykinin, bradykinin B2 receptor polymorphism, serum ACE activity ---------------------------------------------------------------------- WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT• Angiotensin-converting enzyme inhibitor (ACEi)-induced angio-oedema is an underestimated clinical life-threatening problem.• The incidence of this non-allergic, bradykinin-induced drug side-effect is 1 : 4000.• Although most ACEi-treated patients probably have an increased bradykinin plasma concentration, only 0.5% of them develop an angio-oedema and nothing is known about potential risk factors. WHAT THIS STUDY ADDS• In our attempt to elucidate the unpredictable character of ACEi-induced angio-oedema, we investigated bradykinin B2 receptor 2/3 and c.C181T polymorphisms as well as the ACE insertion/deletion polymorphism in combination with serum ACE activity in 65 patients.• ACE insertion/deletion and bradykinin B2 receptor polymorphisms are not involved in the development of ACEi-induced angio-oedema.• Further studies should be carried out to clarify whether a combination of these polymorphisms might be a risk factor for ACEi-induced angio-oedema. AIMSThe pathophysiology of angiotensin-converting enzyme inhibitor (ACEi)-induced angio-oedema remains unclear. We have investigated the impact of ACE insertion/deletion (I/D) polymorphism in combination with serum ACE activity as well as the bradykinin B2 receptor 2/3 and c.C181T polymorphisms. METHODSWe analysed the ACE I/D as well as bradykinin B2 (2/3 and C181T) receptor polymorphisms in 65 patients with documented episodes of ACEi-induced angio-oedema and 65 patients matched for age and sex being under ACEi treatment without history of angio-oedema. Furthermore, we determined serum ACE activity in 47 of the 65 angio-oedema patients 3 months after the angio-oedema attack and compared these values with 51 healthy individuals (control II). ; P = 0.9). RESULTS No risk association was identified between ACE CONCLUSIONSOur data suggest that polymorphism of ACE I/D and the bradykinin B2 receptor polymorphisms are not involved in the development of ACEi-induced angio-oedema when considered individually. Further studies should be carried out to clarify whether a combination of these polymorphisms might be a risk factor for ACEi-induced angio-oedema.

Physical activity induces favourable changes of arterial gene expression and protein activity, although little is known about its effect in venous tissue. Although our understanding of the initiating molecular signals is still incomplete, increased expression of endothelial nitric oxide synthase (eNOS) is considered a key event. This study sought to investigate the effects of two different training protocols on the expression of eNOS and extracellular superoxide dismutase (ecSOD) in venous and lung tissue and to evaluate the underlying molecular mechanisms. C57Bl/6 mice underwent voluntary exercise or forced physical activity. Changes of vascular mRNA and protein levels and activity of eNOS, ecSOD and catalase were determined in aorta, heart, lung and vena cava. Both training protocols similarly increased relative heart weight and resulted in up-regulation of aortic and myocardial eNOS. In striking contrast, eNOS expression in vena cava and lung remained unchanged. Likewise, exercise up-regulated ecSOD in the aorta and in left ventricular tissue but remained unchanged in lung tissue. Catalase expression in lung tissue and vena cava of exercised mice exceeded that in aorta by 6.9- and 10-fold, respectively, suggesting a lack of stimulatory effects of hydrogen peroxide. In accordance, treatment of mice with the catalase inhibitor aminotriazole for 6 weeks resulted in significant up-regulation of eNOS and ecSOD in vena cava. These data suggest that physiological venous catalase activity prevents exercise-induced up-regulation of eNOS and ecSOD. Furthermore, therapeutic inhibition of vascular catalase might improve pulmonary rehabilitation.

Aims: Vascular oxidative stress generated by endothelial NO synthase (eNOS) was observed in experimental and clinical cardiovascular disease, but its relative importance for vascular pathologies is unclear. We investigated the impact of eNOS-dependent vascular oxidative stress on endothelial function and on neointimal hyperplasia. Results: A dimer-destabilized mutant of bovine eNOS where cysteine 101 was replaced by alanine was cloned and introduced into an eNOS-deficient mouse strain (eNOS-KO) in an endothelial-specific manner. Destabilization of mutant eNOS in cells and eNOS-KO was confirmed by the reduced dimer/monomer ratio. Purified mutant eNOS and transfected cells generated less citrulline and NO, respectively, while superoxide generation was enhanced. In eNOS-KO, introduction of mutant eNOS caused a 2.3-3.7-fold increase in superoxide and peroxynitrite formation in the aorta and myocardium. This was completely blunted by an NOS inhibitor. Nevertheless, expression of mutant eNOS in eNOS-KO completely restored maximal aortic endothelium-dependent relaxation to acetylcholine. Neointimal hyperplasia induced by carotid binding was much larger in eNOS-KO than in mutant eNOS-KO and C57BL/6, while the latter strains showed comparable hyperplasia. Likewise, vascular remodeling was blunted in eNOS-KO only. Innovation: Our results provide the first in vivo evidence that eNOS-dependent oxidative stress is unlikely to be an initial cause of impaired endothelium-dependent vasodilation and/or a pathologic factor promoting intimal hyperplasia. These findings highlight the importance of other sources of vascular oxidative stress in cardiovascular disease. Conclusion: eNOS-dependent oxidative stress is unlikely to induce functional vascular damage as long as concomitant generation of NO is preserved. This underlines the importance of current and new therapeutic strategies in improving endothelial NO generation. Antioxid. Redox Signal. 23, 711-723.