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Abstract The relationship between noise annoyance and risk of cardiovascular disease (CVD) still needs to be fully elucidated. Thus, we examined the relationship between noise annoyance and CVD risk in a large population-based cohort study. Cross-sectional (N = 15,010, aged 35–74 years, baseline investigation period 2007–2012) and prospective data (5- and 10-year follow-up from 2012 to 2022) from the Gutenberg Health Study were used to examine the relationship between noise annoyance due to different sources and risk of prevalent and incident CVD comprising atrial fibrillation, coronary artery disease, myocardial infarction, stroke, chronic heart failure, peripheral artery disease, and venous thromboembolism. In cross-sectional analyses, noise annoyance was an independent risk factor for prevalent CVD, with the strongest associations seen for noise annoyance during sleep (e.g., neighborhood noise annoyance: odds ratio 1.20, 95% confidence interval 1.13–1.27, p

The cytoprotective transcription factor NRF2 regulates the expression of several hundred genes in mammalian cells and is a promising therapeutic target in a number of diseases associated with oxidative stress and inflammation. Hence, an ability to monitor basal and inducible NRF2 signalling is vital for mechanistic understanding in translational studies. Due to some caveats related to the direct measurement of NRF2 levels, the modulation of NRF2 activity is typically determined by measuring changes in the expression of one or more of its target genes and/or the associated protein products. However, there is a lack of consensus regarding the most relevant set of these genes/proteins that best represents NRF2 activity across cell types and species. We present the findings of a comprehensive literature search that according to stringent criteria identifies GCLC, GCLM, HMOX1, NQO1, SRXN1 and TXNRD1 as a robust panel of markers that are directly regulated by NRF2 in multiple cell and tissue types. We assess the relevance of these markers in clinically accessible biofluids and highlight future challenges in the development and use of NRF2 biomarkers in humans.

Aims: We examined the cardiovascular effects of celiac disease (CeD) in a humanized mouse model, with a focus on vascular inflammation, endothelial dysfunction, and oxidative stress. Methods and results: NOD.DQ8 mice genetically predisposed to CeD were subjected to a diet regime and oral gavage to induce the disease (gluten group vs. control). We tested vascular function, confirmed disease indicators, and evaluated inflammation and oxidative stress in various tissues. Plasma proteome profiling was also performed.CeD markers were confirmed in the gluten group, indicating increased blood pressure and impaired vascular relaxation. Pro-inflammatory genes were upregulated in this group, with increased CD11b+ myeloid cell infiltration and oxidative stress parameters observed in aortic and heart tissue. However, heart function remained unaffected. Plasma proteomics suggested the cytokine interleukin-17A (IL-17A) as a link between gut and vascular inflammation. Cardiovascular complications were reversed by adopting a gluten-free diet. Conclusion: Our study sheds light in the heightened cardiovascular risk associated with active CeD, revealing a gut-to-cardiovascular inflammatory axis potentially mediated by immune cell infiltration and IL-17A. These findings augment our understanding of the link between CeD and cardiovascular disease providing clinically relevant insight into the underlying mechanism. Furthermore, our discovery that cardiovascular complications can be reversed by a gluten-free diet underscores a critical role for dietary interventions in mitigating cardiovascular risks associated with CeD.

Transportation noise is a ubiquitous urban exposure. In 2018, the World Health Organization concluded that chronic exposure to road traffic noise is a risk factor for ischemic heart disease. In contrast, they concluded that the quality of evidence for a link to other diseases was very low to moderate. Since then, several studies on the impact of noise on various diseases have been published. Also, studies investigating the mechanistic pathways underlying noise-induced health effects are emerging. We review the current evidence regarding effects of noise on health and the related disease-mechanisms. Several high-quality cohort studies consistently found road traffic noise to be associated with a higher risk of ischemic heart disease, heart failure, diabetes, and all-cause mortality. Furthermore, recent studies have indicated that road traffic and railway noise may increase the risk of diseases not commonly investigated in an environmental noise context, including breast cancer, dementia, and tinnitus. The harmful effects of noise are related to activation of a physiological stress response and nighttime sleep disturbance. Oxidative stress and inflammation downstream of stress hormone signaling and dysregulated circadian rhythms are identified as major disease-relevant pathomechanistic drivers. We discuss the role of reactive oxygen species and present results from antioxidant interventions. Lastly, we provide an overview of oxidative stress markers and adverse redox processes reported for noise-exposed animals and humans. This position paper summarizes all available epidemiological, clinical, and preclinical evidence of transportation noise as an important environmental risk factor for public health and discusses its implications on the population level.

Neurodegenerative diseases are often referred to as diseases of old age, and with the aging population, they are gaining scientific and medical interest. Environmental stressors, most notably traffic noise and air pollution, have recently come to the forefront, and have emerged as disease risk factors. The evidence for a connection between environmental risk factors and neurodegenerative disease is growing. In this review, the most common neurodegenerative diseases and their epidemiological association with traffic noise and air pollution are presented. Also, the most important mechanisms involved in neurodegenerative disease development, oxidative stress, and neuroinflammation are highlighted. An overview of the in vivo findings will provide a mechanistic link between noise, air pollution, and neurodegenerative pathology. Finally, the importance of the direct and indirect pathways, by which noise and air pollution cause cerebral damage, is discussed. More high-quality data are still needed from both epidemiological and basic science studies in order to better understand the causal connection between neurodegenerative diseases and environmental risk factors.

During the last two decades, large epidemiological studies have shown that the physical environment, including noise, air pollution or heavy metals, have a considerable impact on human health. It is known that the most common cardiovascular risk factors are all associated with endothelial dysfunction. Vascular tone, circulation of blood cells, inflammation, and platelet activity are some of the most essential functions regulated by the endothelium that suffer negative effects as a consequence of environmental pollution, causing endothelial dysfunction. In this review, we delineate the impact of environmental risk factors in connection to endothelial function. On a mechanistic level, a significant number of studies suggest the involvement of endothelial dysfunction to fundamentally drive the adverse endothelium health effects of the different pollutants. We focus on well-established studies that demonstrate the negative effects on the endothelium, with a focus on air, noise, and heavy metal pollution. This in-depth review on endothelial dysfunction as a consequence of the physical environment aims to contribute to the associated research needs by evaluating current findings from human and animal studies. From a public health perspective, these findings may also help to reinforce efforts promoting the research for adequate promising biomarkers for cardiovascular diseases since endothelial function is considered a hallmark of environmental stressor health effects.

Despite major advances in acute interventions for myocardial infarction (MI), adverse cardiac remodeling and excess fibrosis after MI causing ischemic heart failure (IHF) remain a leading cause of death worldwide. Here we identify a profibrotic coagulation signaling pathway that can be targeted for improved cardiac function following MI with persistent ischemia. Quantitative phosphoproteomics of cardiac tissue revealed an upregulated mitogen-activated protein kinase (MAPK) pathway in human IHF. Intervention in this pathway with trametinib improves myocardial function and prevents fibrotic remodeling in a murine model of non-reperfused MI. MAPK activation in MI requires myeloid cell signaling of protease-activated receptor 2 linked to the cytoplasmic domain of the coagulation initiator tissue factor (TF). They act upstream of pro-oxidant NOX2 NADPH oxidase, ERK1/2 phosphorylation, and activation of profibrotic TGF-β1. Specific targeting with the TF inhibitor nematode anticoagulant protein c2 (NAPc2) starting 1 day after established experimental MI averts IHF. Increased TF cytoplasmic domain phosphorylation in circulating monocytes from patients with subacute MI identifies a potential thromboinflammatory biomarker reflective of increased risk for IHF and suitable for patient selection to receive targeted TF inhibition therapy.

Worldwide, up to 8.8 million excess deaths/year have been attributed to air pollution, mainly due to the exposure to fine particulate matter (PM). Traffic-related noise is an additional contributor to global mortality and morbidity. Both health risk factors substantially contribute to cardiovascular, metabolic and neuropsychiatric sequelae. Studies on the combined exposure are rare and urgently needed because of frequent co-occurrence of both risk factors in urban and industrial settings. To study the synergistic effects of PM and noise, we used an exposure system equipped with aerosol generator and loud-speakers, where C57BL/6 mice were acutely exposed for 3d to either ambient PM (NIST particles) and/or noise (aircraft landing and take-off events). The combination of both stressors caused endothelial dysfunction, increased blood pressure, oxidative stress and inflammation. An additive impairment of endothelial function was observed in isolated aortic rings and even more pronounced in cerebral and retinal arterioles. The increase in oxidative stress and inflammation markers together with RNA sequencing data indicate that noise particularly affects the brain and PM the lungs. The combination of both stressors has additive adverse effects on the cardiovascular system that are based on PM-induced systemic inflammation and noise-triggered stress hormone signaling. We demonstrate an additive upregulation of ACE-2 in the lung, suggesting that there may be an increased vulnerability to COVID-19 infection. The data warrant further mechanistic studies to characterize the propagation of primary target tissue damage (lung, brain) to remote organs such as aorta and heart by combined noise and PM exposure.

The results from epidemiological studies suggest that environmental noise including aircraft, railway, road traffic, wind turbine, and leisure-related noise is a growing public health concern. According to the WHO, at least 100 million people in the European Union are affected by traffic noise levels above the WHO-recommended thresholds. Environmental noise can adversely affect physical and mental health, as well as wellbeing. Chronic low-level noise exposure typical for most environmental sources is associated with psychophysiological stress causing non-auditory or indirect noise effects leading ultimately to cardiovascular diseases. Among all environmental noise sources, aircraft noise is considered the most annoying, and its leading mechanism of action is autonomic system activation such as increases in heart rate and blood pressure. Previously, we observed that long-term exposure to aircraft noise was associated with increased diastolic blood pressure, arterial stiffness (as assessed by pulse wave velocity), and impaired left ventricular diastolic function. All mentioned above effects are early, subclinical, and potentially reversible changes which preceded late noise effects in the cardiovascular system, that is, established cardiovascular diseases such as myocardial infarction, stroke, and heart failure. However, even a short-term reduction in aircraft noise exposure as observed during the COVID-19 lockdown may reverse these negative effects on arterial stiffness and blood pressure and may decrease the prevalence of insomnia. In this review, we aimed to critically discuss our obtained results considering recent studies on the influence of aircraft noise (and other traffic noises) on cardiovascular diseases in the context of the WHO Environmental Noise Guidelines for the European Region.

BackgroundWhile chronic exposure to high levels of noise was demonstrated to increase the risk of various cardiovascular diseases, the association between noise annoyance and risk of cardiovascular disease remains still inconsistent. Recently, we showed that noise annoyance is associated with prevalent atrial fibrillation in the general population. However, the association between noise annoyance and risk of incident atrial fibrillation as well as potential sex-differences remain still elusive.Methods and results15,010 subjects from a German population-based cohort were examined at baseline (2007 to 2012) and follow-up five years later (2012 to 2017) to investigative the association between noise annoyance due to multiple sources and prevalent and incident atrial fibrillation. After multivariable adjustment, the results from logistic regression analyses revealed overall consistent and positive associations between noise annoyance and prevalent and incident atrial fibrillation in men, whereas this association was weaker in women, in particular with respect to incident atrial fibrillation. For instance, industrial noise annoyance was associated with 21% (95% confidence interval (CI) 9–34%) and 18% (8–29%) higher odds of prevalent atrial fibrillation in men and women, respectively. In prospective analysis, this association remained stable in men (odds ratio (OR) 1.25, 1.07–1.44), while in women no association was observed (OR 1.03, 0.89–1.18).ConclusionsThe findings suggest that noise annoyance can increase the risk of incident atrial fibrillation in a large population-based cohort and that men may be more sensitive to the adverse effects of noise annoyance with regard to the risk of atrial fibrillation.

Abstract Background Many patients with acute respiratory distress syndrome (ARDS) suffer from cognitive impairment after hospital discharge. Different mechanisms have been implicated as potential causes for this impairment, inter alia cerebral inflammation. A class of drugs with antioxidant and anti-inflammatory properties are β-HMG-CoA-reductase inhibitors (“statins”). We hypothesized that treatment with rosuvastatin attenuates cerebral cytokine mRNA expression and nitro-oxidative stress in an animal model of acute lung injury. Methods After approval of the institutional and state animal care committee, we performed this prospective randomized controlled animal study in accordance with the international guidelines for the care and use of laboratory animals. Thirty-two healthy male pigs were randomized to one of four groups: lung injury by central venous injection of oleic acid (n = 8), statin treatment before and directly after lung injury (n = 8), statin treatment after lung injury (n = 8), or ventilation-only controls (n = 8). About 18 h after lung injury and standardized treatment, the animals were euthanised, and the brains and lungs were collected for further examinations. We determined histologic lung injury and cerebral and pulmonal cytokine and 3-nitrotyrosine production. Results We found a significant increase in hippocampal IL-6 mRNA after lung injury (p

Nitrosation of critical thiols has been elaborated as reversible posttranslational modification with regulatory function in multiple disorders. Reversibility of S-nitrosation is generally associated with enzyme-mediated one-electron reductions, catalyzed by the thioredoxin system, or by nitrosoglutathione reductase.In the present study, we confirm previous evidence for a non-enzymatic de-nitrosation of nitrosoglutathione (GSNO) by superoxide. The interaction leads to the release of nitric oxide that subsequently interacts with a second molecule of superoxide (O2•−) to form peroxynitrite. Despite the formation of peroxynitrite, approximately 40–70% of GSNO yielded reduced glutathione (GSH), depending on the applied analytical assay. The concept of O2•− dependent denitrosation was then applied to S-nitrosated enzymes. S-nitrosation of isocitrate dehydrogenase (ICDH; NADP+-dependent) was accompanied by an inhibition of the enzyme and could be reversed by dithiothreitol. Treatment of nitrosated ICDH with O2•− indicated ca. 50% recovery of enzyme activity. Remaining inhibition was largely consequence of oxidative modifications evoked either by O2•− or by peroxynitrite. Recovery of activity in S-nitrosated enzymes by O2•− appears relevant only for selected examples. In contrast, recovery of reduced glutathione from the interaction of GSNO with O2•− could represent a mechanism to regain reducing equivalents in situations of excess O2•− formation, e.g. in the reperfusion phase after ischemia.

Cardiovascular diseases (CVD) and cardiovascular risk factors are the leading cause of death in the world today. According to the Global Burden of Disease Study, hypertension together with ischemic heart and cerebrovascular diseases is responsible for approximately 40% of all deaths worldwide. The major pathomechanism underlying almost all CVD is atherosclerosis, an inflammatory disorder of the vascular system. Recent large-scale clinical trials demonstrated that inflammation itself is an independent cardiovascular risk factor. Specific anti-inflammatory therapy could decrease cardiovascular mortality in patients with atherosclerosis (increased markers of inflammation). Inflammation, however, can also be beneficial by conferring so-called resolution, a process that contributes to clearing damaged tissue from cell debris upon cell death and thereby represents an essential step for recovery from, e.g., ischemia/reperfusion damage. Based on these considerations, the present review highlights features of the detrimental inflammatory reactions as well as of the beneficial process of immune cell-triggered resolution. In this context, we discuss the polarization of macrophages to either M1 or M2 phenotype and critically assess the role of the CD40L-CD40-TRAF signaling cascade in atherosclerosis and its potential link to resolution. As CD40L can bind to different cellular receptors, it can initiate a broad range of inflammatory processes that may be detrimental or beneficial. Likewise, the signaling of CD40L downstream of CD40 is mainly determined by activation of TRAF1-6 pathways that again can be detrimental or beneficial. Accordingly, CD40(L)-based therapies may be Janus-faced and require sophisticated fine-tuning in order to promote cardioprotection.

The importance of noise exposure as a major environmental determinant of public health is being increasingly recognized. While in recent years a large body evidence has emerged linking environmental noise exposure mainly to cardiovascular disease, much less is known concerning the adverse health effects of noise on the brain and associated neuropsychiatric outcomes. Despite being a relatively new area of investigation, indeed, mounting research and conclusive evidence demonstrate that exposure to noise, primarily from traffic sources, may affect the central nervous system and brain, thereby contributing to an increased risk of neuropsychiatric disorders such as stroke, dementia and cognitive decline, neurodevelopmental disorders, depression, and anxiety disorder. On a mechanistic level, a significant number of studies suggest the involvement of reactive oxygen species/oxidative stress and inflammatory pathways, among others, to fundamentally drive the adverse brain health effects of noise exposure. This in-depth review on the cerebral consequences of environmental noise exposure aims to contribute to the associated research needs by evaluating current findings from human and animal studies. From a public health perspective, these findings may also help to reinforce efforts promoting adequate mitigation strategies and preventive measures to lower the societal consequences of unhealthy environments.

Ischemia-reperfusion (I/R) events are involved in the development of various ocular pathologies, e.g., retinal artery or vein occlusion. We tested the hypothesis that resveratrol is protective against I/R injury in the murine retina. Intraocular pressure (IOP) was elevated in anaesthetized mice to 110 mm Hg for 45 min via a micropipette placed in the anterior chamber to induce ocular ischemia. In the fellow eye, which served as control, IOP was kept at a physiological level. One group received resveratrol (30 mg/kg/day p.o. once daily) starting one day before the I/R event, whereas the other group of mice received vehicle solution only. On day eight after the I/R event, mice were sacrificed and retinal wholemounts were prepared and immuno-stained using a Brn3a antibody to quantify retinal ganglion cells. Reactivity of retinal arterioles was measured in retinal vascular preparations using video microscopy. Reactive oxygen species (ROS) and nitrogen species (RNS) were quantified in ocular cryosections by dihydroethidium and anti-3-nitrotyrosine staining, respectively. Moreover, hypoxic, redox and nitric oxide synthase gene expression was quantified in retinal explants by PCR. I/R significantly diminished retinal ganglion cell number in vehicle-treated mice. Conversely, only a negligible reduction in retinal ganglion cell number was observed in resveratrol-treated mice following I/R. Endothelial function and autoregulation were markedly reduced, which was accompanied by increased ROS and RNS in retinal blood vessels of vehicle-exposed mice following I/R, whereas resveratrol preserved vascular endothelial function and autoregulation and blunted ROS and RNS formation. Moreover, resveratrol reduced I/R-induced mRNA expression for the prooxidant enzyme, nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2). Our data provide evidence that resveratrol protects from I/R-induced retinal ganglion cell loss and endothelial dysfunction in the murine retina by reducing nitro-oxidative stress possibly via suppression of NOX2 upregulation.

The microbiota has been linked to the development of atherosclerosis, but the functional impact of these resident bacteria on the lesion size and cellular composition of atherosclerotic plaques in the aorta has never been experimentally addressed with the germ-free low-density lipoprotein receptor-deficient (Ldlr−/-) mouse atherosclerosis model. Here, we report that 16 weeks of high-fat diet (HFD) feeding of hypercholesterolemic Ldlr−/- mice at germ-free (GF) housing conditions did not impact relative aortic root plaque size, macrophage content, and necrotic core area. Likewise, we did not find changes in the relative aortic arch lesion size. However, late atherosclerotic GF Ldlr−/- mice had altered inflammatory plasma protein markers and reduced smooth muscle cell content in their atherosclerotic root plaques relative to CONV-R Ldlr−/- mice. Neither absolute nor relative aortic root or aortic arch plaque size correlated with age. Our analyses on GF Ldlr−/- mice did not reveal a significant contribution of the microbiota in late aortic atherosclerosis.

Transportation noise is recognized as an important cardiovascular risk factor. Key mechanisms are noise-triggered vascular inflammation and oxidative stress with subsequent endothelial dysfunction. Here, we test for adaptation or tolerance mechanisms in mice in response to chronic noise exposure. C57BL/6J mice were exposed to aircraft noise for 0, 4, 7, 14 and 28d at a mean sound pressure level of 72 dB(A) and peak levels of 85 dB(A). Chronic aircraft noise exposure up to 28d caused persistent endothelial dysfunction and elevation of blood pressure. Likewise, reactive oxygen species (ROS) formation as determined by dihydroethidium (DHE) staining and HPLC-based measurement of superoxide formation in the aorta/heart/brain was time-dependently increased by noise. Oxidative burst in the whole blood showed a maximum at 4d or 7d of noise exposure. Increased superoxide formation in the brain was mirrored by a downregulation of neuronal nitric oxide synthase (Nos3) and transcription factor Foxo3 genes, whereas Vcam1 mRNA, a marker for inflammation was upregulated in all noise exposure groups. Induction of a pronounced hearing loss in the mice was excluded by auditory brainstem response audiometry. Endothelial dysfunction and inflammation were present during the entire 28d of aircraft noise exposure. ROS formation gradually increases with ongoing exposure without significant adaptation or tolerance in mice in response to chronic noise stress at moderate levels. These data further illustrate health side effects of long-term noise exposure and further strengthen a consequent implementation of the WHO noise guidelines in order to prevent the development of noise-related future cardiovascular disease.

Diabetes mellitus may cause severe damage to retinal blood vessels. The central aim of this study was to test the hypothesis that sulodexide, a mixture of glycosaminoglycans, has a protective effect against hyperglycemia-induced endothelial dysfunction in the retina. Functional studies were performed in isolated porcine retinal arterioles. Vessels were cannulated and incubated with highly concentrated glucose solution (HG, 25 mM D-glucose) +/− sulodexide (50/5/0.5 μg/mL) or normally concentrated glucose solution (NG, 5.5 mM D-glucose) +/− sulodexide for two hours. Endothelium-dependent and endothelium-independent vasodilatation were measured by videomicroscopy. Reactive oxygen species (ROS) were quantified by dihydroethidium (DHE) fluorescence. Using high-pressure liquid chromatography (HPLC), the intrinsic antioxidant properties of sulodexide were investigated. Quantitative PCR was used to determine mRNA expression of regulatory, inflammatory, and redox genes in retinal arterioles, some of which were subsequently quantified at the protein level by immunofluorescence microscopy. Incubation of retinal arterioles with HG caused significant impairment of endothelium-dependent vasodilation, whereas endothelium-independent responses were not affected. In the HG group, ROS formation was markedly increased in the vascular wall. Strikingly, sulodexide had a protective effect against hyperglycemia-induced ROS formation in the vascular wall and had a concentration-dependent protective effect against endothelial dysfunction. Although sulodexide itself had only negligible antioxidant properties, it prevented hyperglycemia-induced overexpression of the pro-oxidant redox enzymes, NOX4 and NOX5. The data of the present study provide evidence that sulodexide has a protective effect against hyperglycemia-induced oxidative stress and endothelial dysfunction in porcine retinal arterioles, possibly by modulation of redox enzyme expression.

Previous studies demonstrated an important role of oxidative stress in the pathogenesis of cardiovascular disease (CVD) in diabetic patients due to hyperglycemia. CVD remains the leading cause of premature death in the western world. Therefore, diabetes mellitus-associated oxidative stress and subsequent inflammation should be recognized at the earliest possible stage to start with the appropriate treatment before the onset of the cardiovascular sequelae such as arterial hypertension or coronary artery disease (CAD). The pathophysiology comprises increased reactive oxygen and nitrogen species (RONS) production by enzymatic and non-enzymatic sources, e.g., mitochondria, an uncoupled nitric oxide synthase, xanthine oxidase, and the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX). Considering that RONS originate from different cellular mechanisms in separate cellular compartments, adequate, sensitive, and compartment-specific methods for their quantification are crucial for early detection. In this review, we provide an overview of these methods with important information for early, appropriate, and effective treatment of these patients and their cardiovascular sequelae.

Environmental exposures represent a significant health hazard, which cumulatively may be responsible for up to 2/3 of all chronic non-communicable disease and associated mortality (Global Burden of Disease Study and The Lancet Commission on Pollution and Health), which has given rise to a new concept of the exposome: the sum of environmental factors in every individual’s experience. Noise is part of the exposome and is increasingly being investigated as a health risk factor impacting neurological, cardiometabolic, endocrine, and immune health. Beyond the well-characterized effects of high-intensity noise on cochlear damage, noise is relatively well-studied in the cardiovascular field, where evidence is emerging from both human and translational experiments that noise from traffic-related sources could represent a risk factor for hypertension, ischemic heart disease, diabetes, and atherosclerosis. In the present review, we comprehensively discuss the current state of knowledge in the field of noise research. We give a brief survey of the literature documenting experiments in noise exposure in both humans and animals with a focus on cardiovascular disease. We also discuss the mechanisms that have been uncovered in recent years that describe how exposure to noise affects physiological homeostasis, leading to aberrant redox signaling resulting in metabolic and immune consequences, both of which have considerable impact on cardiovascular health. Additionally, we discuss the molecular pathways of redox involvement in the stress responses to noise and how they manifest in disruptions of the circadian rhythm, inflammatory signaling, gut microbiome composition, epigenetic landscape and vessel function.

Epidemiological studies showed that traffic noise has a dose-dependent association with increased cardiovascular morbidity and mortality. Whether microvascular dysfunction contributes significantly to the cardiovascular health effects by noise exposure remains to be established. The connection of inflammation and immune cell interaction with microvascular damage and functional impairment is also not well characterized. Male C57BL/6J mice or gp91phox−/y mice with genetic deletion of the phagocytic NADPH oxidase catalytic subunit (gp91phox or NOX-2) were used at the age of 8 weeks, randomly instrumented with dorsal skinfold chambers and exposed or not exposed to aircraft noise for 4 days. Proteomic analysis (using mass spectrometry) revealed a pro-inflammatory phenotype induced by noise exposure that was less pronounced in noise-exposed gp91phox−/y mice. Using in vivo fluorescence microscopy, we found a higher number of adhesive leukocytes in noise-exposed wild type mice. Dorsal microvascular diameter (by trend), red blood cell velocity, and segmental blood flow were also decreased by noise exposure indicating microvascular constriction. All adverse effects on functional parameters were normalized or improved at least by trend in noise-exposed gp91phox−/y mice. Noise exposure also induced endothelial dysfunction in cerebral microvessels, which was associated with higher oxidative stress burden and inflammation, as measured using video microscopy. We here establish a link between a pro-inflammatory phenotype of plasma, activation of circulating leukocytes and microvascular dysfunction in mice exposed to aircraft noise. The phagocytic NADPH oxidase was identified as a central player in the underlying pathophysiological mechanisms.

Aims: Oxidative stress and inflammation contribute to atherogenesis. Rac1 GTPase regulates pro-oxidant NADPH oxidase activity, reactive oxygen species (ROS) formation, actin cytoskeleton organization and monocyte adhesion. We investigated the vascular effects of pharmacological inhibition of Rac1 GTPase in mice.Methods and Results: We treated wild-type and apolipoprotein E-deficient (ApoE−/−) mice with Clostridium sordellii lethal toxin (LT), a Rac1 inhibitor, and assessed vascular oxidative stress, expression and activity of involved proteins, endothelial function, macrophage infiltration, and atherosclerosis development. LT-treated wild-type mice displayed decreased vascular NADPH oxidase activity and ROS production. Therapeutic LT doses had no impact on behavior, food intake, body weight, heart rate, blood pressure, vascular and myocardial function, differential blood count, and vascular permeability. ApoE−/− mice were fed a cholesterol-rich diet and were treated with LT or vehicle. LT treatment led to decreased aortic Rac1 GTPase activity, NADPH oxidase activity and ROS production, but had no impact on expression and membrane translocation of NADPH oxidase subunits and RhoA GTPase activity. LT-treated mice showed improved aortic endothelium-dependent vasodilation, attenuated atherosclerotic lesion formation and reduced macrophage infiltration of atherosclerotic plaques. Concomitant treatment of cholesterol-fed ApoE−/− mice with LT, the specific synthetic Rac1 inhibitor NSC 23766 or simvastatin comparably reduced aortic Rac1 activity, NADPH oxidase activity, oxidative stress, endothelial dysfunction, atherosclerosis development, and macrophage infiltration.Conclusions: These findings identify an important role of the small GTPase Rac1 in atherogenesis and provide a potential target for anti-atherosclerotic therapy.

Oral treatment of apolipoprotein E-knockout (ApoE-KO) mice with the putative sirtuin 1 (SIRT1) activator resveratrol led to a reduction of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in the heart. In contrast, the SIRT1 inhibitor EX527 enhanced the superoxide production in isolated human polymorphonuclear granulocytes. In human monocytic THP-1 cells, phorbol ester-stimulated superoxide production was enhanced by inhibitors of histone deacetylases (HDACs; including quisinostat, trichostatin A (TSA), PCI34051, and tubastatin A) and decreased by inhibitors of histone acetyltransferases [such as garcinol, curcumin, and histone acetyltransferase (HAT) Inhibitor II]. These results indicate that protein acetylation and deacetylation may represent crucial mechanisms regulating NADPH oxidase-mediated superoxide production. In cell-free systems, incubation of recombinant Rac1 with SIRT1 resulted in decreased Rac1 acetylation. Mass spectrometry analyses identified lysine 166 (K166) in Rac1 as a residue targeted by SIRT1. Deacetylation of Rac1 by SIRT1 markedly reduced the interaction of Rac1 with p67phox in in vitro assays. Computational modeling analyses revealed that K166 deacetylation of Rac1 led to a 5-fold reduction in its binding affinity to guanosine-5'-triphosphate, and a 21-fold decrease in its binding potential to p67phox. The latter is crucial for Rac1-mediated recruitment of p67phox to the membrane and for p67phox activation. In conclusion, both SIRT1 and non-sirtuin deacetylases play a role in regulating NADPH oxidase activity. Rac1 can be directly deacetylated by SIRT1 in a cell-free system, leading to an inhibition of Rac1-p67phox interaction. The downstream targets of non-sirtuin deacetylases are still unknown. The in vivo significance of these findings needs to be investigated in future studies.

Global epidemiological studies show that chronic non-communicable diseases such as atherosclerosis and metabolic disorders represent the leading cause of premature mortality and morbidity. Cardiovascular disease such as ischemic heart disease is a major contributor to the global burden of disease and the socioeconomic health costs. Clinical and epidemiological data show an association of typical oxidative stress markers such as lipid peroxidation products, 3-nitrotyrosine or oxidized DNA/RNA bases with all major cardiovascular diseases. This supports the concept that the formation of reactive oxygen and nitrogen species by various sources (NADPH oxidases, xanthine oxidase and mitochondrial respiratory chain) represents a hallmark of the leading cardiovascular comorbidities such as hyperlipidemia, hypertension and diabetes. These reactive oxygen and nitrogen species can lead to oxidative damage but also adverse redox signaling at the level of kinases, calcium handling, inflammation, epigenetic control, circadian clock and proteasomal system. The in vivo footprints of these adverse processes (redox biomarkers) are discussed in the present review with focus on their clinical relevance, whereas the details of their mechanisms of formation and technical aspects of their detection are only briefly mentioned. The major categories of redox biomarkers are summarized and explained on the basis of suitable examples. Also the potential prognostic value of redox biomarkers is critically discussed to understand what kind of information they can provide but also what they cannot achieve.

Tetrahydrobiopterin (BH4) is an essential cofactor of all nitric oxide synthase isoforms, thus determination of BH4 levels can provide important mechanistic insight into diseases. We established a protocol for high-performance liquid chromatography/electrochemical detection (HPLC/ECD)-based determination of BH4 in tissue samples. We first determined the optimal storage and work-up conditions for authentic BH4 and its oxidation product dihydrobiopterin (BH2) under various conditions (pH, temperature, presence of antioxidants, metal chelators, and storage time). We then applied optimized protocols for detection of BH4 in tissues of septic (induced by lipopolysaccharide [LPS]) rats. BH4 standards in HCl are stabilized by addition of 1,4-dithioerythritol (DTE) and diethylenetriaminepentaacetic acid (DTPA), while HCl was sufficient for BH2 standard stabilization. Overnight storage of BH4 standard solutions at room temperature in HCl without antioxidants caused complete loss of BH4 and the formation of BH2. We further optimized the protocol to separate ascorbate and the BH4 tissue sample and found a significant increase in BH4 in the heart and kidney as well as higher BH4 levels by trend in the brain of septic rats compared to control rats. These findings correspond to reports on augmented nitric oxide and BH4 levels in both animals and patients with septic shock.

Aims: Cigarette smoking is one of the most complex and least understood cardiovascular risk factors. Importantly, differences in the tobacco-related pathophysiology of endothelial dysfunction, an early event in atherogenesis, between circulatory beds remain elusive. Therefore, this study evaluated how smoking impacts endothelial function of conduit and resistance arteries in a large population-based cohort.Methods and results: 15,010 participants (aged 35–74 years) of the Gutenberg Health Study were examined at baseline from 2007 to 2012. Smoking status, pack-years of smoking, and years since quitting smoking were assessed by a computer-assisted interview. Endothelial function of conduit and resistance arteries was determined by flow-mediated dilation (FMD) of the brachial artery, reactive hyperemia index (RHI) using peripheral arterial tonometry, as well as by reflection index (RI) derived from digital photoplethysmography, respectively. Among all subjects, 45.8% had never smoked, 34.7% were former smokers, and 19.4% were current smokers. Mean cumulative smoking exposure was 22.1 ± 18.1 pack-years in current smokers and mean years since quitting was 18.9 ± 12.7 in former smokers. In multivariable linear regression models adjusted for typical confounders, smoking status, pack-years of smoking, and years since quitting smoking were independently associated with RHI and RI, while no association was found for FMD. Overall, no clear dose-dependent associations were observed between variables, whereby higher exposure tended to be associated with pronounced resistance artery endothelial dysfunction.Conclusions: Cigarette smoking is associated with altered endothelial function of resistance, but not conduit arteries. The present results suggest that smoking-induced endothelial dysfunction in different circulatory beds may exhibit a differential picture.

This article contains supporting information on data collection for the research article entitled “Aircraft noise exposure drives the activation of white blood cells and induces microvascular dysfunction in mice” by Eckrich et al. We found that noise-induced stress triggered microvascular dysfunction via involvement of innate immune-derived reactive oxygen species. In this article, we present the instrumentation of mice with dorsal skinfold chambers for in vivo microscopic imaging of blood flow, interaction of leukocytes with the vascular wall (also by fluorescent labelling of blood cells) and vessel diameter. In addition, we explain the preparation of cerebral arterioles for measurement of vascular reactivity in vitro. • visualization of noise-dependent effects in dorsal skinfold chamber. • in vivo microscopy of noise-dependent activation of white blood cells. • analysis of noise-dependent microvascular dysfunction in dorsal skinfold chamber and cannulated cerebral arterioles.

The prevalence and clinical importance of arterial hypertension are still growing. Inorganic nitrite (NO2−) represents an attractive dietary antihypertensive agent, but its metabolism and mode of action, which we aimed to investigate with the present study, are not completely understood. Isolated aortic rings from rats were treated ex vivo with oxidants, and rats were infused in vivo with angiotensin-II. Vascular responses to acetylcholine (ACh) and nitrite were assessed by isometric tension recording. The loss of vasodilatory potency in response to oxidants was much more pronounced for ACh as compared to nitrite ex vivo (but not in vivo with angiotensin-II). This effect may be caused by the redox regulation of conversion to xanthine oxidase (XO). Conventionally raised and germ-free mice were treated with nitrite by gavage, which did not improve ACh-mediated vasodilation, but did increase the plasma levels of S-nitros(yl)ated proteins in the conventionally-raised, but not in the germ-free mice. In conclusion, inorganic nitrite represents a dietary drug option to treat arterial hypertension in addition to already established pharmacological treatment. Short-term oxidative stress did not impair the vasodilatory properties of nitrite, which may be beneficial in cardiovascular disease patients. The gastrointestinal microbiome appears to play a key role in nitrite metabolism and bioactivation.

Arterial hypertension is the most important risk factor for the development of cardiovascular disease. Recently, aircraft noise has been shown to be associated with elevated blood pressure, endothelial dysfunction, and oxidative stress. Here, we investigated the potential exacerbated cardiovascular effects of aircraft noise in combination with experimental arterial hypertension. C57BL/6J mice were infused with 0.5 mg/kg/d of angiotensin II for 7 days, exposed to aircraft noise for 7 days at a maximum sound pressure level of 85 dB(A) and a mean sound pressure level of 72 dB(A), or subjected to both stressors. Noise and angiotensin II increased blood pressure, endothelial dysfunction, oxidative stress and inflammation in aortic, cardiac and/or cerebral tissues in single exposure models. In mice subjected to both stressors, most of these risk factors showed potentiated adverse changes. We also found that mice exposed to both noise and ATII had increased phagocytic NADPH oxidase (NOX-2)-mediated superoxide formation, immune cell infiltration (monocytes, neutrophils and T cells) in the aortic wall, astrocyte activation in the brain, enhanced cytokine signaling, and subsequent vascular and cerebral oxidative stress. Exaggerated renal stress response was also observed. In summary, our results show an enhanced adverse cardiovascular effect between environmental noise exposure and arterial hypertension, which is mainly triggered by vascular inflammation and oxidative stress. Mechanistically, noise potentiates neuroinflammation and cerebral oxidative stress, which may be a potential link between both risk factors. The results indicate that a combination of classical (arterial hypertension) and novel (noise exposure) risk factors may be deleterious for cardiovascular health.

Environmental pollution and non-chemical stressors such as mental stress or traffic noise exposure are increasingly accepted as health risk factors with substantial contribution to chronic noncommunicable diseases (e.g. cardiovascular, metabolic and mental). Whereas the mechanisms of air pollution-mediated adverse health effects are well characterized, the mechanisms of traffic noise exposure are not completely understood, despite convincing clinical and epidemiological evidence for a significant contribution of environmental noise to overall mortality and disability. The initial mechanism of noise-induced cardiovascular, metabolic and mental disease is well defined by the „noise reaction model“ and consists of neuronal activation involving the hypothalamic-pituitary-adrenal (HPA) axis as well as the sympathetic nervous system, followed by a classical stress response via cortisol and catecholamines. Stress pathways are initiated by noise-induced annoyance and sleep deprivation/fragmentation. This review highlights the down-stream pathophysiology of noise-induced mental stress, which is based on an induction of inflammation and oxidative stress. We highlight the sources of reactive oxygen species (ROS) involved and the known targets for noise-induced oxidative damage. Part of the review emphasizes noise-triggered uncoupling/dysregulation of endothelial and neuronal nitric oxide synthase (eNOS and nNOS) and its central role for vascular dysfunction.

AimsCurrent antihypertensive therapies cannot cure hypertension and a life-long medication is necessary. Maternal treatment may represent a promising strategy for hypertension treatment. We have previously shown that maternal treatment of spontaneously hypertensive rats (SHR) with pentaerythritol tetranitrate (PETN) leads to a persistent blood pressure reduction in the female offspring. The underlying mechanisms include improved endothelial function resulting from long-lasting epigenetic changes. In the present study, we address the renal effects of maternal PETN treatment.Methods and ResultsF0 parental SHR were fed with either normal chow or PETN-containing (1 g/kg) chow ad libitum from the time point of mating to the end of lactation period. The F1 offspring received normal chow without PETN from the time point of weaning (at the age of 3 weeks). At the age of 16 weeks, female PETN offspring showed lower blood pressure than the control. No difference was observed in male offspring. All following experiments were performed with kidneys from 16-week-old female offspring. Maternal PETN treatment reduced the mRNA and protein expression of angiotensin-converting enzyme (ACE) and basic fibroblast growth factor (FGF2), resulting from epigenetic modifications found at the proximal promoter regions. The expression levels of mineralocorticoid receptor (MR) and factors in the MR signalling pathway (Rac1 and Sgk1) were also reduced by PETN. Major profibrotic cytokines, including Wnt4, TNF-alpha, TGF-beta, and MMP9, were downregulated by PETN, which was associated with reduced collagen deposition and glomerulus sclerosis in the kidney of PETN offspring. In addition, PETN treatment also decreased the markers of inflammation and immune cell infiltration in the kidneys.ConclusionsPETN maternal treatment leads to epigenetic changes in the kidney of female SHR offspring. The reduced renal inflammation, alleviated kidney fibrosis, and decreased MR signalling are potential mechanisms contributing to the observed blood pressure-lowering effect.

Voluntary exercise training is an effective way to prevent cardiovascular disease, since it results in increased NO bioavailability and decreased reactive oxygen species (ROS) production. AMP-activated protein kinase (AMPK), especially its α1AMPK subunit, modulates ROS-dependent vascular homeostasis. Since endothelial cells play an important role in exercise-induced changes of vascular signaling, we examined the consequences of endothelial-specific α1AMPK deletion during voluntary exercise training. We generated a mouse strain with specific deletion of α1AMPK in endothelial cells (α1AMPKflox/flox x TekCre+). While voluntary exercise training improved endothelial function in wild-type mice, it had deleterious effects in mice lacking endothelial α1AMPK indicated by elevated reactive oxygen species production (measured by dihydroethidum fluorescence and 3-nitrotyrosine staining), eNOS uncoupling and endothelial dysfunction. Importantly, the expression of the phagocytic NADPH oxidase isoform (NOX-2) was down-regulated by exercise in control mice, whereas it was up-regulated in exercising α1AMPKflox/flox x TekCre+ animals. In addition, nitric oxide bioavailability was decreased and the antioxidant/protective nuclear factor erythroid 2-related factor 2 (Nrf-2) response via heme oxygenase 1 and uncoupling protein-2 (UCP-2) was impaired in exercising α1AMPKflox/flox x TekCre+ mice. Our results demonstrate that endothelial α1AMPK is a critical component of the signaling events that enable vascular protection in response to exercise. Moreover, they identify endothelial α1AMPK as a master switch that determines whether the effects of exercise on the vasculature are protective or detrimental.

Both exposure to higher levels of polluted air and physical inactivity are crucial risk factors for the development and progression of major noncommunicable diseases and, in particular, of cardiovascular disease. In this context, the World Health Organization estimated 4.2 and 3.2 million global deaths per year in response to ambient air pollution and insufficient physical activity, respectively. While regular physical activity is well known to improve general health, it may also increase the uptake and deposit of air pollutants in the lungs/airways and circulation, due to increased breathing frequency and minute ventilation, thus increasing the risk of cardiovascular disease. Thus, determining the tradeoff between the health benefits of physical activity and the potential harmful effects of increased exposure to air pollution during physical activity has important public health consequences. In the present comprehensive review, we analyzed evidence from human and animal studies on the combined effects of physical activity and air pollution on cardiovascular and other health outcomes. We further report on pathophysiological mechanisms underlying air pollution exposure, as well as the protective effects of physical activity with a focus on oxidative stress and inflammation. Lastly, we provide mitigation strategies and practical recommendations for physical activity in areas with polluted air.

B lymphocytes have been implicated in the development of insulin resistance, atherosclerosis and certain types of hypertension. In contrast to these studies, which were performed under pathological conditions, the present study provides evidence for the protective effect of B lymphocytes in maintaining vascular homeostasis under physiological conditions. In young mice not exposed to any known risk factors, the lack of B cells led to massive endothelial dysfunction. The vascular dysfunction in B cell-deficient mice was associated with an increased number of neutrophils in the circulating blood. Neutrophil depletion in B cell-deficient mice resulted in the complete normalization of vascular function, indicating a causal role of neutrophilia. Moreover, vascular function in B cell-deficient mice could be restored by adoptive transfer of naive B-1 cells isolated from wild-type mice. Interestingly, B-1 cell transfer also reduced the number of neutrophils in the recipient mice, further supporting the involvement of neutrophils in the vascular pathology caused by B cell-deficiency. In conclusion, we report in the present study the hitherto undescribed role of B lymphocytes in regulating vascular function. B cell dysregulation may represent a crucial mechanism in vascular pathology.

Ischemia/reperfusion (I/R) events are involved in the pathophysiology of numerous ocular diseases. The purpose of this study was to test the hypothesis that betulinic acid protects from I/R injury in the mouse retina. Ocular ischemia was induced in mice by increasing intraocular pressure (IOP) to 110 mm Hg for 45 min, while the fellow eye served as a control. One group of mice received betulinic acid (50 mg/kg/day p.o. once daily) and the other group received the vehicle solution only. Eight days after the I/R event, the animals were killed and the retinal wholemounts and optic nerve cross-sections were prepared and stained with cresyl blue or toluidine blue, respectively, to count cells in the ganglion cell layer (GCL) of the retina and axons in the optic nerve. Retinal arteriole responses were measured in isolated retinas by video microscopy. The levels of reactive oxygen species (ROS) were assessed in retinal cryosections and redox gene expression was determined in isolated retinas by quantitative PCR. I/R markedly reduced cell number in the GCL and axon number in the optic nerve of the vehicle-treated mice. In contrast, only a negligible reduction in cell and axon number was observed following I/R in the betulinic acid-treated mice. Endothelial function was markedly reduced and ROS levels were increased in retinal arterioles of vehicle-exposed eyes following I/R, whereas betulinic acid partially prevented vascular endothelial dysfunction and ROS formation. Moreover, betulinic acid boosted mRNA expression for the antioxidant enzymes SOD3 and HO-1 following I/R. Our data provide evidence that betulinic acid protects from I/R injury in the mouse retina. Improvement of vascular endothelial function and the reduction in ROS levels appear to contribute to the neuroprotective effect.

Angiotensin II (Ang II) has been implicated in the pathophysiology of various age-dependent ocular diseases. The purpose of this study was to test the hypothesis that Ang II induces endothelial dysfunction in mouse ophthalmic arteries and to identify the underlying mechanisms. Ophthalmic arteries were exposed to Ang II in vivo and in vitro to determine vascular function by video microscopy. Moreover, the formation of reactive oxygen species (ROS) was quantified and the expression of prooxidant redox genes and proteins was determined. The endothelium-dependent artery responses were blunted after both in vivo and in vitro exposure to Ang II. The Ang II type 1 receptor (AT1R) blocker, candesartan, and the ROS scavenger, Tiron, prevented Ang II-induced endothelial dysfunction. ROS levels and NOX2 expression were increased following Ang II incubation. Remarkably, Ang II failed to induce endothelial dysfunction in ophthalmic arteries from NOX2-deficient mice. Following Ang II incubation, endothelium-dependent vasodilation was mainly mediated by cytochrome P450 oxygenase (CYP450) metabolites, while the contribution of nitric oxide synthase (NOS) and 12/15-lipoxygenase (12/15-LOX) pathways became negligible. These findings provide evidence that Ang II induces endothelial dysfunction in mouse ophthalmic arteries via AT1R activation and NOX2-dependent ROS formation. From a clinical point of view, the blockade of AT1R signaling and/or NOX2 may be helpful to retain or restore endothelial function in ocular blood vessels in certain ocular diseases.

Sepsis causes high mortality in the setting of septic shock. LEADER and other trials revealed cardioprotective and anti-inflammatory properties of glucagon-like peptide-1 (GLP-1) analogs like liraglutide (Lira). We previously demonstrated improved survival in lipopolysaccharide (LPS)-induced endotoxemia by inhibition of GLP-1 degradation. Here we investigate the effects of Lira in the polymicrobial sepsis model of cecal ligation and puncture (CLP). C57BL/6J mice were intraperitoneally injected with Lira (200 µg/kg/d; 3 days) and sepsis induced by CLP after one day of GLP-1 analog treatment. Survival and body temperature were monitored. Aortic vascular function (isometric tension recording), protein expression (immunohistochemistry and dot blot) and gene expression (qRT-PCR) were determined. Endothelium-dependent relaxation in the aorta was impaired by CLP and correlated with markers of inflammation (e.g., interleukin 6 and inducible nitric oxide synthase) and oxidative stress (e.g., 3-nitrotyrosine) was higher in septic mice, all of which was almost completely normalized by Lira therapy. We demonstrate that the GLP-1 analog Lira ameliorates sepsis-induced endothelial dysfunction by the reduction of vascular inflammation and oxidative stress. Accordingly, the findings suggest that the antioxidant and anti-inflammatory effects of GLP-1 analogs may be a valuable tool to protect the cardiovascular system from dysbalanced inflammation in polymicrobial sepsis.

Hyperglycemia associated with inflammation and oxidative stress is a major cause of vascular dysfunction and cardiovascular disease in diabetes. Recent data reports that a selective sodium-glucose co-transporter 2 inhibitor (SGLT2i), empagliflozin (Jardiance®), ameliorates glucotoxicity via excretion of excess glucose in urine (glucosuria) and significantly improves cardiovascular mortality in type 2 diabetes mellitus (T2DM). The overarching hypothesis is that hyperglycemia and glucotoxicity are upstream of all other complications seen in diabetes. The aim of this study was to investigate effects of empagliflozin on glucotoxicity, β-cell function, inflammation, oxidative stress and endothelial dysfunction in Zucker diabetic fatty (ZDF) rats. Male ZDF rats were used as a model of T2DM (35 diabetic ZDF‐Leprfa/fa and 16 ZDF-Lepr+/+ controls). Empagliflozin (10 and 30 mg/kg/d) was administered via drinking water for 6 weeks. Treatment with empagliflozin restored glycemic control. Empagliflozin improved endothelial function (thoracic aorta) and reduced oxidative stress in the aorta and in blood of diabetic rats. Inflammation and glucotoxicity (AGE/RAGE signaling) were epigenetically prevented by SGLT2i treatment (ChIP). Linear regression analysis revealed a significant inverse correlation of endothelial function with HbA1c, whereas leukocyte-dependent oxidative burst and C-reactive protein (CRP) were positively correlated with HbA1c. Viability of hyperglycemic endothelial cells was pleiotropically improved by SGLT2i. Empagliflozin reduces glucotoxicity and thereby prevents the development of endothelial dysfunction, reduces oxidative stress and exhibits anti-inflammatory effects in ZDF rats, despite persisting hyperlipidemia and hyperinsulinemia. Our preclinical observations provide insights into the mechanisms by which empagliflozin reduces cardiovascular mortality in humans (EMPA-REG trial).

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed.

Reactive oxygen and nitrogen species (ROS and RNS, e.g. H2O2, nitric oxide) confer redox regulation of essential cellular signaling pathways such as cell differentiation, proliferation, migration and apoptosis. At higher concentrations, ROS and RNS lead to oxidative stress and oxidative damage of biomolecules (e.g. via formation of peroxynitrite, fenton chemistry). Peripheral artery disease (PAD) is characterized by severe ischemic conditions in the periphery leading to intermittent claudication and critical limb ischemia (end stage). It is well known that redox biology and oxidative stress play an important role in this setting. We here discuss the major pathways of oxidative stress and redox signaling underlying the disease progression with special emphasis on the contribution of inflammatory processes. We also highlight therapeutic strategies comprising pharmacological (e.g. statins, angiotensin-converting enzyme inhibitors, phosphodiesterase inhibition) and non-pharmacological (e.g. exercise) interventions. Both of these strategies induce potent indirect antioxidant and anti-inflammatory mechanisms that may contribute to an improvement of PAD associated complications and disease progression by removing excess formation of ROS and RNS (e.g. by ameliorating primary complications such as hyperlipidemia and hypertension) as well as the normalization of the inflammatory phenotype suppressing the progression of atherosclerosis. Keywords: Oxidative stress, Redox signaling, Peripheral artery (occlusive) disease, Claudication and critical limb ischemia, Walking distance, Antioxidant therapy

Reactive oxygen and nitrogen species (RONS such as H2O2, nitric oxide) confer redox regulation of essential cellular functions (e.g. differentiation, proliferation, migration, apoptosis), initiate and catalyze adaptive stress responses. In contrast, excessive formation of RONS caused by impaired break-down by cellular antioxidant systems and/or insufficient repair of the resulting oxidative damage of biomolecules may lead to appreciable impairment of cellular function and in the worst case to cell death, organ dysfunction and severe disease phenotypes of the entire organism. Therefore, the knowledge of the severity of oxidative stress and tissue specific localization is of great biological and clinical importance. However, at this level of investigation quantitative information may be enough. For the development of specific drugs, the cellular and subcellular localization of the sources of RONS or even the nature of the reactive species may be of great importance, and accordingly, more qualitative information is required. These two different philosophies currently compete with each other and their different needs (also reflected by different detection assays) often lead to controversial discussions within the redox research community. With the present review we want to shed some light on these different philosophies and needs (based on our personal views), but also to defend some of the traditional assays for the detection of RONS that work very well in our hands and to provide some guidelines how to use and interpret the results of these assays. We will also provide an overview on the “new assays” with a brief discussion on their strengths but also weaknesses and limitations. Keywords: Oxidative stress, Redox signaling, Fluorescence and chemiluminescence-based assays, Dihydroethidium oxidative fluorescence microtopography, Lucigenin-enhanced chemiluminescence, L-012-enhanced chemiluminescence

Tyrosine nitration is a post-translational protein modification relevant to various pathophysiological processes. Chemical nitration procedures have been used to generate and study nitrated proteins, but these methods regularly lead to modifications at other amino acid residues. A novel strategy employs a genetic code modification that allows incorporation of 3-nitrotyrosine (3-NT) during ribosomal protein synthesis to generate a recombinant protein with defined 3-NT-sites, in the absence of other post-translational modifications. This approach was applied to study the generation and stability of the 3-NT moiety in recombinant proteins produced in E.coli. Nitrated alpha-synuclein (ASYN) was selected as exemplary protein, relevant in Parkinson's disease (PD). A procedure was established to obtain pure tyrosine-modified ASYN in mg amounts. However, a rapid (t1/2 = 0.4 h) reduction of 3-NT to 3-aminotyrosine (3-AT) was observed. When screening for potential mechanisms, we found that 3-NT can be reduced enzymatically to 3-AT, whilst biologically relevant low molecular weight reductants, such as NADPH or GSH, did not affect 3-NT. A genetic screen for E.coli proteins, involved in the observed 3-NT reduction, revealed the contribution of several, possibly redundant pathways. Green fluorescent protein was studied as an alternative model protein. These data confirm 3-NT reduction as a broadly-relevant pathway in E.coli. In conclusion, incorporation of 3-NT as a genetically-encoded non-natural amino acid allows for generation of recombinant proteins with specific nitration sites. The potential reduction of the 3-NT moiety by E.coli, however, requires attention to the design of the purification strategy for obtaining pure nitrated protein. Keywords: Alpha synuclein, Nitration, 3-Nitrotyrosine, 3-Aminotyrosine, E.coli

While the importance of the iron-load of lipocalin-2 (Lcn-2) in promoting tumor progression is widely appreciated, underlying molecular mechanisms largely remain elusive. Considering its role as an iron-transporter, we aimed at clarifying iron-loaded, holo-Lcn-2 (hLcn-2)-dependent signaling pathways in affecting renal cancer cell viability. Applying RNA sequencing analysis in renal CAKI1 tumor cells to explore highly upregulated molecular signatures in response to hLcn-2, we identified a cluster of genes (SLC7A11, GCLM, GLS), which are implicated in regulating ferroptosis. Indeed, hLcn-2-stimulated cells are protected from erastin-induced ferroptosis. We also noticed a rapid increase in reactive oxygen species (ROS) with subsequent activation of the antioxidant Nrf2 pathway. However, knocking down Nrf2 by siRNA was not sufficient to induce erastin-dependent ferroptotic cell death in hLcn-2-stimulated tumor cells. In contrast, preventing oxidative stress through N-acetyl-l-cysteine (NAC) supplementation was still able to induce erastin-dependent ferroptotic cell death in hLcn-2-stimulated tumor cells. Besides an oxidative stress response, we noticed activation of the integrated stress response (ISR), shown by enhanced phosphorylation of eIF-2α and induction of ATF4 after hLcn-2 addition. ATF4 knockdown as well as inhibition of the ISR sensitized hLcn-2-treated renal tumor cells to ferroptosis, thus linking the ISR to pro-tumor characteristics of hLcn-2. Our study provides mechanistic details to better understand tumor pro-survival pathways initiated by iron-loaded Lcn-2.

Vascular oxidative stress, inflammation, and subsequent endothelial dysfunction are consequences of traditional cardiovascular risk factors, all of which contribute to cardiovascular disease. Environmental stressors, such as traffic noise and air pollution, may also facilitate the development and progression of cardiovascular and metabolic diseases. In our previous studies, we investigated the influence of aircraft noise exposure on molecular mechanisms, identifying oxidative stress and inflammation as central players in mediating vascular function. The present study investigates the role of heme oxygenase-1 (HO-1) as an antioxidant response preventing vascular consequences following exposure to aircraft noise. C57BL/6J mice were treated with the HO-1 inducer hemin (25 mg/kg i.p.) or the NRF2 activator dimethyl fumarate (DMF, 20 mg/kg p.o.). During therapy, the animals were exposed to noise at a maximum sound pressure level of 85 dB(A) and a mean sound pressure level of 72 dB(A). Our data showed a marked protective effect of both treatments on animals exposed to noise for 4 days by normalization of arterial hypertension and vascular dysfunction in the noise-exposed groups. We observed a partial normalization of noise-triggered oxidative stress and inflammation by hemin and DMF therapy, which was associated with HO-1 induction. The present study identifies possible new targets for the mitigation of the adverse health effects caused by environmental noise exposure. Since natural dietary constituents can achieve HO-1 and NRF2 induction, these pathways represent promising targets for preventive measures.

There has been some dispute regarding reaction products formed at physiological peroxynitrite fluxes in the nanomolar range with phenolic molecules, when used to predict the behavior of protein-bound aromatic amino acids like tyrosine. Previous data showed that at nanomolar fluxes of peroxynitrite, nitration of these phenolic compounds was outcompeted by dimerization (e.g. biphenols or dityrosine). Using 3-morpholino sydnonimine (Sin-1), we created low fluxes of peroxynitrite in our reaction set-up to demonstrate that salicylaldehyde displays unique features in the detection of physiological fluxes of peroxynitrite, yielding detectable nitration but only minor dimerization products. By means of HPLC analysis and detection at 380 nm we could identify the expected nitration products 3- and 5-nitrosalicylaldehyde, but also novel nitrated products. Using mass spectrometry, we also identified 2-nitrophenol and a not fully characterized nitrated dimerization product. The formation of 2-nitrophenol could proceed either by primary generation of a phenoxy radical, followed by addition of the NO2-radical to the various resonance structures, or by addition of the peroxynitrite anion to the polarized carbonyl group with subsequent fragmentation of the adduct (as seen with carbon dioxide). Interestingly, we observed almost no 3- and 5-nitrosalicylic acid products and only minor dimerization reaction. Our results disagree with the previous general assumption that nitration of low molecular weight phenolic compounds is always outcompeted by dimerization at nanomolar peroxynitrite fluxes and highlight unique features of salicylaldehyde as a probe for physiological concentrations of peroxynitrite.

Cardiovascular diseases represent the leading cause of global deaths and life years spent with a severe disability. Endothelial dysfunction and vascular oxidative stress are early precursors of atherosclerotic processes in the vascular wall, all of which are hallmarks in the development of cardiovascular diseases and predictors of future cardiovascular events. There is growing evidence that inflammatory processes represent a major trigger for endothelial dysfunction, vascular oxidative stress and atherosclerosis and clinical data identified inflammation as a cardiovascular risk factor on its own. AMP-activated protein kinase (AMPK) is a central enzyme of cellular energy balance and metabolism that has been shown to confer cardio-protection and antioxidant defense which thereby contributes to vascular health. Interestingly, AMPK is also redox-regulated itself. We have previously shown that AMPK largely contributes to a healthy endothelium, confers potent antioxidant effects and prevents arterial hypertension. Recently, we provided deep mechanistic insights into the role of AMPK in cardiovascular protection and redox homeostasis by studies on arterial hypertension in endothelial and myelomonocytic cell-specific AMPK knockout (Cadh5CrexAMPKfl/fl and LysMCrexAMPKfl/fl) mice. Using these cell-specific knockout mice, we revealed the potent anti-inflammatory properties of AMPK representing the molecular basis of the antihypertensive effects of AMPK. Here, we discuss our own findings in the context of literature data with respect to the anti-inflammatory and antioxidant effects of AMPK in the specific setting of arterial hypertension as well as cardiovascular diseases in general.

Reactive oxygen and nitrogen species (RONS) cause oxidative damage, which is associated with endothelial dysfunction and cardiovascular disease, but may also contribute to redox signaling. Therefore, their precise detection is important for the evaluation of disease mechanisms. Here, we compared three different methods for the detection of 3-nitrotyrosine (3-NT), a marker of nitro-oxidative stress, in biological samples. Nitrated proteins were generated by incubation with peroxynitrite or 3-morpholino sydnonimine (Sin-1) and subjected to total hydrolysis using pronase, a mixture of different proteases. The 3-NT was then separated by high performance liquid chromatography (HPLC) and quantified by electrochemical detection (ECD, CoulArray) and compared to classical methods, namely enzyme-linked immunosorbent assay (ELISA) and dot blot analysis using specific 3-NT antibodies. Calibration curves for authentic 3-NT (detection limit 10 nM) and a concentration-response pattern for 3-NT obtained from digested nitrated bovine serum albumin (BSA) were highly linear over a wide 3-NT concentration range. Also, ex vivo nitration of protein from heart, isolated mitochondria, and serum/plasma could be quantified using the HPLC/ECD method and was confirmed by LC-MS/MS. Of note, nitro-oxidative damage of mitochondria results in increased superoxide (O2•–) formation rates (measured by dihydroethidium-based HPLC assay), pointing to a self-amplification mechanism of oxidative stress. Based on our ex vivo data, the CoulArray quantification method for 3-NT seems to have some advantages regarding sensitivity and selectivity. Establishing a reliable automated HPLC assay for the routine quantification of 3-NT in biological samples of cell culture, of animal and human origin seems to be more sophisticated than expected.

Reactive oxygen and nitrogen species (e.g. H2O2, nitric oxide) confer redox regulation of essential cellular signaling pathways such as cell differentiation, proliferation, migration and apoptosis. In addition, classical regulation of gene expression or activity, including gene transcription to RNA followed by translation to the protein level, by transcription factors (e.g. NF-κB, HIF-1α) and mRNA binding proteins (e.g. GAPDH, HuR) is subject to redox regulation. This review will give an update of recent discoveries in this field, and specifically highlight the impact of reactive oxygen and nitrogen species on DNA repair systems that contribute to genomic stability. Emphasis will be placed on the emerging role of redox mechanisms regulating epigenetic pathways (e.g. miRNA, DNA methylation and histone modifications). By providing clinical correlations we discuss how oxidative stress can impact on gene regulation/activity and vise versa, how epigenetic processes, other gene regulatory mechanisms and DNA repair can influence the cellular redox state and contribute or prevent development or progression of disease. Keywords: Oxidative stress, Redox signaling, Gene regulation, Epigenetics, DNA repair