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Superoxide formation is a hallmark of cardiovascular disease with the involvement of different tissues and cell types. Identification of the cellular sources and subcellular localization of superoxide formation is important to understand the underlying disease pathomechanisms. In the present study, we used HPLC quantification of the superoxide-specific oxidation products of hydroethidine (HE or DHE) and its derivative hydropropidine (HPr+) for measurement of intra- and extracellular superoxide formation in isolated leukocytes and tissues of hypertensive rats. Superoxide generation by isolated leukocytes from human subjects as well as tissue samples of hypertensive rats (infusion of angiotensin-II for 7 days) was investigated using HPr+ and HE fluorescent probes with HPLC or plate reader detection. Both fluorescent dyes were used to test for intra- and extracellular superoxide formation using the supernatant or cell/tissue pellet for analysis. We demonstrate the correlation of impaired functional parameters (blood pressure, vascular function, and oxidative burst) and increased superoxide formation in different organ systems of hypertensive rats using the HPr+/HPLC method. In the cell model, the differences between HE and HPr+ and especially the advantage of the extracellular specificity of HPr+, due to its cell impermeability, became evident. Plate reader-based assays showed much higher background signal and were inferior to HPLC based methods. In conclusion, the HPr+/HPLC assay for superoxide determination is highly reliable in isolated immune cells and an animal model of arterial hypertension. In particular, the cell impermeability of HPr+ made it possible to differentiate between intra- and extracellular superoxide formation.

Background: Large epidemiological studies point towards a link between the incidence of arterial hypertension, ischaemic heart disease, metabolic disease and exposure to traffic noise, supporting t...

Aims Electronic (e)-cigarettes have been marketed as a ‘healthy’ alternative to traditional combustible cigarettes and as an effective method of smoking cessation. There are, however, a paucity of data to support these claims. In fact, e-cigarettes are implicated in endothelial dysfunction and oxidative stress in the vasculature and the lungs. The mechanisms underlying these side effects remain unclear. Here, we investigated the effects of e-cigarette vapour on vascular function in smokers and experimental animals to determine the underlying mechanisms. Methods and results Acute e-cigarette smoking produced a marked impairment of endothelial function in chronic smokers determined by flow-mediated dilation. In mice, e-cigarette vapour without nicotine had more detrimental effects on endothelial function, markers of oxidative stress, inflammation, and lipid peroxidation than vapour containing nicotine. These effects of e-cigarette vapour were largely absent in mice lacking phagocytic NADPH oxidase (NOX-2) or upon treatment with the endothelin receptor blocker macitentan or the FOXO3 activator bepridil. We also established that the e-cigarette product acrolein, a reactive aldehyde, recapitulated many of the NOX-2-dependent effects of e-cigarette vapour using in vitro blood vessel incubation. Conclusions E-cigarette vapour exposure increases vascular, cerebral, and pulmonary oxidative stress via a NOX-2-dependent mechanism. Our study identifies the toxic aldehyde acrolein as a key mediator of the observed adverse vascular consequences. Thus, e-cigarettes have the potential to induce marked adverse cardiovascular, pulmonary, and cerebrovascular consequences. Since e-cigarette use is increasing, particularly amongst youth, our data suggest that aggressive steps are warranted to limit their health risks.

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.

Arterial hypertension is one of the major health risk factors leading to coronary artery disease, stroke or peripheral artery disease. Dietary uptake of inorganic nitrite (NO2−) and nitrate (NO3−) via vegetables leads to enhanced vascular NO bioavailability and provides antihypertensive effects. The present study aims to understand the underlying vasoprotective effects of nutritional NO2− and NO3− co-therapy in mice with angiotensin-II (AT-II)-induced arterial hypertension. High-dose AT-II (1 mg/kg/d, 1w, s. c.) was used to induce arterial hypertension in male C57BL/6 mice. Additional inorganic nitrite (7.5 mg/kg/d, p. o.) or nitrate (150 mg/kg/d, p. o.) were administered via the drinking water. Blood pressure (tail-cuff method) and endothelial function (isometric tension) were determined. Oxidative stress and inflammation markers were quantified in aorta, heart, kidney and blood. Co-treatment with inorganic nitrite, but not with nitrate, normalized vascular function, oxidative stress markers and inflammatory pathways in AT-II treated mice. Of note, the highly beneficial effects of nitrite on all parameters and the less pronounced protection by nitrate, as seen by improvement of some parameters, were observed despite no significant increase in plasma nitrite levels by both therapies. Methemoglobin levels tended to be higher upon nitrite/nitrate treatment. Nutritional nitric oxide precursors represent a non-pharmacological treatment option for hypertension that could be applied to the general population (e.g. by eating certain vegetables). The more beneficial effects of inorganic nitrite may rely on superior NO bioactivation and stronger blood pressure lowering effects. Future large-scale clinical studies should investigate whether hypertension and cardiovascular outcome in general can be influenced by dietary inorganic nitrite therapy.

Aircraft noise induces vascular and cerebral inflammation and oxidative stress causing hypertension and cardiovascular/cerebral dysfunction. With the present studies, we sought to determine the role of myeloid cells in the vascular vs. cerebral consequences of exposure to aircraft noise. Toxin-mediated ablation of lysozyme M+ (LysM+) myeloid cells was performed in LysMCreiDTR mice carrying a cre-inducible diphtheria toxin receptor. In the last 4d of toxin treatment, the animals were exposed to noise at maximum and mean sound pressure levels of 85 and 72 dB(A), respectively. Flow cytometry analysis revealed accumulation of CD45+, CD11b+, F4/80+, and Ly6G−Ly6C+ cells in the aortas of noise-exposed mice, which was prevented by LysM+ cell ablation in the periphery, whereas brain infiltrates were even exacerbated upon ablation. Aircraft noise-induced increases in blood pressure and endothelial dysfunction of the aorta and retinal/mesenteric arterioles were almost completely normalized by ablation. Correspondingly, reactive oxygen species in the aorta, heart, and retinal/mesenteric vessels were attenuated in ablated noise-exposed mice, while microglial activation and abundance in the brain was greatly increased. Expression of phagocytic NADPH oxidase (NOX-2) and vascular cell adhesion molecule-1 (VCAM-1) mRNA in the aorta was reduced, while NFκB signaling appeared to be activated in the brain upon ablation. In sum, we show dissociation of cerebral and peripheral inflammatory reactions in response to aircraft noise after LysM+ cell ablation, wherein peripheral myeloid inflammatory cells represent a dominant part of the pathomechanism for noise stress-induced cardiovascular effects and their central nervous counterparts, microglia, as key mediators in stress responses.

Aims Immune cell function involves energy-dependent processes including growth, proliferation, and cytokine production. Since the AMP-activated protein kinase (AMPK) is a crucial regulator of intracellular energy homeostasis, its expression and activity may also affect innate and adaptive immune cell responses. Therefore, we aimed to investigate the consequences of α1AMPK deletion in myelomonocytic cells on vascular function, inflammation, and hypertension during chronic angiotensin II (ATII) treatment. Methods and results We generated a mouse strain with α1AMPK deletion in lysozyme M+ myelomonocytic cells. Compared to controls, chronic ATII infusion (1 mg/kg/day for 7 days) lead to increased vascular oxidative stress and aggravated endothelial dysfunction in LysM-Cre+ x α1AMPKfl/fl mice. This was accompanied by an increased aortic infiltration of CD11b+F4/80+ macrophages and enhanced pro-inflammatory cytokine release (tumour necrosis factor-alpha, interferon-gamma, and interleukin-6). Mechanistically, we found that increased expression of C-C chemokine receptor 2 (CCR2) in α1AMPK deficient myelomonocytic cells facilitated their recruitment to the vascular wall. In addition, expression of the ATII receptor type 1a and the oxidative burst was increased in these cells, indicating an increased susceptibility towards pro-oxidant stimuli. Conclusions In summary, α1AMPK deletion in myelomonocytic cells aggravates vascular oxidative stress and dysfunction by enhancing their recruitment to the vascular wall and increasing their susceptibility towards pro-oxidant stimuli. Our observations suggest that metabolic control in myelomonocytic cells has profound implications for their inflammatory phenotype and may trigger the development of vascular disease.

Aims Doxorubicin (DOX) is an important drug for the treatment of various tumor entities. However, the occurrence of heart failure limits its application. This study investigated differential gene expression profiles in the left and right ventricles of DOX treated mice with either preserved or impaired myocardial function. We provide new mechanistic insights into the pathophysiology of DOX-induced heart failure and have discovered pathways that counteract DOX-induced cardiotoxicity. Main methods We used in total 48 male mice and applied a chronic low dose DOX administration (5 mg/kg per injection, in total 20 mg/kg over 4 weeks) to induce heart failure. Echocardiographic parameters were evaluated one week after the final dose and mice were separated according to functional parameters into doxorubicin responding and non-responding animals. Post mortem, measurements of reactive oxygen species (ROS) and gene expression profiling was performed in separated right and left hearts. Key findings We detected significant ROS production in the left heart of the mice in response to DOX treatment, although interestingly, not in the right heart. We found that transcriptional changes differ between right and left heart correlating with the occurrence of myocardial dysfunction. Significance Doxorubicin induces changes in gene expression in the entire heart of animals without necessarily impairing cardiac function. We identified a set of transcripts that are associated with DOX cardiotoxicity. These might represent promising targets to ameliorate DOX-induced heart failure. Moreover, our results emphasize that parameters of left and right heart function should be evaluated during standardized echocardiography in patients undergoing DOX therapy.

Oxidative stress plays a key role for the development of cardiovascular, metabolic, and neurodegenerative disease. This concept has been proven by using the approach of genetic deletion of reactive oxygen and nitrogen species (RONS) producing, pro-oxidant enzymes as well as by the overexpression of RONS detoxifying, antioxidant enzymes leading to an amelioration of the severity of diseases. Vice versa, the development and progression of cardiovascular diseases is aggravated by overexpression of RONS producing enzymes as well as deletion of RONS detoxifying enzymes. We have previously identified cross talk mechanisms between different sources of RONS, which can amplify the oxidative stress-mediated damage. Here, the pathways and potential mechanisms leading to this cross talk are analyzed in detail and highlighted by selected examples from the current literature and own data including hypoxia, angiotensin II (AT-II)-induced hypertension, nitrate tolerance, aging, and others. The general concept of redox-based activation of RONS sources via “kindling radicals” and enzyme-specific “redox switches” as well as the interaction with redox-sensitive inflammatory pathways are discussed. Here, we present evidence for the existence of such cross talk mechanisms in the setting of diabetes and critically assess their contribution to the severity of diabetic complications.

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&bull, &ndash, ) 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.

Background: Reactive oxygen and nitrogen species (RONS such as H2O2, nitric oxide) are generated within the organism. Whereas physiological formation rates confer redox regulation of essential cellular functions and provide the basis for adaptive stress responses, their excessive formation contributes to impaired cellular function or even cell death, organ dysfunction and severe disease phenotypes of the entire organism. Therefore, quantification of RONS formation and knowledge of their tissue/cell/compartment-specific distribution is of great biological and clinical importance. Methods: Here, we used a high-performance/pressure liquid chromatography (HPLC) assay to quantify the superoxide-specific oxidation product of the mitochondria-targeted fluorescence dye triphenylphosphonium-linked hydroethidium (mitoSOX) in biochemical systems and three animal models with established oxidative stress. Type 1 diabetes (single injection of streptozotocin), hypertension (infusion of angiotensin-II for 7 days) and nitrate tolerance (infusion of nitroglycerin for 4 days) was induced in male Wistar rats. Results: The usefulness of mitoSOX/HPLC for quantification of mitochondrial superoxide was confirmed by xanthine oxidase activity as well as isolated stimulated rat heart mitochondria in the presence or absence of superoxide scavengers. Vascular function was assessed by isometric tension methodology and was impaired in the rat models of oxidative stress. Vascular dysfunction correlated with increased mitoSOX oxidation but also classical RONS detection assays as well as typical markers of oxidative stress. Conclusion: mitoSOX/HPLC represents a valid method for detection of mitochondrial superoxide formation in tissues of different animal disease models and correlates well with functional parameters and other markers of oxidative stress.

International audience; Mice with a global deletion of α1AMPK are characterized by endothelial dysfunction and NADPH oxidase subunit 2 (NOX-2)-mediated vascular oxidative stress. However, the underlying mechanisms are incompletely understood and may involve endothelial NOX-2 upregulation or facilitated vascular infiltration of phagocytic cells. Therefore, the current study was designed to investigate the vascular effects of chronic angiotensin II (AngII) infusion in mice with an endothelial-specific α1AMPK deletion. A mouse strain with endothelial-specific α1AMPK deletion was generated by breeding α1AMPKflox/flox mice with TekCre+ or Cadh5Cre+ mice. Chronic AngII infusion (0.5 mg/kg/day for 7day) caused mild endothelial dysfunction in wild-type mice that was significantly aggravated in endothelial α1AMPK knockout mice. Aortic NOX-2 and CD68 expression were increased, indicating that infiltrating leukocytes may significantly contribute to enhanced vascular oxidative stress. Flow cytometry revealed a higher abundance of aortic CD90.2+ T-cells, CD11b+F4/80+ macrophages and Ly6G-Ly6C+ monocytes. Vascular mRNA expression of monocyte chemoattractant protein 1, CCL5 and vascular cell adhesion molecule 1 was enhanced in AngII-infused mice lacking endothelial α1AMPK, facilitating the recruitment of inflammatory cells to the vessel wall. In addition, AngII-induced upregulation of cytoprotective heme oxygenase 1 (HO-1) was blunted in mice with endothelial α1AMPK deletion, compatible with an impaired antioxidant defense in these animals. In summary, endothelial expressed α1AMPK limits the recruitment of inflammatory cells to the vessel wall and maintains HO-1 mediated antioxidant defense. Both mechanisms reduce vascular oxidative damage and preserve endothelial function during chronic AngII treatment.

Cardiovascular disease is a leading cause of death and reduced quality of life, proven by the latest data of the Global Burden of Disease Study, and is only gaining in prevalence worldwide. Clinical trials have identified chronic inflammatory disorders as cardiovascular risks, and recent research has revealed a contribution by various inflammatory cells to vascular oxidative stress. Atherosclerosis and cardiovascular disease are closely associated with inflammation, probably due to the close interaction of inflammation with oxidative stress. Classical therapies for inflammatory disorders have demonstrated protective effects in various models of cardiovascular disease; especially established drugs with pleiotropic immunomodulatory properties have proven beneficial cardiovascular effects; normalization of oxidative stress seems to be a common feature of these therapies. The close link between inflammation and redox balance was also supported by reports on aggravated inflammatory phenotype in the absence of antioxidant defense proteins (e.g., superoxide dismutases, heme oxygenase-1, and glutathione peroxidases) or overexpression of reactive oxygen species producing enzymes (e.g., NADPH oxidases). The value of immunomodulation for the treatment of cardiovascular disease was recently supported by large-scale clinical trials demonstrating reduced cardiovascular mortality in patients with established atherosclerotic disease when treated by highly specific anti-inflammatory therapies (e.g., using monoclonal antibodies against cytokines). Modern antidiabetic cardiovascular drugs (e.g., SGLT2 inhibitors, DPP-4 inhibitors, and GLP-1 analogs) seem to share these immunomodulatory properties and display potent antioxidant effects, all of which may explain their successful lowering of cardiovascular risk.