Effects of aircraft noise cessation on blood pressure, cardio- and cerebrovascular endothelial function, oxidative stress, and inflammation in an experimental animal model.

Large epidemiological studies have shown that traffic noise promotes the development of cardiometabolic diseases. It remains to be established how long these adverse effects of noise may persist in response to a noise-off period. We investigated the effects of acute aircraft noise exposure (mean sound level of 72 dB(A) applied for 4d) on oxidative stress and inflammation mediating vascular dysfunction and increased blood pressure in male C57BL/6 J mice. 1, 2 or 4d of noise cessation after a 4d continuous noise exposure period completely normalized noise-induced endothelial dysfunction of the aorta (measured by acetylcholine-dependent relaxation) already after a 1d noise pause. Vascular oxidative stress and the increased blood pressure were partially corrected, while markers of inflammation (VCAM-1, IL-6 and leukocyte oxidative burst) showed a normalization within 4d of noise cessation. In contrast, endothelial dysfunction, oxidative stress, and inflammation of the cerebral microvessels of noise-exposed mice did not improve at all. These data demonstrate that the recovery from noise-induced damage is more complex than expected demonstrating a complete restoration of large conductance vessel function but persistent endothelial dysfunction of the microcirculation. These findings also imply that longer noise pauses are required to completely reverse noise-induced vascular dysfunction including the resistance vessels.

Sexual Dimorphism in Cardiometabolic Diseases: The Role of AMPK.

Cardiovascular diseases (CVDs) are the leading cause of mortality and disability among both males and females. The risk of cardiovascular diseases is heightened by the presence of a risk factor cluster of metabolic syndrome, covering obesity and obesity-related cardiometabolic risk factors such as hypertension, glucose, and lipid metabolism dysregulation primarily. Sex hormones contribute to metabolic regulation and make women and men susceptible to obesity development in a different manner, which necessitates sex-specific management. Identifying crucial factors that protect the cardiovascular system is essential to enhance primary and secondary prevention of cardiovascular diseases and should be explicitly studied from the perspective of sex differences. It seems that AMP-dependent protein kinase (AMPK) may be such a factor since it has the protective role of AMPK in the cardiovascular system, has anti-diabetic properties, and is regulated by sex hormones. Those findings highlight the potential cardiometabolic benefits of AMPK, making it an essential factor to consider. Here, we review information about the cross-talk between AMPK and sex hormones as a critical point in cardiometabolic disease development and progression and a target for therapeutic intervention in human disease.

Mitigation of aircraft noise-induced vascular dysfunction and oxidative stress by exercise, fasting, and pharmacological α1AMPK activation: molecular proof of a protective key role of endothelial α1AMPK against environmental noise exposure.

AIMS: Environmental stressors such as traffic noise represent a global threat, accounting for 1.6 million healthy life years lost annually in Western Europe. Therefore, the noise-associated health side effects must be effectively prevented or mitigated. Non-pharmacological interventions such as physical activity or a balanced healthy diet are effective due to the activation of the adenosine monophosphate-activated protein kinase (α1AMPK). Here, we investigated for the first time in a murine model of aircraft noise-induced vascular dysfunction the potential protective role of α1AMPK activated via exercise, intermittent fasting, and pharmacological treatment. METHODS AND RESULTS: Wild-type (B6.Cg-Tg(Cdh5-cre)7Mlia/J) mice were exposed to aircraft noise [maximum sound pressure level of 85 dB(A), average sound pressure level of 72 dB(A)] for the last 4 days. The α1AMPK was stimulated by different protocols, including 5-aminoimidazole-4-carboxamide riboside application, voluntary exercise, and intermittent fasting. Four days of aircraft noise exposure produced significant endothelial dysfunction in wild-type mice aorta, mesenteric arteries, and retinal arterioles. This was associated with increased vascular oxidative stress and asymmetric dimethylarginine formation. The α1AMPK activation with all three approaches prevented endothelial dysfunction and vascular oxidative stress development, which was supported by RNA sequencing data. Endothelium-specific α1AMPK knockout markedly aggravated noise-induced vascular damage and caused a loss of mitigation effects by exercise or intermittent fasting. CONCLUSION: Our results demonstrate that endothelial-specific α1AMPK activation by pharmacological stimulation, exercise, and intermittent fasting effectively mitigates noise-induced cardiovascular damage. Future population-based studies need to clinically prove the concept of exercise/fasting-mediated mitigation of transportation noise-associated disease.

Traffic noise, e.g. from aircraft, significantly contributes to an increased risk of cardiovascular or metabolic diseases in the general population by brain-dependent stress reactions leading to higher levels of circulating stress hormones and vasoconstrictors, all of which cause hypertension, oxidative stress, and inflammation. With the present experimental studies, we provide for the first time molecular mechanisms responsible for successful noise mitigation: Physical exercise, intermittent fasting, and pharmacological activation of the adenosine monophosphate-activated protein kinase (AMPK), a metabolic master regulator protein, prevent cardiovascular damage caused by noise exposure, such as hypertension, endothelial dysfunction, and reactive oxygen species formation (e.g. free radicals) and inflammation.These beneficial mitigation manoeuvers are secondary to an activation of the endothelial AMPK, thereby mimicking the antidiabetic drug metformin.

Mechanistic Insights into Inorganic Nitrite-Mediated Vasodilation of Isolated Aortic Rings under Oxidative/Hypertensive Conditions and S-Nitros(yl)ation of Proteins in Germ-Free Mice.

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.

PGC1α Regulates the Endothelial Response to Fluid Shear Stress via Telomerase Reverse Transcriptase Control of Heme Oxygenase-1.

OBJECTIVE: Fluid shear stress (FSS) is known to mediate multiple phenotypic changes in the endothelium. Laminar FSS (undisturbed flow) is known to promote endothelial alignment to flow, which is key to stabilizing the endothelium and rendering it resistant to atherosclerosis and thrombosis. The molecular pathways responsible for endothelial responses to FSS are only partially understood. In this study, we determine the role of PGC1α (peroxisome proliferator gamma coactivator-1α)-TERT (telomerase reverse transcriptase)-HMOX1 (heme oxygenase-1) during shear stress in vitro and in vivo. Approach and Results: Here, we have identified PGC1α as a flow-responsive gene required for endothelial flow alignment in vitro and in vivo. Compared with oscillatory FSS (disturbed flow) or static conditions, laminar FSS (undisturbed flow) showed increased PGC1α expression and its transcriptional coactivation. PGC1α was required for laminar FSS-induced expression of TERT in vitro and in vivo via its association with ERRα(estrogen-related receptor alpha) and KLF (Kruppel-like factor)-4 on the TERT promoter. We found that TERT inhibition attenuated endothelial flow alignment, elongation, and nuclear polarization in response to laminar FSS in vitro and in vivo. Among the flow-responsive genes sensitive to TERT status, HMOX1 was required for endothelial alignment to laminar FSS. CONCLUSIONS: These data suggest an important role for a PGC1α-TERT-HMOX1 axis in the endothelial stabilization response to laminar FSS.

Comparison of three methods for in vivo quantification of glutathione in tissues of hypertensive rats.

Glutathione (γ-L-glutamyl-L-cysteinyl-glycine, GSH) is a tripeptide that is part of the antioxidant defense system and contributes to numerous redox-regulatory processes. In vivo, reduced GSH and oxidized glutathione disulfide (GSSG) are present in redox equilibrium and their ratio provides important information on the cellular redox state. Here, we compared three different methods for in vivo quantification of glutathione in tissues of hypertensive rats, an accepted animal model of oxidative stress. In the present study, we used hypertensive rats (infusion of 1 mg/kg/d angiotensin-II for 7 days) to determine the levels of reduced GSH and/or GSH/GSSG ratios in different tissue samples. We used an HPLC-based method with direct electrochemical detection (HPLC/ECD) and compared it with Ellman's reagent (DTNB) dependent derivatization of reduced GSH to the GS-NTB adduct and free NTB (UV/Vis HPLC) as well as with a commercial GSH/GSSG assay (Oxiselect). Whereas all three methods indicated overall a decreased redox state in hypertensive rats, the assays based on HPLC/ECD and DTNB derivatization provided the most significant differences. We applied a direct, fast and sensitive method for electrochemical GSH detection in tissues from hypertensive animals, and confirmed its reliability for in vivo measurements by head-to-head comparison with two other established assays. The HPLC/ECD but not DTNB and Oxiselect assays yielded quantitative GSH data but all three assays reflected nicely the qualitative redox changes and functional impairment in hypertensive rats. However, especially our GSH/GSSG values are lower than reported by others pointing to problems in the work-up protocol.

Lack of Endothelial α1AMPK Reverses the Vascular Protective Effects of Exercise by Causing eNOS Uncoupling.

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.

Doxorubicin induces wide-spread transcriptional changes in the myocardium of hearts distinguishing between mice with preserved and impaired cardiac function.

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.

Direct comparison of inorganic nitrite and nitrate on vascular dysfunction and oxidative damage in experimental arterial hypertension.

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.

Noise-Induced Vascular Dysfunction, Oxidative Stress, and Inflammation Are Improved by Pharmacological Modulation of the NRF2/HO-1 Axis.

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.

Detection of extracellular superoxide in isolated human immune cells and in an animal model of arterial hypertension using hydropropidine probe and HPLC analysis.

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.

Long-Term Effects of Aircraft Noise Exposure on Vascular Oxidative Stress, Endothelial Function and Blood Pressure: No Evidence for Adaptation or Tolerance Development.

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.

The AMP-Activated Protein Kinase Plays a Role in Antioxidant Defense and Regulation of Vascular Inflammation.

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.

Environmental aircraft noise aggravates oxidative DNA damage, granulocyte oxidative burst and nitrate resistance in Ogg1-/- mice.

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 the role of noise exposure as an independent cardiovascular risk factor. We characterised the underlying molecular mechanisms leading to noise-dependent adverse effects on the vasculature and myocardium in an animal model of aircraft noise exposure and identified oxidative stress and inflammation as central players in mediating vascular and cardiac dysfunction. Here, we studied the impact of noise-induced oxidative DNA damage on vascular function in DNA-repair deficient 8-oxoguanine glycosylase knockout (Ogg1-/-) mice.Methods and results: Noise exposure (peak sound levels of 85 and mean sound level of 72 dB(A) applied for 4d) caused oxidative DNA damage (8-oxoguanine) and enhanced NOX-2 expression in C57BL/6 mice with synergistic increases in Ogg1-/- mice (shown by immunohistochemistry). A similar pattern was found for oxidative burst of blood leukocytes and other markers of oxidative stress (4-hydroxynonenal, 3-nitrotyrosine) and inflammation (cyclooxygenase-2). We observed additive impairment of noise exposure and genetic Ogg1 deficiency on endothelium-independent relaxation (nitroglycerine), which may be due to exacerbated oxidative DNA damage leading to leukocyte activation and oxidative aldehyde dehydrogenase inhibition.Conclusions: The finding that chronic noise exposure causes oxidative DNA damage in mice is worrisome since these potential mutagenic lesions could contribute to cancer progression. Human field studies have to demonstrate whether oxidative DNA damage is also found in urban populations with high levels of noise exposure as recently shown for workers with high occupational noise exposure.

Exacerbation of adverse cardiovascular effects of aircraft noise in an animal model of arterial hypertension.

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.

Short-term e-cigarette vapour exposure causes vascular oxidative stress and dysfunction: evidence for a close connection to brain damage and a key role of the phagocytic NADPH oxidase (NOX-2).

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.

Comparison of Mitochondrial Superoxide Detection Ex Vivo/In Vivo by mitoSOX HPLC Method with Classical Assays in Three Different Animal Models of Oxidative Stress.

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.

Age-dependent effect of PPARγ agonist pioglitazone on kidney signaling in borderline hypertensive rats.

The peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor and nutrition factor which takes part in the cellular signaling by several agonists such as pioglitazone. PPARγ can serve as potential target in treatments of metabolic syndrome diseases and/or hypertension. In the present study we investigated the effects of pioglitazone, a PPARγ agonist, on hypertension development in young and adult borderline hypertensive rats (BHR). In renal signaling we observed connections between PPARγ and Nrf2, antioxidant in adult animals and differences between young and adult BHR in Nrf2-activated detoxificant outputs (NQO1, HO-1) and NO-synthases. Blood pressure in animals had been detected by cuff plethysmography, cell signaling in the kidney was studied by gene expression determination using qPCR, and nitric oxide synthase (NOS) activity was measured by radioactive detection. Pioglitazone treatment in adult BHR caused no detectable changes in antioxidant and detoxificant responses. The main effects were observed in blood pressure improvement, endothelial NOS expression and NOS activities in both young and adult BHR.

ADMA, homocysteine and redox status improvement affected by 7-nitroindazole in spontaneously hypertensive rats.

Inhibition of nitric oxide (NO) production can influence blood pressure regulation and increase hypertension. Asymmetric dimethylarginine, ADMA, an analogue of L-arginine, can inhibit NO synthesis, impair endothelial function, and is a risk marker of cardiovascular diseases. Homocysteine (Hcy) level affects oxidative stress production of reactive oxygen species (ROS) in hypertension and also influences changes in signaling and cell damage. The present study was focused on experimental effects of exogenous NOS inhibitors and their effect on ADMA, an endogenous NOS inhibitor, homocysteine and ROS production measured as reactive oxidative metabolites (ROM). We compared effects of the two potential exogenous NO-inhibitors: NG-nitro L-arginine methyl ester (L-NAME) and 7-nitroindazole (7-NI). Levels of ADMA, Hcy, ROM and total thiols (TTL) were not changed in the L-NAME group. With 7-NI administration, we observed unchanged NOS activity in the left ventricle and a pronounced decrease of ADMA and Hcy levels, accompanied by ROM over-production in plasma. TTL/ROM ratio was more favorable than in the L-NAME group. We observed that 7-NI, an exogenous NOinhibitor, can decrease and improve the levels of ADMA, Hcy, and ROM, and increase TTL/ROM ratio in the plasma of spontaneously hypertensive rats.

α1AMPK deletion in myelomonocytic cells induces a pro-inflammatory phenotype and enhances angiotensin II-induced vascular dysfunction.

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.