Exhaled nitric oxide, nitrite/nitrate levels, allergy, rhinitis and asthma in the EGEA study.

Although interest in biomarkers in the nitrate-nitrite-NO pathway has recently increased, associations between nitrite (NO2(-)) and nitrate (NO3(-)), and asthma, allergic sensitisation and rhinitis remain unclear. The study aimed to evaluate the associations between NO2(-)/NO3(-) and exhaled fraction of nitric oxide (FeNO) levels with asthma, allergic sensitisation and rhinitis. Plasma and exhaled breath condensate (EBC) NO2(-)/NO3(-) and FeNO levels were measured in 523 adults of the French Epidemiological study on Genetics and Environment of Asthma. Allergic sensitisation was defined by a positive skin prick test for at least one aeroallergen. Subjects were classified as non-sensitised, sensitised and as having allergic rhinitis. Plasma NO2 (-)/NO3(-) level was unrelated to any disease phenotypes. EBC NO2(-)/NO3(-) level was unrelated to any asthma phenotypes. EBC NO2(-)/NO3(-) and FeNO levels were correlated in sensitised subjects only (r = 0.21 ± 0.10, p=0.01). EBC NO2(-)/NO3(-) and FeNO levels were higher in sensitised than in non-sensitised subjects (adjusted geometric mean (95% CI): 2.36 (1.96-2.84) versus 1.72 (1.38-2.14) µmol per mg proteins, p=0.008; and 18.3 (16.7-20.0) versus 14.8 (13.3-16.5) ppb, p=0.0006, respectively), with gradual relationships from sensitised subjects to those with allergic rhinitis (p<0.0001). Results suggest that EBC NO2(-)/NO3(-) and FeNO levels may be considered as biological markers of intensity of allergic sensitisation and rhinitis.

Plasma and exhaled breath condensate nitrite-nitrate level in relation to environmental exposures in adults in the EGEA study.

This study evaluated the associations between biological markers in the nitrate-nitrite-NO pathway and four environmental exposures among subjects examined in the second survey (2003-2007) of the French Epidemiological study on Genetics and Environment of Asthma (EGEA). Total nitrite and nitrate (NO(2)(-) /NO(3)(-)) levels were measured both in plasma and in exhaled breath condensate (EBC) in 949 adults. Smoking, diet and exposure to chlorine products were assessed using standardized questionnaires. Exposure to air pollutants was estimated by using geostatistical models. All estimates were obtained with generalized estimating equations for linear regression models. Median levels of NO(2)(-)/NO(3)(-) were 36.3 µM (1st-3rd quartile: 25.7, 51.1) in plasma and 2.0 µmol/mg proteins (1st-3rd quartile 0.9, 3.9) in EBC. After adjustment for asthma, age, sex and menopausal status, plasma NO(2)(-)/NO(3)(-) level increased with leafy vegetable consumption (above versus below median=0.04 (95%CI: 0.001, 0.07)) and decreased in smokers (versus non/ex-smokers=-0.08 (95%CI: -0.11, -0.04). EBC NO(2)(-)/NO(3)(-) level decreased in smokers (-0.08 (95%CI: -0.16, -0.001)) and with exposure to ambient O(3) concentration (above versus below median=-0.10 (95%CI: -0.17, -0.03)). Cured meat, chlorine products, PM(10) and NO(2) concentrations were not associated with NO(2)(-)/NO(3)(-) levels. Results suggest that potential modifiable environmental and behavioral risk factors may modify NO(2)(-)/NO(3)(-) levels in plasma and EBC according to the route of exposure.

Caspase-dependent protein phosphatase 2A activation contributes to endotoxin-induced cardiomyocyte contractile dysfunction.

OBJECTIVE: Several studies report calcium mishandling, sarcomere disarray, and caspase activation during heart failure. Although active caspases have been shown to cleave myofibrillar proteins, little is known regarding their effects on calcium handling proteins. Therefore, we aimed to explore how endotoxin-induced caspase activation disrupts intracellular calcium regulation. DESIGN: Randomized controlled trial. SETTING: Small animal research laboratory. SUBJECTS: Adult male Sprague-Dawley rats. INTERVENTIONS: Sepsis was induced by injection of endotoxin (10 mg/kg, intravenously). Caspase inhibition was achieved by coinjection with zVAD.fmk (3 mg/kg, intravenously). We first isolated adult rat ventricular myocytes from control, endotoxin, and (endotoxin + zVAD)-treated rats to characterize contractile parameters and cellular calcium homeostasis. Underlying molecular mechanisms responsible for calcium mishandling were explored on sarcoplasmic reticulum vesicles and mitochondria prepared from treated animals. All experiments were performed 4 hrs postendotoxin treatment. MEASUREMENTS AND MAIN RESULTS: zVAD normalized reductions in fractional cell shortening and relaxation rate triggered by endotoxin treatment. Both sarco-/endoplasmic reticulum Ca-ATPase and mitochondria-dependent calcium uptakes were impaired after endotoxin treatment and prevented when myocytes were isolated from zVAD-treated endotoxinic rat hearts. zVAD blocked endotoxin-induced phospholamban dephosphorylation, protein phosphatase 2A activation, and mitochondrial calcium retention capacity reduction. To strengthen these results, control sarcoplasmic reticulum vesicles and mitochondria were incubated with active recombinant caspase-3. Although no effects were observed on mitochondria, caspase-3 directly exerts detrimental effects on sarcoplasmic reticulum calcium uptake capacity by activating protein phosphatase 2A, leading to phospholamban dephosphorylation. CONCLUSIONS: Caspase inhibition protects from endotoxin-induced sarcoplasmic reticulum calcium uptake capacity reduction and mitochondrial dysfunction.

Carbon monoxide rescues mice from lethal sepsis by supporting mitochondrial energetic metabolism and activating mitochondrial biogenesis.

Use of metal carbonyl-based compounds capable of releasing carbon monoxide (CO) in biological systems have emerged as a potential adjunctive therapy for sepsis via their antioxidant, anti-inflammatory, and antiapoptotic effects. The role of CO in regulation of mitochondrial dysfunction and biogenesis associated with sepsis has not been investigated. In the present study, we employed a ruthenium-based water-soluble CO carrier, tricarbonylchoro(glycinato)ruthenium (II) (CORM-3), one of the novel CO-releasing molecules (CO-RMs), to test whether CO can improve cardiac mitochondrial dysfunction and survival in peritonitis-induced sepsis. Peritonitis was performed in mice by cecal ligation and perforation. Tumor necrosis factor-alpha, interleukin-10, and nitrite/nitrate plasma levels were tested to evaluate the systemic inflammatory response. Functional mitochondrial studies included determination of membrane potential, respiration, and redox status. Oxidative stress was evaluated by measurements of mitochondrial hydrogen peroxide, carbonyl protein and GSH levels. Mitochondrial biogenesis was assessed by peroxisome proliferator-activated receptor gamma coactivator (PGC)-1alpha protein expression and mitochondrial DNA (mtDNA) copy number. The systemic inflammatory response elicited by peritonitis was accompanied by mitochondrial energetic metabolism deterioration and reduced PGC-1alpha protein expression. CORM-3 treatment in septic mice restored the deleterious effects of sepsis on mitochondrial membrane potential, respiratory control ratio, and energetics. It is interesting that administration of CORM-3 during sepsis elicited a mild oxidative stress response that stimulated mitochondrial biogenesis with PGC-1alpha protein expression and mtDNA copy number increases. Our results reveal that delivery of controlled amounts of CO dramatically reduced mortality in septic mice, indicating that CO-RMs could be used therapeutically to prevent organ dysfunction and death in sepsis.

Prevention of endotoxin-induced sarcoplasmic reticulum calcium leak improves mitochondrial and myocardial dysfunction.

OBJECTIVE: Growing evidence suggests that mitochondria function is impaired in sepsis. Here, we tested the hypothesis that lipopolysaccharide would induce mitochondrial Ca2+ overload and oxygen utilization abnormalities as consequences of sarcoplasmic reticulum Ca2+ handling derangements that are typically observed in sepsis. As lipopolysaccharide-induced sarcoplasmic reticulum dysfunction was mainly characterized by reduced sarcoplasmic reticulum Ca2+ uptake and Ca2+ leak, we tested whether dantrolene, a sarco(endo)plasmic reticulum calcium ATPase leak inhibitor, would prevent mitochondrial and cardiac contractile dysfunction. DESIGN: Randomized controlled trial. SETTING: Experimental laboratory. SUBJECTS: Male Sprague Dawley rats. INTERVENTIONS: Sepsis was induced by injection of endotoxin lipopolysaccharide (10 mg/kg/intravenously). Assessment of contractile function and Ca2+ handling was performed 4 hr after lipopolysaccharide. The relative contribution of the different Ca2+ transporters to relaxation in intact cardiomyocytes was studied during successive electrically evoked twitches and caffeine stimulation. Sarcoplasmic reticulum vesicles and mitochondria from ventricles of rats treated or not with lipopolysaccharide were prepared to evaluate Ca2+ uptake-release and oxygen fluxes, respectively. Effects of dantrolene (10 mg/kg) treatment in rats were evaluated in sarcoplasmic reticulum vesicles, mitochondria, and isolated hearts. MEASUREMENTS AND MAIN RESULTS: Lipopolysaccharide challenge elicited cardiac contractile dysfunction that was accompanied by severe derangements in sarcoplasmic reticulum function, i.e., reduced Ca2+ uptake and increased sarcoplasmic reticulum Ca2+ leak. Functional sarcoplasmic reticulum changes were associated with modification in the status of phospholamban phosphorylation whereas SERCA was unchanged. Rises in mitochondrial Ca2+ content observed in lipopolysaccharide-treated rats coincided with derangements in mitochondrial oxygen efficacy, i.e., reduced respiratory control ratio. Administration of dantrolene in lipopolysaccharide-treated rats prevented mitochondrial Ca2+ overload and mitochondrial oxygen utilization abnormalities. Moreover, dantrolene treatment in lipopolysaccharide rats improved heart mitochondrial redox state and myocardial dysfunction. CONCLUSION: These experiments suggest that sarcoplasmic reticulum Ca2+ handling dysfunction is an early event during endotoxemia that could be responsible for, or contribute to, mitochondrial Ca2+ overload, metabolic failure, and cardiac dysfunction.

The synthetic pentasaccharide fondaparinux prevents coronary microvascular injury and myocardial dysfunction in the ischemic heart.

Fondaparinux is a synthetic pentasaccharide with powerful anticoagulant properties, which may also reduce ischemia-reperfusion (I/R) injury in vivo. However, the relative contributions of the anticoagulant and anti-inflammatory activities of fondaparinux to the observed protection are unknown. To address this issue, a crystalloid-perfused heart model was used to assess potential effects of fondaparinux on IR-induced heart injury in the absence of blood. Fondaparinux protects the ischemic myocardium independently of its haemostasis effects. Fondaparinux improved post ischemic myocardial contractile performance and tissue damage. These beneficial effects of fondaparinux may be related to the observed reduction in IR-induced oxidative stress and endothelial activation. In addition, fondaparinux altered NADPH oxidase activity and phosphorylated extracellular signal-regulated kinase (ERK) 1/2, suggesting activation of survival signaling pathways. The present study provides novel information by demonstrating that fondaparinux can attenuate inflammatory responses and oxidative stress in connection with IR heart injury. These findings could represent a potential therapeutic strategy for the prevention of myocardial dysfunction.

Endothelial glycocalyx damage during endotoxemia coincides with microcirculatory dysfunction and vascular oxidative stress.

The glycocalyx constitutes the first line of the blood tissue interface and is thus involved in many physiological processes, deregulation of which may lead to microvascular dysfunction. Because administration of LPS is accompanied by severe microvascular dysfunction, the purpose of the study was to investigate microvascular glycocalyx function during endotoxemia. Bolus infusion of LPS (10 mg kg(-1)) to male Sprague-Dawley rats elicited the development of hyporeactivity to vasoactive agents and microvascular derangements, including decreased capillary density and significant increases in intermittent and stopped flow capillaries in the small intestine muscularis layer compared with controls. LPS elicited plasma hyluronan release and reduction in endothelial surface thickness, indicative of glycocalyx degradation. Because endothelial glycocalyx is extremely sensitive to free radicals, oxidative stress was evaluated by oxidation of dihydrorhodamine in microvascular beds and levels of heart malondialdehyde and plasma carbonyl proteins, which were all increased in LPS-treated rats. Activated protein C (240 microg kg(-1) h(-1)) enhanced systemic arterial pressure response to norepinephrine in LPS-treated rats. Activated protein C (240 microg kg(-1) h(-1)) prevented capillary perfusion deficit in the septic microvasculature that were associated with reduced oxidative stress and preservation of glycocalyx. Our findings support the conclusion that LPS induces major microcirculation dysfunction accompanied by microvascular oxidative stress and glycocalyx degradation that may be limited by activated protein C treatment.

Sphingosine impairs mitochondrial function by opening permeability transition pore.

Growing evidence suggest that, in the heart, sphingosine participates to contractile dysfunction by altering calcium transients and mitochondria function. However, mechanisms underlying sphingosine-induced cardiac mitochondria dysfunction are poorly understood. Here, we studied the effects of sphingosine on isolated cardiac mitochondria of either wild-type or Bcl-2 overexpressing transgenic mice. Sphingosine induced reductions in ADP-coupled respiration, membrane potential, mitochondrial cytochrome c content and ATP production, which were partially prevented by cyclosporine A and mitochondrial Bcl-2 overexpression. These data suggest that sphingosine promotes mitochondrial permeability transition pore opening, which may result in uncoupled respiration and participate in cardiac contractile dysfunction.

Macrophage migration inhibitory factor inhibition is deleterious for high-fat diet-induced cardiac dysfunction.

AIMS: Development of metabolic syndrome is associated with impaired cardiac performance, mitochondrial dysfunction and pro-inflammatory cytokine increase, such as the macrophage migration inhibitory factor MIF. Depending on conditions, MIF may exert both beneficial and deleterious effects on the myocardium. Therefore, we tested whether pharmacological inhibition of MIF prevented or worsened metabolic syndrome-induced myocardial dysfunction. METHODS AND RESULTS: C57BL/6J mice were fed for ten weeks with 60% fat-enriched diet (HFD) or normal diet (ND). MIF inhibition was obtained by injecting mice twice a week with ISO-1, for three consecutive weeks. Then, triglycerides, cholesterol, fat mass, glucose intolerance, insulin resistance, ex vivo cardiac contractility, animal energetic substrate utilization assessed by indirect calorimetry and mitochondrial respiration and biogenesis were evaluated. HFD led to fat mass increase, dyslipidemia, glucose intolerance and insulin resistance. ISO-1 did not alter these parameters. However, MIF inhibition was responsible for HFD-induced cardiac dysfunction worsening. Mouse capacity to increase oxygen consumption in response to exercise was reduced in HFD compared to ND, and further diminished in ISO-1-treated HFD group. Mitochondrial respiration was reduced in HFD mice, treated or not with ISO-1. Compared to ND, mitochondrial biogenesis signaling was upregulated in the HFD as demonstrated by mitochondrial DNA amount and PGC-1α expression. However, this increase in biogenesis was blocked by ISO-1 treatment. CONCLUSION: MIF inhibition achieved by ISO-1 was responsible for a reduction in HFD-induced mitochondrial biogenesis signaling that could explain majored cardiac dysfunction observed in HFD mice treated with MIF inhibitor.

Carbon monoxide improves cardiac function and mitochondrial population quality in a mouse model of metabolic syndrome.

AIMS: Metabolic syndrome induces cardiac dysfunction associated with mitochondria abnormalities. As low levels of carbon monoxide (CO) may improve myocardial and mitochondrial activities, we tested whether a CO-releasing molecule (CORM-3) reverses metabolic syndrome-induced cardiac alteration through changes in mitochondrial biogenesis, dynamics and autophagy. METHODS AND RESULTS: Mice were fed with normal diet (ND) or high-fat diet (HFD) for twelve weeks. Then, mice received two intraperitoneal injections of CORM-3 (10 mg x kg(-1)), with the second one given 16 hours after the first. Contractile function in isolated hearts and mitochondrial parameters were evaluated 24 hours after the last injection. Mitochondrial population was explored by electron microscopy. Changes in mitochondrial dynamics, biogenesis and autophagy were assessed by western-blot and RT-qPCR. Left ventricular developed pressure was reduced in HFD hearts. Mitochondria from HFD hearts presented reduced membrane potential and diminished ADP-coupled respiration. CORM-3 restored both cardiac and mitochondrial functions. Size and number of mitochondria increased in the HFD hearts but not in the CORM-3-treated HFD group. CORM-3 modulated HFD-activated mitochondrial fusion and biogenesis signalling. While autophagy was not activated in the HFD group, CORM-3 increased the autophagy marker LC3-II. Finally, ex vivo experiments demonstrated that autophagy inhibition by 3-methyladenine abolished the cardioprotective effects of CORM-3. CONCLUSION: CORM-3 may modulate pathways controlling mitochondrial quality, thus leading to improvements of mitochondrial efficiency and HFD-induced cardiac dysfunction.

Myocardial dysfunction and potential cardiac hypoxia in rats induced by carbon monoxide inhalation.

BACKGROUND: Results from both animal and human being studies provide evidence that inhalation of concentrations of carbon monoxide (CO) at around 100 ppm has antiinflammatory effects. These low levels of CO are incriminated in ischemic heart diseases experienced by cigarette smokers and, in some cases, from air pollution. Although neurologic mechanisms have been investigated, the effects of CO on cardiovascular function are still poorly understood. METHODS AND RESULTS: The effects of CO (250 ppm; 90 min) inhalation on myocardial function were investigated in isolated heart of rats killed immediately, and 3, 24, 48, and 96 h after CO exposure. CO exposure at 250 ppm resulted in an arterial carboxyhemoglobin (HbCO) level of approximately 11%, which was not associated with changes in mean arterial pressure and heart rate. CO exposure induced coronary perfusion pressure increases, which were associated with endothelium-dependent and -independent vascular relaxation abnormalities. CO-induced coronary vascular relaxation perturbations were observed in the presence of increased heart contractility. Spontaneous peak to maximal Ca(2+)-activated left ventricular pressure ratio was markedly increased in CO-exposed rats, indicating increases in myofilament calcium sensitivity. Heart cyclic guanosine monophosphate/cAMP ratio and myocardial permeabilized fiber respiration (complex intravenous activity) were reduced in CO-exposed rats, which lasted after 48 h of reoxygenation in air. CONCLUSIONS: These findings suggest that CO deteriorates heart oxygen supply to utilization and potentially may induce myocardial hypoxia through mechanisms that include increased oxygen demand due to increased contractility, reduced coronary blood flow reserve, and cardiomyocyte respiration inhibition.

Inhibition of mitochondrial permeability transition prevents sepsis-induced myocardial dysfunction and mortality.

OBJECTIVES: The purpose of this study was to test whether mitochondrial dysfunction is causative of sepsis sequelae, a mouse model of peritonitis sepsis induced by cecal ligation and perforation. Inhibition of mitochondrial permeability transition was achieved by means of pharmacological drugs and overexpression of the antiapoptotic protein B-cell leukemia (Bcl)-2. BACKGROUND: Sepsis is the leading cause of death in critically ill patients and the predominant cause of multiple organ failure. Although precise mechanisms by which sepsis leads to multiple organ dysfunction are unknown, growing evidence suggests that perturbations of key mitochondrial functions, including adenosine triphosphate production, Ca2+ homeostasis, oxygen-derived free radical production, and permeability transition, might be involved in sepsis pathophysiology. METHODS: Heart and lung functions were evaluated respectively by means of isolated heart preparation, bronchoalveolar lavage fluid protein concentration, lung wet/dry weight ratio, lung homogenate myeloperoxidase activity, and histopathologic grading. Respiratory fluxes, calcium uptake, and membrane potential were evaluated in isolated heart mitochondria. RESULTS: Peritonitis sepsis induced multiple organ dysfunction, mitochondrial abnormalities, and increased mortality rate, which were reduced by pharmacological inhibition of mitochondrial transition by cyclosporine derivatives and mitochondrial Bcl-2 overexpression. CONCLUSIONS: Our study provides strong evidence that mitochondrial permeability transition plays a critical role in septic organ dysfunction. These studies demonstrate that mitochondrial dysfunction in sepsis is causative rather than epiphenomenal and relevant in terms of vital organ function and outcome. Regarding the critical role of heart failure in the pathophysiology of septic shock, our study also indicates a potentially new therapeutic approach for treatment of sepsis syndrome.