Effect of mitoTEMPO on Redox Reactions in Different Body Compartments upon Endotoxemia in Rats.

Mitochondrial ROS (mitoROS) control many reactions in cells. Biological effects of mitoROS in vivo can be investigated by modulation via mitochondria-targeted antioxidants (mtAOX, mitoTEMPO). The aim of this study was to determine how mitoROS influence redox reactions in different body compartments in a rat model of endotoxemia. We induced inflammatory response by lipopolysaccharide (LPS) injection and analyzed effects of mitoTEMPO in blood, abdominal cavity, bronchoalveolar space, and liver tissue. MitoTEMPO decreased the liver damage marker aspartate aminotransferase; however, it neither influenced the release of cytokines (e.g., tumor necrosis factor, IL-4) nor decreased ROS generation by immune cells in the compartments examined. In contrast, ex vivo mitoTEMPO treatment substantially reduced ROS generation. Examination of liver tissue revealed several redox paramagnetic centers sensitive to in vivo LPS and mitoTEMPO treatment and high levels of nitric oxide (NO) in response to LPS. NO levels in blood were lower than in liver, and were decreased by in vivo mitoTEMPO treatment. Our data suggest that (i) inflammatory mediators are not likely to directly contribute to ROS-mediated liver damage and (ii) mitoTEMPO is more likely to affect the redox status of liver cells reflected in a redox change of paramagnetic molecules. Further studies are necessary to understand these mechanisms.

Cerebral nitric oxide and mitochondrial function in patients suffering aneurysmal subarachnoid hemorrhage-a translational approach.

BACKGROUND: Cerebral ischemia and neuroinflammation following aneurysmal subarachnoid hemorrhage (SAH) are major contributors to poor neurological outcome. Our study set out to investigate in an exploratory approach the interaction between NO and energy metabolism following SAH as both hypoxia and inflammation are known to affect nitric oxide (NO) metabolism and NO in turn affects mitochondria. METHODS: In seven patients under continuous multimodality neuromonitoring suffering poor-grade aneurysmal SAH, cerebral metabolism and NO levels (determined as a sum of nitrite plus nitrate) were determined in cerebral microdialysate for 14 days following SAH. In additional ex vivo experiments, rat cortex homogenate was subjected to the NO concentrations determined in SAH patients to test whether these NO concentrations impair mitochondrial function (determined by means of high-resolution respirometry). RESULTS: NO levels showed biphasic kinetics with drastically increased levels during the first 7 days (74.5 ± 29.9 µM) and significantly lower levels thereafter (47.5 ± 18.7 µM; p = 0.02). Only during the first 7 days, NO levels showed a strong negative correlation with brain tissue oxygen tension (r = - 0.78; p < 0.001) and a positive correlation with cerebral lactate (r = 0.79; p < 0.001), pyruvate (r = 0.68; p < 0.001), glutamate (r = 0.65; p < 0.001), as well as the lactate-pyruvate ratio (r = 0.48; p = 0.01), suggesting mitochondrial dysfunction. Ex vivo experiments confirmed that the increase in NO levels determined in patients during the acute phase is sufficient to impair mitochondrial function (p < 0.001). Mitochondrial respiration was inhibited irrespectively of whether glutamate (substrate of complex I) or succinate (substrate of complex II) was used as mitochondrial substrate suggesting the inhibition of mitochondrial complex IV. The latter was confirmed by direct determination of complex IV activity. CONCLUSIONS: Exploratory analysis of our data suggests that during the acute phase of SAH, NO plays a key role in the neuronal damage impairing mitochondrial function and facilitating accumulation of mitochondrial substrate; further studies are required to understand mechanisms underlying this observation.

Oxygen Tension Strongly Influences Metabolic Parameters and the Release of Interleukin-6 of Human Amniotic Mesenchymal Stromal Cells In Vitro.

The human amniotic membrane (hAM) has been used for tissue regeneration for over a century. In vivo (in utero), cells of the hAM are exposed to low oxygen tension (1-4% oxygen), while the hAM is usually cultured in atmospheric, meaning high, oxygen tension (20% oxygen). We tested the influence of oxygen tensions on mitochondrial and inflammatory parameters of human amniotic mesenchymal stromal cells (hAMSCs). Freshly isolated hAMSCs were incubated for 4 days at 5% and 20% oxygen. We found 20% oxygen to strongly increase mitochondrial oxidative phosphorylation, especially in placental amniotic cells. Oxygen tension did not impact levels of reactive oxygen species (ROS); however, placental amniotic cells showed lower levels of ROS, independent of oxygen tension. In contrast, the release of nitric oxide was independent of the amniotic region but dependent on oxygen tension. Furthermore, IL-6 was significantly increased at 20% oxygen. To conclude, short-time cultivation at 20% oxygen of freshly isolated hAMSCs induced significant changes in mitochondrial function and release of IL-6. Depending on the therapeutic purpose, cultivation conditions of the cells should be chosen carefully for providing the best possible quality of cell therapy.

Thiamine preserves mitochondrial function in a rat model of traumatic brain injury, preventing inactivation of the 2-oxoglutarate dehydrogenase complex.

BACKGROUND AND PURPOSE: Based on the fact that traumatic brain injury is associated with mitochondrial dysfunction we aimed at localization of mitochondrial defect and attempted to correct it by thiamine. EXPERIMENTAL APPROACH: Interventional controlled experimental animal study was used. Adult male Sprague-Dawley rats were subjected to lateral fluid percussion traumatic brain injury. Thiamine was administered 1 h prior to trauma; cortex was extracted for analysis 4 h and 3 d after trauma. KEY RESULTS: Increased expression of inducible nitric oxide synthase (iNOS) and tumor necrosis factor receptor 1 (TNF-R1) by 4 h was accompanied by a decrease in mitochondrial respiration with glutamate but neither with pyruvate nor succinate. Assays of TCA cycle flux-limiting 2-oxoglutarate dehydrogenase complex (OGDHC) and functionally linked enzymes (glutamate dehydrogenase, glutamine synthetase, pyruvate dehydrogenase, malate dehydrogenase and malic enzyme) indicated that only OGDHC activity was decreased. Application of the OGDHC coenzyme precursor thiamine rescued the activity of OGDHC and restored mitochondrial respiration. These effects were not mediated by changes in the expression of the OGDHC sub-units (E1k and E3), suggesting post-translational mechanism of thiamine effects. By the third day after TBI, thiamine treatment also decreased expression of TNF-R1. Specific markers of unfolded protein response did not change in response to thiamine. CONCLUSION AND IMPLICATIONS: Our data point to OGDHC as a major site of damage in mitochondria upon traumatic brain injury, which is associated with neuroinflammation and can be corrected by thiamine. Further studies are required to evaluate the pathological impact of these findings in clinical settings.

Mitochondria-Targeted Antioxidants SkQ1 and MitoTEMPO Failed to Exert a Long-Term Beneficial Effect in Murine Polymicrobial Sepsis.

Mitochondrial-derived reactive oxygen species have been deemed an important contributor in sepsis pathogenesis. We investigated whether two mitochondria-targeted antioxidants (mtAOX; SkQ1 and MitoTEMPO) improved long-term outcome, lessened inflammation, and improved organ homeostasis in polymicrobial murine sepsis. 3-month-old female CD-1 mice (n = 90) underwent cecal ligation and puncture (CLP) and received SkQ1 (5 nmol/kg), MitoTEMPO (50 nmol/kg), or vehicle 5 times post-CLP. Separately, 52 SkQ1-treated CLP mice were sacrificed at 24 h and 48 h for additional endpoints. Neither MitoTEMPO nor SkQ1 exerted any protracted survival benefit. Conversely, SkQ1 exacerbated 28-day mortality by 29%. CLP induced release of 10 circulating cytokines, increased urea, ALT, and LDH, and decreased glucose but irrespectively of treatment. Similar occurred for CLP-induced lymphopenia/neutrophilia and the NO blood release. At 48 h post-CLP, dying mice had approximately 100-fold more CFUs in the spleen than survivors, but this was not SkQ1 related. At 48 h, macrophage and granulocyte counts increased in the peritoneal lavage but irrespectively of SkQ1. Similarly, hepatic mitophagy was not altered by SkQ1 at 24 h. The absence of survival benefit of mtAOX may be due to the extended treatment and/or a relatively moderate-risk-of-death CLP cohort. Long-term effect of mtAOX in abdominal sepsis appears different to sepsis/inflammation models arising from other body compartments.

Impact of mitochondrial nitrite reductase on hemodynamics and myocardial contractility.

Inorganic nitrite (NO2-) can be reduced back to nitric oxide (NO) by several heme proteins called nitrite reductases (NR) which affect both the vascular tonus and hemodynamics. The objective of this study was to clarify the impact of several NRs on the regulation of hemodynamics, for which hemodynamic parameters such as heart rate, blood pressure, arterial stiffness, peripheral resistance and myocardial contractility were characterized by pulse wave analysis. We have demonstrated that NO2- reduced to NO in RBCs predominantly influences the heart rate, while myoglobin (Mb) and mitochondria-derived NO regulates arterial stiffness, peripheral resistance and myocardial contractility. Using ex vivo on-line NO-detection, we showed that Mb is the strongest NR occurring in heart, which operates sufficiently only at very low oxygen levels. In contrast, mitochondrial NR operates under both hypoxia and normoxia. Additional experiments with cardiomyocytes suggested that only mitochondria-derived generation of NO regulates cGMP levels mediating the contractility of cardiomyocytes. Our data suggest that a network of NRs is involved in NO2- mediated regulation of hemodynamics. Oxygen tension and hematocrit define the activity of specific NRs.

EPR analysis of extra- and intracellular nitric oxide in liver biopsies.

PURPOSE: To develop an assay that can enable the quantification of intra- and extracellular nitric oxide (NO) levels in liver biopsies without application of potentially harmful exogenous NO traps. THEORY: Electron paramagnetic resonance (EPR) spectroscopy is currently the most appropriate method of measuring NO in biological samples due to the outstanding specificity resulting from the interaction of NO with exogenous NO traps. Because such traps are not allowed in clinical settings, we tested the reliability of endogenous NO traps for the determination of NO levels in blood and liver compartments. METHODS: Rats were injected with 0-8 mg/kg lipopolysaccharide (LPS) to gradually induce a systemic inflammatory response. Specific features of NO-hemoglobin and NO-Fe EPR signals were quantified using a specifically developed calibration procedure. RESULTS: Whereas both NO-hemoglobin (NO-HbLIVER BLOOD ) and NO-Fe (NO-FeLIVER ) complexes were detected in nonperfused liver tissue, only NO-Fe complexes were detected in perfused tissue and only NO-Hb complexes were detected in blood (NO-HbBLOOD ). The NO concentrations increased in the sequence NO-HbBLOOD < NO-FeLIVER < NO-HbLIVER BLOOD (9.4, 18.5, 27.9 nmol/cm3 , respectively at 2.5 mg/kg LPS). The detection limit of the method was 0.61 nmol/cm3 for NO-Hb and 0.52 nmol/cm3 for NO-Fe. CONCLUSION: The assay reported here does not influence natural NO pathways and enables the quantification of NO distribution in two liver compartments using a single liver biopsy. Magn Reson Med 77:2372-2380, 2017. © 2016 International Society for Magnetic Resonance in Medicine.