Mitochondrial Oxidative Stress Markers Research Articles

Aldehyde Dehydrogenase 2 Preserves Mitochondrial Function in the Ischemic Heart: A Redox-dependent Mechanism for AMPK Activation by Thioredoxin-1.

Aldehyde dehydrogenase 2 (ALDH2) protects the ischemic heart by activating adenosine 5'-monophosphate-activated protein kinase (AMPK) signaling. However, the molecular mechanisms linking ALDH2 and AMPK signaling are not fully understood. This study aimed to explore the potential mechanisms linking ALDH2 and AMPK in myocardial ischemic injury. An ischemic model was established by ligating the left anterior descending coronary artery in rats. The overexpression or knockdown of ALDH2 in H9c2 cells treated with oxygen-glucose deprivation was obtained through lentivirus infection. Transferase-mediated dUTP nick-end labeling was used to evaluate apoptosis in an ischemic rat model and oxygen-glucose deprivation cells. ALDH2 activity, mitochondrial oxidative stress markers, adenosine triphosphate, respiratory control ratio, and cell viability in H9c2 cells were evaluated using a biological kit and 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide. Protein expression of ALDH2 , 4-hydroxynonenal, thioredoxin-1 (Trx-1), and AMPK-proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) signaling pathway was detected through Western blotting. ALDH2 activation reduced ischemic-induced myocardial infarct size and apoptosis. ALDH2 protected mitochondrial function by enhancing mitochondrial respiratory control ratio and adenosine triphosphate production, alleviated mitochondrial oxidative stress, and suppressed myocardial apoptosis. Moreover, ALDH2 attenuated ischemia-induced oxidative stress and maintained Trx-1 levels by reducing 4-hydroxynonenal, thereby promoting AMPK-PGC-1α signaling activation. Inhibiting Trx-1 or AMPK abolished the cardioprotective effect of ALDH2 on ischemia. ALDH2 alleviates myocardial injury through increased mitochondrial biogenesis and reduced oxidative stress, and these effects were achieved through Trx1-mediating AMPK-PGC1-α signaling activation.

Flavoprotein fluorescence elevation is a marker of mitochondrial oxidative stress in patients with retinal disease.

Recent studies of glaucoma, age-related macular degeneration, and diabetic retinopathy have demonstrated that flavoprotein fluorescence (FPF) can be utilized non-invasively as an indicator of mitochondrial oxidative stress in the retina. However, a comprehensive assessment of the validity and reliability of FPF in differentiating between healthy and diseased eyes across multiple disease states is lacking. Here, we evaluate the sensitivity and specificity of FPF in discriminating between healthy and diseased eyes in four leading causes of visual impairment worldwide, one of which has not been previously evaluated using FPF. We also evaluate the association between FPF and visual acuity. A total of 88 eyes [21 eyes of 21 unaffected controls, 20 eyes from 20 retinal vein occlusion (RVO) patients, 20 eyes from 20 diabetic retinopathy (DR) patients, 17 eyes from 17 chronic exudative age-related macular degeneration (exudative AMD) patients, and 10 eyes from 10 central serous retinopathy (CSR) patients] were included in the present cross-sectional observational study. Eyes were imaged non-invasively using a specially configured fundus camera OcuMet Beacon® (OcuSciences, Ann Arbor, MI). The macula was illuminated using a narrow bandwidth blue light (455 - 470 nm) and fluorescence was recorded using a narrow notch filter to match the peak emission of flavoproteins from 520 to 540 nm. AUROC analysis was used to determine the sensitivity of FPF in discriminating between diseased eyes and healthy eyes. Nonparametric Kruskal-Wallis Tests with post-hoc Mann Whitney U tests with the Holm-Bonferroni correction were performed to assess differences in FPF intensity, FPF heterogeneity, and best corrected visual acuity (BCVA) between the five groups. Spearman rank correlation coefficients were calculated to assess the relationship between FPF and BCVA. AUROC analysis indicated that FPF intensity is highly sensitive for detecting disease, particularly for exudative AMD subjects (0.989; 95% CI = 0.963 - 1.000, p=3.0 x 107). A significant difference was detected between the FPF intensity, FPF heterogeneity, and BCVA in all four disease states compared to unaffected controls (Kruskal-Wallis Tests, p = 1.06 x 10-8, p = 0.002, p = 5.54 x 10-8, respectively). Compared to healthy controls, FPF intensity values were significantly higher in RVO, DR, exudative AMD, and CSR (p < 0.001, p < 0.001, p < 0.001, and p = 0.001, respectively). Spearman rank correlation coefficient between FPF intensity and BCVA was ρ = 0.595 (p = 9.62 x 10-10). Despite variations in structural retinal findings, FPF was found to be highly sensitive for detecting retinal disease. Significant FPF elevation were seen in all four disease states, with the exudative AMD patients exhibiting the highest FPF values compared to DR, CSR, and RVO subjects. This is consistent with the hypothesis that there is elevated oxidative stress in all of these conditions as previously demonstrated by blood studies. FPF intensity is moderately correlated with the late-in disease-marker BCVA, which suggests that the degree of FPF elevation can be used as a metabolic indicator of disease severity.

Effect of Aerobic Exercise and Hydroalcoholic Extract of Tribulus Terrestris on Mitochondrial Oxidative Stress Markers in Heart Tissue of Rats Poisoned With Hydrogen Peroxide

Objective: Oxidative stress can cause DNA damage and apoptosis, and leads to cardiovascular disease. This study aims to evaluate the effect of aerobic exercise combined with consumption of hydroalcoholic extract of tribulus terrestris on mitochondrial oxidative stress markers in heart tissue of rats poisoned with hydrogen peroxide. Methods: This is an experimental study conducted on 42 male Wistar rats divided randomly into seven groups of Control (poisoned without supplementation), Aerobic Exercise, Aerobic Exercise + Supplementation with 5mg/kg extract, Aerobic Exercise + Supplementation with 10 mg/kg extract, Supplementation with 5mg/kg extract, Supplementation with 10mg/kg extract, and healthy control. All groups received hydrogen peroxide (100 mg/kg body weight) for 14 days intraperitoneally. The rats in supplementation groups received hydroalcoholic extract of tribulus terrestris with doses of 5 and 10 mg/kg of body weight by gavage. Aerobic exercise was performed on a treadmill at a speed of 23 m/min for 8 weeks, 5 days per week, each for 30 min. Twenty-four hours after the last exercise session, the heart tissues of rats were collected. Data were analyzed by independent t-test, two-way ANOVA, and Bonferroni post hoc test considering a significance level of P&lt;0.05. Results: Consumption of tribulus terrestris extracts alone and in combination with aerobic exercise led to a significant increase in the levels of O6-methylguanine-DNA-methyl transferase, prooxidants-antioxidant balance, and cytochrome C oxidase, and a significant decrease in adenosine triphosphate and malondialdehyde levels compared to the control group (P&lt;0.05). Conclusion: It seems that regular aerobic exercise and consumption of various doses of tribulus terrestris extract is a moderating factor in mitochondrial biogenesis, and is effective in reducing DNA damage in the heart tissue of rats. Lower dosage of tribulus terrestris extract has more benefits.

Mitochondrial Isolevuglandins Contribute to Vascular Oxidative Stress and Mitochondria-Targeted Scavenger of Isolevuglandins Reduces Mitochondrial Dysfunction and Hypertension.

Hypertension remains a major health problem in Western Societies, and blood pressure is poorly controlled in a third of patients despite use of multiple drugs. Mitochondrial dysfunction contributes to hypertension, and mitochondria-targeted agents can potentially improve treatment of hypertension. We have proposed that mitochondrial oxidative stress produces reactive dicarbonyl lipid peroxidation products, isolevuglandins, and that scavenging of mitochondrial isolevuglandins improves vascular function and reduces hypertension. To test this hypothesis, we have studied the accumulation of mitochondrial isolevuglandins-protein adducts in patients with essential hypertension and Ang II (angiotensin II) model of hypertension using mass spectrometry and Western blot analysis. The therapeutic potential of targeting mitochondrial isolevuglandins was tested by the novel mitochondria-targeted isolevuglandin scavenger, mito2HOBA. Mitochondrial isolevuglandins in arterioles from hypertensive patients were 250% greater than in arterioles from normotensive subjects, and ex vivo mito2HOBA treatment of arterioles from hypertensive subjects increased deacetylation of a key mitochondrial antioxidant, SOD2 (superoxide dismutase 2). In human aortic endothelial cells stimulated with Ang II plus TNF (tumor necrosis factor)-α, mito2HOBA reduced mitochondrial superoxide and cardiolipin oxidation, a specific marker of mitochondrial oxidative stress. In Ang II-infused mice, mito2HOBA diminished mitochondrial isolevuglandins-protein adducts, raised Sirt3 (sirtuin 3) mitochondrial deacetylase activity, reduced vascular superoxide, increased endothelial nitric oxide, improved endothelium-dependent relaxation, and attenuated hypertension. Mito2HOBA preserved mitochondrial respiration, protected ATP production, and reduced mitochondrial permeability pore opening in Ang II-infused mice. These data support the role of mitochondrial isolevuglandins in endothelial dysfunction and hypertension. We conclude that scavenging of mitochondrial isolevuglandins may have therapeutic potential in treatment of vascular dysfunction and hypertension.

Abstract P074: Mitochondrial Isolevuglandins Contribute To Vascular Oxidative Stress And Mitochondria-targeted Scavenger Of Isolevuglandins Reduces Mitochondrial Dysfunction And Hypertension

Hypertension remains a major health problem in Western Societies, and blood pressure is poorly controlled in a third of patients despite use of multiple drugs. Mitochondrial dysfunction contributes to hypertension and mitochondria-targeted agents can potentially improve treatment of hypertension. We have proposed that mitochondrial oxidative stress produces reactive dicarbonyl lipid peroxidation products isolevuglandins (isoLGs) and that scavenging of mitochondrial isoLG improves vascular function and reduces hypertension. To test this hypothesis, we have studied the accumulation of mitochondrial isoLG-protein adducts in human patients with essential hypertension and angiotensin II mouse model of hypertension using mass spectrometry and Western blot analysis. The therapeutic potential of targeting mitochondrial isoLG was tested by the novel mitochondria-targeted isoLG scavenger, mito2HOBA. Mitochondrial isoLG in arterioles isolated from hypertensive patients were 250% greater than in arterioles from normotensive subjects, and ex vivo mito2HOBA treatment of arterioles from hypertensive subjects improved deacetylation of a key mitochondrial antioxidant, superoxide dismutase 2 (SOD2). In human aortic endothelial cells, mito2HOBA diminished mitochondrial superoxide and inhibited cardiolipin oxidation, a specific marker of mitochondrial oxidative stress. In angiotensin II-infused mice, mito2HOBA prevented accumulation of mitochondrial isoLG-protein adducts, improved Sirt3 mitochondrial deacetylase activity, reduced vascular superoxide, increased endothelial nitric oxide, improved endothelium-dependent relaxation, and attenuated hypertension. Mito2HOBA preserved mitochondrial respiration, protected ATP production, and reduced mitochondrial permeability pore opening in angiotensin II-infused mice. These data support the role of mitochondrial isoLGs in endothelial dysfunction and hypertension. We conclude that scavenging of mitochondrial isoLGs may have therapeutic potential in treatment of vascular dysfunction and hypertension.

Pyrvinium pamoate attenuates non-alcoholic steatohepatitis: Insight on hedgehog/Gli and Wnt/β-catenin signaling crosstalk

Non‐alcoholic steatohepatitis (NASH) is a devastating form of non‐alcoholic fatty liver disease (NAFLD). Pyrvinium pamoate (PP) has been recently introduced as anti-adipogenic compound. We aimed to investigate the effects of PP on high fat diet (HFD)-induced NASH in rats and examine the underlying mechanisms. NASH was induced by exposing rats to HFD for 16 weeks and a single dose of streptozotocin (STZ) 35 mg/kg at the fifth week. At the tenth week, PP was given orally at a dose of 60 µg/kg, day after day for 6 weeks. HFD/STZ induced significant steatohepatitis and insulin resistance as was evident by the elevated transaminases activity, NAFLD activity score and HOMA-IR level. Also, HFD induced serum hyperlipidemia and hepatic lipid accumulation. In addition, HFD induced an imbalance in the oxidative status of the liver via upregulating lipid peroxides and mitochondrial oxidative stress markers (MnSOD, UCP-2), together with marked decrease in anti-oxidant glutathione level, glutathione peroxidase activity and expression of mitophagy related markers (PINK1, Parkin, ULK1) and increase in SQSTM1/p62 and LC3II/LC3I. Upregulation of inflammatory mediators (TNF-α, IL-6, IL-1β) and apoptotic marker (caspase 3) were observed. Those events all together precipitated in initiation of liver fibrosis as confirmed by elevation of transforming growth factor-β1 (TGF-β1), alpha-smooth muscle actin (α-SMA) and liver collagen content. Co-treatment with PP protected against HFD-induced NASH and liver fibrosis via downregulating the expression of key factors in Hedgehog and Wnt/ β-catenin signaling pathway. These findings imply that PP can attenuate the progression of NASH and its associated sequela of liver fibrosis.

Physiopathological Bases of the Disease Caused by HACE1 Mutations: Alterations in Autophagy, Mitophagy and Oxidative Stress Response.

Recessive HACE1 mutations are associated with a severe neurodevelopmental disorder (OMIM: 616756). However, the physiopathologycal bases of the disease are yet to be completely clarified. Whole-exome sequencing identified homozygous HACE1 mutations (c.240C>A, p.Cys80Ter) in a patient with brain atrophy, psychomotor retardation and 3-methylglutaconic aciduria, a biomarker of mitochondrial dysfunction. To elucidate the pathomechanisms underlying HACE1 deficiency, a comprehensive molecular analysis was performed in patient fibroblasts. Western Blot demonstrated the deleterious effect of the mutation, as the complete absence of HACE1 protein was observed. Immunofluorescence studies showed an increased number of LC3 puncta together with the normal initiation of the autophagic cascade, indicating a reduction in the autophagic flux. Oxidative stress response was also impaired in HACE1 fibroblasts, as shown by the reduced NQO1 and Hmox1 mRNA levels observed in H2O2-treated cells. High levels of lipid peroxidation, consistent with accumulated oxidative damage, were also detected. Although the patient phenotype could resemble a mitochondrial defect, the analysis of the mitochondrial function showed no major abnormalities. However, an important increase in mitochondrial oxidative stress markers and a strong reduction in the mitophagic flux were observed, suggesting that the recycling of damaged mitochondria might be targeted in HACE1 cells. In summary, we demonstrate for the first time that the impairment of autophagy, mitophagy and oxidative damage response might be involved in the pathogenesis of HACE1 deficiency.

Mitochondrial oxidative stress induces leaky ryanodine receptor during mechanical ventilation

RationaleVentilator-induced diaphragm dysfunction (VIDD) increases morbidity and mortality in critical care patients. Although VIDD has been associated with mitochondrial oxidative stress and calcium homeostasis impairment, the underling mechanisms are still unknown. We hypothesized that diaphragmatic mitochondrial oxidative stress causes remodeling of the ryanodine receptor (RyR1)/calcium release channel, contributing to sarcoplasmic reticulum (SR) Ca2+ leak, proteolysis and VIDD. MethodIn mice diaphragms mechanically ventilated for short (6 h) and long (12 h) period, we assessed mitochondrial ROS production, mitochondrial aconitase activity as a marker of mitochondrial oxidative stress, RyR1 remodeling and function, Ca2+ dependent proteolysis, TGFβ1 and STAT3 pathway, muscle fibers cross-sectional area, and diaphragm specific force production, with or without the mitochondrial targeted anti-oxidant peptide d-Arg-2′, 6′-dimethyltyrosine-Lys-Phe-NH2 (SS31). Measurements and main results6 h of mechanical ventilation (MV) resulted in increased mitochondrial ROS production, reduction of mitochondrial aconitase activity, increased oxidation, S-nitrosylation, S-glutathionylation and Ser-2844 phosphorylation of RyR1, depletion of stabilizing subunit calstabin1 from RyR1, increased SR Ca2+ leak. Preventing mROS production by SS31 treatment does not affect the TGFβ1 and STAT3 activation, which suggests that mitochondrial oxidative stress is a downstream pathway to TGFβ1 and STAT3, early involved in VIDD. This is further supported by the fact that SS-31 rescue all the other described cellular events and diaphragm contractile dysfunction induced by MV, while SS20, an analog of SS31 lacking antioxidant properties, failed to prevent these cellular events and the contractile dysfunction.Similar results were found in ventilated for 12 h. Moreover, SS31 treatment prevented calpain1 activity and diaphragm atrophy observed after 12 h of MV.This study emphasizes that mitochondrial oxidative stress during 6 h-MV contributes to SR Ca2+ leak via RyR1 remodeling, and diaphragm weakness, while longer periods of MV (12 h) were also associated with increased Ca2+-dependent proteolysis and diaphragm atrophy.

Abstract LB-368: Mitochondrial DNA and cancer in the normative aging study

Abstract Background: Mitochondrial DNA (mtDNA) is a potentially sensitive marker of damage by reactive oxygen species (ROS). Two biomarkers in mtDNA, copy number (mtDNAcn) and 8-OHdG, have therefore been proposed as potential markers of oxidative stress and therefore carcinogenesis. Prospective human studies subjects of mtDNAcn have found inconsistent results, but many suggest that the interval between mtDNAcn measurement and cancer diagnosis affects the association. Moreover, prior studies suggest that the interplay of mtDNAcn and 8-OHdG reflect ongoing oxidative stress related to carcinogenesis, suggesting their potential use in tandem as an early detection biomarker of cancer. Our objective was to examine longitudinal associations between mtDNAcn and 8-OHdG measured in whole blood in relation to both cancer incidence and mortality. Methods: We studied 526 Normative Aging Study participants who had blood drawn 1-4 times from 1999 through 2012 and who were cancer free at first blood draw (median 9.7 years between blood draw and cancer diagnosis/censoring). mtDNAcn and 8-OHdG were measured using quantitative real-time PCR. We used Cox proportional hazards regression models to examine associations between mtDNAcn and mitochondrial 8-OHdG measured at the first blood draw only as well as time-dependent associations across a total of 1043 blood draws and time to all-cancer diagnosis. We ran a second set of models stratified by time between blood draw and diagnosis/censoring, and a third set that explored possible interaction between mtDNAcn and 8-OHdG via inclusion of a product term. All models adjusted for age, BMI, white blood cell count, proportion neutrophils, race, education, smoking status, pack-years of smoking, and alcohol consumption. Results: We found no significant associations between pre-diagnostic mtDNAcn and time to cancer diagnosis or death. When measured at the first blood draw only, 8-OHdG was inversely associated with all-cancer (HR: 0.17; 95% CI: 0.03-0.87) and prostate cancer incidence (HR: 0.05; 95% CI: 0.00-0.97). In the time-dependent models, mitochondrial 8-OHdG was inversely associated with time to all-cancer diagnosis (HR: 0.31; 95% CI: 0.10-1.01) and positively associated with time to all-cancer death (HR: 1.83; 95% CI: 1.13-2.98), while mtDNAcn was positively and independently associated with time to all-cancer death (HR: 1.59; 95% CI: 1.17-2.15; pinteraction = 0.96). In the stratum of 4-6 years between blood draw and diagnosis, 8-OHdG was associated with cancer incidence (HR: 0.01; 95% CI: 0-1.00). Modeling 8-OHdG and mtDNAcn together produced no noteworthy changes in results. Similarly, the test for interaction was non-significant across all models. Conclusion: Pre-diagnostic mtDNAcn does not appear to be significantly associated with cancer, however mitochondrial 8-OHdG may be a significant predictor of cancer several years prior to diagnosis. Mitochondiral markers of oxidative stress may also be significant predictors of cancer mortality, possibly related to metastasis and/or treatment. Additional prospective studies in larger cohorts with greater racial/ethnic, gender, and socioeconomic diversity are needed to validate the utility of 8-OHdG as an early detection biomarker of cancer. Future studies of mitochondrial oxidative stress markers measurable in blood and cancer mortality are warranted. Citation Format: Brian T. Joyce, Tao Gao, Yinan Zheng, Lei Liu, Andrea Baccarelli, Joel Schwartz, Lifang Hou. Mitochondrial DNA and cancer in the normative aging study. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-368.

Prolonged swimming promotes cellular oxidative stress and p66Shc phosphorylation, but does not induce oxidative stress in mitochondria in the rat heart

Exercise-induced changes in p66Shc-dependent signaling pathway are still not fully understood. The p66Shc protein is one of the key players in cell signaling, particularly in response to oxidative stress. Therefore, the aim of this study was to investigate the effect of prolonged swimming on the phosphorylation of p66Shc as well as the induction of mitochondrial and cellular oxidative stress in rat hearts.Male Wistar rats were divided into a sedentary control group and an exercise group. The exercised rats swam for 3 hours and were burdened with an additional 3% of their body weight. After the cessation of exercise, their hearts were removed immediately for experiments.The exercise protocol caused increased levels of the following oxidative stress parameters in cardiac cells: DNA damage, protein carbonyls, and lipid dienes. There was also increased phosphorylation of p66Shc without any alterations in Akt and extracellular signal-regulated kinases. Changes in the ferritin L levels and the L to H subunit ratio were also observed in the exercised hearts compared with the control hearts. Despite increased phosphorylation of p66Shc, no significant increase was observed in either mitochondrial H2O2 release or mitochondrial oxidative stress markers. Regardless of the changes in phosphorylation of p66Shc, the antioxidant enzyme activities (superoxide dismutase and catalase) and anti-apoptotic (Bcl2), and pro-apoptotic (Bax) protein levels were not affected by prolonged swimming. Further studies are required to investigate whether p66Shc phosphorylation is beneficial or detrimental to cardiac cells after exercise cessation.

Exercise mitigates diclofenac‐induced liver mitochondrial dysfunction

Several strategies have been developed to counteract liver injury as a consequence of nonsteroid anti-inflammatory drugs toxicity. Here, we aimed to determine whether physical exercise results in liver mitochondrial protection against in vitro diclofenac toxicity. Male adult Sprague-Dawley rats were divided into sedentary, 12-week endurance training (ET) and voluntary activity (VPA). In vitro liver mitochondrial function as assessed by oxygen consumption, transmembrane electric potential (ΔΨ) and susceptibility to the mitochondrial permeability transition pore (MPTP) was evaluated in the absence and presence of diclofenac. Mitochondrial oxidative stress markers [MnSOD, aconitase, -SH and MDA, SIRT3, p66shc(Ser36)/p66shc ratio] and apoptotic signalling (caspases 3, 8 and 9, Bax, Bcl-2 and CypD) were assessed. Content of OXPHOS components and qualitative liver morphological evaluation were assessed. Despite no effects of ET and VPA on basal liver mitochondrial oxygen consumption or ΔΨ endpoints, exercised animals showed lower susceptibility to MPTP. Diclofenac-induced decrease in ΔΨ, increased state 4 respiration and susceptibility to MPTP opening were all prevented by exercise. Under untreated conditions, VPA group showed higher aconitase activity, while ET decreased MDA and increased Bax content. VPA decreased p66shc(Ser36), complex III and V OXPHOS subunits. Both ET and VPA increased complex IV OXPHOS subunit, and SIRT3 and Bcl-2 content and decreased caspase 9 activity. Unexpectedly, ET and VPA decreased ANT. Both chronic physical exercise models augmented the resistance to in vitro diclofenac-induced mitochondrial alterations, including increased MPTP susceptibility, possibly by modulating oxidative stress and MPTP regulators.

Sepsis-Induced Cardiac Mitochondrial Dysfunction Involves Altered Mitochondrial-Localization of Tyrosine Kinase Src and Tyrosine Phosphatase SHP2

Our previous research demonstrated that sepsis produces mitochondrial dysfunction with increased mitochondrial oxidative stress in the heart. The present study investigated the role of mitochondria-localized signaling molecules, tyrosine kinase Src and tyrosine phosphatase SHP2, in sepsis-induced cardiac mitochondrial dysfunction using a rat pneumonia-related sepsis model. SD rats were given an intratracheal injection of Streptococcus pneumoniae, 4×106 CFU per rat, (or vehicle for shams); heart tissues were then harvested and subcellular fractions were prepared. By Western blot, we detected a gradual and significant decrease in Src and an increase in SHP2 in cardiac mitochondria within 24 hours post-inoculation. Furthermore, at 24 hours post-inoculation, sepsis caused a near 70% reduction in tyrosine phosphorylation of all cardiac mitochondrial proteins. Decreased tyrosine phosphorylation of certain mitochondrial structural proteins (porin, cyclophilin D and cytochrome C) and functional proteins (complex II subunit 30kD and complex I subunit NDUFB8) were evident in the hearts of septic rats. In vitro, pre-treatment of mitochondrial fractions with recombinant active Src kinase elevated OXPHOS complex I and II-III activity, whereas the effect of SHP2 phosphatase was opposite. Neither Src nor SHP2 affected complex IV and V activity under the same conditions. By immunoprecipitation, we showed that Src and SHP2 consistently interacted with complex I and III in the heart, suggesting that complex I and III contain putative substrates of Src and SHP2. In addition, in vitro treatment of mitochondrial fractions with active Src suppressed sepsis-associated mtROS production and protected aconitase activity, an indirect marker of mitochondrial oxidative stress. On the contrary, active SHP2 phosphatase overproduced mtROS and deactivated aconitase under the same in vitro conditions. In conclusion, our data suggest that changes in mitochondria-localized signaling molecules Src and SHP2 constitute a potential signaling pathway to affect mitochondrial dysfunction in the heart during sepsis.

Oxidative stress, Nrf2 and keratin up-regulation associate with Mallory-Denk body formation in mouse erythropoietic protoporphyria

Mallory-Denk bodies (MDBs) are hepatocyte inclusions commonly seen in steatohepatitis. They are induced in mice by feeding 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) for 12 weeks, which also causes porphyrin accumulation. Erythropoietic protoporphyria (EPP) is caused by mutations in ferrochelatase (fch), and a fraction of EPP patients develop liver disease that is phenocopied in Fech(m1Pas) mutant (fch/fch) mice, which have an inactivating fch mutation. fch/fch mice develop spontaneous MDBs, but the molecular factors involved in their formation and whether they relate to DDC-induced MDBs are unknown. We tested the hypothesis that fch mutation creates a molecular milieu that mimics experimental drug-induced MDBs. In 13- and 20-week-old fch/fch mice, serum alkaline phosphatase, alanine aminotransferase, and bile acids were increased. The 13-week-old fch/fch mice did not develop histologically evident MDBs but manifested biochemical alterations required for MDB formation, including increased transglutaminase-2 and keratin overexpression, with a greater keratin 8 (K8)-to-keratin 18 (K18) ratio, which are critical for drug-induced MDB formation. In 20-week-old fch/fch mice, spontaneous MDBs were readily detected histologically and biochemically. Short-term (3-week) DDC feeding markedly induced MDB formation in 20-week-old fch/fch mice. Under basal conditions, old fch/fch mice had significant alterations in mitochondrial oxidative-stress markers, including increased protein oxidation, decreased proteasomal activity, reduced adenosine triphosphate content, and Nrf2 (redox sensitive transcription factor) up-regulation. Nrf2 knockdown in HepG2 cells down-regulated K8, but not K18. Fch/fch mice develop age-associated spontaneous MDBs, with a marked propensity for rapid MDB formation upon exposure to DDC, and therefore provide a genetic model for MDB formation. Inclusion formation in the fch/fch mice involves oxidative stress which, together with Nrf2-mediated increase in K8, promotes MDB formation.

Interleukin-1 Receptor-Associated Kinase-3 Is a Key Inhibitor of Inflammation in Obesity and Metabolic Syndrome

BackgroundVisceral obesity is associated with the rising incidence of type 2 diabetes and metabolic syndrome. Low-grade chronic inflammation and oxidative stress synergize in obesity and obesity-induced disorders.ObjectiveWe searched a cluster of molecules that support interactions between these stress conditions in monocytes.Methods RNA expressions in blood monocytes of two independent cohorts comprising 21 and 102 obese persons and 46 age-matched controls were determined by microarray and independently validated by quantitative RT-PCR analysis. The effect of three-month weight loss after bariatric surgery was determined. The effect of RNA silencing on inflammation and oxidative stress was studied in human monocytic THP-1 cells.Results Interleukin-1 receptor-associated kinase-3 (IRAK3), key inhibitor of IRAK/NFκB-mediated chronic inflammation, is downregulated in monocytes of obese persons. Low IRAK3 was associated with high superoxide dismutase-2 (SOD2), a marker of mitochondrial oxidative stress. A comparable expression profile was also detected in visceral adipose tissue of the same obese subjects. Low IRAK3 and high SOD2 was associated with a high prevalence of metabolic syndrome (odds ratio: 9.3; sensitivity: 91%; specificity: 77%). By comparison, the odds ratio of high-sensitivity C-reactive protein, a widely used marker of systemic inflammation, was 4.3 (sensitivity: 69%; specificity: 66%). Weight loss was associated with an increase in IRAK3 and a decrease in SOD2, in association with a lowering of systemic inflammation and a decreasing number of metabolic syndrome components. We identified the increase in reactive oxygen species in combination with obesity-associated low adiponectin and high glucose and interleukin-6 as cause of the decrease in IRAK3 in THP-1 cells in vitro.ConclusionIRAK3 is a key inhibitor of inflammation in association with obesity and metabolic syndrome. Our data warrant further evaluation of IRAK3 as a diagnostic and prognostic marker, and as a target for intervention.

Endothelial MnSOD overexpression prevents retinal VEGF expression in diabetic mice

We previously proposed that hyperglycemia-induced mitochondrial ROS overproduction is a key event in the development of diabetic complications. In this study, we established a novel transgenic mouse (eMnSOD-Tg), which specifically expressed MnSOD in endothelial cells, by employing a Tie2 promoter/enhancer, and investigated the impact of mitochondrial ROS production on diabetic retinopathy in vivo. Using immunohistochemistry, overexpression of MnSOD in endothelial cells was confirmed in eMnSOD-Tg mice. By introduction of diabetes by streptozotocin, levels of urinary 8-hydroxydeoxyguanosine, a marker of mitochondrial oxidative stress, and expression of VEGF mRNA and protein and fibronectin mRNA in retinas were increased in wild-type littermates. However, these observations were ameliorated in eMnSOD-Tg mice, although control and eMnSOD-Tg mice showed a comparable level of hyperglycemia. In the present study, we newly developed a line of transgenic mice, which specifically express MnSOD in endothelium. In addition, overexpression of mitochondrial-specific SOD in endothelium could prevent diabetic retinopathy in vivo.

Expression of chaperonin 60 in the hippocampus of the streptozotocin diabetic rat

Mitochondrial dysfunction and oxidative stress are implicated in the pathological changes observed in the diabetic central nervous system. In this study, using the streptozotocin-induced diabetic rat model we document for the first time the over-expression of a mitochondrial specific stress protein (chaperonin 60) in the CA1/CA3 regions of the diabetic hippocampus in the absence of neurodegeneration. The increase in expression of chaperonin 60 was not observed in the cohort treated with insulin, suggesting that the observed effect was not due to streptozotocin per se but due to the hyperglycaemic state induced by the diabetic state. The expression of chaperonin 60 was also positively correlated with a marker of mitochondrial oxidative stress (manganese superoxide dismutase). We suggest that chaperonin 60 could be an early event marker of mitochondrial dysfunction in the diabetic central nervous system and indeed be neuroprotective in the early stages of hyperglycaemic-induced oxidative stress.

Mitochondrial oxidative stress in human hepatoma cells exposed to stavudine

The toxicity of nucleoside reverse transcriptase inhibitors (NRTIs) is linked to altered mitochondrial DNA (mtDNA) replication and subsequent disruption of cellular energetics. This manifests clinically as elevated concentrations of lactate in plasma. The mechanism(s) underlying how the changes in mtDNA replication lead to lactic acidosis remains unclear. It is hypothesized that mitochondrial oxidative stress links the changes in mtDNA replication to mitochondrial dysfunction and ensuing NRTIs toxicity. To test this hypothesis, changes in mitochondrial function, mtDNA amplification efficiency, and oxidative stress were assessed in HepG2-cultured human hepatoblasts treated with the NRTI stavudine (2′,3′-didehydro-2′,3′-deoxythymidine or d4T) for 48 h. d4T produced significant mitochondrial dysfunction with a 1.5-fold increase in cellular lactate to pyruvate ratios. In addition, d4T caused a dose-dependent decrease in mtDNA amplification and a correlative increase in abundance of markers of mitochondrial oxidative stress. Manganese (III) meso-tetrakis (4-benzoic acid) porphyrin, MnTBAP, a catalytic antioxidant, ameliorated or reversed d4T-induced changes in cell injury, energetics, mtDNA amplification, and mitochondrial oxidative stress. In conclusion, d4T treatment elevates mitochondrial reactive oxygen species (ROS), enhances mitochondrial oxidative stress, and contributes mechanistically to NRTI-induced toxicity. These deleterious events may be potentiated in acquired immunodeficiency syndrome (AIDS) by human immunodeficiency virus (HIV) infection itself, coinfection (e.g., viral hepatitis), aging, substance, and alcohol use.

8-oxo-dGTPase, which prevents oxidative stress-induced DNA damage, increases in the mitochondria from failing hearts.

Reactive oxygen species (ROS) can cause an oxidative modification of nucleotides, such as 8-oxo-7,8-dihydrodeoxyguanosine triphosphate (8-oxo-dGTP), which can lead to defects in DNA replication. The misincorporation of 8-oxo-dGTP into DNA is prevented by 8-oxo-dGTPase, which hydrolyzes 8-oxo-dGTP into 8-oxo-dGMP. The changes in this defensive system have not yet been examined in failing hearts, in which the generation of ROS increases. Myocardial infarction (MI) was created in mice by ligating the left coronary artery. Four weeks later, the left ventricle was dilated and contractility was diminished on echocardiography. The generation of ROS, as measured by electron spin resonance spectroscopy with 4-hydroxy-2,2,6,6-tetramethyl-piperidine-N-oxyl, increased in the noninfarcted left ventricle from MI mice. The formation of thiobarbituric acid-reactive substances also increased in the mitochondria from MI mice. 8-Oxo-dGTPase was detected in the mitochondrial fractions isolated from MI mice using a Western blot analysis with an antibody to its human homologue (hMTH1). Immunohistochemistry showed positive staining for hMTH1 was localized in the cardiac myocytes. The level of 8-oxo-dGTPase increased in the mitochondria isolated from post-MI hearts as oxidative stress increased, thus suggesting that a preventive mechanism is activated against ROS-induced DNA damage. As a result, 8-oxo-dGTPase is considered a useful marker of mitochondrial oxidative stress in heart failure.

A Fraction of Yeast Cu,Zn-Superoxide Dismutase and Its Metallochaperone, CCS, Localize to the Intermembrane Space of Mitochondria: A PHYSIOLOGICAL ROLE FOR SOD1 IN GUARDING AGAINST MITOCHONDRIAL OXIDATIVE DAMAGE

Cu,Zn-superoxide dismutase (SOD1) is an abundant, largely cytosolic enzyme that scavenges superoxide anions. The biological role of SOD1 is somewhat controversial because superoxide is thought to arise largely from the mitochondria where a second SOD (manganese SOD) already resides. Using bakers' yeast as a model, we demonstrate that Cu,Zn-SOD1 helps protect mitochondria from oxidative damage, as sod1Delta mutants show elevated protein carbonyls in this organelle. In accordance with this connection to mitochondria, a fraction of active SOD1 localizes within the intermembrane space (IMS) of mitochondria together with its copper chaperone, CCS. Neither CCS nor SOD1 contains typical N-terminal presequences for mitochondrial uptake; however, the mitochondrial accumulation of SOD1 is strongly influenced by CCS. When CCS synthesis is repressed, mitochondrial SOD1 is of low abundance, and conversely IMS SOD1 is very high when CCS is largely mitochondrial. The mitochondrial form of SOD1 is indeed protective against oxidative damage because yeast cells enriched for IMS SOD1 exhibit prolonged survival in the stationary phase, an established marker of mitochondrial oxidative stress. Cu,Zn-SOD1 in the mitochondria appears important for reactive oxygen physiology and may have critical implications for SOD1 mutations linked to the fatal neurodegenerative disorder, amyotrophic lateral sclerosis.