Changes In Superoxide Research Articles

Exercise Training Increases the Contribution of NADPH Oxidase 4 to Endothelium‐Dependent Dilation in Collateral‐Dependent Coronary Arterioles

We have previously reported that exercise training enhances endothelium-dependent dilation in arterioles distal to chronic coronary artery occlusion through an increased contribution of the reactive oxygen species, hydrogen peroxide (H2O2). In the current study, we began to test the hypothesis that exercise training increases the role of NADPH oxidase (NOX) 4 in bradykinin-mediated, endothelium-dependent dilation, which mediates the increase in H2O2, independent of changes in superoxide. Yucatan miniature swine were instrumented with an ameroid constrictor around the proximal left circumflex coronary artery to induce gradual occlusion and create a collateral-dependent vascular bed. Eight weeks postoperatively, swine were assigned to a progressive exercise-training regimen (treadmill run; 5 days/week for 14 weeks) or remained sedentary and limited to normal pen activity. Coronary arterioles were isolated from both nonoccluded and collateral-dependent myocardial regions and cannulated and pressurized to measure vascular reactivity. Bradykinin-mediated dilation was significantly attenuated in the presence of the NOX 1/4 inhibitor, GKT136901 (1 μM), in collateral-dependent arterioles from exercise-trained pigs, which had no significant influence in other arteriole treatment groups. However, immunoblot analysis revealed no effects of occlusion or exercise training on NOX4 protein levels. In addition, superoxide levels in isolated microvascular endothelial cells, measured by HPLC and dihydroethidium (DHE; 50 μM), revealed that bradykinin-stimulated superoxide levels were not altered by occlusion or exercise training. Taken together, our data suggest that exercise training increases the contribution of H2O2 to endothelium-dependent dilation in collateral-dependent arterioles independent of changes in superoxide levels. Additional evidence suggests that increased H2O2 levels may be produced by NOX4, despite no change in NOX4 protein levels.

Potential role of cartilage oligomeric matrix protein in the modulation of pulmonary arterial smooth muscle superoxide by hypoxia.

Changes in reactive oxygen species and extracellular matrix seem to participate in pulmonary hypertension development. Because we recently reported evidence for chronic hypoxia decreasing expression of cartilage oligomeric matrix protein (COMP) and evidence for this controlling loss of pulmonary arterial smooth muscle bone morphogenetic protein receptor-2 (BMPR2) and contractile phenotype proteins, we examined if changes in superoxide metabolism could be an important factor in a bovine pulmonary artery (BPA), organoid cultured under hypoxia for 48 h model. Hypoxia (3% O2) caused a depletion of COMP in BPA, but not in bovine coronary arteries. Knockdown of COMP by small-interfering RNA (siRNA) increased BPA levels of mitochondrial and extra-mitochondrial superoxide detected by MitoSOX and dihydroethidium (DHE) HPLC products. COMP siRNA-treated BPA showed reduced levels of SOD2 and SOD3 and increased levels of NADPH oxidases NOX2 and NOX4. Hypoxia increased BPA levels of MitoSOX-detected superoxide and caused changes in NOX2 and SOD2 expression similar to COMP siRNA, and exogenous COMP (0.5 μM) prevented the effects of hypoxia. In the presence of COMP, BMPR2 siRNA-treated BPA showed increases in superoxide detected by MitoSOX and depletion of SOD2. Superoxide scavengers (0.5 μM TEMPO or mitoTEMPO) maintained the expression of contractile phenotype proteins calponin and SM22α decreased by 48 h hypoxia (1% O2). Adenoviral delivery of BMPR2 to rat pulmonary artery smooth muscle cells prevented the depletion of calponin and SM22α by COMP siRNA. Thus, COMP regulation of BMPR2 appears to have an important role in controlling hypoxia-elicited changes in BPA superoxide and its potential regulation of contractile phenotype proteins.

Partial loss of complex I due to NDUFS4 deficiency augments myocardial reperfusion damage by increasing mitochondrial superoxide/hydrogen peroxide production

Recent work has found that complex I is the sole source of reactive oxygen species (ROS) during myocardial ischemia-reperfusion (IR) injury. However, it has also been reported that heart mitochondria can also generate ROS from other sources in the respiratory chain and Krebs cycle. This study examined the impact of partial complex I deficiency due to selective loss of the Ndufs4 gene on IR injury to heart tissue. Mice heterozygous for NDUFS4 (NDUFS4+/−) did not display any significant changes in overall body or organ weight when compared to wild-type (WT) littermates. There were no changes in superoxide (O2●-)/hydrogen peroxide (H2O2) release from cardiac or liver mitochondria isolated from NDUFS4 ± mice. Using selective ROS release inhibitors, we found that complex III is a major source of ROS in WT and NDUFS4 ± cardiac mitochondria respiring under state 4 conditions. Subjecting hearts from NDUFS4 ± mice to reperfusion injury revealed that the partial loss of complex I decreases contractile recovery and increases myocardial infarct size. These results correlated with a significant increase in O2●-/H2O2 release rates in mitochondria isolated from NDUFS4 ± hearts subjected to an IR challenge. Taken together, these results demonstrate that the partial absence of complex I sensitizes the myocardium towards IR injury and that the main source of ROS following reperfusion is complex III.

SS-31 attenuates TNF-α induced cytokine release from C2C12 myotubes

TNF-α is a key inflammatory mediator and is proposed to induce transcriptional responses via the mitochondrial generation of Reactive Oxygen Species (ROS). The aim of this study was to determine the effect of TNF-α on the production of myokines by skeletal muscle. Significant increases were seen in the release of IL-6, MCP-1/CCL2, RANTES/CCL5 and KC/CXCL1 and this release was inhibited by treatment with Brefeldin A, suggesting a golgi-mediated release of cytokines by muscle cells. An increase was also seen in superoxide in response to treatment with TNF-α, which was localised to the mitochondria and this was also associated with activation of NF-κB. The changes in superoxide, activation of NF-kB and release of myokines were attenuated following pre-treatment with SS-31 peptide indicating that the ability of TNF-α to induce myokine release may be mediated through mitochondrial superoxide, which is, at least in part, associated with activation of the redox sensitive transcription factor NF-kB.

Heme biosynthesis modulation via δ-aminolevulinic acid administration attenuates chronic hypoxia-induced pulmonary hypertension.

This study examines how heme biosynthesis modulation with δ-aminolevulinic acid (ALA) potentially functions to prevent 21-day hypoxia (10% oxygen)-induced pulmonary hypertension in mice and the effects of 24-h organoid culture with bovine pulmonary arteries (BPA) with the hypoxia and pulmonary hypertension mediator endothelin-1 (ET-1), with a focus on changes in superoxide and regulation of micro-RNA 204 (miR204) expression by src kinase phosphorylation of signal transducer and activator of transcription-3 (STAT3). The treatment of mice with ALA attenuated pulmonary hypertension (assessed through echo Doppler flow of the pulmonary valve, and direct measurements of right ventricular systolic pressure and right ventricular hypertrophy), increases in pulmonary arterial superoxide (detected by lucigenin), and decreases in lung miR204 and mitochondrial superoxide dismutase (SOD2) expression. ALA treatment of BPA attenuated ET-1-induced increases in mitochondrial superoxide (detected by MitoSox), STAT3 phosphorylation, and decreases in miR204 and SOD2 expression. Because ALA increases BPA protoporphyrin IX (a stimulator of guanylate cyclase) and cGMP-mediated protein kinase G (PKG) activity, the effects of the PKG activator 8-bromo-cGMP were examined and found to also attenuate the ET-1-induced increase in superoxide. ET-1 increased superoxide production and the detection of protoporphyrin IX fluorescence, suggesting oxidant conditions might impair heme biosynthesis by ferrochelatase. However, chronic hypoxia actually increased ferrochelatase activity in mouse pulmonary arteries. Thus, a reversal of factors increasing mitochondrial superoxide and oxidant effects that potentially influence remodeling signaling related to miR204 expression and perhaps iron availability needed for the biosynthesis of heme by the ferrochelatase reaction could be factors in the beneficial actions of ALA in pulmonary hypertension.

Skeletal muscle contractions induce acute changes in cytosolic superoxide, but slower responses in mitochondrial superoxide and cellular hydrogen peroxide.

Skeletal muscle generation of reactive oxygen species (ROS) is increased following contractile activity and these species interact with multiple signaling pathways to mediate adaptations to contractions. The sources and time course of the increase in ROS during contractions remain undefined. Confocal microscopy with specific fluorescent probes was used to compare the activities of superoxide in mitochondria and cytosol and the hydrogen peroxide content of the cytosol in isolated single mature skeletal muscle (flexor digitorum brevis) fibers prior to, during, and after electrically stimulated contractions. Superoxide in mitochondria and cytoplasm were assessed using MitoSox red and dihydroethidium (DHE) respectively. The product of superoxide with DHE, 2-hydroxyethidium (2-HE) was acutely increased in the fiber cytosol by contractions, whereas hydroxy-MitoSox showed a slow cumulative increase. Inhibition of nitric oxide synthases increased the contraction-induced formation of hydroxy-MitoSox only with no effect on 2-HE formation. These data indicate that the acute increases in cytosolic superoxide induced by contractions are not derived from mitochondria. Data also indicate that, in muscle mitochondria, nitric oxide (NO) reduces the availability of superoxide, but no effect of NO on cytosolic superoxide availability was detected. To determine the relationship of changes in superoxide to hydrogen peroxide, an alternative specific approach was used where fibers were transduced using an adeno-associated viral vector to express the hydrogen peroxide probe, HyPer within the cytoplasmic compartment. HyPer fluorescence was significantly increased in fibers following contractions, but surprisingly followed a relatively slow time course that did not appear directly related to cytosolic superoxide. These data demonstrate for the first time temporal and site specific differences in specific ROS that occur in skeletal muscle fibers during and after contractile activity.

Mitochondrial ROS generation and function in skeletal muscle from older subjects (863.5)

Oxidative damage is reported to be involved in loss of tissue function with aging and the potential role of mitochondria as a source of increased ROS generation has been attributed to the impaired mitochondrial function and oxidative damage to mitochondrial components that occurs with advancing age. Data have predominantly been obtained from rodent studies and there is limited information on the role of mitochondrial ROS generation in muscle from older humans. To determine the role of mitochondrial ROS in the muscle decline that occurs with aging, muscle biopsies from young (20‐30 years) middle‐aged (45‐55 years) and older(>60 years) healthy volunteers were taken from the vastus lateralis muscle. Analyses were performed in small bundles of permeabilized fibers including mitochondrial ROS changes in the presence of various ETC substrates and inhibitors, changes in mitochondrial respiration, membrane potential and changes in maximum calcium‐activated tetanic force. No differences in fiber cross‐sectional area or force generation in response to calcium stimulation were observed in fibers between age groups. Respiratory function, mitochondrial membrane potential and ROS generation, assessed via changes in superoxide and hydrogen peroxide were not significantly different between age groups. Our data challenge the concept that mitochondrial ROS generation plays a key role in human skeletal muscle aging.Grant Funding Source: Funded by the Medical Research Council, UK (Grant No: G1002120)

Mitochondrial superoxide mediates TNF‐α‐induced myokine release from C2C12 myotubes (1153.4)

TNF‐α is a key inflammatory mediator and is proposed to induce transcriptional responses via the generation of Reactive Oxygen Species (ROS). The aim of this study was to determine the role of mitochondrial superoxide generation in TNF‐α ‐ induced generation of myokines by skeletal muscle. Significant elevation in the release of IL‐6 (control untreated: 20.1pg/ml ± 2.3; TNF‐α treated: 73.8pg/ml ± 9.3), CCL2/MCP‐1 (980pg/ml ± 74.8; 31194pg/ml ± 5409), CCL5/RANTES (22.4pg/ml ± 2.09; 604pg/ml ± 93.4) and CXCL1/KC (236pg/ml ± 32.6; 3477pg/ml ± 383) and in superoxide, localised to the mitochondria were seen in response to TNF‐α treatment of muscle cells and this was associated with activation of NFκB. The TNF‐α mediated changes in superoxide, activation of NFκB and increased release of myokines were attenuated following pre‐treatment with the mitochondria targeted antioxidant, SS‐31 peptide (IL‐6, 11.9pg/ml ± 1.83; MCP‐1, 11322pg/ml ± 1124; RANTES, 252pg/ml ± 51.4; KC, 1166pg/ml ± 204) indicating that the ability of TNF‐α to induce myokine release is mediated through mitochondrial superoxide, which is, at least in part, associated with activation of the redox sensitive transcription factor NFκB. These novel findings provide evidence that targeting mitochondrial ROS could provide a potential for therapy for inflammatory myopathies. The authors thank the Medical Research Council and National Institute on Aging (grant AG‐020591) for their generous financial support and Dr M.B Reid (University of Kentucky Center for Muscle Biology) for the kind gift of the SS‐31 peptide.

Albumin fusion renders thioredoxin an effective anti-oxidative and anti-inflammatory agent for preventing cisplatin-induced nephrotoxicity

BackgroundA strategy for preventing cisplatin nephrotoxicity due to enhanced oxidative stress and inflammatory response is highly desirable. Thioredoxin-1 (Trx), an endogenous redox-active protein, has a short retention time in the blood. A long acting form of Trx, human serum albumin-Trx (HSA-Trx), was produced by recombinant HSA fusion and its effectiveness in preventing cisplatin nephrotoxicity was examined. MethodsHSA-Trx was prepared in Pichia expression system. Cisplatin-induced nephropathy mouse model was established by a single administration of cisplatin. ResultsCompared to saline, Trx or N-acetylcysteine, an intravenous administration of HSA-Trx attenuated the cisplatin-induced elevation in serum creatinine, blood urea nitrogen and urinary N-acetyl-β-d-glucosaminidase along with the decrease in creatinine clearance. HSA-Trx caused a substantial reduction in the histological features of renal tubular injuries and the apoptosis-positive tubular cells. Changes in superoxide, 8-OHdG, glutathione and nitrotyrosine levels indicated that HSA-Trx significantly suppressed renal oxidative stress. HSA-Trx also suppressed the elevation of TNF-α, IL-1β and IL-6. Administered fluorescein isothiocyanate-labeled HSA-Trx was found partially localized in the proximal tubular cells whereas majority remained in the blood circulation. Specific cellular uptake and the scavenging of intracellular reactive oxygen species by HSA-Trx were observed in HK-2 cells. ConclusionHSA-Trx could be a novel and effective approach for preventing cisplatin nephrotoxicity due to its prolonged anti-oxidative and anti-inflammatory action not only in extracellular compartment but also inside the proximal tubular cell. General significanceWe report the renoprotective effect of HSA-Trx against cisplatin nephrotoxicity. This work would enhance developing therapeutics against acute kidney injuries including cisplatin nephrotoxicity.

Heme Oxygenase-1 Attenuates Hypoxia-Induced sFlt-1 and Oxidative Stress in Placental Villi through Its Metabolic Products CO and Bilirubin

One of the most prevalent complications of pregnancy is preeclampsia, a hypertensive disorder which is a leading cause of maternal and perinatal morbidity and premature birth with no effective pharmacological intervention. While the underlying cause is unclear, it is believed that placental ischemia/hypoxia induces the release of factors into the maternal vasculature and lead to widespread maternal endothelial dysfunction. Recently, HO-1 has been shown to downregulate two of these factors, reactive oxygen species and sFlt-1, and we have reported that HO-1 induction attenuates many of the pathological factors of placental ischemia experimentally. Here, we have examined the direct effect of HO-1 and its bioactive metabolites on hypoxia-induced changes in superoxide and sFlt-1 in placental vascular explants and showed that HO-1 and its metabolites attenuate the production of both factors in this system. These findings suggest that the HO-1 pathway may be a promising therapeutic approach for the treatment of preeclampsia.

Critical Role for Copper/Zinc–Superoxide Dismutase in Preventing Spontaneous Intracerebral Hemorrhage During Acute and Chronic Hypertension in Mice

Superoxide is associated with spontaneous intracerebral hemorrhage (ICH) during hypertension. The goal of this study was to test the hypothesis that changes in superoxide, in genetically altered mice with deletion and overexpression of copper/zinc-superoxide dismutase (SOD1), modulate susceptibility to ICH. Chronic hypertension was produced by infusion of angiotensin II and an inhibitor of nitric oxide synthase in drinking water in SOD1 transgenic (SOD1Tg) mice, SOD1-deficient (SOD1(-/-)) mice, and their respective wild-type littermates. Acute hypertension was produced by daily injections of angiotensin II in some mice with chronic hypertension to produce ICH. We evaluated susceptibility to ICH, oxidative stress (superoxide, NAD[P]H oxidase activity, SOD activity), gene expression, and activity of matrix metalloproteinases. Incidence, size, and number of ICHs were reduced in SOD1Tg mice and were increased in SOD1(-/-) mice compared with their wild-type littermates. Levels of superoxide increased in the brain even before developing ICH in wild-type littermates, whereas levels of superoxide remained low in SOD1Tg mice. Changes in level of matrix metalloproteinase-9 paralleled oxidative stress in SOD1Tg mice and wild-type littermates. Moreover, levels of superoxide and matrix metalloproteinase-9 were greater in SOD1(-/-) mice than wild-type littermates after induction of ICH. Active matrix metalloproteinases colocalized on cerebral vessels that appeared to lead toward regions with ICH. These results suggest that superoxide contributes to the pathogenesis of spontaneous ICH, possibly through activation of matrix metalloproteinase-9, and that SOD1 protects against spontaneous ICH during hypertension.

20-HETE can act as a nonhypoxic regulator of HIF-1α in human microvascular endothelial cells

20-HETE increases the expression of VEGF in human dermal microvascular endothelial cells (ECs). Since VEGF is regulated by hypoxia inducible factor (HIF)-1, we studied whether 20-HETE also upregulates HIF-1alpha using the stable 20-HETE analog 20-hydroxyeicosa-5(Z),14(Z)dienoic acid (WIT003; 1-10 microM) and found that it induced a marked increase in HIF-1alpha protein levels. The increases in VEGF after the addition of WIT003 preceded the changes in HIF-1alpha, and the increases in HIF-1alpha were prevented by a VEGF neutralizing antibody. This suggests that 20-HETE first causes increases in VEGF, which then, in turn, cause the upregulation of HIF-1alpha. Stimulation with exogenously added VEGF also led to an upregulation of HIF-1alpha. Incubation with the MEK1/ERK1/2 inhibitor U-0126 (10 microM) completely abolished the increases in VEGF and thus HIF-1alpha, suggesting the involvement of ERK1/2 activation. The addition of WIT003 resulted in a rapid and sustained increase in superoxide formation. When WIT003 was added in the presence of the nitric oxide (NO) synthase (NOS) inhibitor N-nitro-L-arginine, no changes in superoxide, VEGF, or HIF-1alpha were observed. This suggests that NOS is responsible for the early changes in superoxide induced by WIT003. Furthermore, WIT003 induced the expression of the NADPH oxidase subunit p47(phox) in ECs before the increases in HIF-1alpha. Incubation with polyethylene glycol-superoxide dismutase (400 U/ml), apocynin (100 microM), diphenylene iodonium (10 microM), or p47(phox) downregulation with small interfering (si)RNA all inhibited the increases in HIF-1alpha expression. This indicates that the early changes in superoxide lead to VEGF increases and thereby NADPH oxidase-dependent superoxide production, which is required for HIF-1alpha upregulation. We also found that the higher HIF-1alpha expression induced by WIT003 was accompanied by higher expression of erythropoietin receptor and angiopoietin-2 proteins. These increases were caused by HIF-1alpha because their levels were markedly decreased by siRNA downregulation of HIF-1alpha. 20-HETE may be a novel nonhypoxic regulator of HIF-1alpha and HIF-1alpha-regulated genes in ECs.

Mitochondrial Effects of Estrogen Are Mediated by Estrogen Receptor α in Brain Endothelial Cells

Mitochondrial reactive oxygen species (ROS) and endothelial dysfunction are key contributors to cerebrovascular pathophysiology. We previously found that 17beta-estradiol profoundly affects mitochondrial function in cerebral blood vessels, enhancing efficiency of energy production and suppressing mitochondrial oxidative stress. To determine whether estrogen specifically affects endothelial mitochondria through receptor mechanisms, we used cultured human brain microvascular endothelial cells (HBMECs). 17beta-Estradiol treatment for 24 h increased mitochondrial cytochrome c protein and mRNA; use of silencing RNA for estrogen receptors (ERs) showed that this effect involved ERalpha, but not ERbeta. Mitochondrial ROS were determined by measuring the activity of aconitase, an enzyme with an iron-sulfur center inactivated by mitochondrial superoxide. 17beta-Estradiol increased mitochondrial aconitase activity in HBMECs, indicating a reduction in ROS. Direct measurement of mitochondrial superoxide with MitoSOX Red showed that 17beta-estradiol, but not 17alpha-estradiol, significantly decreased mitochondrial superoxide production, an effect blocked by the ER antagonist, ICI-182,780 (fulvestrant). Selective ER agonists demonstrated that the decrease in mitochondrial superoxide was mediated by ERalpha, not ERbeta. The selective estrogen receptor modulators, raloxifene and 4-hydroxy-tamoxifen, differentially affected mitochondrial superoxide production, with raloxifene acting as an agonist but 4-hydroxy-tamoxifen acting as an estrogen antagonist. Changes in superoxide by 17beta-estradiol could not be explained by changes in manganese superoxide dismutase. Instead, ERalpha-mediated decreases in mitochondrial ROS may depend on the concomitant increase in mitochondrial cytochrome c, previously shown to act as an antioxidant. Mitochondrial protective effects of estrogen in cerebral endothelium may contribute to sex differences in the occurrence of stroke and other age-related neurodegenerative diseases.

Intracellular Effects of NSAIDs/ASA in Oxidatively Stressed Human Lens Epithelial Cells in Culture

The aim of the study was to examine the effects of nonsteroidal anti-inflammatory drugs (NSAIDs)/acetylsalicylic acid (ASA) on human lens epithelial cells (HLECs) during oxidative stress. HLECs were exposed to H₂O₂ in the absence or presence of indomethacin, diclofenac, celecoxib (NSAIDs) or ASA for 24 h. HLECs were assayed for changes in superoxide and peroxide production and for variations in glutathione. Mitochondrial depolarization was measured using the membrane potential-sensitive dye JC-1. The results of the study include reduction in superoxide and peroxide production as well as reduction in glutathione depletion in oxidatively stressed HLECs incubated with low concentrations of NSAIDs/ASA. However, no protection against H₂O₂-induced mitochondrial depolarization by NSAIDs/ASA could be seen. In conclusion, NSAIDs/ASA display reactive oxygen species-scavenging properties in H₂O₂-exposed HLECs in culture.