Dependent Reactive Oxygen Species Research Articles

Effect of fuel zinc content on toxicological responses of particulate matter from pellet combustion in vitro

Significant amounts of transition metals such as zinc, cadmium and copper can become enriched in the fine particle fraction during biomass combustion with Zn being one of the most abundant transition metals in wood combustion. These metals may have an important role in the toxicological properties of particulate matter (PM). Indeed, many epidemiological studies have found associations between mortality and PM Zn content.The role of Zn toxicity on combustion PM was investigated. Pellets enriched with 170, 480 and 2300mgZn/kg of fuel were manufactured. Emission samples were generated using a pellet boiler and the four types of PM samples; native, Zn-low, Zn-medium and Zn-high were collected with an impactor from diluted flue gas. The RAW 264.7 macrophage cell line was exposed for 24h to different doses (15, 50,150 and 300μgml−1) of the emission samples to investigate their ability to cause cytotoxicity, to generate reactive oxygen species (ROS), to altering the cell cycle and to trigger genotoxicity as well as to promote inflammation.Zn enriched pellets combusted in a pellet boiler produced emission PM containing ZnO. Even the Zn-low sample caused extensive cell cycle arrest and there was massive cell death of RAW 264.7 macrophages at the two highest PM doses. Moreover, only the Zn-enriched emission samples induced a dose dependent ROS response in the exposed cells. Inflammatory responses were at a low level but macrophage inflammatory protein 2 reached a statistically significant level after exposure of RAW 264.7 macrophages to ZnO containing emission particles.ZnO content of the samples was associated with significant toxicity in almost all measured endpoints. Thus, ZnO may be a key component producing toxicological responses in the PM emissions from efficient wood combustion. Zn as well as the other transition metals, may contribute a significant amount to the ROS responses evoked by ambient PM.

CD28 Regulates Mitochondrial Metabolism in Long Lived Plasma Cells for Survival

Sustained humoral immunity is dependent upon the ability of plasma cells to produce antigen specific antibody titers over a long period of time. Whether fighting against pathogens such as Ebola, Influenza, or the common cold, the continual presence of neutralizing antibodies in circulation is critical for an effective humoral immune response. When activated by antigen, B cells differentiate into a short lived plasma cell (SLPC) pool and reside in secondary lymphoid organs such as the spleen for days to weeks before dying by apoptosis. However, in the absence of constant antigenic presence there are examples of continual production of antigen-specific antibodies in the human body. To explain this, a different subset of long lived plasma cells (LLPC) has been proposed wherein the plasma cells compete for space in specialized bone marrow niches. These cells are not intrinsically long lived, but rather depend upon extrinsic survival factors for their persistence. Many of the survival factors for LLPCs in the bone marrow are shared with their malignant counterpart, e.g. multiple myeloma (MM). Our work centers on the elucidation of mechanisms by which both MM and LLPCs survive in the bone marrow microenvironment.Recently our lab has demonstrated a cell intrinsic role for CD28 signaling in the survival of both LLPCs and MM. CD28 is known as the canonical T cell co-stimulatory molecule and is required for effector T cell metabolic fitness. Under nutrient deprivation and chemotherapeutic challenge, CD28 is able to induce survival of LLPCs and MM cells respectively. However, the molecular and metabolic pathways that govern this prosurvival effect are not well understood.Here we demonstrate that CD28 induces mitochondrial respiration in bone marrow resident LLPCs but not in splenic SLPCs by staining with a mitochondrial-specific dye that is taken up in proportion to the mitochondrial membrane potential. A major byproduct of mitochondrial respiration is the production of reactive oxygen species (ROS). As a result of increased mitochondrial metabolism through the electron transport chain, CD28 is able to induce ROS in LLPCs but not in SLPCs. This is somewhat counterintuitive, in that ROS are well-characterized as cell damaging agents. Fascinatingly, mitochondrial respiration dependent ROS production downstream of CD28 is required for the prosurvival effect seen in LLPCs. Mechanistically, CD28-mediated production of ROS drive NFkB translocation as seen by ImageStream technology, which goes on to drive Blimp1 expression, the master transcriptional regulator of plasma cell identity. Utilizing a luciferase expressing plasmid and including different lengths of the Blimp1 promoter, we show that the CD28 responsive element lies from 4500 to 7500 base pairs from the transcriptional start site. Furthermore, we are able to demonstrate by CHIP that NFkB binds directly to the Blimp1 promoter.In order to understand why this occurs in MM and bone marrow resident LLPCs but not splenic derived SLPCs, we made use of in silico and genetic approaches to discover how the cells differentially signal through CD28. We demonstrate that the Grb2-Vav binding domain in the cytoplasmic tail of CD28 is critical for its prosurvival signal. Vav is known to bind the major adaptor molecule Slp-76. Using transcriptomic analysis we demonstrate that in humans, the major adaptor molecule Slp-76 is highly expressed in LLPCs but not SLPCs. A major downstream target of Slp-76 is PLC-g1 which is phosphorylated by CD28 activation in LLPCs but not SLPCs. To demonstrate that signals emanating from Slp-76 drive LLPC survival, we made use of Slp-76 mutant mice wherein the Vav binding domain is mutated. These mice had lower LLPC numbers and could not transduce a CD28 prosurvival signal. Furthermore, genetic knockdown of Slp-76 diminished the ability of CD28 to induce Blimp1 upregulation. Altogether these data suggest that CD28, through a Grb2-Vav-Slp-76 signal induces mitochondrial respiration dependent production of ROS. These ROS go on to activate NFkB mediated induction of Blimp1, thereby reinforcing the plasma cell phenotype for survival and function. This knowldedge will augment our ability to create effective vaccines as well as disrupt antibody-mediated autoimmunity and multiple myeloma progression in patients. DisclosuresNo relevant conflicts of interest to declare.

Control of vascular smooth muscle function by Src-family kinases and reactive oxygen species in health and disease.

Reactive oxygen species (ROS) are now recognised as second messenger molecules that regulate cellular function by reversibly oxidising specific amino acid residues of key target proteins. Amongst these are the Src-family kinases (SrcFKs), a multi-functional group of non-receptor tyrosine kinases highly expressed in vascular smooth muscle (VSM). In this review we examine the evidence supporting a role for ROS-induced SrcFK activity in normal VSM contractile function and in vascular remodelling in cardiovascular disease. VSM contractile responses to G-protein-coupled receptor stimulation, as well as hypoxia in pulmonary artery, are shown to be dependent on both ROS and SrcFK activity. Specific phosphorylation targets are identified amongst those that alter intracellular Ca(2+) concentration, including transient receptor potential channels, voltage-gated Ca(2+) channels and various types of K(+) channels, as well as amongst those that regulate actin cytoskeleton dynamics and myosin phosphatase activity, including focal adhesion kinase, protein tyrosine kinase-2, Janus kinase, other focal adhesion-associated proteins, and Rho guanine nucleotide exchange factors. We also examine a growing weight of evidence in favour of a key role for SrcFKs in multiple pro-proliferative and anti-apoptotic signalling pathways relating to oxidative stress and vascular remodelling, with a particular focus on pulmonary hypertension, including growth-factor receptor transactivation and downstream signalling, hypoxia-inducible factors, positive feedback between SrcFK and STAT3 signalling and positive feedback between SrcFK and NADPH oxidase dependent ROS production. We also discuss evidence for and against the potential therapeutic targeting of SrcFKs in the treatment of pulmonary hypertension.

Iron and ferritin dependent ROS distribution impact Arabidopsis root system architecture.

Iron (Fe) homeostasis is integrated with the production of Reactive Oxygen Species (ROS) whose distribution at the root tip participates in the control of root growth. Excess Fe increases ferritin abundance, enabling the storage of Fe which contributes to protection of plants against Fe-induced oxidative stress. AtFer1 and AtFer3 are the two ferritin genes expressed in the meristematic zone, pericycle and endodermis of the Arabidopsis thaliana (Arabidopsis) root, and it is in these regions that we observe Fe stained dots. This staining disappears in the triple fer1-3-4 ferritin mutant. Fe excess decreases primary root length in the same way in wild-type and in fer1-3-4 mutant. In contrast, the Fe mediated decrease of lateral root (LR) length and density is enhanced in fer1-3-4 plants due to a defect in LR emergence. We observe that this interaction between excess Fe, ferritin and RSA is in part mediated by the H2O2/O2.- balance between the root cell proliferation and differentiation zones regulated by the UPB1 transcription factor. Further, meristem size is also decreased in response to Fe excess in ferritin mutant plants, implicating cell cycle arrest mediated by the ROS-activated SMR5/SMR7 cyclin-dependent kinase inhibitors pathway in the interaction between Fe and RSA.

Mcl-1 promotes lung cancer cell migration by directly interacting with VDAC to increase mitochondrial Ca2+ uptake and reactive oxygen species generation.

Mcl-1 is an antiapoptotic member of the Bcl-2 family frequently upregulated in non-small cell lung carcinoma (NSCLC). We now report the physiological significance of an interaction between Mcl-1 and the mitochondrial outer membrane-localized voltage-dependent anion channel (VDAC) in NSCLC cell lines. Mcl-1 bound with high affinity to VDAC1 and 3 isoforms but only very weakly to VDAC2 and binding was disrupted by peptides based on the VDAC1 sequence. In A549 cells, reducing Mcl-1 expression levels or application of VDAC-based peptides limited Ca2+ uptake into the mitochondrial matrix, the consequence of which was to inhibit reactive oxygen species (ROS) generation. In A549, H1299 and H460 cells, both Mcl-1 knockdown and VDAC-based peptides attenuated cell migration without affecting cell proliferation. Migration was rescued in Mcl-1 knockdown cells by experimentally restoring ROS levels, consistent with a model in which ROS production drives increased migration. These data suggest that an interaction between Mcl-1 and VDAC promotes lung cancer cell migration by a mechanism that involves Ca2+-dependent ROS production.

Pilin SpaC‐mediated epithelial adhesion is required for Lactobacillus rhamnosus GG‐induced probiotic effects (60.9)

Lactobacillus rhamnosus GG (LGG) is a widely used probiotic, and the strain’s salutary effects on the intestine have been extensively documented. We have demonstrated this strain can modulate inflammatory signaling, as well as epithelial migration and proliferation via stimulation of Nox1 dependent reactive oxygen species. However, how this strain mediates these effects is largely unknown. Here, we report that LGG’s probiotic benefits are dependent on the SpaC pilin subunit. By comparing LGG to an isogenic mutant that lacks SpaC (LGGΩSpaC) in a variety of in vitro and in vivo assays, we establish that SpaC is necessary for LGG to adhere to gut mucosa, that SpaC contributes to LGG’s ability to induce the epithelial generation of ROS, and that SpaC plays a role in LGG’s capacity to stimulate the phosphorylation of ERK in enterocytes. In addition, we show that SpaC is required for LGG‐mediated stimulation of cell proliferation, and protection against radiologically‐inflicted intestinal injury. The identification of a critical surface protein required for LGG to mediate its probiotic influence furthers our understanding of the molecular basis for the symbiotic relationship between some commensal bacteria of the gut lumen and enterocytes. Further insights into this relationship are critical for the development of novel approaches to treat inflammatory bowel diseases and other intestinal disorders.Grant Funding Source: Supported by NIH RO1 AI 64462

MP20-14 UNCOUPLED ENDOTHELIAL NITRIC OXIDE SYNTHASE CONTRIBUTES TO OXALATE-INDUCED OXIDATIVE CELL INJURY IN RENAL EPITHELIAL CELLS: EFFECT OF ANTIOXIDANTS

You have accessJournal of UrologyStone Disease: Basic Research I1 Apr 2014MP20-14 UNCOUPLED ENDOTHELIAL NITRIC OXIDE SYNTHASE CONTRIBUTES TO OXALATE-INDUCED OXIDATIVE CELL INJURY IN RENAL EPITHELIAL CELLS: EFFECT OF ANTIOXIDANTS Vijayalakshmi Thamilselvan, Mani Menon, and Sivagnanam Thamilselvan Vijayalakshmi ThamilselvanVijayalakshmi Thamilselvan More articles by this author , Mani MenonMani Menon More articles by this author , and Sivagnanam ThamilselvanSivagnanam Thamilselvan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.736AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Hyperoxaluria is one of the major risk factors for calcium oxalate urolithiasis and studies support the concept that cellular injury is a critical factor in calcium oxalate crystal nucleation and development of kidney stones. We have previously shown that oxalate-induced reactive oxygen species (ROS) mediates oxidative renal injury via NADPH oxidase (NOX) activation and mitochondrial dysfunction in renal epithelial cells. Here we demonstrate that Uncoupling of Endothelial Nitric Oxide Synthase (eNOS) due to increased NOX and mitochondrial dependent ROS generation also mediates oxalate-induced oxidative cell injury in renal epithelial cells. METHODS LLC-PK1 cells were exposed to oxalate (0.5 mM-1.0 mM) for different time periods in the presence or absence of inhibitors to NOX (Diphenyleneiodonium), mitochondrial electron transport chain (ETC) (rotenone and antimycin A), Nitric oxide synthase (L-NAME) and antioxidants (vitamin E and ascorbic acid). The ROS production, LDH release, NOX activity (lucigenin chemiluminescence), and nitric oxide (NO) production were determined. Mitochondrial ROS was determined by MitoSox oxidation. Protein expression levels of eNOS and nitrotyrosine were determined by western analysis. RESULTS Treatment of LLC-PK1 cells with oxalate increased superoxide, H2O2 generation, LDH release, NOX activity and mitochondrial superoxide generation in a dose and time dependent manner. Pretreatment of LLC-PK1 cells with inhibitors of NOX, mitochondria, and nitric oxide synthase significantly decreased oxalate-induced ROS and LDH release. Oxalate-induced uncoupling of eNOS in LLC-PK1 cells was confirmed by decreased levels of nitric oxide production, eNOS protein expression and increased levels of nitrotyrosine. NOX and mitochondrial inhibitors significantly attenuated oxalate-induced eNOS uncoupling and NO-dependent damage. Vitamin E and ascorbic acid treatment effectively prevented oxalate-induced eNOS uncoupling mediated renal cell injury. CONCLUSIONS We demonstrate that oxalate-induced eNOS uncoupling contributes to additional formation of ROS and subsequent induction of oxidative renal cell injury. Combination of vitamin E and ascorbic acid is more efficient in preventing NOX/mitochondria/eNOS uncoupling mediated renal cell injury. These novel findings suggest that a combination of vitamin E and ascorbic acid should be considered when treating patients with recurrent calcium oxalate kidney stones. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e201 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Vijayalakshmi Thamilselvan More articles by this author Mani Menon More articles by this author Sivagnanam Thamilselvan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Ros in Cardiac Calcium Signaling

ROS in cardiac calcium signaling Reactive oxygen species (ROS) play important roles in physiologic and pathophysiologic signaling within diverse cells including cardiac myocytes. Here we examine the rapid application of extracellular H2O2 (100 uM) to examine how this ROS reagent affects Ca2+ signaling in single isolated cardiac ventricular myocytes. Single mouse ventricular myocytes were examined with a confocal microscope and found to have a robust, reversible increase in the Ca2+ spark rate. In quiescent cells this increase in the Ca2+ spark rate was associated with a decrease in the SR Ca2+ content. This is consistent with the increase in the SR Ca2+ leak (as evidenced by the increase in the Ca2+ spark rate) that followed the H2O2 application. Since ROS has been shown to activate other signaling systems in heart (e.g. CaMKII), the interactions between H2O2 dependent ROS elevation and both CaMKII and PKA were examined. While significant interactions between rapid, transient ROS elevation and CaMKII and PKA were observed, it was also determined that the actions of these ROS elevations on Ca2+ sparks was not mediated by either CaMKII or PKA. How ROS may affect EC coupling under these conditions is also examined and discussed.

Moderate Hypoxia Followed by Reoxygenation Results in Blood-Brain Barrier Breakdown via Oxidative Stress-Dependent Tight-Junction Protein Disruption

Re-canalization of cerebral vessels in ischemic stroke is pivotal to rescue dysfunctional brain areas that are exposed to moderate hypoxia within the penumbra from irreversible cell death. Goal of the present study was to evaluate the effect of moderate hypoxia followed by reoxygenation (MHR) on the evolution of reactive oxygen species (ROS) and blood-brain barrier (BBB) integrity in brain endothelial cells (BEC). BBB integrity was assessed in BEC in vitro and in microvessels of the guinea pig whole brain in situ preparation. Probes were exposed to MHR (2 hours 67-70 mmHg O2, 3 hours reoxygenation, BEC) or towards occlusion of the arteria cerebri media (MCAO) with or without subsequent reperfusion in the whole brain preparation. In vitro BBB integrity was evaluated using trans-endothelial electrical resistance (TEER) and transwell permeability assays. ROS in BEC were evaluated using 2′,7′-dichlorodihydrofluorescein diacetate (DCF), MitoSox and immunostaining for nitrotyrosine. Tight-junction protein (TJ) integrity in BEC, stainings for nitrotyrosine and FITC-albumin extravasation in the guinea pig brain preparation were assessed by confocal microscopy. Diphenyleneiodonium (DPI) was used to investigate NADPH oxidase dependent ROS evolution and its effect on BBB parameters in BEC. MHR impaired TJ proteins zonula occludens 1 (ZO-1) and claudin 5 (Cl5), decreased TEER, and significantly increased cytosolic ROS in BEC. These events were blocked by the NADPH oxidase inhibitor DPI. MCAO with or without subsequent reoxygenation resulted in extravasation of FITC-albumin and ROS generation in the penumbra region of the guinea pig brain preparation and confirmed BBB damage. BEC integrity may be impaired through ROS in MHR on the level of TJ and the BBB is also functionally impaired in moderate hypoxic conditions followed by reperfusion in a complex guinea pig brain preparation. These findings suggest that the BBB is susceptible towards MHR and that ROS play a key role in this process.

Symbiotic lactobacilli stimulate gut epithelial proliferationviaNox-mediated generation of reactive oxygen species

The resident prokaryotic microbiota of the metazoan gut elicits profound effects on the growth and development of the intestine. However, the molecular mechanisms of symbiotic prokaryotic-eukaryotic cross-talk in the gut are largely unknown. It is increasingly recognized that physiologically generated reactive oxygen species (ROS) function as signalling secondary messengers that influence cellular proliferation and differentiation in a variety of biological systems. Here, we report that commensal bacteria, particularly members of the genus Lactobacillus, can stimulate NADPH oxidase 1 (Nox1)-dependent ROS generation and consequent cellular proliferation in intestinal stem cells upon initial ingestion into the murine or Drosophila intestine. Our data identify and highlight a highly conserved mechanism that symbiotic microorganisms utilize in eukaryotic growth and development. Additionally, the work suggests that specific redox-mediated functions may be assigned to specific bacterial taxa and may contribute to the identification of microbes with probiotic potential.

An Arabidopsis Soil-Salinity–Tolerance Mutation Confers Ethylene-Mediated Enhancement of Sodium/Potassium Homeostasis

High soil Na concentrations damage plants by increasing cellular Na accumulation and K loss. Excess soil Na stimulates ethylene-induced soil-salinity tolerance, the mechanism of which we here define via characterization of an Arabidopsis thaliana mutant displaying transpiration-dependent soil-salinity tolerance. This phenotype is conferred by a loss-of-function allele of ethylene overproducer1 (ETO1; mutant alleles of which cause increased production of ethylene). We show that lack of ETO1 function confers soil-salinity tolerance through improved shoot Na/K homeostasis, effected via the ethylene resistant1-constitutive triple response1 ethylene signaling pathway. Under transpiring conditions, lack of ETO1 function reduces root Na influx and both stelar and xylem sap Na concentrations, thereby restricting root-to-shoot delivery of Na. These effects are associated with increased accumulation of respiratory burst oxidase homolog F (RBOHF)-dependent reactive oxygen species in the root stele. Additionally, lack of ETO1 function leads to significant enhancement of tissue K status by an RBOHF-independent mechanism associated with elevated high-affinity K(+) TRANSPORTER5 transcript levels. We conclude that ethylene promotes soil-salinity tolerance via improved Na/K homeostasis mediated by RBOHF-dependent regulation of Na accumulation and RBOHF-independent regulation of K accumulation.

Oxidative Stress and Atrial Fibrillation

Atrial fibrillation (AF), one of the most prevalent cardiac arrhythmias, afflicts >3 million people in the United States.1 Because the risk of developing AF increases with age, the prevalence of AF is projected to be 7.6 million in 2050,1 when 1 in 5 Americans will be over the age of 65.2 AF not only causes debilitating symptoms and functional impairments owing to worsened hemodynamics and embolic stroke, it also increases the risk of mortality up to 1.5- to 1.9-fold.3 Despite the significant morbidity and mortality burden incurred with AF, there are limited therapeutic options that may improve the outcomes of AF patients, and these options are associated with significant AF recurrence rates.4 Article see p 1748 One possible explanation for the limitations of current therapies is that they do not address effectively or completely the underlying causes of AF. Evidence has been mounting that AF is associated with systemic and cardiac oxidation.4,5 Risk factors for AF are similar to those of atherosclerosis, a disease known to be perpetuated by oxidative stress. These risk factors, such as hypertension, aging, diabetes mellitus, and coronary artery bypass surgery, have each been associated with increases in the systemic markers of oxidation.4 In addition, there is also evidence of increased cardiac oxidation of myofibrillar protein5 and membrane lipids6 with AF or with risk factors linked to AF. Although it is not clear whether cardiac oxidation leads to systemic markers of oxidation or whether systemic oxidation leads to cardiac oxidation, the association of oxidative stress and AF is robust and suggests that AF is possibly a manifestation of a systemic disease. Despite the link between oxidative stress and AF, systemic antioxidant therapy for arrhythmias has not met with much success in clinical trials.7, …

Molecular Steps in the Immune Signaling Pathway Evoked by Plant Elicitor Peptides: Ca2+-Dependent Protein Kinases, Nitric Oxide, and Reactive Oxygen Species Are Downstream from the Early Ca2+ Signal

Endogenous plant elicitor peptides (Peps) can act to facilitate immune signaling and pathogen defense responses. Binding of these peptides to the Arabidopsis (Arabidopsis thaliana) plasma membrane-localized Pep receptors (PEPRs) leads to cytosolic Ca(2+) elevation, an early event in a signaling cascade that activates immune responses. This immune response includes the amplification of signaling evoked by direct perception of pathogen-associated molecular patterns by plant cells under assault. Work included in this report further characterizes the Pep immune response and identifies new molecular steps in the signal transduction cascade. The PEPR coreceptor BRASSINOSTEROID-INSENSITIVE1 Associated Kinase1 contributes to generation of the Pep-activated Ca(2+) signal and leads to increased defense gene expression and resistance to a virulent bacterial pathogen. Ca(2+)-dependent protein kinases (CPKs) decode the Ca(2+) signal, also facilitating defense gene expression and enhanced resistance to the pathogen. Nitric oxide and reduced nicotinamide adenine dinucleotide phosphate oxidase-dependent reactive oxygen species generation (due to the function of Respiratory Burst Oxidase Homolog proteins D and F) are also involved downstream from the Ca(2+) signal in the Pep immune defense signal transduction cascade, as is the case with BRASSINOSTEROID-INSENSITIVE1 Associated Kinase1 and CPK5, CPK6, and CPK11. These steps of the pathogen defense response are required for maximal Pep immune activation that limits growth of a virulent bacterial pathogen in the plant. We find a synergism between function of the PEPR and Flagellin Sensing2 receptors in terms of both nitric oxide and reactive oxygen species generation. Presented results are also consistent with the involvement of the secondary messenger cyclic GMP and a cyclic GMP-activated Ca(2+)-conducting channel in the Pep immune signaling pathway.

P.1.8 Genetic ablation of p66Shc rescues functional and morphological abnormalities in collagen VI dystrophic mice

Several studies documented the key role of abnormal production of reactive oxygen species (ROS) in the pathophysiology of muscular dystrophies (MDs) that are contributed also by mitochondrial dysfunction. The sources of ROS, however, are still controversial as well as their major molecular targets. This study investigated whether ROS produced in mitochondria by p66 Shc contributes to MD pathogenesis. p66 Shc is a growth factor adapter that is phosphorylated upon oxidative stress. In this form, a fraction of p66shc localizes to mitochondria, where it binds to cytochrome c and acts as an oxidoreductase, generating ROS and leading to organelle dysfunction and cell death. We provide clear evidence that inactivation of the gene encoding for p66 Shc reduced ROS accumulation along with a beneficial effect on the dystrophic phenotype of Col6a1 −/− mice, a model of Bethlem myopathy and Ullrich congenital MD. Based upon our previous observations on oxidative damage of myofibrillar proteins in heart failure and MDs, we hypothesized that p66 Shc -dependent ROS formation might impair contractile function in dystrophic muscles. Indeed, oxidation of myofibrillar proteins, as probed by formation of disulphide cross-bridges in tropomyosin, was detected in Col6a1 −/− mice. Notably, genetic ablation of p66 Shc significantly reduced myofiber apoptosis and ameliorated muscle strength in Col6a1 −/− mice. These findings demonstrate a novel and determinant role of p66 Shc in MDs, adding evidence of the pivotal role of mitochondria and suggesting p66 Shc as a novel pharmacological target for the therapy of human ColVI myopathies.

188: Dengue infection generates an early NADPH-oxidase dependent ROS production required for antiviral and inflammatory responses

The rapid host response to virus infection is crucial for establishment of the antiviral state and for the release of pro-inflammatory mediators that coordinate the adaptive immune response. Induction of antiviral and inflammatory genes is achieved largely through recognition of viral nucleic acid motifs by pattern recognition receptors. Dengue virus (DENV) is a significant human pathogen that causes a wide spectrum of clinical symptoms, ranging from mild dengue fever to severe hemorrhagic fever and shock syndrome. The cytosolic receptors RIG-I/MDA5 as well as the membrane-bound TLR3/TLR7 localized in the endosomes are important sensors of DENV infection. The role of reactive oxygen species (ROS) in the activation of biological processes such as autophagy, necrosis, and inflammasome activation adds another layer of complexity to the regulation of immune sensing. Transcriptome analysis of DENV-infected human monocyte-derived dendritic cells (Mo-DC) revealed the activation of distinct patterns of gene transcription, with involvement of pathways including the antiviral response, the TNF superfamily and late activation of death receptor signaling. Interestingly, a dominant early gene network associated with the Nrf2-dependent antioxidant response was also identified; DENV infection induced an early production of ROS that was dependent on NADPH-oxidase, and was essential for the activation of inflammatory and antiviral programs, through NF-κB and IRF-3 transcription factors, respectively. Using a high throughput qPCR assay, we demonstrated that interfering with NADPH-oxidase inhibited DENV-induced antiviral and inflammatory responses. We also demonstrated that the antioxidant pathway controlled by the transcription factor Nrf2 is a pivotal upstream link to the regulation of DENV-induced antiviral and inflammatory responses under oxidative conditions. In conclusion, these findings demonstrate that ROS production is crucial for effective activation of pathways leading to innate immune responses against DENV infection in human Mo-DC.

Antihypertensive effects of inducible nitric oxide synthase inhibition in experimental pre‐eclampsia

Upregulation of inducible nitric oxide synthase (iNOS) has been reported in both experimental and clinical hypertension. However, although pro-inflammatory cytokines that up-regulate iNOS contribute to pre-eclampsia, no previous study has tested the hypothesis that a selective iNOS inhibitor (1400 W) could exert antihypertensive effects associated with decreased iNOS expression and nitrosative stress in pre-eclampsia. This study examined the effects of 1400 W in the reduced uteroplacental perfusion pressure (RUPP) placental ischaemia animal model and in normal pregnant rats. Sham-operated and RUPP rats were treated with daily vehicle or 1 mg/kg/day N-[3-(Aminomethyl) benzyl] acetamidine (1400 W) subcutaneously for 5 days. Plasma 8-isoprostane levels, aortic reactive oxygen species (ROS) levels and nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ROS production were evaluated by ELISA, dihydroethidium fluorescence microscopy and lucigenin chemiluminescence respectively. Inducible nitric oxide synthase expression was assessed by western blotting analysis and aortic nitrotyrosine was evaluated by immunohistochemistry. Mean arterial blood pressure increased by ∼30 mmHg in RUPP rats, and 1400 W attenuated this increase by ∼50% (P < 0.05). While RUPP increased plasma 8-isoprostane levels, aortic ROS levels, and NADPH-dependent ROS production (P < 0.05), treatment with 1400 W blunted these alterations (P < 0.05). Moreover, while RUPP increased iNOS expression and aortic nitrotyrosine levels (P < 0.05), treatment with 1400 W blunted these alterations (P < 0.05). These results clearly implicate iNOS in the hypertension associated with RUPP. Our findings may suggest that iNOS inhibitors could be clinically useful in the therapy of pre-eclampsia, especially in particular groups of patients genetically more prone to express higher levels of iNOS. This issue deserves further confirmation.

A low temperature-inducible protein AtSRC2 enhances the ROS-producing activity of NADPH oxidase AtRbohF

Reactive oxygen species (ROS) produced by NADPH oxidases play critical roles in plant environmental responses. Arabidopsis thaliana NADPH oxidase AtRbohF-mediated ROS-production is involved in abiotic stress responses. Because overproduction of ROS is highly toxic to cells, the activity of AtRbohF needs to be tightly regulated in response to diverse stimuli. The ROS-producing activity of AtRbohF is activated by Ca2+ and protein phosphorylation, but other regulatory factors for AtRbohF are mostly unknown. In this study, we screened for proteins that interact with the N-terminal cytosolic region of AtRbohF by a yeast two-hybrid screen, and isolated AtSRC2, an A. thaliana homolog of SRC2 (soybean gene regulated by cold-2). A co-immunoprecipitation assay revealed that AtSRC2 interacts with the N-terminal region of AtRbohF in plant cells. Intracellular localization of GFP-tagged AtSRC2 was partially overlapped with that of GFP-tagged AtRbohF at the cell periphery. Co-expression of AtSRC2 enhanced the Ca2+-dependent ROS-producing activity of AtRbohF in HEK293T cells, but did not affect its phosphorylation-dependent activation. Low-temperature treatment induced expression of the AtSRC2 gene in Arabidopsis roots in proportion to levels of ROS production that was partially dependent on AtRbohF. Our findings suggest that AtSRC2 is a novel activator of Ca2+-dependent AtRbohF-mediated ROS production and may play a role in cold responses.

Peroxisome Proliferator-activated Receptor γ, Coactivator 1α Deletion Induces Angiotensin II–Associated Vascular Dysfunction by Increasing Mitochondrial Oxidative Stress and Vascular Inflammation

Peroxisome proliferator-activated receptor γ, coactivator 1α (PGC-1α) is an important mediator of mitochondrial biogenesis and function. Because dysfunctional mitochondria might be involved in the pathogenesis of vascular disease, the current study was designed to investigate the effects of in vivo PGC-1α deficiency during chronic angiotensin II (ATII) treatment. Although ATII infusion at subpressor doses (0.1 mg/kg per day for 7 days) did not cause vascular dysfunction in wild-type mice, it led to impaired endothelial-dependent and endothelial-independent relaxation in PGC-1α knockout mice. In parallel, oxidative stress was increased in aortic rings from ATII-treated PGC-1α knockout mice, whereas no change in nitric oxide production was observed. By using the mitochondrial-specific superoxide dye MitoSox and complex I inhibitor rotenone, we identified the mitochondrial respiratory chain as the major PGC-1α-dependent reactive oxygen species source in vivo, accompanied by increased vascular inflammation and cell senescence. In vivo treatment with the mitochondria-targeted antioxidant Mito-TEMPO partially corrected endothelial dysfunction and prevented vascular inflammation in ATII-treated PGC-1α mice, suggesting a causative role of mitochondrial reactive oxygen species in this setting. PGC-1α deletion induces vascular dysfunction and inflammation during chronic ATII infusion by increasing mitochondrial reactive oxygen species production.

Overexpression of Smad7 suppressed ROS/MMP9-dependent collagen synthesis through regulation of heme oxygenase-1

We previously reported that AngiotensinII receptor blocker effectively inhibited TGF-β1-mediated epithelial-to-mesenchymal transition progress through regulating Smad7. However, the underlying mechanism by which Smad7 exerted in regulating MMP9 and fibrogenic response has not been fully elucidated. In the current study, we proved that NADPH p47(phox)-dependent reactive oxygen species (ROS) production contributed to MMP9 activation and collagen expression, which was suppressed by transfecting pcDNA3-Smad7 in cardiac fibroblasts. The effect of Smad7 overexpression on MMP9 activity and collagen expression was further reversed by adding H2O2 (10μmol/L). In contrast, knockdown of Smad7 caused the enhanced collagen synthesis in cardiac fibroblasts, which was also reversed by treating cells with a ROS inhibitor, YCG063 (2μmol/L). Further investigation showed that Smad7 regulated NADPH-mediated ROS production through activating Heme oxygenase-1 (HO-1). Meanwhile, the intercellular level of bilirubin (product of hemin) and nitric oxide (NO) in cell supernatant were not significantly increased in cells treated with AngII or transfected with Smad7. Knockdown of HO-1 in Smad7-overexpressed cardiac fibroblasts or cells pretreated with SnPP IX, a competitive inhibitor of HO-1 activity, resulted in increased productions of ROS and NADPH p47(phox), and abolished the inhibitory effects of Smad7 on MMP9 activity and collagen expression. Our results indicated that HO-1 might be critically involved in Smad7-mediated regulation of MMP9 activity and fibrogenic genes expression via antagonizing the enhanced myocardial oxidative stress.

Hepatocytes produce TNF-α following hypoxia-reoxygenation and liver ischemia-reperfusion in a NADPH oxidase- and c-Src-dependent manner

Cell line studies have previously demonstrated that hypoxia-reoxygenation (H/R) leads to the production of NADPH oxidase 1 and 2 (NOX1 and NOX2)-dependent reactive oxygen species (ROS) required for the activation of c-Src and NF-κB. We now extend these studies into mouse models to evaluate the contribution of hepatocytes to the NOX- and c-Src-dependent TNF-α production that follows H/R in primary hepatocytes and liver ischemia-reperfusion (I/R). In vitro, c-Src-deficient primary hepatocytes produced less ROS and TNF-α following H/R compared with controls. In vivo, c-Src-KO mice also had impaired TNF-α and NF-κB responses following partial lobar liver I/R. Studies in NOX1 and p47phox knockout primary hepatocytes demonstrated that both NOX1 and p47phox are partially required for H/R-mediated TNF-α production. To further investigate the involvement of NADPH oxidases in the production of TNF-α following liver I/R, we performed additional in vivo experiments in knockout mice deficient for NOX1, NOX2, p47phox, Rac1, and/or Rac2. Cumulatively, these results demonstrate that NOX2 and its activator subunits (p47phox and Rac) control the secretion of TNF-α by the liver following I/R. Interestingly, in the absence of Kupffer cells and NOX2, NOX1 played a dominant role in TNF-α production following hepatic I/R. However, NOX1 deletion alone had little effect on I/R-induced TNF-α. Thus Kupffer cell-derived factors and NOX2 act to suppress hepatic NOX1-dependent TNF-α production. We conclude that c-Src and NADPH oxidase components are necessary for redox-mediated production of TNF-α following liver I/R and that hepatocytes play an important role in this process.