Shift In Redox State Research Articles

Abstract Tu138: Tandem Mass Tagging (TMT)-based Identification of Proteome Signatures for Ischemia-Reperfusion Injury in Swine

Background: The composition of proteins in blood plasma mirrors the physical changes occurring in the course of disease pathogenesis. Currently available clinical markers are insufficient to predict the actual damage and the redox changes in response to ischemia. Hence, we identified novel redox markers for ischemia/reperfusion (I/R) in swine using TMT-proteomics. Methodology: We employed TMT-10plex isobaric labeling-based quantitative proteomics to track the plasma proteome in Sus Scorfa pigs subjected to surgical I/R at different time points. Plasma samples were collected before ischemia, at 1-hr after ischemia (baseline), and 4-hr, 24-hrs, and 7 days following reperfusion. Results: An average of 4853 spectra and a total of 457 proteins were identified, ensuring a 1% false discovery rate for peptides and proteins with at least two distinct peptides. Pre-ischemia plasma samples served as the control group in all the analyses. TMT analysis revealed temporal changes in the plasma proteome with a prominent shift at 4 hours after I/R insult. To investigate the significantly changed proteins (>1.5 fold), a temporal analysis was carried out. Notably, endopin-1, a serpin protein family member, and fibronectin B were detectable in the plasma following 4 hours of ischemia-reperfusion, indicating the release of these proteins in the circulation during the ischemia-reperfusion. A positive correlation was observed between the redox proteins (i.e., catalase and glutathione peroxidase) with the serpin proteins in the plasma. Biochemical analysis of circulating glutathione, a small-molecular antioxidant, revealed a reductive redox after 60 minutes of Ischemia (hypoxic condition) but this was shifted to an oxidative milieu at 4hrs following reperfusion (hyperoxia) in the adult pigs subjected to I/R insult. Interestingly, we noticed a significant increase in GSH levels and a decrease in catalase levels at 24 hours following I/R, suggesting a compensatory biochemical response in association with repair after I/R insult/injury in the heart. These results correlated with the increased circulating LDH levels indicating the rate of tissue damage following I/R. Conclusion: The present study reveals the presence of a previously unexplored Serpin protein member, endopin-1, during I/R. Understanding the kinetics of endopin-1 expression along with the shift in redox state can help unravel its role in the progression and resolution phases of I/R injury.

A28 A SERUM REDOX STATUS-RELATED INDICATOR IS ASSOCIATED WITH THE RISK OF ONSET OF CROHN'S DISEASE

Abstract Background Crohn's disease (CD) is a gastrointestinal disorder characterized by chronic inflammation. While increased oxidative stress is observed in established CD patients, it remains unknown whether a shift in redox status is present before the diagnosis of CD and whether it is correlated with changes in immune response and microbial composition. Our hypothesis is that oxidative stress plays a role in the development of CD, and it could be detected before the diagnosis of CD. Furthermore, it is likely to be correlated with systemic inflammation and alterations in gut microbiota composition. Aims We aimed to assess the relationship between the serum redox status-related indicators, specifically amino acids ratios, with risk of CD onset and if this is further association with systemic inflammation marker, and gut microbiota composition. Methods In the Genetic Environment Microbial Project (CCC-GEM), a cohort of first-degree relatives (FDRs) of CD patients was prospectively followed. Among them, we identified subjects who later developed CD, defined as pre-CD (n=69), and matched them at a 1:4 ratio with FDRs who remained disease-free (n=276). Serum levels at enrollment of cysteineglycine (reduced form) and cystineglycine (oxidized form) were quantified by mass spectrometry, and the cysteineglycine/cystineglycine ratio was used as an indicator of redox status. Conditional logistic regression assessed the association with CD, while partial Spearman regression evaluated its correlation with systemic inflammation, as indicated by c-reactive protein (CRP), and gut microbiota composition (determined by fecal 16S rRNA sequencing). Results A decrease in the ratio indicates a shift in redox status toward oxidative stress. The cysteineglycine/cystineglycine ratio was negatively associated with the likelihood of developing CD (coefficient = -0.6188; p =0.0146), and it was also negatively correlated with CRP levels (coefficient = -0.177; p =0.00095). A list of taxa belonging to the phyla Firmicutes and Actinobacteriota were positively correlated with cysteineglycine/cystineglycine ratio (p≤0.05). Conclusions This study is the first to report that when redox status shifts towards oxidative stress, as indicated by the cysteineglycine/cystineglycine ratio, the likelihood of CD increases. Furthermore, these markers also correlate with CRP levels and gut microbiota composition, indicating a loss of various taxa when the redox status shifts towards oxidative stress. Submitted on behalf of the CCC-GEM consortium. Funding Agencies CCC, CIHRHCT

Coupling of the recovery of earliest Silurian sponges and ocean redox conditions: Evidence from South China

Many aspects of the Late Ordovician Mass Extinction (LOME) aftermath and recovery have been puzzling due to heterogeneities in tempo and triggering mechanisms. Benthic fossil groups, which are the most severely affected by oxidative stress, offer the best opportunities for understanding both biological and ecological recovery after the LOME. In recent studies, deep-water sponge assemblages (which may have had high physiological tolerance to oxygen deviations) have been reported widely across South China in the immediate aftermath of the extinction interval. In order to further explore the lateral and temporal distributions of sponges, and ecological effects of benthic recovery during this critical interval, this study presents new Llandovery sponge assemblages recovered from two sections in Hunan Province, South China, accompanied by geochemical analyses. The sponge communities are preserved by pyritic spicule replacements and shows a relatively deep-water affinity (estimated around 60–150 m in depth) comparable to several previously reported assemblages in South China, and consistent with the observed graptolite ecology. Geochemical analysis of total organic carbon (TOC) and major and trace element composition in both sections show similar trends, indicating a shift in redox state of the bottom-water from persistent anoxia or intermittent euxinia in the earliest Rhuddanian, becoming oxygenated in the early Aeronian. Based on the present study and previous biological and geochemical data from South China, the distribution of early Silurian sponge assemblages in South China demonstrates a gradual expansion towards deeper regions when ocean redox conditions ameliorated. This study confirms the continuity of the end-Ordovician sponge faunas and taxa, both laterally across South China, and temporally through the early Silurian. The pioneering colonization of sponges in low-oxygen environments after the LOME may have set the stage for the subsequent recovery of other benthic organisms.

Salinity induced redox metabolic shift influence hormonal profile and germination performance of two contrasting indica rice cultivars

The role of redox deviations under salinity on metabolic dysfunction associated with progression of seed germination is well documented. However, the correlative evaluation of the salinity induced changes in the redox system and hormonal profile in regulating germination are least studied and hence is the subject of present investigation. Imposition of post imbibitional salinity stress (PISS) to two contrasting rice genotypes differing in sensitivity towards salinity (Oryza sativa L., Cultivars Patnai and IR29) caused differential and significant redox-metabolic shift and germination performances. Biomarkers of oxidative stress like, accumulation of total ROS, in situ localization of hydrogen peroxide, radical scavenging property, and lipid peroxidation are assessed for the determination of salinity induced differential changes in redox status of both the experimental cultivars. Salt resistant cultivar Patnai exhibiting better redox regulatory property under PISS in terms of controlled generation of ROS (DCFDA oxidation, H2O2 content) with greater elicitation of total antioxidant capacity (DPPH radical scavenging property), contends lipid peroxidation (accumulation of TBARS) as compared to the salt-sensitive cultivar IR 29. RP-HPLC based estimation of PISS-induced alteration in hormonal pools showed strong correlation between altered redox status (assessed in terms of redox biomarkers) and hormonal profile (endogenous titer of gibberellic acid (GA3), abscisic acid (ABA) and jasmonic acid (JA)) and germination and other physiological phenotypes (t50 value, allocation index, relative water content, and Na+ / K+ ratio) of the experimental rice germplasms, suggesting the influence of differential shift in redox status on germination hormones and early growth performances. Taken as a whole, the work proposes close connection between salinity induced changes in oxidative windows and hormonal profile of germinating seeds, necessary for better management of salinity stress in agriculture.

Effect of redox imbalance on protein modifications in lymphocytes of psoriatic patients.

Lymphocytes are one of the most important cells involved in the pathophysiology of psoriasis; therefore, the aim of this study was to assess the redox imbalance and protein modifications in the lymphocytes of patients with psoriasis vulgaris (PsV) or psoriatic arthritis (PsA). The results show a stronger shift in redox status to pro-oxidative conditions (observed as an increased reactive oxygen species level, a decrease in catalase activity and lower levels of glutathione peroxidase and vitamin E compared with healthy controls) in the lymphocytes of PsA than PsV patients. It is also favoured by the enhanced level of activators of the Nrf2 transcription factor in lymphocytes of PsV compared with decreased of these proteins level in PsA. Moreover, the differential modifications of proteins by lipid peroxidation products 4-oxononenal (mainly binding proteins) and malondialdehyde (mainly catalytic proteins with redox activity), promoted a pro-apoptotic pathway in lymphocytes of PsV, which was manifested by enhanced expression of pro-apoptotic caspases, particularly caspase 3. Taken together, differences in Nrf2 pathway activation may be responsible for the differential level of redox imbalance in lymphocytes of patients with PsV and PsA. This finding may enable identification of a targeted therapy to modify the metabolic pathways disturbed in psoriasis.

Senescent neurophysiology: Ca2+ signaling from the membrane to the nucleus.

The current review provides a historical perspective on the evolution of hypothesized mechanisms for senescent neurophysiology, focused on the CA1 region of the hippocampus, and the relationship of senescent neurophysiology to impaired hippocampal-dependent memory. Senescent neurophysiology involves processes linked to calcium (Ca2+) signaling including an increase in the Ca2+-dependent afterhyperpolarization (AHP), decreasing pyramidal cell excitability, hyporesponsiveness of N-methyl-D-aspartate (NMDA) receptor function, and a shift in Ca2+-dependent synaptic plasticity. Dysregulation of intracellular Ca2+ and downstream signaling of kinase and phosphatase activity lies at the core of senescent neurophysiology. Ca2+-dysregulation involves a decrease in Ca2+ influx through NMDA receptors and an increase release of Ca2+ from internal Ca2+ stores. Recent work has identified changes in redox signaling, arising in middle-age, as an initiating factor for senescent neurophysiology. The shift in redox state links processes of aging, oxidative stress and inflammation, with functional changes in mechanisms required for episodic memory. The link between age-related changes in Ca2+ signaling, epigenetics and gene expression is an exciting area of research. Pharmacological and behavioral intervention, initiated in middle-age, can promote memory function by initiating transcription of neuroprotective genes and rejuvenating neurophysiology. However, with more advanced age, or under conditions of neurodegenerative disease, epigenetic changes may weaken the link between environmental influences and transcription, decreasing resilience of memory function.

Hypoxia‐induced depolarization of pulmonary arterial smooth muscle cells relies on simultaneous inhibition of K+‐currents and redox state alterations

Hypoxic pulmonary vasoconstriction (HPV) is a physiological response to localized alveolar hypoxia that is intrinsic to the pulmonary circulation. By hypoxia‐induced contraction of pulmonary arterial smooth muscle cells (PASMCs), pulmonary capillary blood flow is redirected to alveolar areas of high oxygen partial pressure (pO2), thus maintaining the ventilation‐perfusion ratio. Although the principle of HPV was recognized decades ago the underlying pathway still remains elusive.Voltage gated K+‐channels (Kv‐channels) are known to be redox‐sensitive and crucial for mediating membrane potential in PASMCs ‐ thereby controlling Ca2+‐entry and subsequently vascular tone. Here, we investigated whether acute hypoxia alters the redox state of isolated PASMCs from mice and if this impacts Kv‐channel activity, membrane potential and intracellular Ca2+‐ level.Kv‐currents and membrane potential in response to hypoxia or the oxidizing agent H2O2 were investigated by whole cell patch clamp experiments (voltage and current clamp, resp.) on freshly isolated murine PASMCs. Simultaneously, pO2 was recorded using an optical oxygen sensor and intracellular Ca2+ was measured by ratiometric FURA‐2 imaging. The redox state was monitored by Raman spectroscopy using an excitation wavelength that was in resonance with the hemeproteins (532 nm).Raman‐spectra values for reduced myoglobin (1607 cm−1) and cytochrome c (1622 cm−1) both increased upon exposure of PASMCs to hypoxia (4 % O2) and returned to the initial value upon reverting to normoxia (21 % O2), thus indicating a shift in the cells' redox state. In accordance, Kv‐currents were dose‐dependently inhibited by hypoxia and H2O2, resulting in a depolarization from −37,5 ± 2 mV (before) to −28.09 ± 2 mV after exposure to hypoxia (Mean ± SEM; n = 23; p = < 0,001; paired t‐test). The oxidizing agent H2O2 (124 nM) mimicked this effect under normoxic conditions by elevating the membrane potential of the PASMCs from −37.4 ± 3 mV (before) to −25,2 ± 4 mV upon application of 124 nM H2O2 (Mean ± SEM; n = 5, p = 0,005, paired t‐test) ‐ thereby triggering a rise in intracellular Ca2+.In conclusion, hypoxia induced a shift in redox state in PASMCs, thereby mediating the inhibition of KV‐channels. The resulting depolarization and Ca2+‐entry may account for the contraction of PASMCs – thus initiating HPV.Support or Funding InformationThis study was funded by the German Research Foundation (DFG, CRC 1213) and the Swedish Research Council (Grant 2016‐04220).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Bioelectrical brain cortex activity in intellectually gifted children of primary and middle school age in solving cognitive tasks

The current review provides a historical perspective on the evolution of hypothesized mechanisms for senescent neurophysiology, focused on the CA1 region of the hippocampus, and the relationship of senescent neurophysiology to impaired hippocampal-dependent memory. Senescent neurophysiology involves processes linked to calcium (Ca2+) signaling including an increase in the Ca2+-dependent afterhyperpolarization (AHP), decreasing pyramidal cell excitability, hyporesponsiveness of N-methyl-D-aspartate (NMDA) receptor function, and a shift in Ca2+-dependent synaptic plasticity. Dysregulation of intracellular Ca2+ and downstream signaling of kinase and phosphatase activity lies at the core of senescent neurophysiology. Ca2+-dysregulation involves a decrease in Ca2+ influx through NMDA receptors and an increase release of Ca2+ from internal Ca2+ stores. Recent work has identified changes in redox signaling, arising in middle-age, as an initiating factor for senescent neurophysiology. The shift in redox state links processes of aging, oxidative stress and inflammation, with functional changes in mechanisms required for episodic memory. The link between age-related changes in Ca2+ signaling, epigenetics and gene expression is an exciting area of research. Pharmacological and behavioral intervention, initiated in middle-age, can promote memory function by initiating transcription of neuroprotective genes and rejuvenating neurophysiology. However, with more advanced age, or under conditions of neurodegenerative disease, epigenetic changes may weaken the link between environmental influences and transcription, decreasing resilience of memory function.

Redox Paradox: A Novel Approach to Therapeutics-Resistant Cancer.

Cancer cells that are resistant to radiation and chemotherapy are a major problem limiting the success of cancer therapy. Aggressive cancer cells depend on elevated intracellular levels of reactive oxygen species (ROS) to proliferate, self-renew, and metastasize. As a result, these aggressive cancers maintain high basal levels of ROS compared with normal cells. The prominence of the redox state in cancer cells led us to consider whether increasing the redox state to the condition of oxidative stress could be used as a successful adjuvant therapy for aggressive cancers. Recent Advances: Past attempts using antioxidant compounds to inhibit ROS levels in cancers as redox-based therapy have met with very limited success. However, recent clinical trials using pro-oxidant compounds reveal noteworthy results, which could have a significant impact on the development of strategies for redox-based therapies. The major objective of this review is to discuss the role of the redox state in aggressive cancers and how to utilize the shift in redox state to improve cancer therapy. We also discuss the paradox of redox state parameters; that is, hydrogen peroxide (H2O2) as the driver molecule for cancer progression as well as a target for cancer treatment. Based on the biological significance of the redox state, we postulate that this system could potentially be used to create a new avenue for targeted therapy, including the potential to incorporate personalized redox therapy for cancer treatment.

Mercury exposure and a shift toward oxidative stress in avid seafood consumers

Mechanisms of mercury (Hg) toxicity at low doses from seafood consumption, the most common exposure route, are not well understood. We tested the hypothesis that seafood Hg exposure is related to a shift in redox status, indicated by a decrease in the ratio of reduced to oxidized glutathione (GSH:GSSG) in blood, or increase in redox potential (Eh). We also examined whether key seafood nutrients (selenium (Se), omega-3 fatty acids) confound or modify this shift. We measured blood concentrations of total Hg, Se, GSH, GSSG, and the Omega-3 Index (% omega-3s of total fatty acids in red blood cell membranes) in seafood consumers in Long Island, NY. We examined relationships between Hg, GSH:GSSG ratio and Eh. Elevated blood Hg (>5.8µgL−1) was associated with lower GSH:GSSG (β=−116.73, p=0.01), with no evidence of confounding by Se or Omega-3 Index. However, in models stratified by Omega-3 Index levels, Hg-GSH:GSSG associations were weakened among those with high Omega-3 Index levels (>6% of fatty acids, β=−63.46, p=0.28), and heightened among those with low Omega-3 Index (β=−182.53, p<0.01). We observed comparable patterns for Eh in relation to Hg. These results support the hypothesis that Hg exposure from seafood is linked to a shift in redox status toward oxidative stress, modified by omega-3 fatty acids in this population. Further work should examine the role of different seafood nutrients and Hg-induced shifts in redox status in the diverse health effects associated with elevated Hg exposure.

The behavior of biologically important trace elements across the oxic/euxinic transition of meromictic Fayetteville Green Lake, New York, USA

Trace elements are central components of enzymes that catalyze many of the essential reactions mediated by life. The redox sensitive nature of trace elements also permits their use as a record of ancient ocean conditions preserved in the geologic record. Trace element geochemistry in modern stratified systems is often used as a proxy for the redox state of the ancient oceans, which are thought to have been largely anoxic. In the present study, we examined trace element behavior of simultaneously collected samples at a heretofore unprecedented depth resolution (1–0.25m intervals) throughout the redox-stratified water column of Fayetteville Green Lake, N.Y. (FGL), a 53m deep meromictic lake under euxinic conditions similar to those thought to have been prevalent in Proterozoic oceans. Among characterized Proterozoic ocean analogs, FGL represents an understudied proxy in terms of trace elements, with characteristics of low salinity and high sulfate. In the FGL water column, spikes in the concentration of dissolved Mn, Fe and Co are coincident with the transition from oxic to euxinic conditions, and are associated with a decrease in dissolved Mo concentration. In contrast, the concentration of dissolved Ni did not vary across this transition despite the dramatic shift in redox state. From these data we present a one dimensional model for element transport and cycling through the water column to the sediments. Collectively, this comprehensive analysis of water column geochemistry provides insight into the effects of biogeochemical cycling in stratified systems on dissolved trace element concentrations in the water column. This study, in concert with characterization of other early Earth analogs, will greatly enhance the use of trace elements in interpreting the geologic record.

High mitochondrial respiration and glycolytic capacity represent a metabolic phenotype of human tolerogenic dendritic cells.

Human dendritic cells (DCs) regulate the balance between immunity and tolerance through selective activation by environmental and pathogen-derived triggers. To characterize the rapid changes that occur during this process, we analyzed the underlying metabolic activity across a spectrum of functional DC activation states, from immunogenic to tolerogenic. We found that in contrast to the pronounced proinflammatory program of mature DCs, tolerogenic DCs displayed a markedly augmented catabolic pathway, related to oxidative phosphorylation, fatty acid metabolism, and glycolysis. Functionally, tolerogenic DCs demonstrated the highest mitochondrial oxidative activity, production of reactive oxygen species, superoxide, and increased spare respiratory capacity. Furthermore, assembled, electron transport chain complexes were significantly more abundant in tolerogenic DCs. At the level of glycolysis, tolerogenic and mature DCs showed similar glycolytic rates, but glycolytic capacity and reserve were more pronounced in tolerogenic DCs. The enhanced glycolytic reserve and respiratory capacity observed in these DCs were reflected in a higher metabolic plasticity to maintain intracellular ATP content. Interestingly, tolerogenic and mature DCs manifested substantially different expression of proteins involved in the fatty acid oxidation (FAO) pathway, and FAO activity was significantly higher in tolerogenic DCs. Inhibition of FAO prevented the function of tolerogenic DCs and partially restored T cell stimulatory capacity, demonstrating their dependence on this pathway. Overall, tolerogenic DCs show metabolic signatures of increased oxidative phosphorylation programing, a shift in redox state, and high plasticity for metabolic adaptation. These observations point to a mechanism for rapid genome-wide reprograming by modulation of underlying cellular metabolism during DC differentiation.

Don't mess with stress: vitamin supplementation and the role of oxidative stress for aerobic exercise adaptation.

The effect of chronic antioxidant and anti-inflammatory supplementation on the adaptive process to exercise training is an emerging area of research, with training implications that have yet to be elucidated fully. The use of these agents has become more popular amongst athletes and recreationally active individuals, thus the long-term consequence of their chronic use for muscle and cardiovascular adaptation is an important consideration for this population. Usage of these supplements relates to the common belief that exercise-induced inflammation and oxidative stress play a crucial role in muscle soreness and weakness in the hours and days following a strenuous bout of exercise. Hence, anti-inflammatory and antioxidant supplements are used with the expectation that reducing exercise-induced stress in fatigued muscles will reduce soreness and promote quicker recovery.

Distinct redox behaviors of chloroplast thiol enzymes and their relationships with photosynthetic electron transport in Arabidopsis thaliana.

The thiol/disulfide redox network mediated by the thioredoxin (Trx) system in chloroplasts ensures light-responsive control of diverse crucial functions. Despite the suggested importance of this system, the working dynamics against changing light environments remains largely unknown. Thus, we directly assessed the in vivo redox behavior of chloroplast Trx-targeted thiol enzymes in Arabidopsis thaliana. In a time-course analysis throughout a day period that was artificially mimicked to natural light conditions, thiol enzymes showed a light-dependent shift in redox state, but the patterns were distinct among thiol enzymes. Notably, the ATP synthase CF(1-γ) subunit was rapidly reduced even under low-light conditions, whereas the stromal thiol enzymes fructose 1,6-bisphosphatase, sedoheptulose 1,7-bisphosphatase, and NADP-malate dehydrogenase were gradually reduced/re-oxidized along with the increase/decrease in light intensity. Photo-reduction of thiol enzymes was suppressed by the impairment of photosynthetic linear electron transport using DCMU and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, but sensitivity to the impairment was uneven between CF(1-γ) and other stromal thiol enzymes. These different dependencies of photo-reduction on electron transport, rather than the redox state of Trx and the circadian clock, could readily explain the distinct diurnal redox behaviors of thiol enzymes. In addition, our results indicate that the cyclic electron transport around PSI is also involved in redox regulation of some thiol enzymes. Based on these findings, we propose an in vivo working model of the redox regulation system in chloroplasts.

Effect of Maternal Diabetes on Pre- and Post-Natal Redox Status of F1 Rat Offspring

Background: The oxidative stress and disturbed redox signaling during gestation my play an important role in the fetal programming of adult diabetes. Objective: The study aimed to investigate the effects of maternal diabetes on pre- and post-natal pancreatic and peripheral tissues redox and oxidative status in order to clarify their role in the diabetogenic programming of F1 offspring. Methods: Two groups of female Wistar rats were used (diabetic and control); diabetes was neonatally induced by STZ injection to 5-day old rats. 10 pregnancies of each group were terminated at GD 17 to obtain placentas and fetal pancreas, liver, muscle and adipose tissues for prenatal measurements. The rest of pregnancies were completed to term and the offspring were weaned to control diet or high-caloric (HCD) diet and followed up for 30 weeks. Every 5 weeks 10 male rats were sacrificed and serum and tissues were obtained for assessment of fasting blood glucose, tissues content of 8-oxo-dG, TBARS, GSH, GSSG, antioxidant enzymes and caspase-3. Results: The results indicated that, prenatally redox status of the foetuses of diabetic mothers is shifted toward more oxidizing environment which results from elevated oxidative stress and impaired antioxidants as indicated by elevated fetal tissues content of 8-oxo-dG, TBARS and GSSG and decreased GSH and GSH/GSSG ratio. All of these induce the apoptotic pathway in fetal pancreas. Postnatally, impaired glucose tolerance in the offspring of diabetic mothers is detected at 15th week of age and no hyperglycemia was detected until age of 30 weeks in the offspring under CD while some of offspring under HCD at age 25 weeks and most of them at 30 weeks have developed hyperglycemia. The pancreas of the offspring of diabetic mothers suffers from oxidative stress from the 5th week of age as indicated by elevated levels of nuclear and mitochondrial 8-oxo-dG contents and TBARS. Also, GSH level showed depletion with age and the activity of glutathione reductase was lower in the β-cell and hepatic tissues of the offspring of diabetic mothers. The prenatal shift of redox status persists postnatal and exaggerates with age which may explain the significant induction of the apoptotic pathway in the pancreatic β-cell. Conclusion: Maternal diabetes can crucially affect the redox status in fetal tissues prenatally and these effects can persist postnatal which may play an important role in the programming of the metabolic state of the offspring. Postnatal diet play important role in accelerating development of metabolic derangements and aggravates oxidative stress in the tissues of F1 offspring.

Traumatic brain injury in aged animals increases lesion size and chronically alters microglial/macrophage classical and alternative activation states

Traumatic brain injury (TBI) causes chronic microglial activation that contributes to subsequent neurodegeneration, with clinical outcomes declining as a function of aging. Microglia/macrophages (MG/Mɸ) have multiple phenotypes, including a classically activated, proinflammatory (M1) state that might contribute to neurotoxicity, and an alternatively activated (M2) state that might promote repair. In this study we used gene expression, immunohistochemical, and stereological analyses to show that TBI in aged versus young mice caused larger lesions associated with an M1/M2 balance switch and increased numbers of reactive (bushy and hypertrophic) MG/Mɸ in the cortex, hippocampus, and thalamus. Chitinase3-like 3 (Ym1), an M2 phenotype marker, displayed heterogeneous expression after TBI with amoeboid-like Ym1-positive MG/Mɸ at the contusion site and ramified Ym1-positive MG/Mɸ at distant sites; this distribution was age-related. Aged-injured mice also showed increased MG/Mɸ expression of major histocompatibility complex II and NADPH oxidase, and reduced antioxidant enzyme expression which was associated with lesion size and neurodegeneration. Thus, altered relative M1/M2 activation and an nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase)-mediated shift in redox state might contribute to worse outcomes observed in older TBI animals by creating a more proinflammatory M1 MG/Mɸ activation state.

Mitochondrial redox signalling at a glance

Redox signalling occurs when a biological system alters in response to a change in the level of a particular reactive oxygen species (ROS) or the shift in redox state of a responsive group such as a dithiol–disulphide couple ([D'Autreaux and Toledano, 2007][1]; [Finkel, 2011][2]; [Fourquet et al

Abstract P315: S-Glutathiolation of Low-Molecular-Weight PTP Regulates VEGF-Angiogenic Signal

While reparative angiogenesis improves outcome of stroke and myocardial infarction; pathological angiogenesis aggravates tumors, psoriasis and diabetic retinopathy. We have previously shown that low levels of peroxynitrite (PN, 1µM) are required to mediate VEGF’s angiogenic signal. Prior studies showed that low molecular weight protein tyrosine phosphatase (LMW-PTP) can regulate activation of VEGFR2 and focal adhesion kinase (FAK), key proteins involved in VEGF angiogenic signal. Here, we test the hypothesis that VEGF-induced peroxynitrite regulates LMW-PTP activity via reversible S-glutathiolation. Our results using human microvascular endothelial (HME) cells showed that VEGF (20 ng/ml) caused immediate (1,5 min) and transient negative shift in redox-state assessed by reduced-glutathione (GSH) level that was restored back to baseline by 15–30 minutes. In parallel, total free thiols of LMW-PTP; assessed by 5-iodoacetamide; were immediately oxidized (1, 5 min) and recovered after 15–30 minutes. PN (1μM) mimicked VEGF’ actions by inducing oxidative inhibition of LMW-PTP and FAK phosphorylation that peaked at 15 min. Studies using anti-GSH antibody demonstrated strong s-glutathiolation of LMW-PTP thiols that peaked at (5–10min) under non-reducing conditions and were completely abrogated in DTT-treated samples. These effects were associated with impaired tyrosine phosphorylation of LMW-PTP. PN caused concentration-dependent significant inhibition of LMW-PTP phosphatase activity reaching maximum at 0.5mM. Under pathological conditions recapitulated by combining with peroxynitrite (0.5mM), VEGF caused immediate yet permanent negative shift in cellular redox-state, LMW-PTP inactivation and sustained FAK activation in HME cells. Modulating cellular redox-state to reductive stress using N-acetyl cysteine (NAC, 1mM) or FeTPPS (2.5μM) prevented VEGF-induced angiogenesis in vitro and in vivo using hypoxia-induced neovascularization mouse model. Taken together, shifting redox-state to oxidative stress or reductive stress can impair VEGF’s signal and achieving a balanced redox-state is critical to facilitate VEGF’ angiogenic signal.

Inhibition of NADPH oxidase promotes alternative and anti‐inflammatory microglial activation during neuroinflammation

Like macrophages, microglia are functionally polarized into different phenotypic activation states, referred as classical and alternative. The balance of the two phenotypes may be critical to ensure proper brain homeostasis, and may be altered in brain pathological states, such as Alzheimer's disease. We investigated the role of NADPH oxidase in microglial activation state using p47(phox) and gp91(phox) -deficient mice as well as apocynin, a NADPH oxidase inhibitor during neuroinflammation induced by an intracerebroventricular injection of LPS or Aβ₁₋₄₂. We showed that NADPH oxidase plays a critical role in the modulation of microglial phenotype and subsequent inflammatory response. We demonstrated that inhibition of NADPH oxidase or gene deletion of its functional p47(phox) subunit switched microglial activation from a classical to an alternative state in response to an inflammatory challenge. Moreover, we showed a shift in redox state towards an oxidized milieu and that subpopulations of microglia retain their detrimental phenotype in Alzheimer's disease brains. Microglia can change their activation phenotype depending on NADPH oxidase-dependent redox state of microenvironment. Inhibition of NADPH oxidase represents a promising neuroprotective approach to reduce oxidative stress and modulate microglial phenotype towards an alternative state.

Thioredoxin Reductase-2 Is Essential for Keeping Low Levels of H2O2 Emission from Isolated Heart Mitochondria

Respiring mitochondria produce H(2)O(2) continuously. When production exceeds scavenging, H(2)O(2) emission occurs, endangering cell functions. The mitochondrial peroxidase peroxiredoxin-3 reduces H(2)O(2) to water using reducing equivalents from NADPH supplied by thioredoxin-2 (Trx2) and, ultimately, thioredoxin reductase-2 (TrxR2). Here, the contribution of this mitochondrial thioredoxin system to the control of H(2)O(2) emission was studied in isolated mitochondria and cardiomyocytes from mouse or guinea pig heart. Energization of mitochondria by the addition of glutamate/malate resulted in a 10-fold decrease in the ratio of oxidized to reduced Trx2. This shift in redox state was accompanied by an increase in NAD(P)H and was dependent on TrxR2 activity. Inhibition of TrxR2 in isolated mitochondria by auranofin resulted in increased H(2)O(2) emission, an effect that was seen under both forward and reverse electron transport. This effect was independent of changes in NAD(P)H or membrane potential. The effects of auranofin were reproduced in cardiomyocytes; superoxide and H(2)O(2) levels increased, but similarly, there was no effect on NAD(P)H or membrane potential. These data show that energization of mitochondria increases the antioxidant potential of the TrxR2/Trx2 system and that inhibition of TrxR2 results in increased H(2)O(2) emission through a mechanism that is independent of changes in other redox couples.