Redox-sensitive Gene Expression Research Articles

ROS-hormone interaction in regulating integrative d閒ense signaling of plant cell

The elaborate redox network of the cell, comprising of events like turnover of reactive oxygen species (ROS), redox sensing, signaling, expression of redox-sensitive genes, etc., often orchestrates with other bonafide hormonal signaling pathways through their synergistic or antagonistic action in the plant cell. The redox cue generated in plant cells under fluctuating environmental conditions can significantly influence other hormonal biosynthetic or signaling mechanisms, thereby modulating physiology towards stress acclimation and defense. There is also strong evidence of the recruitment of ROS as a ‘second messenger’ in different hormonal signaling pathways under stress. Moreover, the retrograde signaling initiated by ROS also found to strongly influence hormonal homeostasis and signaling. The present review, in this aspect, is an effort towards understanding the regulatory roles of ROS in integrating and orchestrating other hormonal signaling pathways or vice versa so as to unfold the relationship between these two signaling episodes of plant cells under environmental odds. We also accentuate the significance of understanding the utterly complex interactions, which occur both at metabolic and genetic levels between ROS and phytohormones during stress combinations. Furthermore, the significant and decisive role of ROS turnover, particularly the contribution of RBOH (respiratory burst oxidase homologs) in the synergism of redox and hormone signaling during systemic acquired acclimation under stress is also discussed.

Methods for Measurements of Oxidized LDL, Homocysteine and Nitric Oxide as Clinical Parameters of Oxidative Stress and Endothelial Dysfunction

Abstract: Timely and accurate evaluation of clinical parameters associated with endothelial dysfunction is critical in diagnosing and treating atherosclerosis, which represents a severe health problem, accounting for at least 30% of deaths globally. A critical early event in the pathogenesis of atherosclerosis is the oxidative modification of low-density lipoprotein [LDL]. Oxidized LDL [OxLDL] represents numerous changes in lipid and apolipoprotein B [apo B] fractions of LDLs generated by lipid peroxidation. Another critical indicator of perturbed vascular homeostasis is homocysteine [Hcy], an amino acid containing sulfhydryl-group, an intermediate methionine and cysteine biosynthesis product. The total level of Hcy in plasma correlates better than cholesterol with the risk of cardiovascular disease. In addition, nitric oxide [NO] plays an essential role in regulating vascular physiological homeostasis due to its involvement in intravascular free radical and oxidant reactions. Reduced NO decreases oxidative stress in the vascular wall, which reduces the rate of LDL oxidation and the expression of redox-sensitive genes involved in atherogenesis. Endothelial dysfunction is typically associated with increased levels of OxLDL, decreased nitric oxide [NO], and hyperhomocysteinemia. Thus, OxLDL, Hcy, and NO are representative parameters of oxidative stress and endothelial dysfunction. Considering the important role of oxLDL, Hcy and NO in oxidative stress, atherogenesis and accompanying endothelial dysfunction, the challenge of the present work was to systematically present available methods for reliable measurement of these parameters and assess their potential for the use in the clinical setting. Here we present a comprehensive overview of analytical methods for measuring OxLDL, HCy, and NO in biological samples and discuss their advantages and potential problems regarding their application in clinical settings.

The role of nitric oxide (NO) levels in patients with obstructive sleep apnea-hypopnea syndrome: a meta-analysis.

The pathogenesis of cardiovascular disease (CVD) in patients with obstructive sleep apnea (OSA) is unclear. Several studies have suggested that CVD may be caused by oxidative stress from chronic intermittent hypoxia and associated vascular endothelial dysfunction. Oxidative stress in patients with OSA can induce endothelial cell apoptosis, aggravate vascular endothelial damage, and promote the expression of redox-sensitive genes and adhesion molecules. No meta-analysis has explored whether or not OSA is related to nitric oxide (NO). To assess the association between serum/plasma NO levels and OSA, we performed a meta-analysis of the literature on the subject to grade the strength of evidence. OSA was significantly related to decreased serum or plasma NO levels (WMD = - 11.66, 95% CI - 17.21 to - 6.11; P< 0.01). Among the studies analyzed, there was high degree of heterogeneity (I2 = 79%, P< 0.01). Sensitivity analysis showed that after omitting any single study or converting a random effects model (REM) to a fixed effects model (FEM), the main results still held. This meta-analysis suggests a strong correlation between OSA and serum or plasma NO levels which may explain the link between intermittent hypoxia of OSA and risk of CVD. The strength of this finding may spur further basic and clinical research into vascular endothelial dysfunction in patients with OSA.

The influence of changes in expression of redox-sensitive genes on the development of retinopathy in rats

Age-related macular degeneration (AMD) is a complex multifactorial disease of the elderly, with unclear pathogenesis; AMD is the leading cause of blindness. One of the destructive processes in AMD is oxidative stress, which leads to an imbalance in the processes responsible for production and detoxification of reactive oxygen species. The aryl hydrocarbon receptor (AhR) signaling pathway can participate in the development of oxidative stress, but the main regulator of antioxidant defense is nuclear factor, erythroid derived 2 (Nrf2). AhR-dependent oxidative stress can be attenuated by activation of Nrf2, and defects in the Nrf2 signaling pathway can increase sensitivity of the cell to oxidative stress. Our aim was to determine the role of the pro-oxidant (AhR-dependent) and antioxidant (Nrf2-dependent) systems in the pathogenesis of AMD using rats of OXYS strain and of OXYSb substrain with signs of AMD-like retinopathy of varying severity. We compared the retinal levels of mRNA expression of Nrf2- and AhR-dependent redox-sensitive systems between 1-, 3-, and 12- month-old senescence-accelerated OXYS rats (have been shown to be a valid experimental model of AMD) and the rat substrain OXYSb, which shows low morbidity of AMD. We uncovered interstrain differences in the expression of Nrf2 and Nrf2-dependent genes (glutathione S-reductase [Gsr] and heme oxygenase 1 [Hmox1]), in the expression of AhR-dependent genes (cytochrome P450 1A2 [Cyp1a2] and cytochrome P450 1B1 [Cyp1b1]), and in the NADPH-quinone oxidoreductase (Nqo1) expression, which is controlled by both AhR and Nrf2. Binding of AhR and Nrf2 proteins to the regulatory regions of AhR and Nrf2 genes, respectively, was detected by chromatin immunoprecipitation in the retina of 1-, 3-, and 12-month-old OXYS, OXYSb, and Wistar (control) rats. We compared the strength of DNA-protein interactions of AhR and Nrf2 with regulatory sequences and found that the level of autoupregulation of the AhR gene was higher in the retina of 1-month-old OXYSb rats in comparison with OXYS rats. An imbalance between pro-oxidant (AhR-dependent) and antioxidant (Nrf2-dependent) systems may play a crucial role in the onset and/or progression of AMD.

An evaluation of the antioxidant protein α1-microglobulin as a renal tubular cytoprotectant.

α1-Microglobulin (A1M) is a low-molecular-weight heme-binding antioxidant protein that is readily filtered by the glomerulus and reabsorbed by proximal tubules. Given these properties, recombinant A1M (rA1M) has been proposed as a renal antioxidant and therapeutic agent. However, little direct evidence to support this hypothesis exists. Hence, we have sought "proof of concept" in this regard. Cultured proximal tubule (HK-2) cells or isolated mouse proximal tubule segments were challenged with a variety of prooxidant insults: 1) hemin, 2) myoglobin; 3) "catalytic" iron, 4) H2O2/Fenton reagents, 5) a Ca(2+) ionophore, 6) antimycin A, or 7) hypoxia (with or without rA1M treatment). HK-2 injury was gauged by the percent lactate dehydrogenase release and 4,5-(dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide uptake. In vivo protection was sought in rA1M-treated mice subjected to 1) graded myohemoglobinura (2, 4, 8, or 9 ml/kg glycerol injection), 2) purified myoglobinemia/uria, or 3) endotoxemia. In vivo injury was assessed by blood urea nitrogen, creatinine, and the expression of redox-sensitive genes (heme oxygenase-1, neutrophil gelatinase-associated lipocalin, and monocyte chemoattractant protein-1 mRNAs). Although rA1M totally blocked in vitro hemin toxicity, equimolar albumin (another heme binder) or 10% serum induced equal protection. rA1M failed to mitigate any nonhemin forms of either in vitro or in vivo injury. A1M appeared to be rapidly degraded within proximal tubules (by Western blot analysis). Surprisingly, rA1M exerted select injury-promoting effects (increased in vitro catalytic iron/antimycin toxicities and increased in vivo monocyte chemoattractant protein-1/neutrophil gelatinase-associated lipocalin mRNA expression after glycerol or endotoxin injection). We conclude that rA1M has questionable utility as a renal antioxidant/cytoprotective agent, particularly in the presence of larger amounts of competitive free heme (e.g., albumin) binders.

Lipidomic evidence that lowering the typical dietary palmitate to oleate ratio in humans decreases the leukocyte production of proinflammatory cytokines and muscle expression of redox-sensitive genes

We recently reported that lowering the high, habitual palmitic acid (PA) intake in ovulating women improved insulin sensitivity and both inflammatory and oxidative stress. In vitro studies indicate that PA can activate both cell membrane toll-like receptor-4 and the intracellular nucleotide oligomerization domain-like receptor protein (NLRP3). To gain further insight into the relevance to human metabolic disease of dietary PA, we studied healthy, lean and obese adults enrolled in a randomized, crossover trial comparing 3-week, high-PA (HPA) and low-PA/high-oleic-acid (HOA) diets. After each diet, both hepatic and peripheral insulin sensitivities were measured, and we assessed cytokine concentrations in plasma and in supernatants derived from lipopolysaccharide-stimulated peripheral blood mononuclear cells (PBMCs) as well as proinflammatory gene expression in skeletal muscle. Insulin sensitivity was unaffected by diet. Plasma concentration of tumor necrosis factor-α was higher during the HPA diet. Lowering the habitually high PA intake by feeding the HOA diet resulted in lower secretion of interleukin (IL)-1β, IL-18, IL-10, and tumor necrosis factor-α by PBMCs, as well as lower relative mRNA expression of cJun and NLRP3 in muscle. Principal components analysis of 156 total variables coupled to analysis of covariance indicated that the mechanistic pathway for the differential dietary effects on PBMCs involved changes in the PA/OA ratio of tissue lipids. Our results indicate that lowering the dietary and tissue lipid PA/OA ratio resulted in lower leukocyte production of proinflammatory cytokines and muscle expression of redox-sensitive genes, but the relevance to diabetes risk is uncertain.

Protective effect of nuclear factor E2-related factor 2 on inflammatory cytokine response to brominated diphenyl ether-47 in the HTR-8/SVneo human first trimester extravillous trophoblast cell line

Polybrominated diphenyl ethers (PBDEs) are widely used flame retardants, and BDE-47 is a prevalent PBDE congener detected in human tissues. Exposure to PBDEs has been linked to adverse pregnancy outcomes in humans. Although the underlying mechanisms of adverse birth outcomes are poorly understood, critical roles for oxidative stress and inflammation are implicated. The present study investigated antioxidant responses in a human extravillous trophoblast cell line, HTR-8/SVneo, and examined the role of nuclear factor E2-related factor 2 (Nrf2), an antioxidative transcription factor, in BDE-47-induced inflammatory responses in the cells. Treatment of HTR-8/SVneo cells with 5, 10, 15, and 20μM BDE-47 for 24h increased intracellular glutathione (GSH) levels compared to solvent control. Treatment of HTR-8/SVneo cells with 20μM BDE-47 for 24h induced the antioxidant response element (ARE) activity, indicating Nrf2 transactivation by BDE-47 treatment, and resulted in differential expression of redox-sensitive genes compared to solvent control. Pretreatment with tert-butyl hydroquinone (tBHQ) or sulforaphane, known Nrf2 inducers, reduced BDE-47-stimulated IL-6 release with increased ARE reporter activity, reduced nuclear factor kappa B (NF-κB) reporter activity, increased GSH production, and stimulated expression of antioxidant genes compared to non-Nrf2 inducer pretreated groups, suggesting that Nrf2 may play a protective role against BDE-47-mediated inflammatory responses in HTR-8/SVneo cells. These results suggest that Nrf2 activation significantly attenuated BDE-47-induced IL-6 release by augmentation of cellular antioxidative system via upregulation of Nrf2 signaling pathways, and that Nrf2 induction may be a potential therapeutic target to reduce adverse pregnancy outcomes associated with toxicant-induced oxidative stress and inflammation.

Mono-2-ethylhexyl phthalate induces oxidative stress responses in human placental cells in vitro

Di-2-ethylhexyl phthalate (DEHP) is an environmental contaminant commonly used as a plasticizer in polyvinyl chloride products. Exposure to DEHP has been linked to adverse pregnancy outcomes in humans including preterm birth, low birth-weight, and pregnancy loss. Although oxidative stress is linked to the pathology of adverse pregnancy outcomes, effects of DEHP metabolites, including the active metabolite, mono-2-ethylhexyl phthalate (MEHP), on oxidative stress responses in placental cells have not been previously evaluated. The objective of the current study is to identify MEHP-stimulated oxidative stress responses in human placental cells. We treated a human placental cell line, HTR-8/SVneo, with MEHP and then measured reactive oxygen species (ROS) generation using the dichlorofluorescein assay, oxidized thymine with mass-spectrometry, redox-sensitive gene expression with qRT-PCR, and apoptosis using a luminescence assay for caspase 3/7 activity. Treatment of HTR-8 cells with 180μM MEHP increased ROS generation, oxidative DNA damage, and caspase 3/7 activity, and resulted in differential expression of redox-sensitive genes. Notably, 90 and 180μM MEHP significantly induced mRNA expression of prostaglandin-endoperoxide synthase 2 (PTGS2), an enzyme important for synthesis of prostaglandins implicated in initiation of labor. The results from the present study are the first to demonstrate that MEHP stimulates oxidative stress responses in placental cells. Furthermore, the MEHP concentrations used were within an order of magnitude of the highest concentrations measured previously in human umbilical cord or maternal serum. The findings from the current study warrant future mechanistic studies of oxidative stress, apoptosis, and prostaglandins as molecular mediators of DEHP/MEHP-associated adverse pregnancy outcomes.

Impact of Dietary Soy Isoflavones in Pregnancy on Fetal Programming of Endothelial Function in Offspring

Epidemiological evidence suggests that soy-based diets containing phytoestrogens (isoflavones) afford protection against cardiovascular diseases (CVDs); however, supplementation trials have largely reported only marginal health benefits. The molecular mechanisms by which the isoflavones genistein, daidzein, and equol afford protection against oxidative stress remain to be investigated in large scale clinical trials. Isoflavones are transferred across the placenta in both rodents and humans, yet there is limited information on their actions in pregnancy and the developmental origins of disease. Our studies established that feeding a soy isoflavone-rich diet during pregnancy, weaning, and postweaning affords cardiovascular protection in aged male rats. Notably, rats exposed to a soy isoflavone-deficient diet throughout pregnancy and adult life exhibited increased oxidative stress, diminished antioxidant enzyme and eNOS levels, endothelial dysfunction, and elevated blood pressure in vivo. The beneficial effects of refeeding isoflavones to isoflavone-deficient rats include an increased production of nitric oxide and EDHF, an upregulation of antioxidant defense enzymes and lowering of blood pressure in vivo. This review focuses on the role that isoflavones in the fetal circulation may play during fetal development in affording protection against CVD in the offspring via their ability to activate eNOS, EDHF, and redox-sensitive gene expression.

Role of LOX‐1 in monocyte adhesion‐triggered redox, Akt/eNOS and Ca2+ signaling pathways in endothelial cells

This study was conducted to examine the role of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in monocyte adhesion-induced redox-sensitive, Akt/eNOS and Ca2+ signaling pathways in endothelial cells (ECs). LOX-1 was blocked by an antibody-neutralizing LOX-1 TS92 or small interfering RNA. In cultured human aortic ECs, monocyte adhesion activated Rac1 and p47(phox), and increased NADPH oxidase activity and reactive oxygen species (ROS) generation within 30 min and NF-kappaB phosphorylation within 1 h, resulting in redox-sensitive gene expression. Akt and eNOS phosphorylation was induced 15 min after adding monocytes and returned to control level after 30 min, whereas NO production was not altered by monocyte adhesion. Blockade of LOX-1 blunted the monocyte adhesion-triggered redox-sensitive signaling pathway and Akt/eNOS phosphorylation in ECs. Both endothelial intracellular Ca2+ mobilization and Ca2+ influx caused by monocyte attachment were markedly attenuated by pretreatment of ECs with TS92. This suggests that LOX-1 is involved in redox-sensitive, Akt/eNOS and Ca2+ signaling pathways in monocyte adhesion to ECs independent of oxidized low-density lipoprotein (ox-LDL). Furthermore, blockade of Ca2+ inhibited monocyte adhesion-triggered Rac1 and p47(phox) activation and ROS generation in ECs, whereas Ca2+ signaling was suppressed by blockade of NADPH oxidase and ROS generation. Finally, TS92 blocked the monocyte adhesion to ECs stimulated with or without tumor necrosis factor-alpha or ox-LDL. We provide evidence that LOX-1 plays a role in redox-sensitive, Akt/eNOS and Ca2+ signaling pathways in monocyte adhesion to ECs independent of the ox-LDL-LOX-1 axis.

Protein Nitration in Placenta – Functional Significance

Crucial roles of the placenta are disrupted in early and mid-trimester pregnancy loss, preeclampsia, eclampsia and intrauterine growth restriction. The pathophysiology of these disorders includes a relative hypoxia of the placenta, ischemia/reperfusion injury, an inflammatory response and oxidative stress. Reactive oxygen species including nitric oxide (NO), carbon monoxide and superoxide have been shown to participate in trophoblast invasion, regulation of placental vascular reactivity and other events. Superoxide, which regulates expression of redox sensitive genes, has been implicated in up-regulation of transcription factors, antioxidant production, angiogenesis, proliferation and matrix remodeling. When superoxide and nitric oxide are present in abundance, their interaction yields peroxynitrite a potent pro-oxidant, but also alters levels of nitric oxide, which in turn affect physiological functions. The peroxynitrite anion is extremely unstable thus evidence of its formation in vivo has been indirect via the occurrence of nitrated moieties including nitrated lipids and nitrotyrosine residues in proteins. Formation of 3-nitrotyrosine (protein nitration) is a “molecular fingerprint” of peroxynitrite formation. Protein nitration has been widely reported in a number of pathological states associated with inflammation but is reported to occur in normal physiology and is thought of as a prevalent, functionally relevant post-translational modification of proteins. Nitration of proteins can give either no effect, a gain or a loss of function. Nitration of a range of placental proteins is found in normal pregnancy but increased in pathologic pregnancies. Evidence is presented for nitration of placental signal transduction enzymes and transporters. The targets and extent of nitration of enzymes, receptors, transporters and structural proteins may markedly influence placental cellular function in both physiologic and pathologic settings.

LOX-1 Mediates Redox-Sensitive, Akt/eNOS and Ca2+ Signaling Pathways Induced in Endothelial Cells by Monocyte Adhesion

Background: Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), a major receptor for oxidized LDL, plays a pivotal role in endothelial dysfunction. Monocyte adhesion to endothelial cells (ECs) is crucial for the initiation and development of atherosclerosis. This study was conducted to examine the role of LOX-1 in monocyte adhesion-induced redox-sensitive Akt/eNOS and Ca2+ signaling pathways in ECs. Methods and Results: Isolated human monocytes were added to cultured human aortic ECs. Monocyte adhesion increased Rac1 and p47phox activity, and NADPH oxidase activity and reactive oxygen species generation within 15 minutes, and NF-κB phosphorylation within 1 hour in ECs, resulting in redox-sensitive gene expression. Akt and eNOS phosphorylation was induced 15 minutes after adding monocytes and returned to control level after 30 minutes with no alteration of NO production. Inhibition of LOX-1 blocked monocyte adhesion-triggered activation of redox-sensitive signaling and Akt/eNOS pathways. Both endothelial intracellular Ca2+ mobilization and Ca2+ influx caused by monocyte attachment were also markedly attenuated by inhibition of LOX-1. This suggests that LOX-1 is involved in redox-sensitive, Akt/eNOS and Ca2+ signaling pathways in monocyte adhesion to ECs. Conclusions: We provide evidence that LOX-1 plays a role in redox-sensitive signaling, Akt/eNOS signaling and Ca2+ signaling pathways in monocyte adhesion to ECs independent of an ox-LDL-LOX-1 axis.

Nutritional control of HIV-AIDS- A case for Selenium

Selenium (Se) is a micronutrient that is incorporated into proteins. Some of the selenoenzymes such as GPXs and thioredoxin reductases exhibit glutathione peroxidase-dependent activities towards organic and inorganic peroxides. Thus, selenoenzymes play a pivotal role in mitigating oxidative stress caused by accumulation of peroxides. In this respect, selenoproteins may interfere with the transcription-driven by oxidative stress-dependent signaling mechanisms to influence the expression of redox sensitive genes, including inflammatory cytokines and HIV provirus. Several epidemiological studies support our view that Se supplementation affects transcription of HIV; however, the underlying mechanism is not elucidated. In this mini review, we consider the relationship between Se and HIV and propose possible mechanisms by which Se controls HIV transcription.

Conclusions, perspectives et recommandations

Understanding the notion of oxidative stress implies a good knowledge of the reactivity of the different reactive oxygen species (ROS). Cell damage can be induced by an overproduction of these species and/or by a deficiency in the protective antioxidant systems. Nevertheless, ROS do not display only deleterious effects and play key-roles in cell signalisation and regulation of the expression of redox sensitive genes. Besides ROS, reactive nitrogen species (RNS) with nitric oxide (*NO) as leader element, are widely involved in biology and lead to the term "nitrosative stress" that particularly describes the damage induced by peroxynitrite, a species formed by reaction between superoxide anion and degrees NO. Nutritional strategies have been based on antioxidant-rich diets, or on supplementation with antioxidants; they could constitute adjunct therapies of interest. Given all these data, radical biochemistry must be considered as a specific discipline.

Sublethal concentrations of diverse gold compounds inhibit mammalian cytosolic thioredoxin reductase (TrxR1)

Thioredoxin reductase (TrxR) reduces thioredoxin (Trx), thereby contributing to cellular redox balance, facilitating the synthesis of deoxy-ribose sugars for DNA synthesis, and regulating redox-sensitive gene expression. Auranofin is a gold compound that potently inhibits TrxR. This inhibition is one suspected mechanism of auranofin’s therapeutic benefit in the treatment of rheumatoid arthritis. The use of other gold compounds to treat cancer or inflammatory disease may rely on their ability to inhibit TrxR. In the current study, we tested the hypothesis that a variety of gold compounds may inhibit TrxR. Methods: We exposed rat-TrxR1 to auranofin, gold sodium thiomalate, sodium aurothiosulfate, triphenyl phosphine gold chloride, or gold acetate, and measured TrxR activity ex vivo. We then compared TrxR1 inhibitory levels of gold compounds to those that inhibited mitochondrial activity of THP1 monocytes and OSC2 epithelial cells, estimated by succinate dehydrogenase activity. Results: All gold compounds inhibited TrxR1 at concentrations ranging from 5 to 4000 nM (50% inhibitory concentration). The oxidation state of gold did not correlate with inhibitory potency, but ligand configuration was important. Au(I)-phosphine compounds (triphenyl phosphine gold chloride and auranofin) were the most potent inhibitors of TrxR. All TrxR1 inhibitory concentrations were sublethal to mitochondrial activity in both THP1 and OSC2 cells. Conclusions: Diverse types of gold compounds may be effective inhibitors of TrxR1 at concentrations that do not suppress cellular mitochondrial function. Inhibition may be optimized to some degree by altering the ligand configuration of the compounds. These results support future study of a variety of Au compounds for therapeutic development as inhibitors of TrxR1.

The acute-phase response of the liver in relation to thyroid hormone-induced redox signaling

Recently, we reported that 3,3′,5-triiodothyronine (T 3) induces the expression of redox-sensitive genes as a nongenomic mechanism of T 3 action. In this study, we show that T 3 administration to rats (daily doses of 0.1 mg/kg ip for 3 consecutive days) induced a calorigenic response and liver glutathione depletion as an indication of oxidative stress, with higher levels of interleukin (IL)-6 in serum (ELISA) and hepatic STAT3 DNA binding (EMSA), which were maximal at 48–72 h after treatment. Under these conditions, the protein expression of the acute-phase proteins haptoglobin and β-fibrinogen is significantly augmented, a change that is suppressed by pretreatment with α-tocopherol (100 mg/kg ip) or gadolinium chloride (10 mg/kg iv) before T 3. It is concluded that T 3 administration induces the acute-phase response in rat liver by a redox mechanism triggered at the Kupffer cell level, in association with IL-6 release and activation of the STAT3 cascade, a response that may contribute to reestablishing homeostasis in the liver and extrahepatic tissues exhibiting oxidative stress.

Chronic iron overload enhances inducible nitric oxide synthase expression in rat liver

Iron is an essential micronutrient promoting oxidative stress in the liver of overloaded animals and human, which may trigger the expression of redox-sensitive genes. We have tested the hypothesis that chronic iron overload (CIO) enhances inducible nitric oxide synthase (iNOS) expression in rat liver by extracellular signal-regulated kinase (ERK1/2) and NF-kappaB activation. CIO (diet enriched with 3%(wt/wt) carbonyl-iron for 12 weeks) increased liver protein carbonylation and decreased reduced glutathione (GSH) content and the GSH/GSSG ratio after 6 weeks, parameters that are normalized after 8-12 weeks of treatment. These changes are paralleled by higher phosphorylated-ERK1/2 to non-phosphorylated-ERK1/2 ratios at 6 and 8 weeks, increased NF-kappaB DNA binding to the iNOS gene promoter at 8-12 weeks, and higher iNOS mRNA expression and activity at 8 and 12 weeks. It is concluded that CIO triggers liver oxidative stress at early times, with upregulation of iNOS expression involving the ERK/NF-kappaB pathway at later times, a finding that may represent a hepatoprotective mechanism against CIO toxicity in addition to the recovery of GSH homeostasis.

Redox regulation of thyroid hormone-induced Kupffer cell-dependent IκB-α phosphorylation in relation to inducible nitric oxide synthase expression

Thyroid hormone-induced calorigenesis promotes oxidative stress in the liver with higher respiratory burst activity in Kupffer cells, which could increase the expression of redox-sensitive genes. Our aim was to test the hypothesis that L-3,3′,5-triiodothyronine (T3) triggers inducible nitric oxide synthase (iNOS) expression in rat liver by upstream mechanisms involving the inhibitor of κB (IκB) kinase activation. T3 administration (daily doses of 0.1 mg/kg for three consecutive days) induced a calorigenic response, with maximal increases in the content of hepatic thiobarbituric acid reactants or protein carbonyls and NOS activity at 48–72 h after treatment, compared to control values. In this time interval, the serum levels of tumor necrosis factor-α (TNF-α; ELISA) are enhanced, concomitantly with higher liver IκB-α phosphorylation (Western blot analysis), NF-κB DNA binding (electrophoretic mobility shift assay), and iNOS mRNA expression (reverse transcription-polymerase chain reaction). These changes and the increase in hepatic NOS activity are abolished by the administration of either α-tocopherol (100 mg/kg) or the Kupffer cell inactivator gadolinium chloride (10 mg/kg) prior to T3. It is concluded that T3-induced oxidative stress triggers the redox upregulation of liver iNOS expression through a cascade initiated by TNF-α produced by Kupffer cells and involving IκB-α phosphorylation and NF-κB activation, a response that may represent a defense mechanism by protecting the liver from cytokine-mediated lethality and ROS toxicity.

Adrenomedullin Inhibits Angiotensin II-Induced Oxidative Stress and Gene Expression in Rat Endothelial Cells

Adrenomedullin (AM), a potent vasodilator peptide, has recently been suggested to function as an endogenous antioxidant. However, its potential site of action at the cellular level has not been clarified. The present study was undertaken to investigate whether AM directly inhibits intracellular reactive oxygen species (ROS) generation and redox-sensitive gene expression stimulated by angiotensin (Ang) II in rat aortic endothelial cells (ECs). Ang II (10(-7) mol/l) significantly increased intracellular ROS levels in ECs as measured by dichlorofluorescein (DCF) fluorescence. AM inhibited Ang II-stimulated ROS generation in a dose-dependent manner and this effect was abolished by a superoxide radical scavenger, NAD(P)H oxidase inhibitor, and a protein kinase A (PKA) inhibitor, and mimicked by a cell-permeable cAMP analog. A real-time reverse transcription-polymerase chain reaction (RT-PCR) study showed that Ang II significantly upregulated a set of redox-sensitive genes (ICAM-1, VCAM-1, PAI-1, tissue factor, MCP-1, osteopontin), and these effects were blocked by an antioxidant, N-acetyl cysteine (NAC). AM similarly and dose-dependently inhibited the Ang II-induced upregulation of the entire set of these genes via a receptor-mediated and PKA-dependent pathway, and the degrees of inhibition were similar to those by NAC. In conclusion, the present study demonstrated that AM potently blocked the Ang II-stimulated intracellular ROS generation from NAD(P)H oxidase and the subsequent redox-sensitive gene expression via a cAMP-dependent mechanism in ECs, suggesting that AM has vasculoprotective effects against pro-oxidant stimuli.

Antioxidant effect of adrenomedullin on angiotensin II-induced reactive oxygen species generation in vascular smooth muscle cells.

Recent adrenomedullin (AM) gene-targeting studies have proposed a novel concept that AM plays a protective role against oxidative stress in vivo. The present study was undertaken to explore the underlying molecular mechanism of the putative antioxidant action of AM against angiotensin II (Ang II)induced reactive oxygen species (ROS) generation in rat vascular smooth muscle cells (VSMCs). Intracellular ROS levels were measured by dichlorofluoroscein fluorescence. Redox-sensitive c-Jun amino-terminal kinase (JNK) and ERK1/2 activation and gene expression induced by Ang II in VSMCs were also studied. AM dose-relatedly (10(-8)-10(-7) m) inhibited intracellular ROS generation stimulated by Ang II (10(-7) m), as mimicked by dibutyl-cAMP, the effect of which was inhibited by the pretreatment with N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride, a protein kinase A inhibitor, and calcitonin gene-related peptide(8-37), an AM/calcitonin gene-related peptide receptor antagonist. Ang II induced JNK and ERK1/2 activation via a redox-sensitive manner, whereas AM inhibited JNK, but not ERK1/2, activation by Ang II. Furthermore, AM inhibited Ang II-induced redox-sensitive gene expression (plasminogen activator inhibitor-1 and monocyte chemoattractant protein-1) in the same manner as N-acetyl-l-cysteine, a potent antioxidant. AM also inhibited Ang II-induced up-regulation of Nox1, a critical membrane-bound component of reduced nicotinamide adenine dinucleotide phosphate oxidase in VSMCs, in the same degree as N-acetyl-l-cysteine. Our study demonstrates for the first time that AM directly inhibits intracellular ROS generation via an AM receptor-mediated and c-AMP-protein kinase A-dependent mechanism in VSMCs and that AM with its potent antioxidant action inhibits redox-sensitive JNK activation and gene expression induced by Ang II. These data suggest that AM plays a protective role as an endogenous antioxidant in Ang II-induced vascular injury.