Oxidative stress has been implicated in the pathogenesis of cardiovascular diseases, including myocardial hypertrophy and infarction. Although impairment of antioxidant defense mechanisms has been thought to provoke oxidative stress-induced myocardial dysfunction, it has been difficult to clearly demonstrate. Nuclear erythroid 2 p45-related factor 2 (Nrf2) is a redox-sensitive, basic leucine zipper protein that regulates the transcription of several antioxidant genes. We previously reported that sustained activation of Nrf2 upregulates transcription of a number of endogenous antioxidants in the heart. Here, we show that acute exercise stress (AES) results in activation of Nrf2/ARE (antioxidant response element) signaling and subsequent enhancement of antioxidant defense pathways in wild-type (WT) mouse hearts, while oxidative stress, along with blunted defense mechanisms, was observed in Nrf2−/− mice. We also find that AES is associated with increased trans-activation of ARE-containing genes in exercised animals when compared to age-matched sedentary WT mice. However, enhanced oxidative stress in response to AES was observed in Nrf2−/− mice due to lower basal expression and marked attenuation of the transcriptional induction of several antioxidant genes. Thus, AES induces ROS and promotes Nrf2 function, but disruption of Nrf2 increases susceptibility of the myocardium to oxidative stress. Our findings suggest the basis for a nonpharmacological approach to activate Nrf2/ARE signaling, which might be a potential therapeutic target to protect the heart from oxidative stress-induced cardiovascular complications.
Nrf2; Keap1; Exercise; Oxidative stress; ROS; AES
Acute lung injury (ALI) initiates protective responses involving genes downstream of the Nrf2 (Nfe2l2) transcription factor, including heme oxygenase-1 (HO-1), which stimulates mitochondrial biogenesis and related anti-inflammatory processes. We examined mitochondrial biogenesis during Staphylococcus aureus pneumonia in mice and the effect of Nrf2 deficiency on lung mitochondrial biogenesis and resolution of lung inflammation. S. aureus pneumonia established by nasal insufflation of live bacteria was studied in mitochondrial reporter (mt-COX8-GFP) mice, wild-type (WT) mice, and Nrf2−/− mice. Bronchoalveolar lavage, wet/dry ratios, real-time RT-PCR and Western analysis, immunohistochemistry, and fluorescence microscopy were performed on the lung at 0, 6, 24, and 48 h. The mice survived S. aureus inoculations at 5 × 108 CFU despite diffuse lung inflammation and edema, but the Nrf2−/− lung showed increased ALI. In mt-COX8-GFP mice, mitochondrial fluorescence was enhanced in bronchial and alveolar type II (AT2) epithelial cells. WT mice displayed rapid HO-1 upregulation and lower proinflammatory TNF-α, IL-1β, and CCL2 and, especially in AT2 cells, higher anti-inflammatory IL-10 and suppressor of cytokine signaling-3 than Nrf2−/− mice. In the alveolar region, WT but not Nrf2−/− mice showed strongly induced nuclear respiratory factor-1, PGC-1α, mitochondrial transcription factor-A, SOD2, Bnip3, mtDNA copy number, and citrate synthase. These findings indicate that S. aureus pneumonia induces Nrf2-dependent mitochondrial biogenesis in the alveolar region, mainly in AT2 cells. Absence of Nrf2 suppresses the alveolar transcriptional network for mitochondrial biogenesis and anti-inflammation, which worsens ALI. The findings link redox activation of mitochondrial biogenesis to ALI resolution.
Acute lung injury; IL-10; Inflammation; Mitochondria; Oxidative stress; NRF-1; Nrf2; Free radicals
With the advent of genetic engineering, zebrafish (Danio rerio) were recognized as an attractive model organism to study many biological processes. Remarkably, the small size and optical transparency of zebrafish larvae enable high-resolution imaging of live animals. Zebrafish respond to various environmental and pathological factors with robust oxidative stress. In this article, we provide an overview of molecular mechanisms involved in oxidative stress and antioxidant response in zebrafish. Existing applications of generically-encoded fluorescent sensors allow imaging, in real time, production of H2O2 and studying its involvement in inflammatory responses, as well as activation of oxidation-sensitive transcription factors HIF and NRF2. Oxidative stress, combined with hypelipidemia, leads to oxidation of lipoproteins, the process that contributes significantly to development of human atherosclerosis. Recent work found that feeding zebrafish a high-cholesterol diet results in hypercholesterolemia, vascular lipid accumulation and extreme lipoprotein oxidation. Generation of a transgenic zebrafish expressing a GFP-tagged human antibody to malondialdehyde (MDA)-modified LDL makes possible in vivo visualization of MDA epitopes in the vascular wall and testing the efficacy of antioxidants and dietary interventions. Thus, using zebrafish as a model organism provides important advantages in studying the role of ROS and lipid oxidation in basic biologic and pathologic processes.
The epithelial-to-mesenchymal transition (EMT) is the development of increased cell plasticity that occurs normally during wound healing and embryonic development and can be coopted for cancer invasion and metastasis. TGF-beta induces EMT but the mechanism is unclear. Our studies suggest Nox4, a member of the NADPH oxidase (Nox) family, is a source of reactive oxygen species (ROS) affecting cell migration and fibronectin expression, an EMT marker, in normal and metastatic breast epithelial cells. We found TGF-beta induces Nox4 expression (mRNA and protein) and ROS generation in normal (MCF10A) and metastatic (MDA-MB-231) human breast epithelial cells. Conversely, cells expressing a dominant-negative form of Nox4 or Nox4-targeted shRNA showed significantly lower ROS production upon TGF-beta treatment. Expression of a constitutively active TGF-beta receptor type I significantly increased Nox4 promoter activity, mRNA and protein expression, and ROS generation. Nox4 transcriptional regulation by TGF-beta was SMAD3-dependent based on the effect of constitutively active SMAD3 increasing Nox4 promoter activity, whereas dominant-negative SMAD3 or SIS3, a SMAD3-specific inhibitor, had the opposite effect. Furthermore, Nox4 knockdown, dominant-negative Nox4 or SMAD3, or SIS3 blunted TGF-beta induced wound healing and cell migration, whereas cell proliferation was not effected. Our experiments further indicate Nox4 plays a role in TGF-beta regulation of fibronectin mRNA expression, based on the effects of dominant-negative Nox4 in reducing fibronectin mRNA in TGF-beta treated MDA-MB-231and MCF10A cells. Collectively, these data indicate Nox4 contributes to NADPH oxidase-dependent ROS production that may be critical for progression of the EMT in breast epithelial cells, and thereby has therapeutic implications.
NADPH oxidase 4 (Nox4); Cell Migration; TGF-beta signaling; Epithelial-to-Mesenchymal Transition (EMT)
Cl2 gas toxicity is complex and occurs during, and post exposure leading to acute lung injury (ALI) and reactive airway syndrome (RAS). Moreover, Cl2 exposure can occur in diverse situations encompassing mass casualty scenarios underscoring the need for post-exposure therapies that are efficacious and amenable to rapid and easy administration. In this study, we compared the efficacy of a single dose, post (30min) Cl2 exposure administration of nitrite (1mg/kg) via intraperitoneal (IP) or intramuscular (IM) injection in rats, to decrease ALI. Exposure of rats to Cl2 gas (400ppm, 30min) significantly increased ALI and caused RAS 6–24h post exposure as indexed by BAL sampling of lung surface protein, PMN and increased airway resistance and elastance prior to and post methacholine challenge. IP nitrite decreased Cl2 - dependent increases in BAL protein but not PMN. In contrast IM nitrite decreased BAL PMN levels without decreasing BAL protein in a xanthine oxidoreductase independent manner. Histological evaluation of airways 6h post exposure showed significant bronchial epithelium exfoliation and inflammatory injury in Cl2 exposed rats. Both IP and IM nitrite improved airway histology compared to Cl2 gas alone, but more coverage of the airway by cuboidal or columnar epithelium was observed with IM compared to IP nitrite. Airways were rendered more sensitive to methacholine induced resistance and elastance after Cl2 gas exposure. Interestingly, IM nitrite, but not IP nitrite, significantly decreased airway sensitivity to methacholine challenge. Further evaluation and comparison of IM and IP therapy showed a two-fold increase in circulating nitrite levels with the former, which was associated with reversal of post-Cl2 exposure dependent increases in circulating leukocytes. Halving the IM nitrite dose resulted in no effect in PMN accumulation but significant reduction of of BAL protein levels indicating distinct nitrite dose dependence for inhibition of Cl2 dependent lung permeability and inflammation. These data highlight the potential for nitrite as a post-exposure therapeutic for Cl2 gas induced lung injury and also suggest that administration modality is a key consideration in nitrite therapeutics.
inhaled irritants; nitric oxide; inflammation; lung; nitrite
Ceruloplasmin (ferroxidase) is a copper-binding protein known to promote Fe2+ oxidation in plasma of mammals. Besides its classical ferroxidase activity, ceruloplasmin is known to catalyze the oxidation of various substrates, such as amines and catechols. Assays based on cyclic hydroxylamine oxidation are used to quantify and detect free radicals in biological samples ex vivo and in vitro. We show here that human ceruloplasmin promotes the oxidation of the cyclic hydroxylamine 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine hydrochloride (CPH) and related probes in Chelex-treated phosphate buffer and rat serum. The reaction is suppressed by the metal chelators DTPA, EDTA and Desferal, while heparin and bathocuproine have no effect. Catalase or SOD additions do not interfere with the CPH-oxidation yield, demonstrating that free radicals are not involved in the CPH oxidation mediated by ceruloplasmin. Plasma samples immunodepleted of ceruloplasmin have lower levels of CPH oxidation, which confirms the role of ceruloplasmin (ferroxidase) as a biological oxidizing agent of cyclic hydroxylamines. In conclusion, we show that the ferroxidase activity of ceruloplasmin is a possible biological source of artifacts in the cyclic hydroxylamine-oxidation assay used for ROS detection and quantification.
Ceruloplasmin; Free radicals; Electron Spin and Paramagnetic Resonance; Spin probes
Nitrite, a dietary constituent and endogenous signaling molecule, mediates a number of physiological responses including modulation of ischemia/reperfusion injury, glucose tolerance and vascular remodeling. While the exact molecular mechanisms underlying nitrite’s actions are unknown, current paradigm suggests that these effects depend on the hypoxic reduction of nitrite to nitric oxide (NO). Mitochondrial biogenesis is a fundamental mechanism of cellular adaptation and repair. However, the effect of nitrite on mitochondrial number has not been explored. Herein, we report that nitrite stimulates mitochondrial biogenesis through a mechanism distinct from NO. We demonstrate that nitrite significantly increases cellular mitochondrial number by augmenting the activity of adenylate kinase, resulting in AMP kinase phosphorylation, downstream activation of sirtuin-1, and de-acetylation of PGC1, the master regulator of mitochondrial biogenesis. Unlike NO, nitrite-mediated biogenesis does not require the activation of soluble guanylate cyclase and results in the synthesis of more functionally efficient mitochondria. Further, we provide evidence that nitrite mediates biogenesis in vivo. In a rat model of carotid injury, two weeks of continuous oral nitrite treatment post-injury prevents the hyperproliferative response of smooth muscle cells. This protection is accompanied by a nitrite-dependent upregulation of PGC1 and increased mitochondrial number in the injured artery. These data are the first to demonstrate that nitrite mediates differential signaling than NO. They show that nitrite is a versatile regulator of mitochondrial function and number both in vivo and in vitro, and suggest that nitrite-mediated biogenesis may play a protective role in the setting of vascular injury.
mitochondria; nitrite; nitric oxide; hypoxia; AMP kinase; biogenesis
Nitrosothiols are increasingly regarded as important participants in a range of physiological processes, yet little is known about their biological generation. Nitrosothiols can be formed from the corresponding thiols by nitric oxide in a reaction that requires the presence of oxygen and is mediated by reactive intermediates (NO2 or N2O3) formed in the course of NO autoxidation. Because the autoxidation of NO is second order in NO, it is extremely slow at submicromolar NO concentrations, casting doubt on its physiological relevance. In this paper we present evidence that at submicromolar NO concentrations the aerobic nitrosation of glutathione does not involve NO autoxidation but a reaction that is first order in NO. We show that this reaction produces nitrosoglutathione efficiently in a reaction that is strongly stimulated by physiological concentrations of Mg2+. These observations suggest that direct aerobic nitrosation may represent a physiologically relevant pathway of nitrosothiol formation.
•The mechanism of aerobic nonenzymatic S-nitrosation is revisited.•Nitrosothiol formation is not autoxidation-mediated at physiological NO levels.•Direct S-nitrosation is stimulated by divalent cations such as Mg2+.•Direct nonenzymatic S-nitrosation is fast enough to be physiologically relevant.
GSNO, S-nitrosoglutathione; DTPA, diethylenetriaminepentaacetic acid; DEA/NO, diethylamine NONOate (diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate); PROLI/NO, proline NONOate (1-(hydroxyl-N,N,O-azoxy)-l-proline, disodium salt); DNIC, dinitrosyl–iron complex; SOD, superoxide dismutase; oxy-Hb, oxyhemoglobin; met-Hb, methemoglobin; TEA, triethanolamine; DAN, 2,3-diaminonaphthalene; DTT, dithiothreitol; NAC, N-acetylcysteine; β-ME, β-mercaptoethanol; S-nitrosation; Nitrosoglutathione; Nitric oxide; Free radicals
Deteriorating oocyte quality is a critical hurdle in the management of infertility, especially one associated with advancing age. In this study, we explore the role of nitric oxide (NO) on the sustenance of oocyte quality post-ovulation. Sibling oocytes from superovulated mice were subjected to intracytoplasmic sperm injection (ICSI) with cauda-epididymal spermatozoa following exposure to either the NO donor, S-nitroso N-acetyl penicillamine (SNAP, 0.23 μM/min); an NO synthase (NOS) inhibitor, Nω-nitro-L-arginine methyl ester (L-NAME, 1 mM), or an inhibitor of soluble guanylyl cyclase (sGC), 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ, 100 μM) and their sibling oocytes were subjected to ICSI either before (young) or after culture for the corresponding period of time (old). Outcomes of normal fertilization, cleavage and development to the morula and blastocyst stages were compared. Embryos from each subgroup were also subjected to TUNEL assay for apoptosis. A significant deterioration in the ability of the oocytes to undergo normal fertilization and development to morula and blastocyst stages occurred among oocytes aged in culture medium compared to their sibling cohorts subjected to ICSI immediately after ovulation (P<0.05). This deterioration was prevented in oocytes exposed to SNAP. In contrast, exposure to L-NAME or ODQ resulted in a significant compromise in fertilization and development to the morula and blastocyst stages (P<0.05). Finally, apoptosis was noted in embryos derived from aged oocytes and those exposed to L-NAME or ODQ, but not in embryos derived from young oocytes or oocytes exposed to SNAP. Thus, NO is essential for sustenance of oocyte quality post-ovulation.
Apoptosis; fertilization; intracytoplasmic sperm injection (ICSI); nitric oxide; oocyte postovulatory aging; oocyte quality; oocyte temporal window
Overproduction of hypochlorous acid (HOCl) has been associated with the development of variety of disorders such as inflammation, heart disease, pulmonary fibrosis and cancer through its ability to modify different biomolecules. HOCl is a potent oxidant generated by the myeloperoxidase-hydrogen peroxide-chloride system. Recently, we have provided evidence to support the important link between higher levels of HOCl with heme destruction and free iron release from hemoglobin and RBCs. Our current finding extend this work and show the ability of HOCl to mediate the destruction of metal-ion derivatives of tetrapyrrole macrocyclic rings, such as cyanocobalamin (Cobl) a common pharmacological form of vitamin B12. Cyanocobalamin is a water soluble vitamin which plays an essential role as an enzyme cofactor and antioxidant, modulating nucleic acid metabolism and gene regulation. It is widely used as a therapeutic agent and supplement, because of its efficacy and stability. In this report, we demonstrate that while Cobl can be an excellent antioxidant, exposure to high levels of HOCl can overcome the beneficial effects of Cobl and generate proinflammatory reaction products. Our rapid kinetic, HPLC and mass spectrometric analyses showed that HOCl can mediate corrin ring destruction and liberate cyanogen chloride (CNCl) through a mechanism that initially involves α-axial ligand replacement in Cobl to form a chlorinated derivative, hydrolysis, and cleavage of the phosphor-nucleotide moiety. Additionally, it can liberate free Co which can perpetuate metal-ion induced oxidant stress. Taken together, this is the first report of generation of toxic molecular products through the interaction of Cobl with HOCl.
Free cobalt; vitamin B12 deficiency; vitamin B12 degradation; mammalian peroxidases; mass spectrometry; oxidative stress; corrin; porphyrin; stopped-flow; inflammation; aging; free metal toxicity; cyanogens chloride
Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death in the United States and the incidence is increasing as the population ages. Cigarette smoking is the primary risk factor; however, only a minority of smokers develop the disease. Inhalation of cigarette smoke introduces an abundance of free radicals into the lungs, causing oxidative stress and inflammation. We hypothesized that after the initial burst of oxidative stress associated with cigarette smoke exposure, a sustained source of endogenous free radical production is modulated by the antioxidant enzyme extracellular superoxide dismutase (ECSOD) and the superoxide-generating complex NADPH oxidase (NOX). Primary mouse macrophages exposed to cigarette smoke extract exhibited increased oxidative stress as indicated by fluorogenic dyes and isoprostane concentration, which was suppressed in the presence of both a superoxide dismutase mimetic and a NOX inhibitor. Similarly, primary macrophages isolated from ECSOD-overexpressing mice or NOX-deficient mice showed reduced oxidative stress in response to cigarette smoke treatment. In addition, both reduced glutathione and cytokines (MIP2 and IFNγ) were increased in bronchoalveolar lavage fluid of wild-type mice exposed to cigarette smoke but not in ECSOD-overexpressing or NOX-deficient mice. These data suggest that the mechanisms underlying the host defense against cigarette smoke-induced oxidative damage and subsequent development of COPD may include endogenous oxidases and antioxidant enzymes.
Extracellular superoxide dismutase; NADPH oxidase; Smoke; Macrophages; ROS; Antioxidants; Oxidative stress; GSH; Cytokines; Lung; 4-HNE; BALF; COPD; Free radicals
8-Oxo-7,8 dihydroguanine (8-oxoG) accumulates in the genome over time and is believed to contribute to the development of aging characteristics of skeletal muscle and various aging-related diseases. Here, we show a significantly increased level of intrahelical 8-oxoG and 8-oxoguanine DNA glycosylase (OGG1) expression in aged human skeletal muscle compared to that of young individuals. In response to exercise, the 8-oxoG level was found to be lastingly elevated in sedentary young and old subjects, but returned rapidly to pre-exercise levels in the DNA of physically active individuals independent of age. 8-OxoG levels in DNA were inversely correlated with the abundance of acetylated OGG1 (Ac-OGG1), but not with total OGG1, apurinic/apyrimidinic endonuclease (AP)-1 or Ac-APE1. The actual Ac-OGG1 level was linked to exercise-induced oxidative stress, as shown by changes in lipid peroxide levels and expression of Cu,Zn-SOD, Mn-SOD and SIRT3, as well as the balance between acetyl transferase p300/CBP and the deacetylase SIRT1, but not SIRT6 expression. Together these data suggest that that acetylated form of OGG1, and not OGGl itself, correlates inversely with the 8-oxoG level in the DNA of human skeletal muscle, and the Ac-OGG1 level is dependent on adaptive cellular responses to physical activity, but is age independent.
Cytochrome c Oxidase (CcO) is the terminal oxidase of the mitochondrial electron transport chain. This bigenomic enzyme in mammals contains 13 subunits, of which, three catalytic subunits are encoded by the mitochondrial genes. The remaining ten subunits with suspected roles in the regulation, and/or, assembly are coded by the nuclear genome. The enzyme contains two heme groups (heme a and a3) and two Cu2+ centers (Cu2+ A and Cu2+ B) as catalytic centers and handles more than 90% of molecular O2 respired by the mammalian cells and tissues. CcO is a highly regulated enzyme which is believed to be the pace setter for mitochondrial oxidative metabolism and ATP synthesis. The structure and function of the enzyme is affected in a wide variety of diseases including cancer, neurodegenerative diseases, myocardial ischemia/reperfusion, bone and skeletal diseases and diabetes. Despite handling a high O2 load the role of CcO in the production of reactive oxygen species still remains a subject of debate. However, a volume of evidence suggests that CcO dysfunction is invariably associated with increased mitochondrial reactive oxygen species production and cellular toxicity. In this article we review literature on mechanisms of multimodal regulation of CcO activity by a wide spectrum of physiological and pathological factors. We also review an array of literature on the direct or indirect roles of CcO in reactive oxygen species production.
Cytochrome C Oxidase; mitochondrial dysfunction; oxidative stress; alcohol toxicity; drug effects; myocardial ischemia; regulation of enzyme activity; phosphorylation; Protein kinse A; cAMP; respirasome assembly; mtDNA mutations
The goal of the current study was to evaluate whether CYP2E1 plays a role in binge-ethanol induced steatosis and if autophagy impacts CYP2E1-mediated hepatotoxicity, oxidative stress and fatty liver formation produced by ethanol. Wild type (WT), CYP2E1 knockin (KI) and CYP2E1 knockout (KO) mice were gavaged with 3g/kg body wt ethanol twice a day for four days. This treatment caused fatty liver, elevation of CYP2E1 and oxidative stress in WT and KI mice but not KO mice. Autophagy was impaired in ethanol-treated KI mice compared to KO mice as reflected by a decline in the LC3-II/LC3-I ratio and lower total LC-3 and Beclin-1 levels coupled to increases in P62, pAKT/AKT and mTOR. Inhibition of macroautophagy by administration of 3-methyladenine enhanced the binge ethanol hepatotoxicity, steatosis and oxidant stress in CYP2E1 KI, but not CYP2E1 KO mice. Stimulation of autophagy by rapamycin blunted the elevated steatosis produced by binge ethanol. Treatment of HepG2 E47 cells which express CYP2E1 with 100 mM ethanol for 8 days increased fat accumulation and oxidant stress but decreased autophagy. Ethanol had no effect on these reactions in HepG2 C34 cells which do not express CYP2E1. Inhibition of autophagy elevated ethanol toxicity, lipid accumulation and oxidant stress in the E47, but not C34 cells. The antioxidant N-acetylcysteine, and CYP2E1 inhibitor chlormethiazole blunted these effects of ethanol. These results indicate that CYP2E1 plays an important role in binge ethanol-induced fatty liver. We propose that CYP2E1-derived reactive oxygen species inhibit autophagy, which subsequently causes accumulation of lipid droplets. Inhibition of autophagy promotes binge ethanol induced hepatotoxicity, steatosis and oxidant stress via CYP2E1.
Binge ethanol; Autophagy; Steatosis; hepatotoxicity; CYP2E1; Oxidative Stress
Methionine residues in protein can be oxidized by reactive oxygen or nitrogen species to generate methionine sulfoxide. This covalent modification has been implicated in processes ranging from normal cell signaling to neurodegenerative diseases. A general method for detecting methionine sulfoxide in proteins would be of great value in studying these processes, but development of a chemical or immunochemical technique has been elusive. Recently, an antiserum raised against an oxidized corn protein, DZS18, was reported to be specific for methionine sulfoxide in proteins (Arch. Biochem. Biophys. 485:35–40 2009.) However, data included in that report indicate that the antiserum is not specific. Utilizing well-characterized native and methionine-oxidized glutamine synthetase and aprotinin, we confirm that the antiserum does not possess specificity for methionine sulfoxide.
Methionine sulfoxide; Anti-methionine sulfoxide antibody; Oxidative stress
The blood-brain-barrier, formed by specialized brain endothelial cells that are interconnected by tight junctions, strictly regulates paracellular permeability to maintain an optimal extracellular environment for brain homeostasis. Diabetes is known to compromise the blood-brain-barrier although the underlying mechanism remains unknown.
The aim of this study was to elucidate the molecular mechanisms underlying disruption of the blood-brain-barrier in diabetes and to determine whether activation of AMP-activated protein kinase prevents diabetes-induced blood-brain-barrier dysfunction
Methods and Results
Exposure of human brain microvascular endothelial cells to high glucose (25 mmol/L D-glucose), but not to high osmotic conditions (20 mmol/L L-glucose plus 5 mmol/L D-glucose), for 2 h to 1 week significantly increased the permeability of blood-brain-barrier in parallel with lowered expression levels of zonula occludens-1, occludin, and claudin-5, three proteins that are essential to maintain endothelial cell tight junctions. In addition, high glucose significantly increased generation of superoxide anions. Adenoviral overexpression of superoxide dismutase or catalase significantly attenuated the high glucose-induced reduction of endothelial cell tight junction proteins. Furthermore, administration of apocynin reversed the effects of high glucose on endothelial cell tight junction proteins. Finally, activation of AMP-activated protein kinase with 5-amino-4-imidazole carboxamide riboside (AICAR) or adenoviral overexpression of constitutively active AMP-activated protein kinase mutants (Ad-AMPK-CA) abolished both induction of NAD(P)H oxidase-derived superoxide anions and tight junction protein degradation induced by high glucose.
We conclude that high glucose increases blood-brain-barrier dysfunction in diabetes through induction of superoxide anions and that the activation of AMP-activated protein kinase protects the integrity of the blood-brain-barrier by suppressing the induction of NAD(P)H oxidase-derived superoxide anions.
AMPK; NAD(P)H oxidase; blood brain barrier; tight junctions; diabetes
The genetic links between p53 and metabolic processes such as oxidative phosphorylation are being studied with increasing interest as cellular metabolism appears to play an important role in tumorigenesis. This review focuses on how p53 regulation of various metabolic genes may influence redox homeostasis as the genome is constantly susceptible to oxidative damage, a consequence of living in an aerobic environment. As p53-like genetic sequences are also found in life forms that may not necessarily benefit from tumor suppression, an evolutionary introduction is given in an attempt to understand why p53 might regulate a basic cellular activity such as metabolism. The presented epidemiologic and experimental data suggest that one reason may be for the homeostatic regulation of oxygen, the essential substrate for reactive oxygen species (ROS) generation.
p53; oxygen; oxidative stress; redox; antioxidant; metabolism; cancer; mitochondria; altitude
Lipid aldehydes generated by lipid peroxidation induce cell damage and inflammation. Recent evidence indicates that gamma-ketoaldehydes (isolevuglandins, IsoLG) form inflammatory mediators by modifying the ethanolamine headgroup of phosphatidyl ethanolamine (PEs). To determine if other species of aldehyde-modified PE (al-PEs) with inflammatory bioactivity were generated by lipid peroxidation, we oxidized liposomes containing arachidonic acid and characterized the resulting products. We detected PE modified by IsoLG, malondialdehyde (MDA), and 4-hydroxynonenal (HNE), as well as novel series of N-acyl-PEs and N-carboxyacyl-PEs in these oxidized liposomes. These al-PEs were also detected in HDL exposed to myeloperoxidase. When we tested the ability of al-PEs to induce THP-1 monocyte adhesion to cultured endothelial cells, we found that PE modified by MDA, HNE, and 4-oxononenal induced adhesion with potencies similar to PE modified by IsoLG (~2 μM). A commercially available medium chain N-carboxyacyl-PE (C11:0CAPE) also stimulated adhesion, while C4:0CAPE and N-acyl PEs did not. PE modified by acrolein or by glucose were only partial agonists for adhesion. These studies indicate that lipid peroxidation generates a large family of al-PEs, many of which have the potential to drive inflammation.
Oxidative stress; lipid peroxidation; lipid aldehydes; phosphatidylethanolamines
Lipopolysaccharide (LPS) from Gram-negative bacteria is a major initiator of sepsis, leading to cardiovascular collapse. Accumulating evidence has indicated a role of reactive oxygen species (ROS) in cardiovascular complication in sepsis. This study was designed to examine the effect of cardiac-specific overexpression of catalase in LPS-induced cardiac contractile dysfunction and the underlying mechanism(s) with a focus on autophagy. Catalase transgenic and wild-type FVB mice were challenged with LPS (6 mg/kg) and cardiac function was evaluated. Levels of oxidative stress, autophagy, apoptosis and protein damage were examined using fluorescence microscopy, Western blot, TUNEL assay, caspase-3 activity and carbonyl formation. Kaplan-Meier curve was constructed for survival following LPS treatment. Our results revealed a lower mortality in catalase mice compared with FVB mice following LPS challenge. LPS injection led to depressed cardiac contractile capacity as evidenced by echocardiography and cardiomyocyte contractile function, the effect of which was ablated by catalase overexpression. LPS treatment induced elevated TNF-α level, autophagy, apoptosis (TUNEL, caspase-3 activation, cleaved caspase-3), production of ROS and O2−, and protein carbonyl formation, the effects of which were significantly attenuated by catalase overexpression. Electron microscopy revealed focal myocardial damage characterized by mitochondrial injury following LPS treatment, which was less severe in catalase mice. Interestingly, LPS-induced cardiomyocyte contractile dysfunction was prevented by antioxidant NAC and the autophagy inhibitor 3-methyladenine. Taken together, our data revealed that catalase protects against LPS-induced cardiac dysfunction and mortality, which may be associated with inhibition of oxidative stress and autophagy.
lipopolysaccharide; cardiac; contractile function; oxidative stress; autophagy
Over the past decade immuno-spin trapping (IST) has been used to detect and identify protein radical sites in numerous heme and metalloproteins. To date, however, the technique has had little application toward non-metalloproteins. In this study, we demonstrate the successful application of IST in a system free of transition metals and present the first conclusive evidence of ·NO-mediated protein radical formation in the HRas GTPase. HRas is a non-metalloprotein that plays a critical role in regulating cell growth control. Protein radical formation in Ras GTPases has long been suspected of initiating premature release of bound guanine nucleotide. This action results in altered Ras activity both in vitro and in vivo. As described herein, successful application of IST may provide a means for detecting and identifying radical-mediated Ras activation in many different cancers and disease states where Ras GTPases play an important role.
Ras GTPase; radical-mediated activation; protein radical; immuno-spin trapping
Oxidative stress has been implicated in the development of vascular disease and in the promotion of endothelial dysfunction via the reduction in bioavailable nitric oxide (NO•). Glutathione (GSH) is a tripeptide thiol antioxidant that is utilized by glutathione peroxidase (GPx) to scavenge reactive oxygen species (ROS) such as hydrogen peroxide and phospholipid hydroperoxides. Relatively frequent single nucleotide polymorphisms (SNPs) within the 5’ promoters of the GSH synthesis genes GCLC and GCLM are associated with impaired vasomotor function as measured by decreased acetylcholine-stimulated coronary artery dilation and with increased risk of myocardial infarction. Although the influence of genetic knockdown of GPx on vascular function has been investigated in mice, no work to date has been published on the role of genetic knock down of GSH synthesis genes on vascular reactivity. We therefore investigated the effects of targeted disruption of Gclm in mice and the subsequent depletion of GSH on vascular reactivity, NO• production, aortic nitrotyrosine protein modification, and whole genome transcriptional responses as measured by DNA microarray. Gclm−/+ and Gclm−/− mice had 72% and 12%, respectively, of WT aortic GSH content. Gclm−/+ mice had a significant impairment in acetylcholine (ACh)-induced relaxation in aortic rings as well as increased aortic nitrotyrosine protein modification. Surprisingly, Gclm−/− aortas showed enhanced relaxation compared to Gclm−/+ aortas, as well as increased NO• production. Although aortic rings from Gclm−/− mice had enhanced ACh-relaxation, they have a significantly increased sensitivity to phenylephrine (PE)-induced contraction. Alternatively, the PE response of Gclm−/+ aortas was nearly identical to that of their WT littermates. In order to examine the role of NO• or other potential endothelium derived factors in differentially regulating vasomotor activity, we incubated aortic rings with the NO• synthase inhibitor L-NAME or physically removed the endothelium prior to PE treatment. L-NAME treatment and endothelium removal enhanced PE-induced contraction in WT and Gclm−/+ mice, but this effect was severely diminished in Gclm−/− mice, indicating a potentially unique role for GSH in mediating vessel contraction. Whole genome assessment of aortic mRNA in Gclm−/− and WT mice revealed altered expression of genes within the canonical Ca2+ signaling pathway, which may have a role in mediating these observed functional effects. These findings provide additional evidence that the de novo synthesis of GSH can influence vascular reactivity and provide insights regarding possible mechanisms by which SNPs within GCLM and GCLC influence the risk of developing vascular diseases in humans.
Glutathione; glutamate cysteine ligase; GCLM knockout mouse; nitric oxide; vascular reactivity
Oxidative damage and inflammation are related to the pathogenesis of age-related macular degeneration (AMD). Epidemiologic studies suggest that insufficient dietary lutein and zeaxanthin intake or lower serum zeaxanthin levels are associated with increased risk for AMD. The objective of this work is to test the protective effects of lutein and zeaxanthin against photo-oxidative damage to retinal pigment epithelial cells (RPE) and oxidation-induced changes in expression of inflammation-related genes. To mimic lipofuscin-mediated photo-oxidation in vivo, we used ARPE-19 cells that accumulated A2E, a lipofuscin fluorophore and photosensitizer, as a model system to investigate the effects of lutein and zeaxanthin supplementation. The data show that supplementation with lutein or zeaxanthin in the medium resulted in accumulation of lutein or zeaxanthin in the RPE cells. The concentrations of lutein and zeaxanthin in the cells were 2–14-fold of that detected in the medium, indicating that ARPE-19 cells actively take up lutein or zeaxanthin. As compared with untreated cells, exposure of A2E-containing RPE to blue light resulted in a 40–60% decrease in proteasome activity, a 50–80% decrease in expression of CFH and MCP-1, and an ~ 20-fold increase in expression of IL-8. The photo-oxidation-induced changes in expression of MCP-1, IL-8 and CFH were similar to those caused by chemical inhibition of the proteasome, suggesting that inactivation of the proteasome is involved in the photo-oxidation-induced alteration in expression of these inflammation-related genes. Incubation of the A2E-containing RPE with lutein or zeaxanthin prior to blue light exposure significantly attenuated the photo-oxidation-induced inactivation of the proteasome and photo-oxidation induced changes in expression of MCP-1, IL-8, and CFH. Together, these data indicate that lutein or zeaxanthin modulates inflammatory responses in cultured RPE in response to photo-oxidation. Protecting the proteasome from oxidative inactivation appears to be one of the mechanisms by which lutein and zeaxanthin modulate the inflammatory response. Similar mechanisms may explain salutary effects of lutein and zeaxanthin in reducing the risk for AMD.
Lutein; zeaxanthin; RPE; photo-oxidation; proteasome; inflammation
Hypoxia, or low oxygen tension, is a unique environmental stress that induces global changes in a complex regulatory network of transcription factors and signaling proteins in order to coordinate cellular adaptations in metabolism, proliferation, DNA repair, and apoptosis. Several lines of evidence now establish microRNAs (miRNAs), which are short non-coding RNAs that regulate gene expression through post-transcriptional mechanisms, as key elements in this response to hypoxia. Oxygen deprivation induces a distinct shift in a specific group of miRNAs, termed hypoxamirs, and emerging evidence indicates that hypoxia regulates several facets of hypoxamir transcription, maturation, and function. Transcription factors such as hypoxia-inducible factor (HIF) are upregulated under conditions of low oxygen availability and directly activate the transcription of a subset of hypoxamirs. Conversely, hypoxia selectively represses other hypoxamirs through less well characterized mechanisms. In addition, oxygen deprivation has been directly implicated in epigenetic modifications such as DNA demethylation that control specific miRNA transcription. Finally, hypoxia also modulates the activity of key proteins that control posttranscriptional events in the maturation and activity of miRNAs. Collectively, these findings establish hypoxia as an important proximal regulator of miRNA biogenesis and function. It will be important for future studies to address the relative contributions of transcriptional and posttranscriptional events in the regulation of specific hypoxamirs and how such miRNAs are coordinated order to integrate into the complex hierarchical regulatory network induced by hypoxia.
Hypoxia; MicroRNA; Hypoxamir; HIF
The harmful consequences of carcinogenic metals, such as nickel, arsenic and chromium, are thought to be in part due to their ability to induce oxidative stress. The ubiquity of oxidative stress in biological systems has made it a fairly obvious culprit in causing cellular damage and/or development of disease. However, the full extent of oxidative stress-induced damage is not limited to its direct effects on cellular components, such as lipids, proteins and DNA, but may extend to its ability to alter gene expression. Gene expression regulation is an important component of cellular and/or tissue homeostasis, and its alteration can have detrimental consequences. Therefore, a growing amount of interest is being paid to understanding how oxidative stress can influence gene expression. Oxidative stress-induced epigenetic dysregulation in the form of post-translational histone modifications, in particular, is a popular topic of research. This review will therefore primarily focus on discussing the role of oxidative stress and hypoxia on histone methylation and/or gene expression alterations. The sources of oxidative stress discussed here are carcinogenic metals, such as, nickel, arsenic and chromium.
Metals; histones; hypoxia; oxidative stress; gene expression
Proline metabolism has an underlying role in apoptotic signaling that impacts tumorigenesis. Proline is oxidized to glutamate in the mitochondria with the rate limiting step catalyzed by proline dehydrogenase (PRODH). PRODH expression is inducible by p53 leading to increased proline oxidation, reactive oxygen species (ROS) formation, and induction of apoptosis. Paradoxical to its role in apoptosis, proline also protects cells against oxidative stress. Here we explore the mechanism of proline protection against hydrogen peroxide stress in melanoma WM35 cells. Treatment of WM35 cells with proline significantly increased cell viability, diminished oxidative damage of cellular lipids and proteins, and retained ATP and NADPH levels after exposure to hydrogen peroxide. Inhibition or siRNA-mediated knockdown of PRODH abolished proline protection against oxidative stress whereas knockdown of Δ1-pyrroline-5-carboxylate reductase, a key enzyme in proline biosynthesis, had no impact on proline protection. Potential linkages between proline metabolism and signaling pathways were explored. The combined inhibition of the mammalian target of rapamycin complex 1 (mTORC1) and mTORC2 eliminated proline protection. A significant increase in Akt activation was observed in proline treated cells after hydrogen peroxide stress along with a corresponding increase in the phosphorylation of the fork head transcription factor class O3a (FoxO3a). The role of PRODH in proline mediated protection was validated in the prostate carcinoma cell line, PC3. Knockdown of PRODH in PC3 cells attenuated phosphorylated levels of Akt and FoxO3a and decreased cell survival during hydrogen peroxide stress. The results provide evidence that PRODH is essential in proline protection against hydrogen peroxide mediated cell death and that proline/PRODH helps activate Akt in cancer cells.
Proline; proline dehydrogenase; oxidative stress; pyrroline-5-carboxylate reductase; Akt