Reactive oxygen intermediates induce monocyte chemotactic protein-1 in vascular endothelium after brief ischemia.

Chemokine expression is associated with reperfusion of infarcted myocardium in the setting of tissue necrosis, intense inflammation, and inflammatory cytokine release. The specific synthesis of monocyte chemotactic protein (MCP)-1 mRNA by cardiac venules in reperfused infarcts corresponded to the region where leukocytes normally localize. MCP-1 could be induced by exogenous tumor necrosis factor (TNF)-alpha or by postischemic cardiac lymph containing TNF-alpha. However, the release of TNF-alpha during early reperfusion did not explain the venular localization of MCP-1 induction. To better understand the factors mediating MCP-1 induction, we examined the role of ischemia/reperfusion in a model of brief coronary occlusion in which no necrosis or inflammatory response is seen. Adult mongrel dogs were subjected to 15 minutes of coronary occlusion and 5 hours of reperfusion. Ribonuclease protection assay revealed up-regulation of MCP-1 mRNA only in ischemic segments of reperfused canine myocardium. Pretreatment with the reactive oxygen scavenger N-(2-mercaptopropionyl)-glycine completely inhibited MCP-1 induction. In situ hybridization localized MCP-1 message to small venular endothelium in ischemic areas without myocyte necrosis. Gel shift analysis of nuclear extracts from the ischemic area showed enhanced DNA binding of the transcription factors AP-1 and nuclear factor (NF)-kappaB, crucial for MCP-1 expression, in ischemic myocardial regions. Immunohistochemical staining demonstrated reperfusion-dependent nuclear translocation of c-Jun and NF-kappaB (p65) in small venular endothelium, only in the ischemic regions of the myocardium, that was inhibited by N-(2-mercaptopropionyl)-glycine. In vitro, treatment of cultured canine jugular vein endothelial cells with the reactive oxygen intermediate H2O2 induced a concentration-dependent increase in MCP-1 mRNA levels, which was inhibited by the antioxidant N-acetyl-L-cysteine, a precursor of glutathione, but not pyrrolidine dithiocarbamate, an inhibitor of NF-kappaB and activator of AP-1. In contrast to our studies with infarction, incubation of canine jugular vein endothelial cells with postischemic cardiac lymph did not induce MCP-1 mRNA expression suggesting the absence of cytokine-mediated MCP-1 induction after a sublethal ischemic period. These results suggest that reactive oxygen intermediate generation, after a brief ischemic episode, is capable of inducing MCP-1 expression in venular endothelium through AP-1 and NF-kappaB. Short periods of ischemia/reperfusion, insufficient to produce a myocardial infarction, induce MCP-1 expression, potentially mediating angiogenesis in the ischemic noninfarcted heart.

Thrombin causes vascular endothelial growth factor expression in vascular smooth muscle cells: role of reactive oxygen species.

Vascular endothelial growth factor (VEGF) has been implicated in the reendothelialization of the vascular wall after balloon injury. This study investigated whether thrombin, which is formed during activation of the coagulation cascade at sites of vascular injury, upregulates VEGF expression in vascular smooth muscle cells (VSMCs). VEGF expression was assessed in native and cultured VSMCs by Northern blot analysis and reverse transcription-polymerase chain reaction and the release of VEGF protein by immunoassay. alpha-Thrombin time- and concentration-dependently increased VEGF mRNA levels, mainly that mRNA coding for the soluble splice variant VEGF(164/165), and stimulated the release of VEGF protein. These effects required the proteolytic activity of thrombin and were mimicked by a thrombin receptor activating-peptide. Upregulation of VEGF expression was also induced by conditioned medium from alpha-thrombin-stimulated VSMCs. Both the early and the delayed alpha-thrombin-induced VEGF expressions were attenuated by antioxidants and by diphenyleneiodonium. alpha-Thrombin-induced VEGF release was significantly reduced by a platelet-derived growth factor (PDGF)-, a transforming growth factor (TGF)-beta-, and a basic fibroblast growth factor (bFGF)-neutralizing antibody. Thrombin caused a redox-sensitive upregulation of expression of VEGF in VSMCs through a direct and an indirect effect, which was dependent on the endogenous formation of PDGF, TGF-beta, and bFGF. Upregulation of VEGF expression may represent an important mechanism by which the coagulation cascade contributes to the regeneration of the endothelial lining at sites of balloon injury.

The influence of very low doses of N-nitrosodimethylamine (NDMA) on the apoptosis of rat neutrophils in vivo. The role of reactive oxygen species.

N-nitrosodimethylamine (NDMA) causes the apoptosis of neutrophils in vitro experiments. This compound also has the ability to stimulate neutrophils for the production of reactive oxygen species. It has been decided to examine more closely whether the apoptosis of neutrophils by NDMA is caused by the influence of the radicals produced by these cells and whether the stimulation to undergo apoptosis of neutrophils is caused by NDMA in either the original form or by its metabolites. The experiment was conducted on rats. The animals were administered a one-time dose of NDMA intragastrically, 1.5 mg/kg. The research was conducted 1,2,4,12 h consecutively following NDMA administration. The concentration of NDMA in blood was evaluated by means of the gas chromatography method. The neutrophils were isolated from blood by means of differential centrifugation. Respiratory burst was assessed in cells, by means of the cytochrome c reduction method. The percentage of cells revealing morphological properties of apoptosis was determined under the fluorescent microscope. It has been observed that the activation of the respiratory burst is caused mainly by non-metabolised NDMA. Probably the non-metabolised molecules of this compound also have a decisive role in the initiation of apoptosis of neutrophils. It can be assumed that the main factor responsible for the apoptosis of neutrophil rats following a one-time NDMA administration is the induction of respiratory burst in neutrophils by this compound.

Antioxidant properties of green and black tea, and their potential ability to retard the progression of eye lens cataract.

Aqueous extracts of green and black tea are shown to quench reactive oxygen species such as singlet oxygen, superoxide and hydroxyl radicals, prevent the oxidative cross-linking of test proteins and inhibit single strand breakage of DNA in whole cells. They are also seen to be able to counteract the oxidative insult mounted by cigarette smoke. In rats in which cataract was induced by subcutaneous injection of selenite, administration of green or black tea extracts led to a retardation of the progression of lens opacity, suggesting the potential cataracto-static ability of tea.

Multiple role of reactive oxygen species in the arterial wall.

Increased oxidative stress plays an important role in vascular dysfunction and atherogenesis. Both systemic factors, such as hypercholesterolemia and hyperglycemia, and local factors, such as activation of macrophages and T cells, may contribute to oxidative stress. Oxidation of lipids in lipoproteins and cell membranes leads to functionally important modifications of proteins that affect their recognition by cell surface receptors and protein-protein interactions within the cell, including DNA binding. Oxidized LDL and extracellular oxidation modulate oxidation-sensitive signaling pathways, but it is not clear to what extent this results from receptor-mediated activation or from direct effects on the intracellular redox-balance. Extensive evidence indicates that reactive oxygen species (ROS) regulate gene expression by modulating a large number of transcription factors, including the nuclear transcription factor kappa B (NFkappaB), the peroxisome proliferator activated receptorgamma (PPARgamma), and pathways linked to apoptosis. It is also increasingly recognized that cell differentiation and proliferation, cytokine expression, and programmed cell death are determined by the interactions between oxidation-sensitive regulatory pathways previously thought to lead to distinct outcomes. Because hypercholesterolemia exerts pro-oxidant effects both intra- and extracellularly and because increased ROS formation affects vascular reactivity and atherogenesis by modulating multiple signaling pathways and transcriptional events, future investigations of its atherogenic mechanisms should place greater emphasis on the net effect of such modulation on the expression of a large spectrum of genes. One way of doing this will be by defining clusters of genes responding to hypercholesterolemic stimuli--or interventions with structurally unrelated antioxidants--in analogous ways, irrespective of what regulatory pathway they are controlled by. Microarray technologies that allow simultaneous assessment of large numbers of genes may provide a tool for this approach.

Antimutagenesis and anticarcinogenesis, from the past to the future.

Observations on cancer causation are some 150 years old, but actual detailed research on elements bearing on cancer started at the beginning of the twentieth century. Rapid progress, however, is only some 40 years old. Studies in humans documented certain lifestyle related factors to lead to cancer, and research in animal models strengthened this information. With the realization that there are carcinogens that in a metabolically activated attack DNA, in contrast to other agents that act by promoting, enhancing processes through totally distinct mechanisms, it became possible to develop and apply tests for DNA reactivity, in a prokaryotic organism, the widely used Salmonella typhimurium test by Ames and in a eukaryotic system, namely freshly explanted liver cells displaying evidence of DNA repair by Williams. A battery of these two tests are over 90% accurate in defining genotoxicity. Virtually all documented human carcinogens are genotoxic. With advances in molecular biology, mutational events are traced to changes in tumor suppressor genes or in oncogenes, that can serve as markers of risk. In addition, reactive oxygen systems (ROS) are involved in both the early steps in cancer and in the developmental aspects. Thus, foods containing antioxidants such as vegetables, fruits, soy products, cocoa and tea that counteract ROS are protective in cancer causation and development. Worldwide application of current knowledge and mechanisms to cancer prevention, the definitive means of cancer control, is likely to lower not only cancer but also heart disease risk in the current century.

Repair of oxidative DNA damage--an important factor reducing cancer risk. Minireview.

Oxygen free radicals formed during normal aerobic cellular metabolism generate a variety of DNA lesions including modified bases, abasic sites and single strand breaks with blocked 3' termini. If left unrepaired, these damages may contribute to a number of degenerative processes, including cancer and aging. In most organisms, the repair of oxidative DNA lesions is supposed to be handled by the base excision repair (BER) pathway. BER is a multistep process that involves the sequential activity of several proteins, many of them were isolated and functionally characterized using the simple prokaryotic and lower eukaryotic model systems, Escherichia coli and Saccharomyces cerevisiae, respectively. As the amino acid sequence of DNA repair proteins is often well conserved from bacteria to man, our understanding of BER in higher eukaryotes drives extensively from the microbial models, namely from the yeast S. cerevisiae. Thus, results obtained on a simple yeast model are a source of new information, which can be used as a paradigm for all eukaryotic cells.

Reactive oxygen species mediate angiotensin II-induced leukocyte-endothelial cell interactions in vivo.

Chronically elevated angiotensin II (Ang-II)-induced hypertension is partly mediated by superoxide production. In this study, we have investigated whether the leukocyte-endothelial cell interactions elicited by Ang-II involve reactive oxygen species (ROS) generation. Intravital microscopy within the rat mesenteric microvessels was used. Superfusion (60 min) with Ang-II (1 nM) induced significant increases in leukocyte rolling flux, adhesion, and emigration, which were inhibited by pretreatment with superoxide dismutase or catalase. Dihydrorhodamine-123 oxidation indicated that ROS are primarily produced by the vessel wall. Administration of dimethylthiourea, desferrioxamine, or N-acetylcysteine provoked significant reductions in Ang-II-induced leukocyte-endothelial cell interactions. In addition, a blockade of platelet-activating factor or leukotrienes also attenuated such responses significantly. The results presented indicate that in vivo Ang-II-induced leukocyte recruitment is dependent on the generation of intra- and extracellular ROS. Therefore, the use of anti-oxidants might constitute an alternative therapy for the control of the subendothelial leukocyte infiltration associated with hypertension and atherosclerosis.

Redox regulation of vascular smooth muscle cell differentiation.

Experiments were performed to determine the role of reactive oxygen species (ROS) in regulating vascular smooth muscle cell (VSMC) phenotype. After quiescence, cultured human VSMCs increased their expression of differentiation proteins (alpha-actin, calponin, and SM1 and SM2 myosin), but not beta-actin. ROS activity, determined using the H(2)O(2)-sensitive probe dichlorodihydrofluorescein (DCF), remained high in quiescent cells and was inhibited by catalase (3000 U/mL) or by N-acetylcysteine (NAC, 2 to 20 mmol/L). A superoxide dismutase mimic (SOD; MnTMPyP, 25 micromol/L) or SOD plus low concentrations of NAC (SODNAC2, 2 mmol/L) increased DCF fluorescence, which was inhibited by catalase or by NAC (10 to 20 mmol/L). Inhibition of ROS activity (by catalase or NAC) decreased the baseline expression of differentiation proteins, whereas elevation of ROS (by SOD or SODNAC2) increased expression of the differentiation markers. The latter effect was blocked by catalase or by NAC (10 to 20 mmol/L). None of the treatments altered beta-actin expression. SODNAC2-treated cells demonstrated contractions to endothelin that were absent in proliferating cells. p38 Mitogen-activated protein kinase (MAPK) activity was decreased when ROS activity was reduced (NAC, 10 mmol/L) and was augmented when ROS activity was increased (SODNAC2). Inhibition of p38 MAPK with pyridyl imidazole compound (SB202190, 2 to 10 micromol/L) reduced expression of differentiation proteins occurring under basal conditions and in response to SODNAC2. Transduction of VSMCs with an adenovirus encoding constitutively active MKK6, an activator of p38 MAPK, increased expression of differentiation proteins, whereas transduction with an adenovirus encoding dominant-negative p38 MAPK decreased expression of the differentiation proteins. These findings demonstrate that ROS can increase VSMC differentiation through a p38 MAPK-dependent pathway.

Thrombin activates the hypoxia-inducible factor-1 signaling pathway in vascular smooth muscle cells: Role of the p22(phox)-containing NADPH oxidase.

The heterodimeric transcription factor hypoxia-inducible factor-1 (HIF-1) is activated under hypoxic conditions, resulting in the upregulation of its target genes plasminogen activator inhibitor-1 (PAI-1) and vascular endothelial growth factor (VEGF). PAI-1 and VEGF are also induced in response to vascular injury, which is characterized by the activation of platelets and the coagulation cascade as well as the generation of reactive oxygen species (ROS). However, it is not known whether HIF-1 is also stimulated by thrombotic factors. We investigated the role of thrombin, platelet-associated growth factors, and ROS derived from the p22(phox)-containing NADPH oxidase in the activation of HIF-1 and the induction of its target genes PAI-1 and VEGF in human vascular smooth muscle cells (VSMCs). Thrombin, platelet-derived growth factor-AB (PDGF-AB), and transforming growth factor-beta(1) (TGF-beta(1)) upregulated HIF-1alpha protein in cultured and native VSMCs. This response was accompanied by nuclear accumulation of HIF-1alpha as well as by increased HIF-1 DNA-binding and reporter gene activity. The thrombin-induced expression of HIF-1alpha, PAI-1, and VEGF was attenuated by antioxidant treatment as well as by transfection of p22(phox) antisense oligonucleotides. Inhibition of p38 mitogen-activated protein kinase and phosphatidylinositol-3-kinase significantly decreased thrombin-induced HIF-1alpha, PAI-1, and VEGF expression. These findings demonstrate that the HIF-1 signaling pathway can be stimulated by thrombin and platelet-associated growth factors and that a redox-sensitive cascade activated by ROS derived from the p22(phox)-containing NADPH oxidase is crucially involved in this response.

1,2-bis(2-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid induces caspase-mediated apoptosis and reactive oxygen species-mediated necrosis in cultured cortical neurons.

Sustained alteration in [Ca(2+)]i triggers neuronal death. We examined morphological and signaling events of Ca(2+)-deficiency-induced neuronal death. Cortical cell cultures exposed to 20 microM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM), an intracellular calcium chelator, underwent neuronal apoptosis within 12 h that was evident by shriveled cell bodies, aggregated and condensed nuclear chromatin, and disrupted nuclear membrane. Thereafter, surviving neurons revealed typical necrosis, accompanied by swelling of cell body and mitochondria, over 24 h. Both apoptosis and necrosis were prevented by inclusion of 1 microg/mL cycloheximide, a protein synthesis inhibitor. Treatment with BAPTA-AM induced translocation of Bax into mitochondria within 4 h and release of cytochrome c from mitochondria over 4-12 h. An active fragment of caspase-3, a downstream mediator of cytochrome c, was observed within 8 h and cleaved PHF-1-positive tau. Administration of zVAD-fmk, a broad inhibitor of caspases, or DEVD-amc, a selective inhibitor of caspase-3, selectively prevented the apoptosis component of BAPTA-AM neurotoxicity. In contrast, BAPTA-AM-induced necrosis was propagated through sequential production of superoxide, mitochondrial and cytoplasmic reactive oxygen species. Combined treatment with caspase inhibitors and antioxidants blocked BAPTA-AM neurotoxicity. The present study suggests that neurons deficient in [Ca(2+)]i undergo caspase-3-mediated apoptosis and reactive oxygen species (ROS)-mediated necrosis.

Mechanisms of ageing.

Recent experimental work from a variety of biological systems, ranging from yeast to human beings, lends increasing support to the view that stochastic damage inflicted to biological macromolecules is the driving force for the ageing process. The damage is derived from small reactive molecules, most prominently reactive oxygen intermediates (ROI), that arise during normal cellular metabolism and are associated with important if not essential cellular functions. The major classes of macromolecules at risk are proteins, lipids and DNA, but damage to DNA (both nuclear and mitochondrial) may entail particularly severe consequences. Cellular dysfunction resulting from macromolecular damage can be detected as a variety of expressions, such as genomic instability, inappropriate cell differentiation events or cell death. While for post-mitotic cell types replacement of the dead cell by another cell of the same lineage is not possible, mitotic cell types may initially replace dead cells via cell proliferation. But exhaustion of the self-renewal capacity of the respective lineage, by either replication-associated or damage-associated telomere shortening, will ultimately also lead to loss of parenchymal cell mass and functional impairment of tissues, the latter being a typical feature of ageing of tissues and organs. It has been demonstrated in various experimental systems that the rate ageing of can be retarded by lowering the production of endogenous ROI or by improving cellular anti-oxidative defences. Whether augmentation of cellular DNA repair capacity will have the same effect remains to be seen.

Leukotoxicity of pyoverdin, production of reactive oxygen species, and effect of UV radiation.

Pyoverdin was purified by solvent extraction, gel filtration, and ionic exchange chromatography. Assays of cytotoxic of pyoverdin were done with human leukocytes and macrophages from the peritoneum of mice. Both cell quantities showed a significant reduction. Death was followed by lysis in a dose-dependent form. The mechanism of action of pyoverdin involved the stimulation of reactive oxygen species (ROS) measured by Nitroblue Tetrazolium (NBT) reaction and chemiluminescence (CL). UV radiation at 368 nm increased the leukotoxicity; expositions of 5 min were enough to photostimulate the effect of pyoverdin on cellular oxydative metabolism, which increased between 35.4 and 53.2%. Genestein, an inhibitor of tyrosine kinases, counteracted the ROS stimuli of pyoverdin, suggesting endocytic mechanism of action for this pigment. The little chloroquine interference on oxydative stress indicated that intraphagosomal pH and the stimuli of reactive nitrogen intermediaries (RNI) seem to be of less importance than ROS in pyoverdin action on leukocytes.

[Cytotoxicity of beta-lapachone, an naphthoquinone with possible therapeutic use]. / Citotoxicidad de la beta-lapachona: una o-naftoquinona con posibles usos terapéuticos.

beta-lapachone (beta-lap) is a lipophilic o-naphthoquinone isolated from the bark of the lapacho tree. Initial observations proved its capability for inhibiting growth of Yoshida tumor and Walker 256 carcinosarcoma. beta-Lap redox-cycling in the presence of reductants and oxygen yields "reactive oxygen species" (ROS: O2-, OH and H2O2) which cytotoxicity led to assume its role in beta-lap activity in cells. beta-Lap inhibited DNA synthesis in Trypanosoma cruzi as well as topoisomerases I and II, poly(ADP-ribose) polymerase (PARP) in different cells. These enzymes are essential for maintaining DNA structure. beta-Lap inhibited growth of a large variety of tumor cells including epidermoid laringeal cancer, prostate, colon, ovary and breast cancer and also different types of leukemia cells. Advances in knowledge of apoptosis ("programmed cell death") and necrosis provided useful information for understanding the mechanism of beta-lap cytotoxicity. Thiol-dependent proteases (Calpaine), kinases (e.g. c-JUN NH2-terminal kinase), caspases and nucleases are involved in beta-lap cytotoxicity. These enzymes activity, as well as ROS production by beta-lap redox-cycling, would be essential for beta-lap cytotoxicity. Diaphorase and NAD(P)H-quinone reductase, which catalyse beta-lap redox-cycling and ROS production, seem to play an essential role in beta-lap activity. On these grounds, clinical applications of beta-lap have been suggested.

Histone H3 phosphorylation is coupled to poly-(ADP-ribosylation) during reactive oxygen species-induced cell death in renal proximal tubular epithelial cells.

Although the cellular response to chemical-induced stress is relatively well characterized, particularly the response to DNA damage, factors that govern the outcome of the stress response (cell survival or cell death) are less clearly defined. In this context, the mitogen-activated protein kinase (MAPK) family responds to a variety of physical and chemical stresses. The activation of MAPKs, especially the extracellular-regulated protein kinase subfamily, seems to play a causal role in death of renal proximal tubular epithelial cells (LLC-PK1) induced by reactive oxygen species (ROS). In this study, we show that extracellular signal receptor-activated kinase (ERK) activation may be coupled with LLC-PK1 cell death via changes in chromatin structure, which is mediated by increases in the phosphorylation of histone H3 (a post-translational modification required for both chromosome condensation and segregation during mitosis) and premature chromatin/chromosomal condensation, leading to cell death. In support of this view, 2,3,5-tris-(glutathione-S-yl)hydroquinone (TGHQ)-induced phosphorylation of histone H3 is accompanied by increases in chromatin condensation, as observed with the use of 4,6-diamidino-2-phenylindole-fluorescent staining, and by decreases in the sensitivity of chromatin to digestion by micrococcal nuclease. Changes in chromatin structure precede cell death. TGHQ-induced histone H3 phosphorylation and chromatin condensation are inhibited by PD098059, which selectively inhibits MAPK kinase, an upstream regulator of ERKs. Moreover, histone phosphorylation is modulated by poly(ADP-)ribosylation. Thus, the inhibition of poly(ADP-ribose)polymerase with 3-aminobenzamide prevents histone H3 phosphorylation and increases cell survival, suggesting that ADP-ribosylation and histone H3 phosphorylation are coupled in this model of ROS-induced DNA damage and cell death. The coupling of histone phosphorylation with ribosylation has not been previously demonstrated.

Silent repair accounts for cell cycle specificity in the signaling of oxidative DNA lesions.

Reactive oxygen species are the most important source of DNA lesions in aerobic organisms, but little is known about the activation of the DNA checkpoints in response to oxidative stress. We show that treatment of yeast cells with sublethal concentrations of hydrogen peroxide induces a Mec1-dependent phosphorylation of Rad53 and a Rad53-dependent cell cycle delay specifically during S phase. The lack of Rad53 phosphorylation after hydrogen peroxide treatment in the G1 and G2 phases is due to the silent repair of oxidative DNA lesions produced at these stages by the base excision repair (BER) pathway. Only the disruption of the BER pathway and the accumulation and/or treatment of DNA intermediates by alternative repair pathways reveal the existence of primary DNA lesions induced at all phases of the cell cycle by hydrogen peroxide. Our data illustrate both the concept of silent repair of DNA damage and the high sensitivity of S-phase cells to hydrogen peroxide.

Redox control of AP-1-like factors in yeast and beyond.

Cells have evolved complex and efficient strategies for dealing with variable and often-harsh environments. A key aspect of these stress responses is the transcriptional activation of genes encoding defense and repair proteins. In yeast members of the AP-1 family of proteins are required for the transcriptional response to oxidative stress. This sub-family of AP-1 (called yAP-1) proteins are sensors of the redox-state of the cell and are activated directly by oxidative stress conditions. yAP-1 proteins are bZIP-containing factors that share homology to the mammalian AP-1 factor complex and bind to very similar DNA sequence sites. The generation of reactive oxygen species and the resulting potential for oxidative stress is common to all aerobically growing organisms. Furthermore, many of the features of this response appear to be evolutionarily conserved and consequently the study of model organisms, such as yeast, will have widespread utility. The important structural features of these factors, signaling pathways controlling their activity and the nature of the target genes they control will be discussed.