Biphasic regulation of H2O2 on angiogenesis implicated NADPH oxidase

ROS (reactive oxygen species) take an important signalling role in angiogenesis. Although there are several ways to produce ROS in cells, multicomponent non‐phagocytic NADPH oxidase is an important source of ROS that contribute to angiogenesis. In the present work, we examined the effects of H₂O₂ on angiogenesis including proliferation and migration in HUVECs (human umbilical vein endothelial cells), new vessel formation in chicken embryo CAM (chorioallantoic membrane) and endothelial cell apoptosis, which is closely related to anti‐angiogenesis. Our results showed that H₂O₂ dose‐dependently increased the generation of O₂ ^? (superoxide anion) in HUVECs, which was suppressed by DPI (diphenylene iodonium) and APO (apocynin), two inhibitors of NADPH oxidase. H₂O₂ at low concentrations (10 µM) stimulated cell proliferation and migration, but at higher concentrations, inhibited both. Similarly, H₂O₂ at 4 nmol/cm² strongly induced new vessel formation in CAM, while it suppressed at high concentrations (higher than 4 nmol/cm²). Also, H₂O₂ (200～500 µM) could stimulate apoptosis in HUVECs. All the effects of H₂O₂ on angiogenesis could be suppressed by NADPH oxidase inhibitors, which suggests that NADPH oxidase acts downstream of H₂O₂ to produce O₂ ^? and then to regulate angiogenesis. In summary, our results suggest that H₂O₂ as well as O₂ ^? mediated by NADPH oxidase have biphasic effects on angiogenesis in vitro and in vivo.     

The generation of H2O2 in the xylem of Zinnia elegans is mediated by an NADPH-oxidase-like enzyme

The nature of the enzymatic system responsible for the generation of H₂O₂ in the lignifying xylem of Zinnia elegans (L.) was studied using the starch/KI method for monitoring H₂O₂ production and the nitroblue tetrazolium method for monitoring superoxide production. The results showed that lignifying xylem tissues are able to accumulate H₂O₂ and to sustain H₂O₂ production. Hydrogen peroxide production in the xylem of Z. elegans was sensitive to pyridine, imidazole, quinacrine and diphenylene iodonium, which are inhibitors of phagocytic plasma-membrane NADPH oxidase. The sensitivity of H₂O₂ production to the inhibitor of phospholipase C, neomycin, and to the inhibitor of protein kinase, staurosporine, and its reversion by the inhibitor of protein phosphatases, cantharidin, pointed to the analogies existing between the mechanism of H₂O₂ production in lignifying xylem and the oxidative burst observed during the hypersensitive plant cell response. A further support for the participation of an NADPH-oxidase-like activity in H₂O₂ production in lignifying xylem was obtained from the observation that areas of H₂O₂ production were superimposed on areas producing superoxide anion, the suspected product of NADPH oxidase, although attempts to demonstrate the existence of superoxide dismutase activity in intercellular washing fluid from Z. elegans were unsuccessful. Even so, the levels of NADPH-oxidase-like activity in microsomal fractions, and of peroxidase in intercellular washing fluids, are consistent with a role for NADPH oxidase in the delivery of H₂O₂ which may be further used by xylem peroxidases for the synthesis of lignins. This hypothesis was further confirmed through a direct histochemical probe based on the H₂O₂-dependent oxidation of tetramethylbenzidine by xylem cell wall peroxidases. These results are the first evidence for the existence of an NADPH oxidase responsible for supplying H₂O₂ to peroxidase in the lignifying xylem of Z. elegans. Received: 6 February 1998 / Accepted: 14 August 1998     

Lethality of hydrogen peroxide in wild type and superoxide dismutase mutants of Escherichia coli. (A hypothesis on the mechanism of H2O2-induced inactivation of Escherichia coli)

The toxicity of H₂O₂ in Escherichia coli wild type and superoxide dismutase mutants was investigated under different experimental conditions. Cells were either grown aerobically, and then treated in M9 salts or K medium, or grown anoxically, and then treated in K medium. Results have demonstrated that the wild type and superoxide dismutase mutants display a markedly different sensitivity to both modes of lethality produced by H₂O₂ (i.e. mode one killing, which is produced by concentrations of H₂O₂ lower than 5 mM, and mode two killing which results from the insult generated by concentrations of H₂O₂ higher than 10 mM). Although the data obtained do not clarify the molecular basis of H₂O₂ toxicity and/or do not explain the specific function of superoxide ions in H₂O₂-induced bacterial inactivation, they certainly demonstrate that the latter species plays a key role in both modes of H₂O₂ lethality. A mechanism of H₂O₂ toxicity in E. coli is proposed, involving the action of a hypothetical enzyme which should work as an O₂^-• generating system. This enzyme should be active at low concentrations of H₂O₂ (<5 mM) and high concentrations of the oxidant (>5 mM) should inactivate the same enzyme. Superoxide ions would then be produced and result in mode one lethality. The resistance at intermediate H₂O₂ concentrations may be dependent on the inactivation of such enzyme with no superoxide ions being produced at levels of H₂O₂ in the range 5–10 mM. Mode two killing could be produced by the hydroxyl radical in concert with superoxide ions, chemically produced via the reaction of high concentrations of H₂O₂ (>10 mM) with hydroxyl radicals. The rate of hydroxyl radical production may be increased by the higher availability of Fe²⁺ since superoxide ions may also reduce trivalent iron to the divalent form.     

The effects of superoxide dismutase on H2O2 formation

Numerous reports of the effects of overproduction of SODs have been explained on the basis of increased H2O2 production by the catalyzed dismutation of O2-. In this review we consider the effects of increasing [SOD] on H2O2 formation and question this explanation.     

Effect of acute vs chronic H2O2-induced oxidative stress on antioxidant enzyme activities

H₂O₂ can freely crosses membranes and in the presence of Fe²⁺ (or Cu⁺) it is prone to participate in Fenton reaction. This study evaluated the concentration and time-dependent effects of H₂O₂-induced oxidative stress on MnSOD, Se:GPx and catalase and on aconitase. Acute and chronic H₂O₂ treatments were able to induce oxidative stress in HeLa cells as they significantly decreased aconitase activity and also caused a very significant decrease on antioxidant enzyme activities. The inhibition of enzyme activities was time- and concentration-dependent. Chronic treatment with 5 µM H₂O₂/h after 24 h was able to decrease all enzyme activities almost at the same level as the acute treatment. Acute and chronic treatments on antioxidant enzyme activities were prevented by cell treatment with ascorbic acid or N-acetylcysteine. These results indicate that antioxidant enzymes can also be affected by the same ROS they produce or neutralize if the time of exposure is long enough.     

Production of superoxide/H2O2 by dihydroorotate dehydrogenase in rat skeletal muscle mitochondria

Dehydrogenases that use ubiquinone as an electron acceptor, including complex I of the respiratory chain, complex II, and glycerol-3-phosphate dehydrogenase, are known to be direct generators of superoxide and/or H₂O₂. Dihydroorotate dehydrogenase oxidizes dihydroorotate to orotate and reduces ubiquinone to ubiquinol during pyrimidine metabolism, but it is unclear whether it produces superoxide and/or H₂O₂ directly or does so only indirectly from other sites in the electron transport chain. Using mitochondria isolated from rat skeletal muscle we establish that dihydroorotate oxidation leads to superoxide/H₂O₂ production at a fairly high rate of about 300 pmol H₂O₂·min⁻¹·mg protein⁻¹ when oxidation of ubiquinol is prevented and complex II is uninhibited. This H₂O₂ production is abolished by brequinar or leflunomide, known inhibitors of dihydroorotate dehydrogenase. Eighty percent of this rate is indirect, originating from site II_F of complex II, because it can be prevented by malonate or atpenin A5, inhibitors of complex II. In the presence of inhibitors of all known sites of superoxide/H₂O₂ production (rotenone to inhibit sites in complex I (site I_Q and, indirectly, site I_F), myxothiazol to inhibit site III_Qo in complex III, and malonate plus atpenin A5 to inhibit site II_F in complex II), dihydroorotate dehydrogenase generates superoxide/H₂O₂, at a small but significant rate (23 pmol H₂O₂·min⁻¹·mg protein⁻¹), from the ubiquinone-binding site. We conclude that dihydroorotate dehydrogenase can generate superoxide and/or H₂O₂ directly at low rates and is also capable of indirect production at higher rates from other sites through its ability to reduce the ubiquinone pool.     

A new type of O(2)(-)-generating tool for oxidative stress studies by remodeling neutrophil NADPH oxidase

The effects of reactive oxygen species on cells have attracted much attention in relation to redox regulation and oxidative stress-related diseases. Superoxide (O₂⁻) is the reactive oxygen species primarily formed in biological systems. However, no convenient O₂⁻-generating device has been available for use in cell or tissue culture. The neutrophil NADPH oxidase, a professional enzyme for killing bacteria, has a high ability to produce O₂⁻. However, the cell-free activation process requires several protein factors and an anionic amphiphile, and moreover, the activation is transient. To utilize the enzyme as an O₂⁻ generator, we improved the cell-free activation method by remodeling regulatory components, optimizing lipid composition, and modifying the mixing conditions. We established a new method to produce an active enzyme that is stable, efficient, and preservable. As an application, we examined the effect of the device on cultured HEK293 cells and observed that it caused cell death. This system has several advantages over the xanthine oxidase system often used. The new device will be useful for studies of oxidative stress and related diseases.     

NOX5 is expressed at the plasma membrane and generates superoxide in response to protein kinase C activation

NOX5 is a ROS-generating NADPH oxidase which contains an N-terminal EF-hand region and can be activated by cytosolic Ca(2+) elevations. However the C-terminal region of NOX5 also contains putative phosphorylation sites. In this study we used HEK cells stably expressing NOX5 to analyze the size and subcellular localization of the NOX5 protein, its mechanisms of activation, and the characteristics of the ROS released. We demonstrate that NOX5 can be activated both by the protein kinase C activating phorbol esther PMA and by the Ca(2+) ionophore ionomycin. The PMA- but not the ionomycin-dependent activation can be inhibited by protein kinase C inhibitors. NOX5 activity is inhibited by submicromolar concentrations of diphenyl iodonium (DPI), but not by apocynin. Western blot analysis showed a lower ( approximately 70 kDa) than expected (82 kDa) molecular mass. Two arguments suggest that NOX5 is at least partially expressed on the plasma membrane: (i) the membrane-impermeant superoxide was readily detected by extracellular probes, and (ii) immunofluorescent labeling of NOX5 detected a fraction of the NOX5 protein at the plasma membrane. In summary, we demonstrate that NOX5 can be found intracellularly and at the cell surface. We also describe that it can be activated through protein kinase C, in addition to its Ca(2+) activation.     

Role of H2O2 dynamics in brassinosteroid‐induced stomatal closure and opening in Solanum lycopersicum

Brassinosteroids (BRs) are essential for plant growth and development; however, their roles in the regulation of stomatal opening or closure remain obscure. Here, the mechanism underlying BR‐induced stomatal movements is studied. The effects of 24‐epibrassinolide (EBR) on the stomatal apertures of tomato (Solanum lycopersicum) were measured by light microscopy using epidermal strips of wild type (WT), the abscisic acid (ABA)‐deficient notabilis (not) mutant, and plants silenced for SlBRI1, SlRBOH1 and SlGSH1. EBR induced stomatal opening within an appropriate range of concentrations, whereas high concentrations of EBR induced stomatal closure. EBR‐induced stomatal movements were closely related to dynamic changes in H₂O₂ and redox status in guard cells. The stomata of SlRBOH1‐silenced plants showed a significant loss of sensitivity to EBR. However, ABA deficiency abolished EBR‐induced stomatal closure but did not affect EBR‐induced stomatal opening. Silencing of SlGSH1, the critical gene involved in glutathione biosynthesis, disrupted glutathione redox homeostasis and abolished EBR‐induced stomatal opening. The results suggest that transient H₂O₂ production is essential for poising the cellular redox status of glutathione, which plays an important role in BR‐induced stomatal opening. However, a prolonged increase in H₂O₂ facilitated ABA signalling and stomatal closure.     

Adrenaline stimulates H2O2 generation in liver via NADPH oxidase

It is known that adrenaline promotes hydroxyl radical generation in isolated rat hepatocytes. The aim of this work was to investigate a potential role of NADPH oxidase (Nox) isoforms for an oxidative stress signal in response to adrenaline in hepatocytes. Enriched plasma membranes from isolated rat liver cells were prepared for this purpose. These membranes showed catalytic activity of Nox isoforms, probably Nox 2 based on its complete inhibition with specific antibodies. NADPH was oxidized to convert O₂ into superoxide radical, later transformed into H₂O₂. This enzymatic activity requires previous activation with either 3 mM Mn²⁺ or guanosine 5′-0-(3-thiotriphosphate) (GTPγS) plus adrenaline. Experimental conditions for activation and catalytic steps were set up: ATP was not required; S_0.5 for NADPH was 44 μM; S_0.5 for FAD was 8 μM; NADH up to 1 mM was not substrate, and diphenyleneiodonium was inhibitory. Activation with GTPγS plus adrenaline was dose- and Ca²⁺-dependent and proceeded through α₁-adrenergic receptors (AR), whereas β-AR stimulation resulted in inhibition of Nox activity. These results lead us to propose H₂O₂ as additional transduction signal for adrenaline response in hepatic cells.     

NADPH oxidase is involved in H2O2-induced differentiation of human promyelocytic leukaemia HL-60 cells

The expression and activity of NADPH oxidase increase when HL‐60 cells are induced into terminally differentiated cells. However, the function of NADPH oxidase in differentiation is not well elucidated. With 150–500 μM H₂O₂ inducing differentiation of HL‐60 cells, we measured phagocytosis of latex beads and investigated cell electrophoresis. Two inhibitors of NADPH oxidase, DPI (diphenyleneiodonium) and APO (apocynin), blocked the differentiation potential of cells induced by 200 μM H₂O₂. However, H₂O₂ stimulated the generation of intracellular superoxide (O₂ ^{? ?}), which decreased in the presence of the two inhibitors. DPI also inhibited H₂O₂‐induced ERK (extracellular‐signal‐regulated kinase) activation, as detected by Western blotting. Furthermore, PD98059, the inhibitor of the ERK pathway, inhibited the differentiation of HL‐60 cells induced by H₂O₂. This shows that H₂O₂ can activate NADPH oxidase, leading to O₂ ^{? ?} production, followed by ERK activation and ultimately resulting in the differentiation of HL‐60 cells. The data indicate that NADPH oxidase is an important cell signal regulating cell differentiation.     

Mediation of H2O2-induced vascular relaxation by endothelium-derived relaxing factor

We investigated the effects of H₂O₂ generated by glucose (G) and glucose oxidase (GO) on the isolated rabbit aorta suspended in Krebs-Ringer solution. H₂O₂ produced contraction in small concentration and relaxation followed by contraction in large concentration. Contraction produced by large concentration was smaller than that produced by small concentration of H₂O₂. Relaxation was prevented by deendothelialization or N^G-monomethyl-L-arginine, an inhibitor of nitric oxide synthesis. These results suggest that H₂O₂ in large concentrations produces relaxation followed by contraction, and that the relaxation is endothelium-dependent and is mediated by nitric oxide, an endothelium-derived relaxing factor.     

The ubiquitin/proteasome pathway: friend or foe in zinc-, cadmium-, and H2O2-induced neuronal oxidative stress

One of the hallmarks of neurodegeneration is the accumulation of ubiquitinated proteins in intraneuronal inclusions in the cytosol, endosomes/lysosomes and nuclei of affected cells. The relationship between inclusion production and cell viability is not well understood. On the one hand inclusions may be beneficial and result from an attempt of the cell to isolate a subclass of ubiquitinated proteins that are not effectively degraded. On the other hand, the inclusions may impede normal cell function contributing to cell death. To address this issue we treated mouse neuronal HT4 cells with three toxic agents cadmium, zinc and H2O2, and investigated their effects on glutathione homeostasis, on accumulation of ubiquitinated proteins and on cell viability. The three treatments induce oxidative stress manifested by decreases in glutathione (GSH) and/or increases in protein mixed disulfides (PrSSG). After an overnight recovery period in the absence of treatment, GSH and PrSSG were restored to almost normal levels. However, the levels of ubiquitinated proteins were significantly increased, and cell viability was sharply reduced. These results suggest that the ubiquitin-proteasome pathway is recruited for removal of proteins that are oxidatively modified. However, if the ubiquitinated proteins are not efficiently degraded, they accumulate in the cell and contribute to a decrease in cell viability.     

Antioxidants Inhibit Angiogenesis In Vivo through Down-regulation of Nitric Oxide Synthase Expression and Activity

Although reactive oxygen species (ROS) participate in many cellular mechanisms, only few data exist concerning their involvement in physiological angiogenesis. The aim of the present work was to elucidate possible mechanisms through which ROS affect angiogenesis in vivo, using the model of the chicken embryo chorioallantoic membrane (CAM). Superoxide dismutase (SOD) and its membrane permeable mimetic tempol, dose dependently decreased angiogenesis and down-regulated inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production. The NADPH oxidase inhibitors, 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF) and apocynin, but not allopurinol, also had a dose dependent inhibitory effect on angiogenesis and NO production in vivo. Catalase and the intracellular hydrogen peroxide (H²O²) scavenger sodium pyruvate decreased, while H²O² increased in a dose-dependent manner the number of CAM blood vessels, as well as the expression and activity of iNOS. Dexamethasone, which down-regulated NO production by iNOS and l-NAME, but not d-NAME, dose dependently decreased angiogenesis in vivo. These data suggest that antioxidants affect physiological angiogenesis in vivo, through regulation of NOS expression and activity.     

Metformin protects PC12 cells and hippocampal neurons from H2O 2-induced oxidative damage through activation of AMPK pathway

Metformin, a first line anti type 2 diabetes drug, has recently been shown to extend lifespan in various species, and therefore, became the first antiaging drug in clinical trial. Oxidative stress due to excess reactive oxygen species (ROS) is considered to be an important factor in aging and related disease, such as Alzheimer's disease (AD). However, the antioxidative effects of metformin and its underlying mechanisms in neuronal cells is not known. In the present study, we showed that metformin, in clinically relevant concentrations, protected neuronal PC12 cells from H₂O₂-induced cell death. Metformin significantly ameliorated cell death due to H₂O₂ insult by restoring abnormal changes in nuclear morphology, intracellular ROS, lactate dehydrogenase, and mitochondrial membrane potential induced by H₂O₂. Hoechst staining assay and flow cytometry analysis revealed that metformin significantly reduced the apoptosis in PC12 cells exposed to H₂O₂. Western blot analysis further demonstrated that metformin stimulated the phosphorylation and activation of AMP-activated protein kinase (AMPK) in PC12 cells, while application of AMPK inhibitor compound C, or knockdown of the expression of AMPK by specific small interfering RNA or short hairpin RNA blocked the protective effect of metformin. Similar results were obtained in primary cultured hippocampal neurons. Taken together, these results indicated that metformin is able to protect neuronal cells from oxidative injury, at least in part, via the activation of AMPK. As metformin is comparatively cheaper with much less side effects in clinic, our findings support its potential to be a drug for prevention and treatment of aging and aging-related diseases.     

Effect of H2O2-induced oxidative stress on some yeast membrane functions

This work was supported by the NATO Linkage Grant H TECH.LG 930 686 and by grant no. 204/93/2224 of the Grant Agency of the Czech Republic.     

Sphingosine 1-phosphate attenuates H2O2-induced apoptosis in endothelial cells

Reactive oxygen species including H₂O₂ lead vascular endothelial cells (EC) to undergo apoptosis. Sphingosine 1-phosphate (S1P) is a platelet-derived sphingolipid mediator that elicits various EC responses. We aimed to explore whether and how S1P modulates EC apoptosis induced by H₂O₂. Treatment of cultured bovine aortic EC (BAEC) with H₂O₂ (750 μM for 6 h) led to DNA fragmentation (ELISA), DNA nick formation (TUNEL staining), and cleavage of caspase-3, key features of EC apoptosis. These responses elicited by H₂O₂ were alike markedly attenuated by pretreatment with S1P (1 μM, 30 min). H₂O₂ induced robust phosphorylation of both p38 and JNK MAP kinases. However, pretreatment with S1P decreased phosphorylation of only p38 MAP kinase, but not that of JNK; conversely, an inhibitor of p38 MAP kinase, but not that of JNK, attenuated H₂O₂-induced caspase-3 activation. Thus S1P attenuates H₂O₂-induced apoptosis of cultured BAEC, involving p38 MAP kinase.     

Signaling responses in plants to heavy metal stress

Heavy metal toxicity is one of the major abiotic stresses leading to hazardous health effects in animals and plants. Because of their high reactivity they can directly influence growth, senescence and energy synthesis processes. In this review a new indirect mechanism of heavy metal action is proposed. This mechanism is connected with the generation of reactive oxygen species (especially H₂O₂) and jasmonate and ethylene signaling pathways and shows that toxicity symptoms observed in plants may result from direct heavy metal influence as well as the activity of some signaling molecules induced by the stress action.     

H2O2 is involved in the dormancy-breaking effect of hydrogen cyanamide in grapevine buds

Hydrogen cyanamide (HC) is widely used to induce the breakage of endodormancy (ED) in grape and other deciduous fruit crop, though its mechanism of action is poorly understood. Applications of HC to grapevine buds produce oxidative stress and transient respiratory disturbances which are related to the breakage of ED. Moreover, since the expression and activity of catalase (Cat) is inhibited by HC, enhancements in the levels of H₂O₂ have also been associated to the breakage of ED in grapevine buds. Here, we reported that increases in H₂O₂ level in HC-treated grapevine buds are due to the inhibition of Cat activity and enhancement of the respiratory activity of buds. In addition, exogenous applications of H₂O₂ partially reproduced the inducing effect of HC in the breakage of ED, thus providing further support for the hypothesis that H₂O₂ mediates the effects of HC. On the other hand, Mit isolated from both control and HC-treated buds respired equally well when NADH was used as a respiratory substrate, but when succinate was used as an electron donor Mit respiration was non-detected, suggesting that the stimulatory effect of HC on bud respiration is related to metabolic alterations leading to increase of the concentration of NADH rather than to changes in Mit functionality.