Major Source Of Reactive Oxygen Species Generation Research Articles

Delineating the NOX-Mediated Promising Therapeutic Strategies for the Management of Various Cardiovascular Disorders: A Comprehensive Review.

Cardiovascular disorders (CVDs) are reported to occur with very high rates of incidence and exhibit high morbidity and mortality rates across the globe. Therefore, research is focused on searching for novel therapeutic targets involving multiple pathophysiological mechanisms. Oxidative stress plays a critical role in the development and progression of various CVDs, such as hypertension, pulmonary hypertension, heart failure, arrhythmia, atherosclerosis, ischemia-reperfusion injury, and myocardial infarction. Among multiple pathways generating reactive oxygen species (ROS), NADPH defines all abbreviations oxidases of the NOX family as the major source of ROS generation and plays an intricate role in the development and progression of CVDs. Therefore, exploring the role of different NADPH oxidase isoforms in various cardiovascular pathologies has attracted attention to current cardiovascular research. Focusing on NADPH oxidases to reduce oxidative stress in managing diverse CVDs may offer unique therapeutic approaches to prevent and treat various heart conditions. The current review article highlights the role of different NADPH oxidase isoforms in the pathophysiology of various CVDs. Moreover, the focus is also to emphasize different experimental studies that utilized various NADPH oxidase isoform modulators to manage other disorders. The present review article considers new avenues for researchers/scientists working in the field of cardiovascular pharmacology utilizing NADPH oxidase isoform modulators.

P-300 - Reactive oxygen species production by brain mitochondria depends linearly on oxygen level

Reactive oxygen species (ROS) are by-products of physiological mitochondrial metabolism involved in several cellular signaling pathways and pathophysiological processes, including brain ischemia-reperfusion injury. The mitochondrial respiratory chain is considered a major source of ROS; however, there is a lack of agreement on how ROS release depends on oxygen concentration. We determined how the rate of H2O2 release by intact mouse brain mitochondria oxidizing different substrates depends on oxygen concentrations. The highest rate of H2O2 release occurs in conditions of reverse electron transfer, with complex I flavin as a major source of ROS generation. Our data indicate that the rate of H2O2 release by respiring mitochondria is linearly dependent on oxygen concentration. We also found that complex II can significantly contribute to ROS generation when the quinone pool is reduced even in the absence of its substrate succinate. Our results underscore the critical importance of reverse electron transfer in brain, where a significant accumulation of succinate during ischemia favors a backflow of electrons to complex I at the early stages of reperfusion. Our study also demonstrates that in brain tissue mitochondria ROS generation under hypoxic conditions is lower than in normoxia.

Influence of hydrogen peroxide induced oxidative stress on survival rate of early chick embryo development

Reactive oxygen species (ROS) induced oxidative stress influences embryonic growth and development. Hydrogen peroxide (H2O2) is a major source of ROS generation that induces oxidative stress by altering redox status. In present study, direct effect of H2O2 generated oxidative stress on survival rate and associated toxicity in chick embryo was studied during early development. Chick embryos at 96 hrs of incubation were treated with single doses of 2.5μg, 5.0μg, 10μg, 50μg, 100μg, 300μg and 500μg H2O2 concentrations per embryo. The toxic effects recorded after 144 hrs of development. The results showed that treatments of 2.5 and 5.0μg H2O2 doses did not affect survival rate and embryo development. 10μg and 50μg H2O2 doses treatment exhibited slightly reduced survival rate without affecting normal morphology. Administration of 100 and 300μg H2O2 doses caused predominant decrease in the survival rate in comparison with the normal and PBS treated control embryos with deformities such as growth retardation, defected brain, limbs and vascular development with hemorrhage. Treatment of 500μg H2O2 exhibited no survival of embryos. These results indicated that post-omphalomesentric stages of early chick embryos are more susceptible to elevated level of H2O2 induced oxidative stress leading to significant reduction in survival rate with associated deformities. These results are discussed with probable reasons.

Biochemical and Molecular Alterations Following Arsenic-Induced Oxidative Stress and Mitochondrial Dysfunction in Rat Brain.

Oxidative stress is associated with the generation of reactive oxygen species (ROS), which is supposed to be one of the mechanisms of arsenic-induced neurodegeneration. Mitochondria, being the major source of ROS generation may present an important target of arsenic-mediated neurotoxicity. Hence, we planned the study to elucidate the possible biochemical and molecular alterations induced by arsenic exposure in rat brain mitochondria. Chronic sodium arsenite treatment (25ppm for 12weeks) resulted in decreased activity of mitochondrial complexes I, II, and IV followed by increased ROS generation. There was decrease in mitochondrial superoxide dismutase (MnSOD) activity in arsenic-treated rat brain further showing increased superoxide radical generation in mitochondria. The decrease in MnSOD activity might be responsible for the increased protein and lipid oxidation as observed in our study. Protein and messenger RNA (mRNA) levels of MnSOD and mitochondrial uncoupling protein 2 (UCP-2) were downregulated suggesting decreased removal of ROS in rat brain. Fourier transform infrared (FTIR) spectroscopy analysis revealed significant decrease in amide A, amide I, amide II, and Olefinic = CH stretching band area suggesting molecular alteration in proteins and lipids after arsenic treatment. The results of present study indicate that arsenic-induced disturbed mitochondrial metabolism, decreased removal of ROS, decrease in protein synthesis, and altered membrane lipid polarity and fluidity may be responsible for the mitochondrial oxidative damage in rat brain that may further be implicated as contributing factor in arsenic-induced neurodegeneration.

Mitochondrial ROS govern the LPS-induced pro-inflammatory response in microglia cells by regulating MAPK and NF-κB pathways

Activation of microglia cells in the brain contributes to neurodegenerative processes promoted by many neurotoxic factors such as pro-inflammatory cytokines and nitric oxide (NO). Reactive oxygen species (ROS) actively affect microglia-associated neurodegenerative diseases through their role as pro-inflammatory molecules and modulators of pro-inflammatory processes. Although the ROS which involved in microglia activation are thought to be generated primarily by NADPH oxidase (NOX) and involved in the immune response, mitochondrial ROS have also been proposed as important regulators of the inflammatory response in the innate immune system. However, the role of mitochondrial ROS in microglial activation has yet to be fully elucidated. In this study, we demonstrate that inhibition of mitochondrial ROS by treatment with Mito-TEMPO effectively suppressed the level of mitochondrial and intracellular ROS. Mito-TEMPO treatment also significantly prevented LPS-induced increase in the TNF-α, IL-1β, IL-6, iNOS and Cox-2 in BV-2 and primary microglia cells. Furthermore, LPS-induced suppression of mitochondrial ROS generation not only affected LPS-stimulated activation of MAPKs, including ERK, JNK, and p38, but also regulated IκB activation and NF-κB nuclear localization. These results indicate that mitochondria constitute a major source of ROS generation in LPS-mediated activated microglia cells. Additionally, suppression of LPS-induced mitochondrial ROS plays a role in modulating the production of pro-inflammatory mediators by preventing MAPK and NF-κB activation in microglia cells. Our findings suggest that a potential strategy in the development of therapy for inflammation-associated degenerative neurological diseases involves targeting the regulation of mitochondrial ROS in microglial cells.

Whole-cell and nuclear NADPH oxidases levels and distribution in human endocardial endothelial, vascular smooth muscle, and vascular endothelial cells

The results of our study show that whole-cell and nuclear levels of NADPH oxidase-1 (NOX1) are similar in human vascular endothelial cells (hVECs) and smooth muscle cells (hVSMCs), but lower in human endocardial endothelial cells (hEECs). NOX2 levels were higher in hVECs and lower in hVSMCs. NOX3 levels were the same in hVECs and hVSMCs, but lower in hEECs. NOX4 levels were similar in all of the cell types. NOX4 levels were higher in hVECs than in hVSMCs. NOX5 was also present throughout the 3 cell types, including their nuclei, in the following order: hEECs > hVSMCs > hVECs. The level of basal reactive oxygen species (ROS) was highest in hVECs and lowest in hVSMCs. However, the Ca(2+) level was highest in hVSMCs and lowest in hVECs. These findings suggest that all types of NOXs exist in hEECs, hVECs, and hVSMCs, although their density and distribution are cell-type dependent. The density of the different NOXs correlated with the ROS level, but not with the Ca(2+) level. In conclusion, NOXs, including NOX3, exist in cardiovascular cells and their nuclei. The nucleus is a major source of ROS generation. The nuclear NOXs may contribute to ROS and Ca(2+) homeostasis, which may affect cell remodeling, including the formation of nuclear T-tubules in vascular diseases and aging.

Characterization of Rubus fruticosus mitochondria and salicylic acid inhibition of reactive oxygen species generation at Complex III/Q cycle: potential implications for hypersensitive response in plants

In addition to adenosine triphosphate (ATP) production, mitochondria have been implicated in the regulation of several physiological responses in plants, such as programmed cell death (PCD) activation. Salicylic acid (SA) and reactive oxygen species (ROS) are essential signaling molecules involved in such physiological responses; however, the mechanisms by which they act remain unknown. In non-photosynthesizing tissues, mitochondria appear to serve as the main source of ROS generation. Evidence suggests that SA and ROS could regulate plant PCD through a synergistic mechanism that involves mitochondria. Herein, we isolate and characterize the mitochondria from non-photosynthesizing cell suspension cultures of Rubus fruticosus. Furthermore, we assess the primary site of ROS generation and the effects of SA on isolated organelles. Mitochondrial Complex III was found to be the major source of ROS generation in this model. In addition, we discovered that SA inhibits the electron transport chain by inactivating the semiquinone radical during the Q cycle. Computational analyses confirmed the experimental data, and a mechanism for this action is proposed.

Distinct role of Hsp70 in Drosophila hemocytes during severe hypoxia

Severe hypoxia can lead to injury and mortality in vertebrate or invertebrate organisms. Our research is focused on understanding the molecular mechanisms that lead to injury or adaptation to hypoxic stress using Drosophila as a model system. In this study, we employed the UAS-Gal4 system to dissect the protective role of Hsp70 in specific tissues in vivo under severe hypoxia. In contrast to overexpression in tissues such as muscles, heart, and brain, we found that overexpression of Hsp70 in hemocytes of flies provides a remarkable survival benefit to flies exposed to severe hypoxia for days. Furthermore, these flies were tolerant not only to severe hypoxia but also to other stresses such as oxidant stress (e.g., paraquat feeding or hyperoxia). Interestingly we observed that the better survival with Hsp70 overexpression in hemocytes under hypoxia or oxidant stress is causally linked to reactive oxygen species (ROS) reduction in whole flies. We also show that hemocytes are a major source of ROS generation, leading to injury during hypoxia, and their elimination results in a better survival under hypoxia. Hence, our study identified a protective role for Hsp70 in Drosophila hemocytes, which is linked to ROS reduction in the whole flies and thus helps in their remarkable survival during oxidant or hypoxic stress.

Peroxiredoxin 6 Phosphorylation and Subsequent Phospholipase A2 Activity Are Required for Agonist-mediated Activation of NADPH Oxidase in Mouse Pulmonary Microvascular Endothelium and Alveolar Macrophages

Peroxiredoxin 6 (Prdx6), a bifunctional enzyme with glutathione peroxidase and phospholipase A2 (PLA(2)) activities, participates in the activation of NADPH oxidase 2 (NOX2) in neutrophils, but the mechanism for this effect is not known. We now demonstrate that Prdx6 is required for agonist-induced NOX2 activation in pulmonary microvascular endothelial cells (PMVEC) and that the effect requires the PLA(2) activity of Prdx6. Generation of reactive oxygen species (ROS) in response to angiotensin II (Ang II) or phorbol 12-myristate 13-acetate was markedly reduced in perfused lungs and isolated PMVEC from Prdx6 null mice. Rac1 and p47(phox), cytosolic components of NOX2, translocated to the endothelial cell membrane after Ang II treatment in wild-type but not Prdx6 null PMVEC. MJ33, an inhibitor of Prdx6 PLA(2) activity, blocked agonist-induced PLA(2) activity and ROS generation in PMVEC by >80%, whereas inhibitors of other PLA(2)s were ineffective. Transfection of Prx6 null cells with wild-type and C47S mutant Prdx6, but not with mutants of the PLA(2) active site (S32A, H26A, and D140A), "rescued" Ang II-induced PLA(2) activity and ROS generation. Ang II treatment of wild-type cells resulted in phosphorylation of Prdx6 and its subsequent translocation from the cytosol to the cell membrane. Phosphorylation as well as PLA(2) activity and ROS generation were markedly reduced by the MAPK inhibitor, U0126. Thus, agonist-induced MAPK activation leads to Prdx6 phosphorylation and translocation to the cell membrane, where its PLA(2) activity facilitates assembly of the NOX2 complex and activation of the oxidase.

Mitochondrial metabolism and ROS generation are essential for Kras-mediated tumorigenicity

Otto Warburg's theory on the origins of cancer postulates that tumor cells have defects in mitochondrial oxidative phosphorylation and therefore rely on high levels of aerobic glycolysis as the major source of ATP to fuel cellular proliferation (the Warburg effect). This is in contrast to normal cells, which primarily utilize oxidative phosphorylation for growth and survival. Here we report that the major function of glucose metabolism for Kras-induced anchorage-independent growth, a hallmark of transformed cells, is to support the pentose phosphate pathway. The major function of glycolytic ATP is to support growth under hypoxic conditions. Glutamine conversion into the tricarboxylic acid cycle intermediate alpha-ketoglutarate through glutaminase and alanine aminotransferase is essential for Kras-induced anchorage-independent growth. Mitochondrial metabolism allows for the generation of reactive oxygen species (ROS) which are required for Kras-induced anchorage-independent growth through regulation of the ERK MAPK signaling pathway. We show that the major source of ROS generation required for anchorage-independent growth is the Q(o) site of mitochondrial complex III. Furthermore, disruption of mitochondrial function by loss of the mitochondrial transcription factor A (TFAM) gene reduced tumorigenesis in an oncogenic Kras-driven mouse model of lung cancer. These results demonstrate that mitochondrial metabolism and mitochondrial ROS generation are essential for Kras-induced cell proliferation and tumorigenesis.

Sex Differences in the Phosphorylation of Mitochondrial Proteins Result in Reduced Production of Reactive Oxygen Species and Cardioprotection in Females

Although premenopausal females have a lower risk for cardiovascular disease, the mechanism(s) are poorly understood. We tested the hypothesis that cardioprotection in females is mediated by altered mitochondrial protein levels and/or posttranslational modifications. Using both an in vivo and an isolated heart model of ischemia and reperfusion (I/R), we found that females had less injury than males. Using proteomic methods we found that female hearts had increased phosphorylation and activity of aldehyde dehydrogenase (ALDH)2, an enzyme that detoxifies reactive oxygen species (ROS)-generated aldehyde adducts, and that an activator of ALDH2 reduced I/R injury in males but had no significant effect in females. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, blocked the protection and the increased phosphorylation of ALDH2 in females, but had no effect in males. Furthermore, we found an increase in phosphorylation of alpha-ketoglutarate dehydrogenase (alphaKGDH) in female hearts. alphaKGDH is a major source of ROS generation particularly with a high NADH/NAD ratio which occurs during I/R. We found decreased ROS generation in permeabilized female mitochondria given alphaKGDH substrates and NADH, suggesting that increased phosphorylation of alphaKGDH might reduce ROS generation by alphaKGDH. In support of this hypothesis, we found that protein kinase C-dependent phosphorylation of purified alphaKGDH reduced ROS generation. Additionally, myocytes from female hearts had less ROS generation following I/R than males and addition of wortmannin increased ROS generation in females to the same levels as in males. These data suggest that posttranslational modifications can modify ROS handling and play an important role in female cardioprotection.

The Combination of PEITC (Phenehyl Isothiocyanate) with a Histone Deacetylase Inhibitor (HDACi) Has Synergistic Antileukemia Activity by Overcoming a Redox-Mediated Resistance Pathway.

Abstract 1739Poster Board I-765 IntroductionHistone deacetylase inhibitors (HDACI) have limited but well established clinical activity in human leukemia. Results of a phase 1 trial of vorinostat in AML indicate that a gene signature composed mainly of antioxidants was associated with clinical resistance to vorinostat (Blood 2008;111:1060-60). This study suggested that generation of reactive oxygen species (ROS) appears to be a mechanism of action of vorinostat whereas increase of antioxidants may correlate with vorinostat resistance. The aims of this study were to further investigate the underlying molecular mechanisms and test the combination effect of vorinostat and redox modulation agents. Methods and resultsThe parental HL60 and the pan-HDACI resistant HL60/LR were used to compare the redox parameters in this pair of cell lines. Real time PCR and western blot analysis demonstrated that a variety of glutathione related antioxidant defense enzymes were substantially increased in HL60/LR compared to its parental HL60, which is consistent with the clinical findings cited above. Most importantly, Nrf2, a master transcription factor that activates the transcription of cellular defense and antioxidant genes was also upregulated in HL60/LR. Confocal microscopy study showed that vorinostat treatment of HL60 cells caused translocation of Nrf2 from cytosol to nucleus. Furthermore, its downstream antioxidant genes including GST (glutathione S- Transferase), GSR (glutathione reductase), GCLC (glutathione synthase) and SOD (superoxide dismutase) were upregulated, demonstrating that the cellular defense against oxidative stress was induced by vorinostat. Overexpression of Nrf2 in HEK293 cells prevented ROS generation induced by vorinostat. Knock-down of Nrf2 by siRNA in colon cancer cell HCT116 caused increase of ROS production and cytotoxicity induced by vorinostat. These findings further demonstrated the role of Nrf2 in protecting cells from oxidative stress caused by vorinostat. We also observed that vorinostat substantially activated a ROS generating enzyme NADPH Oxidase (NOX) in various AML cell lines including HL60, U937 and ML1. Vorinostat induced ROS in both HL60 and the mitochondrial deficient cell line HL60-C6F. This indicates that NOX is a major source of ROS generation induced by vorinostat. As a result, modulation of antioxidant response may potentiate the cytotoxic activity of vorinostat. In order to modulate cellular redox balance and overcome the resistance to vorinostat, PEITC, a compound known to deplete cellular glutathione was used to test its combination effect with vorinostat. We found that a subtoxic concentration of PEITC (1-2.5 uM) substantially potentiated cytotoxicity of vorinostat in a dose-dependent manner in various AML cell lines, as demonstrated by Annexin-PI assay after 48 hrs and MTT assay after 72 hrs. Treatment with subtoxic concentrations of vorinostat (1.5 uM) and PEITC (1-2.5 uM) for 48 hrs also resulted in synergistic cytotoxicity in primary leukemia cells obtained from AML patient samples as demonstrated by Annexin-PI assay. Parallel results were also obtained with other HDACI such as MGCD0103. ConclusionsOur study indicates that NADPH Oxidase is likely a major source of ROS generation induced by vorinostat. Nrf2, a master transcription factor and its downstream antioxidant genes, which protect cells from oxidative stress, contributes to leukemia cellular resistance to vorinostat. Modulation of cellular redox balance such as depletion of glutathione by PEITC significantly potentiates the anti-leukemia activity of vorinostat. Our study provides important information for further development of a mechanism-based combination strategy to maximize the potential of vorinostat and other HDACI and provides an alternative mechanism of the anti-leukemia activity of HDACI. DisclosuresNo relevant conflicts of interest to declare.

Vascular NAD(P)H oxidase activation in diabetes: a double-edged sword in redox signalling

Oxidative stress mediated by hyperglycaemia-induced generation of reactive oxygen species (ROS) contributes significantly to the development and progression of diabetes and related vascular complications. NAD(P)H oxidase has been implicated as the major source of ROS generation in the vasculature in response to high glucose and advanced glycation end-products. Sustained activation of NAD(P)H oxidase in diabetes may diminish intracellular levels of NADPH, an essential cofactor for endothelial NO synthase (eNOS) and several antioxidant systems. Recent evidence suggests that basal ROS production via NAD(P)H oxidase may upregulate antioxidant enzyme defenses via redox signalling. Thus, NAD(P)H oxidase may serve as a double-edged sword, with transient activation providing a feedback defense against excessive ROS generation through the activation of receptor tyrosine kinases and the redox-sensitive Nrf2-Keap1 signalling pathway. Overproduction of ROS leads to eNOS uncoupling, mitochondrial dysfunction, and impaired antioxidant defenses owing to depletion of intracellular NADPH. Given the largely negative outcome of antioxidant therapy in the treatment of diabetic complications, targeting the redox-sensitive transcription factor Nfr2 may provide an effective strategy to restore antioxidant defenses in diabetes.

Diabetic Cardiomyopathy in OVE26 Mice Shows Mitochondrial ROS Production and Divergence Between In Vivo and In Vitro Contractility.

Many diabetic patients suffer from a cardiomyopathy that cannot be explained solely by poor coronary perfusion. This cardiomyopathy may be due to either organ-based damage like fibrosis, or to direct damage to cardiomyocytes. Mitochondrial-derived reactive oxygen species (ROS) have been proposed to contribute to this cardiomyopathy. To address these questions, we used the OVE26 mouse model of severe type 1 diabetes to measure contractility in isolated cardiomyocytes by edge detection and in vivo with echocardiography. We also assessed the source of ROS generation using both a general and a mitochondrial specific indicator. When contractility was assayed in freshly isolated myocytes, contraction was much stronger in control myocytes. However, contractility of normal myocytes became weaker during 24 hours of in vitro culture. In contrast, contractility of diabetic OVE26 myocytes remains stable during culture. Echocardiography revealed normal or hyperdynamic function in OVE26 hearts under basal conditions but with a sharply reduced response to isoproterenol, a beta-adrenergic agonist. For ROS generation, we found that ROS production in diabetic myocytes was elevated after exposure to either high glucose or angiotensin II (AngII). Superoxide detection with the mitochondrial sensor MitoSOX Red confirmed that mitochondria are a major source of ROS generation in diabetic myocytes. These results show that contractile deficits in OVE26 diabetic hearts are due primarily to cardiomyocyte impairment and that ROS from mitochondria are a cause of that impairment.

Oxidation-induced changes in human lens epithelial cells 2. Mitochondria and the generation of reactive oxygen species

The relationships among reactive oxygen species (ROS) generation, lipid compositional changes, antioxidant power, and mitochondrial membrane potential were determined in a human lens epithelial cell line, HLE-B3. Cells grown in a hyperoxic atmosphere grew linearly for about 3 days, and then progressively died. Total antioxidant power and ROS generation increased by 50 and 43%, respectively, in cells grown in a hyperoxic atmosphere compared to those cultured in a normoxic atmosphere. By specifically uncoupling the mitochondrial proton gradient, we determined that the mitochondria are most likely the major source of ROS generation. ROS generation correlated inversely with mitochondrial membrane potential and the amount of cardiolipin, factors likely to contribute to loss of cell viability. Our results support the idea that hyperoxic damage to HLE-B3 cells derives from enhanced generation of ROS from the mitochondrial electron transport chain resulting in the oxidation of cardiolipin. With extended hyperoxic insult, the oxidants overwhelm the antioxidant defense system and eventually cell death ensues.

ROS stress in cancer cells and therapeutic implications.

Reactive oxygen species (ROS) are constantly generated and eliminated in the biological system, and play important roles in a variety of normal biochemical functions and abnormal pathological processes. Growing evidence suggests that cancer cells exhibit increased intrinsic ROS stress, due in part to oncogenic stimulation, increased metabolic activity, and mitochondrial malfunction. Since the mitochondrial respiratory chain (electron transport complexes) is a major source of ROS generation in the cells, the vulnerability of the mitochondrial DNA to ROS-mediated damage appears to be a mechanism to amplify ROS stress in cancer cells. The escalated ROS generation in cancer cells serves as an endogenous source of DNA-damaging agents that promote genetic instability and development of drug resistance. Malfunction of mitochondria also alters cellular apoptotic response to anticancer agents. Despite the negative impacts of increased ROS in cancer cells, it is possible to exploit this biochemical feature and develop novel therapeutic strategies to preferentially kill cancer cells through ROS-mediated mechanisms. This article reviews ROS stress in cancer cells, its underlying mechanisms and relationship with mitochondrial malfunction and alteration in drug sensitivity, and suggests new therapeutic strategies that take advantage of increased ROS in cancer cells to enhance therapeutic activity and selectivity.

Oxidative stress and cardiovascular injury: Part II: animal and human studies.

This review so far has considered the role of vascular cells in the generation of reactive oxygen species (ROS) and novel approaches to their detection in integrated systems such as animal models of vascular disease and humans. We now turn attention to evidence consistent with a role for ROS in models of human disease and in discrete patient populations. We also briefly comment on the outcomes of clinical trials of antioxidants. Mainly, these have been disappointing. However, it is premature to conclude that the oxidation hypothesis of disease causality has been adequately tested. Animal studies, for the most part, support a fundamental role for ROS in cardiovascular disease. Any controversy could in part reflect the use of ineffective antioxidants and the selection of models in which ROS generation is of marginal relevance to the measured outcome. Both of these issues require a quantitatively accurate measurement of drug effect (ie, antioxidant capacity) before hypotheses relating to the role of oxidant stress can be addressed rationally. These issues pertain to clinical trials also, as we will discuss. However, animal studies permit administration of much more powerful antioxidants (eg, rate constant for interaction of superoxide dismutase (SOD) with O2·− ≈1.6×109 · mol/L−1 · s−1) than is possible in humans (eg, rate constant for vitamin E ≈0.59 mol/L−1 · s−1) or, like in the case of vitamin E, doses of the antioxidant in excess of those usually applied in clinical research. ### Atherosclerosis Animal models of atherosclerosis have documented that all the constituents of the plaque produce and use ROS. Lesion formation is associated with the accumulation of lipid peroxidation products1,2 and induction of inflammatory genes,3 inactivation of NO·resulting in endothelial dysfunction, 4,5 activation of matrix metalloproteinases,6 and increased smooth muscle cell growth.7 …

Activation of NADPH oxidase during progression of cardiac hypertrophy to failure.

Increased reactive oxygen species (ROS) production is implicated in the pathophysiology of left ventricular (LV) hypertrophy and heart failure. However, the enzymatic sources of myocardial ROS production are unclear. We examined the expression and activity of phagocyte-type NADPH oxidase in LV myocardium in an experimental guinea pig model of progressive pressure-overload LV hypertrophy. Concomitant with the development of LV hypertrophy, NADPH-dependent O2- production in LV homogenates, measured by lucigenin (5 micro mol/L) chemiluminescence or cytochrome c reduction assays, significantly and progressively increased (by approximately 40% at the stage of LV decompensation; P<0.05). O2- production was fully inhibited by diphenyleneiodonium (100 micromol/L). Immunoblotting revealed a progressive increase in expression of the NADPH oxidase subunits p22(phox), gp91(phox), p67(phox), and p47(phox) in the LV hypertrophy group, whereas immunolabeling studies indicated the presence of oxidase subunits in cardiomyocytes and endothelial cells. In parallel with the increase in O2- production, there was a significant increase in activation of extracellular signal-regulated kinase 1/2, extracellular signal-regulated kinase 5, c-Jun NH2-terminal kinase 1/2, and p38 mitogen-activated protein kinase. These data indicate that an NADPH oxidase expressed in cardiomyocytes is a major source of ROS generation in pressure overload LV hypertrophy and may contribute to pathophysiological changes such as the activation of redox-sensitive kinases and progression to heart failure.

Relationship between transmembrane ion movements, production of reactive oxygen species and the hypersensitive response during the challenge of tobacco suspension cells by zoospores of Phytophthora nicotianae

We have examined the responses of suspension cultured tobacco cells after inoculation with zoospores from compatible or incompatible races of the Oomycete pathogen Phytophthora nicotianae. The incompatible interaction, characterized by the production of reactive oxygen species (ROS) (specifically superoxide (<$>{\\rm HO^{\\,\\cdot}_{2}/O_{2}^{\\minus}}<$>) and hydrogen peroxide (H2O2)), and by a hypersensitive response (HR), was not dependent upon the presence of exogenous calcium. However, perturbation of calcium ion movements by EGTA or LaCl3suppressed both ROS production and the extent of cell death. The Ca2+-specific ionophore, A23187, slightly enhanced ROS production during the incompatible interaction but had no effect on the responses of susceptible or unchallenged cells. These results confirm that both ROS production and the HR are potentiated by movement of endogenous calcium across the plasmalemma. While there was no evidence of a transmembrane K+/H+exchange immediately following incompatible zoospore challenge, a later potassium efflux from cells coincided with the onset of the HR. Unexpectedly, medium pH drifted downwards during all host cell responses suggesting that alkaline peroxidases are not the major source of ROS generation during an incompatible tobacco cell/zoospore interaction.