H(2)O(2)-induced O(2) production by a non-phagocytic NAD(P)H oxidase causes oxidant injury

Non-phagocytic NAD(P)H oxidases have been implicated as major sources of reactive oxygen species in blood vessels. These oxidases can be activated by cytokines, thereby generating O(2), which is subsequently converted to H(2)O(2) and other oxidant species. The oxidants, in turn, act as important second messengers in cell signaling cascades. We hypothesized that reactive oxygen species, themselves, can activate the non-phagocytic NAD(P)H oxidases in vascular cells to induce oxidant production and, consequently, cellular injury. The current report demonstrates that exogenous exposure of non-phagocytic cell types of vascular origin (smooth muscle cells and fibroblasts) to H(2)O(2) activates these cell types to produce O(2) via an NAD(P)H oxidase. The ensuing endogenous production of O(2) contributes significantly to vascular cell injury following exposure to H(2)O(2). These results suggest the existence of a feed-forward mechanism, whereby reactive oxygen species such as H(2)O(2) can activate NAD(P)H oxidases in non-phagocytic cells to produce additional oxidant species, thereby amplifying the vascular injury process. Moreover, these findings implicate the non-phagocytic NAD(P)H oxidase as a novel therapeutic target for the amelioration of the biological effects of chronic oxidant stress.     

Homocysteine enhances cell proliferation in vascular smooth muscle cells: role of p38 MAPK and p47phox

Elevation of blood homocysteine levels (hyperhomocysteinemia) is a risk factor for cardiovascular disorders. One of the mechanisms by which homocysteine induces atherosclerosis is to promote the proliferation of vascular smooth muscle cells (VSMCs) in a reactive oxygen species (ROS)-dependent manner. It has been shown that homocysteine induces the production of ROS through the activation of NAD(P)H oxidases in VSMCs. In this study, we investigated the signal transduction pathways involved in the activation of NAD(P)H oxidases. Homocysteine promoted DNA synthesis in VSMCs. Inhibition of ROS by N-acetyl-L-cysteine (an antioxidant) and apocynin (an inhibitor of NAD(P)H oxidases) significantly blocked homocysteine-induced proliferation in VSMCs. Homocysteine induced a rapid increase in the phosphorylation of p38-mitogen-activated protein kinase (p38 MAPK). p38 MAPK in turn activated NAD(P)H oxidases by inducing the phosphorylation of p47phox, resulting in the generation of ROS. ROS induced the phosphorylation of Akt, which was probably responsible for proliferation in VSMCs. These findings demonstrate that homocysteine induces an increase in the activity of NAD(P)H oxidases in VSMCs by activating p38 MAPK and enhancing the phosphorylation of p47phox.     

Regulation of NAD(P)H oxidases by AMPK in cardiovascular systems

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are ubiquitously produced in cardiovascular systems. Under physiological conditions, ROS/RNS function as signaling molecules that are essential in maintaining cardiovascular function. Aberrant concentrations of ROS/RNS have been demonstrated in cardiovascular diseases owing to increased production or decreased scavenging, which have been considered common pathways for the initiation and progression of cardiovascular diseases such as atherosclerosis, hypertension, (re)stenosis, and congestive heart failure. NAD(P)H oxidases are primary sources of ROS and can be induced or activated by all known cardiovascular risk factors. Stresses, hormones, vasoactive agents, and cytokines via different signaling cascades control the expression and activity of these enzymes and of their regulatory subunits. But the molecular mechanisms by which NAD(P)H oxidase is regulated in cardiovascular systems remain poorly characterized. Investigations by us and others suggest that adenosine monophosphate-activated protein kinase (AMPK), as an energy sensor and modulator, is highly sensitive to ROS/RNS. We have also obtained convincing evidence that AMPK is a physiological suppressor of NAD(P)H oxidase in multiple cardiovascular cell systems. In this review, we summarize our current understanding of how AMPK functions as a physiological repressor of NAD(P)H oxidase.     

Differential regulation of dual NADPH oxidases/peroxidases, Duox1 and Duox2, by Th1 and Th2 cytokines in respiratory tract epithelium

Partially reduced metabolites of molecular oxygen, superoxide (O2-) and hydrogen peroxide (H2O2), are detected in respiratory tract lining fluid, and it is assumed that these are key components of innate immunity. Whether these reactive oxygen species (ROS) are produced specifically by the respiratory epithelium in response to infection, or are a non-specific by-product of oxidant-producing inflammatory cells is not well characterized. Increasing evidence supports the hypothesis that the dual function NAD(P)H oxidases/peroxidases, Duox1 and Duox2, are important sources of regulated H2O2 production in respiratory tract epithelium. However, no studies to date have characterized the regulation of Duox gene expression. Accordingly, we examined Duox1 and Duox2 mRNA expression by real-time PCR in primary respiratory tract epithelial cultures after treatment with multiple cytokines. Herein, we determined that Duox1 expression was increased several-fold by treatment with the Th2 cytokines IL-4 and IL-13, whereas Duox2 expression was highly induced following treatment with the Th1 cytokine IFN-gamma. Duox2 expression was also elevated by polyinosine-polycytidylic acid (poly(I:C)) and rhinovirus infection. Diphenyleneiodonium (DPI)-inhibitable apical H2O2 production was similarly increased by the addition of Th1 or Th2 cytokines. These results demonstrate for the first time the regulation of Duox expression by immunomodulatory Th1 and Th2 cytokines, and suggest a mechanism by which ROS production can be regulated in the respiratory tract as part of the host defense response.     

A novel protein, Romo1, induces ROS production in the mitochondria

The majority of endogenous reactive oxygen species (ROS) are produced in the mitochondrial respiratory chain. An imbalance in ROS production alters the intracellular redox homeostasis, triggers DNA damage, and contributes to cancer development and progression. This study identified a novel protein, reactive oxygen species modulator 1 (Romo1), which is localized in the mitochondria. Romo1 was found to increase the level of ROS in the cells. Increased Romo1 expression was observed in various cancer cell lines. This suggests that the increased Romo1 expression during cancer progression may cause persistent oxidative stress to tumor cells, which can increase their malignancy.     

LMP1 Increases Expression of NADPH Oxidase (NOX) and Its Regulatory Subunit p22 in NP69 Nasopharyngeal Cells and Makes Them Sensitive to a Treatment by a NOX Inhibitor

Oxidative stress is thought to contribute to cancer development. Epstein–Barr virus (EBV) and its encoded oncoprotein, latent membrane protein 1 (LMP1), are closely associated with the transformation of nasopharyngeal carcinoma (NPC) and Burkitt’s lymphoma (BL). In this study, we used LMP1-transformed NP cells and EBV-related malignant cell lines to assess the effects of LMP1 on reactive oxygen species (ROS) accumulation and glycolytic activity. Using NPC tissue samples and a tissue array to address clinical implications, we report that LMP1 activates NAD(P)H oxidases to generate excessive amount of ROS in EBV-related malignant diseases. By evaluating NAD(P)H oxidase (NOX) subunit expression, we found that the expression of the NAD(P)H oxidase regulatory subunit p22^phox was significantly upregulated upon LMP1-induced transformation. Furthermore, this upregulation was mediated by the c-Jun N-terminal kinase (JNK) pathway. In addition, LMP1 markedly stimulated anaerobic glycolytic activity through the PI3K/Akt pathway. Additionally, in both NPC cells and tissue samples, p22^phox expression correlated with LMP1 expression. The NAD(P)H oxidase inhibitor diphenyleneiodonium (DPI) also exerted a marked cytotoxic effect in LMP1-transformed and malignant cells, providing a novel strategy for anticancer therapy.     

Induction of heme oxygenase-1 inhibits NAD(P)H oxidase activity by down-regulating cytochrome b558 expression via the reduction of heme availability

Heme-oxygenase-1 (HO-1), the rate-limiting enzyme of heme degradation, has powerful anti-oxidant properties related to the production of the reactive oxygen species scavenger bilirubin. However, some data suggest that HO-1 could also inhibit the cellular production of reactive oxygen species. Therefore, we investigated whether the anti-oxidant properties of HO-1 could be mediated by modulation of the activity and/or expression of the heme-containing NAD(P)H oxidase, the main source of the superoxide anion (O(2)(-)) in phagocytic cells. Increasing HO-1 expression in RAW 264.7 macrophages effectively decreased NAD(P)H oxidase activity and expression of gp91(phox), its heme-containing catalytic component, because of deficient protein maturation and increased degradation. Loading cells with heme reversed the decrease in O(2)(-) production and gp91(phox) expression induced by HO-1 overexpression. Similar results were obtained in vivo in rat alveolar macrophages after pharmacological modulation of HO-1 expression or activity. These results show that a decrease in heme content due to HO-1 activation limits heme availability for maturation of the gp91(phox) subunit and assembly of the functional NAD(P)H oxidase. This study provides a new mechanism to explain HO-1 anti-oxidant properties.     

Cytotoxic effects of menadione on normal and cytochrome c-deficient yeast cells cultivated aerobically or anaerobically

Cytotoxic effects of menadione on normal and cytochrome c-deficient yeast cells were examined on the basis of the cell growth rate, NAD(P)H concentration, reactive oxygen production, plasma membrane H⁺-ATPase activity, and ethanol production. In aerobically or anaerobically cultured yeast cells, NAD(P)H concentration decreased with increasing concentration of menadione, and the recovery of NAD(P)H concentration was proportional to the cell growth rate. However, there was no relationship among the inhibition of the cell growth and reactive oxygen production, plasma membrane H⁺-ATPase activity, and ethanol production. Among them, ethanol production showed resistance to the cytotoxicity of menadione, suggesting the resistance of glycolysis to menadione. The growth inhibitory effect of menadione depended on the rapid decrease and the recovery of NAD(P)H rather than production of reactive oxygen species regardless of aerobic culture or anaerobic culture and presence or absence of mitochondrial function. The recovery of NAD(P)H concentration after the addition of menadione might depend on menadione-resistant glycolytic enzymes.     

ROS production and Glut1 activity in two human megakaryocytic cell lines

Reactive oxygen species (ROS) has been increasingly recognised as intracellular messengers in signal transduction following receptor activation by a variety of bioactive peptides including growth factors, cytokines and hormones. In this study ROS production and glucose transport activity were evaluated in the growth factor dependent M07e cells and in B1647 cells, not requiring additional hematopoietic cytokines for growth: the aim was to investigate whether ROS could be involved in the regulation of Glut1-mediated glucose uptake in both cell lines. The effect of the synthetic superoxide and hydrogen peroxide scavenger EUK-134 on DOG uptake activity and intracellular ROS formation supports the concept of reactive oxygen species as signalling molecules. In order to investigate ROS generation sources, diphenyleneiodonium, an inhibitor of flavoprotein centres and apocynin, an inhibitor of NAD(P)H oxidase, were used: they inhibit both ROS production and glucose uptake activation. All these data support the hypothesis that ROS can contribute to the regulation of glucose transport, not only in M07e cells but also in B1647 cells; we could speculate that one possible source of ROS, linked somehow with Glut1 activity, can be a NAD(P)H oxidase similar to that one present in phagocytic cells.     

GroEL1, a heat shock protein 60 of Chlamydia pneumoniae, induces lectin-like oxidized low-density lipoprotein receptor 1 expression in endothelial cells and enhances atherogenesis in hypercholesterolemic rabbits

Lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) plays a major role in oxidized low-density lipoprotein-induced vascular inflammation. Chlamydia pneumoniae has been found in atherosclerotic lesions and is related to atherosclerotic pathogenesis, although its specific mechanism remains unknown. This study was conducted to investigate the mechanisms of LOX-1 expression in GroEL1 (a heat shock protein from C. pneumoniae)-administered human coronary artery endothelial cells (HCAECs) and atherogenesis in hypercholesterolemic rabbits. We demonstrated that in the hypercholesterolemic rabbit model, GroEL1 administration enhanced fatty streak and macrophage infiltration in atherosclerotic lesions, which may be mediated by elevated LOX-1 expression. In in vitro study using HCAECs, stimulation with GroEL1 increased TLR4 and LOX-1 expression. Increased LOX-1 expression was downregulated by Akt activation and PI3K-mediated endothelial NO synthase activation. PI3K inhibitor and NO synthase inhibitor induced LOX-1 mRNA production, whereas the NO donor ameliorated the increasing effect of LOX-1 mRNA in GroEL1-stimulated HCAECs. LOX-1 expression was regulated by NADPH oxidase, which mediates reactive oxygen species production and intracellular MAPK signaling pathway in GroEL1-stimulated HCAECs. Treatment with polyethylene-glycol-conjugated superoxide dismutase, apocynin, or diphenylene iodonium significantly decreased GroEL1-induced LOX-1 expression, as did the knockdown of Rac1 gene expression by RNA interference. In conclusion, the GroEL1 protein may induce LOX-1 expression in endothelial cells and atherogenesis in hypercholesterolemic rabbits. The elevated level of LOX-1 in vitro may be mediated by the PI3K-Akt signaling pathway, endothelial NO synthase activation, NADPH oxidase-mediated reactive oxygen species production, and MAPK activation in GroEL1-stimulated HCAECs. The GroEL1 protein of C. pneumoniae may contribute to vascular inflammation and cardiovascular disorders.     

Sulfonylurea as well as elevated glucose levels stimulate reactive oxygen species production in the pancreatic beta-cell line, MIN6-a role of NAD(P)H oxidase in beta-cells

Increased oxidative stress may play a key role in the progressive deterioration of pancreatic beta-cells and the development of diabetes. However, the underlying mechanism is not well understood. Exposure of pancreatic beta-cell line, MIN6 cells, to elevated glucose level for 2h induced an increase in reactive oxygen species (ROS) production, as evaluated by the staining of 2',7'-dichlorofluorescein diacetate. This effect was completely blocked by NAD(P)H oxidase inhibitor (diphenylene iodonium) and protein kinase C (PKC) inhibitor (calphostin C), but not affected by other flavoprotein inhibitors (rotenone, oxypurinol, or l-N-monomethyl arginine). Glibenclamide also stimulated ROS production in a dose-dependent manner. This effect was again blocked by diphenylene iodonium and calphostin C. In conclusion, insulin secretagogues, both glibenclamide and elevated glucose level, stimulated ROS production in beta-cells through a PKC-dependent activation of NAD(P)H oxidase. This mechanism may be a novel therapeutic target for preventing the progression of beta-cell deterioration.     

Ragweed Subpollen Particles of Respirable Size Activate Human Dendritic Cells

Ragweed (Ambrosia artemisiifolia) pollen grains, which are generally considered too large to reach the lower respiratory tract, release subpollen particles (SPPs) of respirable size upon hydration. These SPPs contain allergenic proteins and functional NAD(P)H oxidases. In this study, we examined whether exposure to SPPs initiates the activation of human monocyte-derived dendritic cells (moDCs). We found that treatment with freshly isolated ragweed SPPs increased the intracellular levels of reactive oxygen species (ROS) in moDCs. Phagocytosis of SPPs by moDCs, as demonstrated by confocal laser-scanning microscopy, led to an up-regulation of the cell surface expression of CD40, CD80, CD86, and HLA-DQ and an increase in the production of IL-6, TNF-α, IL-8, and IL-10. Furthermore, SPP-treated moDCs had an increased capacity to stimulate the proliferation of naïve T cells. Co-culture of SPP-treated moDCs with allogeneic CD3⁺ pan-T cells resulted in increased secretion of IFN-γ and IL-17 by T cells of both allergic and non-allergic subjects, but induced the production of IL-4 exclusively from the T cells of allergic individuals. Addition of exogenous NADPH further increased, while heat-inactivation or pre-treatment with diphenyleneiodonium (DPI), an inhibitor of NADPH oxidases, strongly diminished, the ability of SPPs to induce phenotypic and functional changes in moDCs, indicating that these processes were mediated, at least partly, by the intrinsic NAD(P)H oxidase activity of SPPs. Collectively, our data suggest that inhaled ragweed SPPs are fully capable of activating dendritic cells (DCs) in the airways and SPPs'' NAD(P)H oxidase activity is involved in initiation of adaptive immune responses against innocuous pollen proteins.     

Reduced antioxidant capacity and diet-induced atherosclerosis in uncoupling protein-2-deficient mice

Vascular dysfunction in response to reactive oxygen species (ROS) plays an important role in the development and progression of atherosclerotic lesions. In most cells, mitochondria are the major source of cellular ROS during aerobic respiration. Under most conditions the rates of ROS formation and elimination are balanced through mechanisms that sense relative ROS levels. However, a chronic imbalance in redox homeostasis is believed to contribute to various chronic diseases, including atherosclerosis. Uncoupling protein-2 (UCP2) is a mitochondrial inner membrane protein shown to be a negative regulator of macrophage ROS production. In response to a cholesterol-containing atherogenic diet, C57BL/6J mice significantly increased expression of UCP2 in the aorta, while mice lacking UCP2, in the absence of any other genetic modification, displayed significant endothelial dysfunction following the atherogenic diet. Compared with wild-type mice, Ucp2(-/-) mice had decreased endothelial nitric oxide synthase, an increase in vascular cell adhesion molecule-1 expression, increased ROS production, and an impaired ability to increase total antioxidant capacity. These changes in Ucp2(-/-) mice were associated with increased aortic macrophage infiltration and more numerous and larger atherosclerotic lesions. These data establish that in the vasculature UCP2 functions as an adaptive antioxidant defense to protect against the development of atherosclerosis in response to a fat and cholesterol diet.     

Interaction of myeloperoxidase with vascular NAD(P)H oxidase-derived reactive oxygen species in vasculature: implications for vascular diseases

Vascular NAD(P)H oxidase-derived reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) have emerged as important molecules in the pathogenesis of atherosclerosis, hypertension, and diabetic vascular complications. Additionally, myeloperoxidase (MPO), a transcytosable heme protein that is derived from leukocytes, is also believed to play important roles in the above-mentioned inflammatory vascular diseases. Previous studies have shown that MPO-induced vascular injury responses are H2O2 dependent. It is well known that MPO can use leukocyte-derived H2O2; however, it is unknown whether the vascular-bound MPO can use vascular nonleukocyte oxidase-derived H2O2 to induce vascular injury. In the present study, ANG II was used to stimulate vascular NAD(P)H oxidases and increase their H2O2 production in the vascular wall, and vascular dysfunction was used as the vascular injury parameter. We demonstrated that vascular-bound MPO has sustained activity in the vasculature. MPO could use the vascular NAD(P)H oxidase-derived H2O2 to produce hypochlorus acid (HOCl) and its chlorinating species. More importantly, MPO derived HOCl and chlorinating species amplified the H2O2-induced vascular injury by additional impairment of endothelium-dependent relaxation. HOCl-modified low-density lipoprotein protein (LDL), a specific biomarker for the MPO-HOCl-chlorinating species pathway, was expressed in LDL and MPO-bound vessels with vascular NAD(P)H oxidase-derived H2O2. MPO-vascular NAD(P)H oxidase-HOCl-chlorinating species may represent a common pathogenic pathway in vascular diseases and a new mechanism involved in exacerbation of vascular diseases under inflammatory conditions.