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Leukocyte Nox2 is recognized to have a fundamental microbicidal function in sepsis but the specific role of Nox2 in endothelial cells (EC) remains poorly elucidated. Here, we tested the hypothesis that endothelial Nox2 participates in the pathogenesis of systemic inflammation and hypotension induced by LPS. LPS was injected intravenously in mice with Tie2-targeted deficiency or transgenic overexpression of Nox2. Mice with Tie2-targeted Nox2 deficiency had increased circulating levels of TNF-α, enhanced numbers of neutrophils trapped in lungs, and aggravated hypotension after LPS injection, as compared to control LPS-injected animals. In contrast, Tie2-driven Nox2 overexpression attenuated inflammation and prevented the hypotension induced by LPS. Because Tie2-Cre targets both EC and myeloid cells we generated bone marrow chimeric mice with Nox2 deletion restricted to leukocytes or ECs. Mice deficient in Nox2 either in leukocytes or ECs had reduced LPS-induced neutrophil trapping in the lungs and lower plasma TNF-α levels as compared to control LPS-injected mice. However, the pronounced hypotensive response to LPS was present only in mice with EC-specific Nox2 deletion. Experiments in vitro with human vein or aortic endothelial cells (HUVEC and HAEC, respectively) treated with LPS revealed that EC Nox2 controls NF-κB activation and the transcription of toll-like receptor 4 (TLR4), which is the recognition receptor for LPS. In conclusion, these results suggest that endothelial Nox2 limits NF-κB activation and TLR4 expression, which in turn attenuates the severity of hypotension and systemic inflammation induced by LPS.

Glutathione is the major intracellular redox buffer in the liver and is critical for hepatic detoxification of xenobiotics and other environmental toxins. Hepatic glutathione is also a major systemic store for other organs and thus impacts on pathologies such as Alzheimer's disease, Sickle Cell Anaemia and chronic diseases associated with aging. Glutathione levels are determined in part by the availability of cysteine, generated from homocysteine through the transsulfuration pathway. The partitioning of homocysteine between remethylation and transsulfuration pathways is known to be subject to redox-dependent regulation, but the underlying mechanisms are not known. An association between plasma Hcy and a single nucleotide polymorphism within the NADPH oxidase 4 locus led us to investigate the involvement of this reactive oxygen species- generating enzyme in homocysteine metabolism. Here we demonstrate that NADPH oxidase 4 ablation in mice results in increased flux of homocysteine through the betaine-dependent remethylation pathway to methionine, catalysed by betaine-homocysteine-methyltransferase within the liver. As a consequence NADPH oxidase 4-null mice display significantly lowered plasma homocysteine and the flux of homocysteine through the transsulfuration pathway is reduced, resulting in lower hepatic cysteine and glutathione levels. Mice deficient in NADPH oxidase 4 had markedly increased susceptibility to acetaminophen-induced hepatic injury which could be corrected by administration of N-acetyl cysteine. We thus conclude that under physiological conditions, NADPH oxidase 4-derived reactive oxygen species is a regulator of the partitioning of the metabolic flux of homocysteine, which impacts upon hepatic cysteine and glutathione levels and thereby upon defence against environmental toxins., Highlights • Ablation of Nox4 results in lowered plasma homocysteine levels in mice. • This results from increased folate-independent remethylation of homocysteine. • As a consequence mice display reduced hepatic cysteine and glutathione. • Reduced glutathione levels render the mice susceptible to hepatotoxic agents. • Nox4 is a physiological regulator of partitioning of homocysteine metabolic flux.

The transcription factor nuclear factor erythroid–derived 2-like 2 (Nrf2) controls a network of cytoprotective genes. Neither how Nrf2 is activated in the heart under hemodynamic overload nor its role and mechanism of action are known. This study aimed to investigate the activation and role of Nrf2 during chronic cardiac pressure overload. We first compared the responses of Nrf2 −/− mice and wild-type littermates to chronic pressure overload. Hearts of Nrf2 −/− mice showed impaired antioxidant gene expression, increased hypertrophy, and worse function compared with those of wild-type littermates after overload. Hearts of Nrf2 −/− mice had increased mitochondrial DNA damage, a caspase 8/BH3-interacting domain death agonist–related cleavage of mitochondrial apoptosis–inducing factor, nuclear DNA damage, and cell death. Nrf2 activation was under the control of the endogenous reactive oxygen species–generating enzyme nicotinamide adenine dinucleotide phosphate oxidase-4, both in vivo and in vitro. In mice with cardiac-specific overexpression of nicotinamide adenine dinucleotide phosphate oxidase-4, Nrf2 deletion significantly attenuated their protective phenotype during chronic pressure overload. This study identifies nicotinamide adenine dinucleotide phosphate oxidase-4–dependent upregulation of Nrf2 as an important endogenous protective pathway that limits mitochondrial damage and apoptosis-inducing factor–related cell death in the heart under hemodynamic overload.

Adult mammalian cardiomyocytes have a very limited capacity to proliferate, and consequently the loss of cells after cardiac stress promotes heart failure. Recent evidence suggests that administration of hydrogen peroxide (H2O2), can regulate redox-dependent signalling pathway(s) to promote cardiomyocyte proliferation in vitro, but the potential relevance of such a pathway in vivo has not been tested. We have generated a transgenic (Tg) mouse model in which the H2O2-generating enzyme, NADPH oxidase 4 (Nox4), is overexpressed within the postnatal cardiomyocytes, and observed that the hearts of 1–3 week old Tg mice pups are larger in comparison to wild type (Wt) littermate controls. We demonstrate that the cardiomyocytes of Tg mouse pups have increased cell cycling capacity in vivo as determined by incorporation of 5-bromo-2′-deoxyuridine. Further, microarray analyses of the transcriptome of these Tg mouse hearts suggested that the expression of cyclin D2 is significantly increased. We investigated the molecular mechanisms which underlie this more proliferative phenotype in isolated neonatal rat cardiomyocytes (NRCs) in vitro, and demonstrate that Nox4 overexpression mediates an H2O2-dependent activation of the ERK1/2 signalling pathway, which in turn phosphorylates and activates the transcription factor c-myc. This results in a significant increase in cyclin D2 expression, which we show to be mediated, at least in part, by cis-acting c-myc binding sites within the proximal cyclin D2 promoter. Overexpression of Nox4 in NRCs results in an increase in their proliferative capacity that is ablated by the silencing of cyclin D2. We further demonstrate activation of the ERK1/2 signalling pathway, increased phosphorylation of c-myc and significantly increased expression of cyclin D2 protein in the Nox4 Tg hearts. We suggest that this pathway acts to maintain the proliferative capacity of cardiomyocytes in Nox4 Tg pups in vivo and so delays their exit from the cell cycle after birth., Highlights • Cardiomyocyte cell cycling is under redox control in vivo. • Nox4 is a potential source of ROS regulating this process. • Redox activation of ERK1/2 promotes cyclin D2 transcription via c-myc activation. • Promotion of proliferation via increased cyclin D2 may be useful therapeutically.

During heart development, the progression from a pluripotent, undifferentiated embryonic stem cell to a functional cardiomyocyte in the adult mammalian heart is characterised by profound changes in gene expression, cell structure, proliferative capacity and metabolism. Whilst the precise causal relationships between these processes are not fully understood, it is clear that the availability and cellular ability to utilise oxygen are critical effectors of cardiomyocyte differentiation and function during development. In particular, cardiomyocytes switch from a largely glycolytic-based production of ATP to predominantly β-oxidation of long-chain fatty acids to generate the cellular energy requirements. Whilst this transition occurs progressively during embryonic and foetal development, it is particularly abrupt over the period of birth. In the adult heart, many cardiopathologies are accompanied by a reversal to a more foetal-like metabolic profile. Understanding the mechanistic causes and consequences of the normal metabolic changes that occur during heart development and those in the pathological heart setting is crucial to inform future potential therapeutic interventions. It is becoming clear that reactive oxygen species (ROS) play critical roles in the regulation of redox-mediated molecular mechanisms that control cellular homoeostasis and function. ROS are generated as a consequence of metabolic processes in aerobic organisms. An overproduction of ROS, when not balanced by the cell's antioxidant defence mechanisms (termed "oxidative stress"), results in non-specific oxidation of proteins, lipids and DNA and is cytotoxic. However, the tightly regulated temporal and spatial production of ROS such as H₂O₂ acts to control the activity of proteins through specific post-translational oxidative modifications and is crucial to cellular function. We describe here the metabolic changes that occur in the developing heart and how they can revert in cardiopathologies. They are discussed in the light of what is currently known about the regulation of these processes by changes in the cellular redox state and levels of ROS production.

Background: The epidermal growth factor receptor (EGFR) is overexpressed in colorectal cancer (CRC), and is correlated with poor prognosis, making it an attractive target for monoclonal antibody (mAb) therapy. A component of the therapeutic efficacy of IgG1 mAbs is their stimulation of antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells bearing the CD16 receptor. As NK cells are functionally impaired in cancer patients and may be further compromised upon chemotherapy, it is crucial to assess whether immunotherapeutic strategies aimed at further enhancing ADCC are viable. Methods: CRC patients before, during and after chemotherapy were immunophenotyped by flow cytometry for major white blood cell populations. ADCC-independent NK cell functionality was assessed in cytotoxicity assays against K562 cells. ADCC-dependent killing of EGFR+ A431 cancer cells by NK cells was measured with a degranulation assay where ADCC was induced by GA201, an anti-EGFR mAb glyco-engineered to enhance ADCC. Results: Here, we confirm the observation that NK cells in cancer patients are dysfunctional. However, GA201 was able to induce robust NK cell-dependent cytotoxicity in CRC patient NK cells, effectively overcoming their impairment. Conclusions: These findings support the evaluation of the therapeutic potential of GA201 in combination with chemotherapy in CRC patients.

Significance: Aerobic organisms must exist between the dueling biological metabolic processes for energy and respiration and the obligatory generation of reactive oxygen species (ROS) whose deleterious consequences can reduce survival. Wide fluctuations in harmful ROS generation are circumvented by endogenous countermeasures (i.e., enzymatic and nonenzymatic antioxidants systems) whose capacity decline with aging and are enhanced by disease states. Recent Advances: Substantial efforts on the cellular and molecular underpinnings of oxidative stress has been complemented recently by the discovery that reductive stress similarly predisposes to inheritable cardiomyopathy, firmly establishing that the biological extremes of the redox spectrum play essential roles in disease pathogenesis. Critical Issues: Because antioxidants by nutritional or pharmacological supplement to prevent or mitigate disease states have been largely disappointing, we hypothesize that lack of efficacy of antioxidants might be related to adverse outcomes in responders at the reductive end of the redox spectrum. As emerging concepts, such as reductive, as opposed, oxidative stress are further explored, there is an urgent and critical gap for biochemical phenotyping to guide the targeted clinical applications of therapeutic interventions. Future Directions: New approaches are vitally needed for characterizing redox states with the long-term goal to noninvasively assess distinct clinical states (e.g., presymptomatic, end-stage) with the diagnostic accuracy to guide personalized medicine. Antioxid. Redox Signal. 18, 1114–1127.

NADPH oxidase 4 (Nox4) generates reactive oxygen species (ROS) that can modulate cellular phenotype and function in part through the redox modulation of the activity of transcription factors. We demonstrate here the potential of Nox4 to drive cardiomyocyte differentiation in pluripotent embryonal carcinoma cells, and we show that this involves the redox activation of c-Jun. This in turn acts to up-regulate GATA-4 expression, one of the earliest markers of cardiotypic differentiation, through a defined and highly conserved cis-acting motif within the GATA-4 promoter. These data therefore suggest a mechanism whereby ROS act in pluripotential cells in vivo to regulate the initial transcription of critical tissue-restricted determinant(s) of the cardiomyocyte phenotype, including GATA-4. The ROS-dependent activation, mediated by Nox4, of widely expressed redox-regulated transcription factors, such as c-Jun, is fundamental to this process.

Caspases, a family of cysteine proteases most often investigated for their roles in apoptosis, have also been demonstrated to have functions that are vital for the efficient execution of cell differentiation. One such role that has been described is the requirement of caspase-3 for the differentiation of skeletal myoblasts into myotubes but, as yet, the mechanism leading to caspase-3 activation in this case remains elusive. Here, we demonstrate that caspase-9, an initiator caspase in the mitochondrial death pathway, is responsible for the activation of caspase-3 in differentiating C2C12 cells. Reduction of caspase-9 levels, using an shRNA construct, prevented caspase-3 activation and inhibited myoblast fusion. Myosin-heavy-chain expression, which accompanies myoblastic differentiation, was not caspase-dependent. Overexpression of Bcl-xL, a protein that inhibits caspase-9 activation, had the same effect on muscle differentiation as knockdown of caspase-9. These data suggest that the mitochondrial pathway is required for differentiation; however, the release of cytochrome c or Smac (Diablo) could not be detected, raising the possibility of a novel mechanism of caspase-9 activation during muscle differentiation.

NADPH oxidase-4 (Nox4) is an important modulator of redox signaling that is inducible at the level of transcriptional expression in multiple cell types. By contrast to other Nox enzymes, Nox4 is continuously active without requiring stimulation. We reported recently that expression of Nox4 is induced in the adult heart as an adaptive stress response to pathophysiological insult. To elucidate the potential downstream target(s) regulated by Nox4, we performed a microarray screen to assess the transcriptomes of transgenic (tg) mouse hearts in which Nox4 was overexpressed. The screen revealed a significant increase in the expression of many antioxidant and detoxifying genes regulated by Nrf2 in tg compared to wild-type (wt) mouse hearts, and this finding was subsequently confirmed by Q-PCR. Expression of glutathione biosynthetic and recycling enzymes was increased in tg hearts and associated with higher levels of both GSH and the ratio of reduced:oxidised GSH, compared to wt hearts. The increases in expression of the antioxidant genes and the changes in glutathione redox effected by Nox4 were ablated in an Nrf2-null genetic background. These data therefore demonstrate that Nox4 can activate the Nrf2-regulated pathway, and suggest a potential role for Nox4 in the regulation of GSH redox in cardiomyocytes.