The lectin-like domain of tumor necrosis factor improves lung function after rat lung transplantation--potential role for a reduction in reactive oxygen species generation

To test the hypothesis that the lectin-like domain of tumor necrosis factor, mimicked by the TIP peptide, can improve lung function after unilateral orthotopic lung isotransplantation. Because of a lack of a specific treatment for ischemia reperfusion-mediated lung injury, accompanied by a disrupted barrier integrity and a dysfunctional alveolar liquid clearance, alternative therapies restoring these parameters after lung transplantation are required.

Reactive oxygen species mediate functional differences in human radial and internal thoracic arteries from smokers

Smoking not only increases the risk that coronary heart disease will develop but also morbidity and mortality in patients with known coronary atherosclerosis and after coronary artery bypass grafting. Excessive generation of reactive oxygen species (ROS) has been implicated as the final common pathway for the development of endothelial dysfunction in various cardiovascular risk factors. This study assessed the influence of smoking on two different human arteries routinely used as coronary artery bypass graft conduits.

Dystrophic cardiomyopathy: amplification of cellular damage by Ca2+ signalling and reactive oxygen species-generating pathways

AIMS: Cardiac myopathies are the second leading cause of death in patients with Duchenne and Becker muscular dystrophy, the two most common and severe forms of a disabling striated muscle disease. Although the genetic defect has been identified as mutations of the dystrophin gene, very little is known about the molecular and cellular events leading to progressive cardiac muscle damage. Dystrophin is a protein linking the cytoskeleton to a complex of transmembrane proteins that interact with the extracellular matrix. The fragility of the cell membrane resulting from the lack of dystrophin is thought to cause an excessive susceptibility to mechanical stress. Here, we examined cellular mechanisms linking the initial membrane damage to the dysfunction of dystrophic heart. METHODS AND RESULTS: Cardiac ventricular myocytes were enzymatically isolated from 5- to 9-month-old dystrophic mdx and wild-type (WT) mice. Cells were exposed to mechanical stress, applied as osmotic shock. Stress-induced cytosolic and mitochondrial Ca(2+) signals, production of reactive oxygen species (ROS), and mitochondrial membrane potential were monitored with confocal microscopy and fluorescent indicators. Pharmacological tools were used to scavenge ROS and to identify their possible sources. Osmotic shock triggered excessive cytosolic Ca(2+) signals, often lasting for several minutes, in 82% of mdx cells. In contrast, only 47% of the WT cardiomyocytes responded with transient and moderate intracellular Ca(2+) signals. On average, the reaction was 6-fold larger in mdx cells. Removal of extracellular Ca(2+) abolished these responses, implicating Ca(2+) influx as a trigger for abnormal Ca(2+) signalling. Our further experiments revealed that osmotic stress in mdx cells produced an increase in ROS production and mitochondrial Ca(2+) overload. The latter was followed by collapse of the mitochondrial membrane potential, an early sign of cell death. CONCLUSION: Overall, our findings reveal that excessive intracellular Ca(2+) signals and ROS generation link the initial sarcolemmal injury to mitochondrial dysfunctions. The latter possibly contribute to the loss of functional cardiac myocytes and heart failure in dystrophy. Understanding the sequence of events of dystrophic cell damage and the deleterious amplification systems involved, including several positive feed-back loops, may allow for a rational development of novel therapeutic strategies.

Nitric oxide induces cell death in canine cruciate ligament cells by activation of tyrosine kinase and reactive oxygen species

BACKGROUND: There is increasing evidence suggesting that development of progressive canine cranial cruciate ligament (CCL) rupture involves a gradual degeneration of the CCL itself, initiated by a combination of factors, ranging from mechanical to biochemical. To date, knowledge is lacking to what extent cruciate disease results from abnormal biomechanics on a normal ligament or contrary how far preliminary alterations of the ligament due to biochemical factors provoke abnormal biomechanics. This study is focused on nitric oxide (NO), one of the potential biochemical factors. The NO-donor sodium nitroprusside (SNP) has been used to study NO-dependent cell death in canine cranial and caudal cruciate ligament cells and to characterize signaling mechanisms during NO-stimulation. RESULTS: Sodium nitroprusside increased apoptotic cell death dose- and time-dependently in cruciate ligamentocytes. Cells from the CCL were more susceptible to apoptosis than CaCL cells. Caspase-3 processing in response to SNP was not detected. Testing major upstream and signal transducing pathways, NO-induced cruciate ligament cell death seemed to be mediated on different levels. Specific inhibition of tyrosine kinase significantly decreased SNP-induced cell death. Mitogen activated protein kinase ERK1 and 2 are activated upon NO and provide anti-apoptotic signals whereas p38 kinase and protein kinase C are not involved. Moreover, data showed that the inhibition reactive oxygen species (ROS) significantly reduced the level of cruciate ligament cell death. CONCLUSIONS: Our data support the hypothesis that canine cruciate ligamentocytes, independently from their origin (CCL or CaCL) follow crucial signaling pathways involved in NO-induced cell death. However, the difference on susceptibility upon NO-mediated apoptosis seems to be dependent on other pathways than on these tested in the present study. In both, CCL and CaCL, the activation of the tyrosine kinase and the generation of ROS reveal important signaling pathways. In perspective, new efforts to prevent the development and progression of cruciate disease may include strategies aimed at reducing ROS.

Loss of sphingosine kinase-1 in carcinoma cells increases formation of reactive oxygen species and sensitivity to doxorubicin-induced DNA damage

Sphingosine kinases (SK) catalyse the formation of sphingosine 1-phosphate, which is a key lipid mediator regulating cell responses such as proliferation, survival and migration. Here we have investigated the effect of targeted inhibition of SK-1 on cell damage and elucidated the mechanisms involved.

Arginase II Promotes Macrophage Inflammatory Responses Through Mitochondrial Reactive Oxygen Species, Contributing to Insulin Resistance and Atherogenesis

Macrophage-mediated chronic inflammation is mechanistically linked to insulin resistance and atherosclerosis. Although arginase I is considered antiinflammatory, the role of arginase II (Arg-II) in macrophage function remains elusive. This study characterizes the role of Arg-II in macrophage inflammatory responses and its impact on obesity-linked type II diabetes mellitus and atherosclerosis.

Up-regulation of the peroxiredoxin-6 related metabolism of reactive oxygen species in skeletal muscle of mice lacking neuronal nitric oxide synthase

Although neuronal nitric oxide synthase (nNOS) plays a substantial role in skeletal muscle physiology, nNOS-knockout mice manifest an only mild phenotypic malfunction in this tissue. To identify proteins that might be involved in adaptive responses in skeletal muscle of knockout mice lacking nNOS, 2D-PAGE with silver-staining and subsequent tandem mass spectrometry (LC-MS/MS) was performed using extracts of extensor digitorum longus muscle (EDL) derived from nNOS-knockout mice in comparison to C57Bl/6 control mice. Six proteins were significantly (P < or = 0.05) more highly expressed in EDL of nNOS-knockout mice than in that of C57 control mice, all of which are involved in the metabolism of reactive oxygen species (ROS). These included prohibitin (2.0-fold increase), peroxiredoxin-3 (1.9-fold increase), Cu(2+)/Zn(2+)-dependent superoxide dismutase (SOD; 1.9-fold increase), heat shock protein beta-1 (HSP25; 1.7-fold increase) and nucleoside diphosphate kinase B (2.6-fold increase). A significantly higher expression (4.1-fold increase) and a pI shift from 6.5 to 5.9 of peroxiredoxin-6 in the EDL of nNOS-knockout mice were confirmed by quantitative immunoblotting. The concentrations of the mRNA encoding five of these proteins (the exception being prohibitin) were likewise significantly (P < or = 0.05) higher in the EDL of nNOS-knockout mice. A higher intrinsic hydrogen peroxidase activity (P < or = 0.05) was demonstrated in EDL of nNOS-knockout mice than C57 control mice, which was related to the presence of peroxiredoxin-6. The treatment of mice with the chemical NOS inhibitor L-NAME for 3 days induced a significant 3.4-fold up-regulation of peroxiredoxin-6 in the EDL of C57 control mice (P < or = 0.05), but did not alter its expression in EDL of nNOS-knockout mice. ESR spectrometry demonstrated the levels of superoxide to be 2.5-times higher (P < or = 0.05) in EDL of nNOS-knockout mice than in C57 control mice while an in vitro assay based on the emission of 2,7-dichlorofluorescein fluorescence disclosed the concentration of ROS to be similar in both strains of mice. We suggest that the up-regulation of proteins that are implicated in the metabolism of ROS, particularly of peroxiredoxin-6, within skeletal muscles of nNOS-knockout mice functionally compensates for the absence of nNOS in scavenging of superoxide.

Impact of reactive oxygen species (ROS) on the control of parasite loads and inflammation in Leishmania amazonensis infection

Background: Reactive oxygen species (ROS) protect the host against a large number of pathogenic microorganisms. ROS have different effects on parasites of the genus Leishmania: some parasites are susceptible to their action, while others seem to be resistant. The role of ROS in L. amazonensis infection in vivo has not been addressed to date. Methods: In this study, C57BL/6 wild-type mice (WT) and mice genetically deficient in ROS production by phagocytes (gp91phox−/− ) were infected with metacyclic promastigotes of L. amazonensis to address the effect of ROS in parasite control. Inflammatory cytokines, parasite loads and myeloperoxidase (MPO) activity were evaluated. In parallel, in vitro infection of peritoneal macrophages was assessed to determine parasite killing, cytokine, NO and ROS production. Results: In vitro results show induction of ROS production by infected peritoneal macrophages, but no effect in parasite killing. Also, ROS do not seem to be important to parasite killing in vivo, but they control lesion sizes at early stages of infection. IFN-γ, TNF-α and IL-10 production did not differ among mouse strains. Myeloperoxidase assay showed augmented neutrophils influx 6 h and 72 h post - infection in gp91phox−/− mice, indicating a larger inflammatory response in gp91phox−/− even at early time points. At later time points, neutrophil numbers in lesions correlated with lesion size: larger lesions in gp91phox−/− at earlier times of infection corresponded to larger neutrophil infiltrates, while larger lesions in WT mice at the later points of infection also displayed larger numbers of neutrophils. Conclusion: ROS do not seem to be important in L. amazonensis killing, but they regulate the inflammatory response probably by controlling neutrophils numbers in lesions.

Role of complement and perspectives for intervention in ischemia-reperfusion damage

Reperfusion of an organ following prolonged ischemia instigates the pro-inflammatory and pro-coagulant response of ischemia / reperfusion (IR) injury. IR injury is a wide-spread pathology, observed in many clinically relevant situations, including myocardial infarction, stroke, organ transplantation, sepsis and shock, and cardiovascular surgery on cardiopulmonary bypass. Activation of the classical, alternative, and lectin complement pathways and the generation of the anaphylatoxins C3a and C5a lead to recruitment of polymorphonuclear leukocytes, generation of radical oxygen species, up-regulation of adhesion molecules on the endothelium and platelets, and induction of cytokine release. Generalized or pathway-specific complement inhibition using protein-based drugs or low-molecular-weight inhibitors has been shown to significantly reduce tissue injury and improve outcome in numerous in-vitro, ex-vivo, and in-vivo models. Despite the obvious benefits in experimental research, only few complement inhibitors, including C1-esterase inhibitor, anti-C5 antibody, and soluble complement receptor 1, have made it into clinical trials of IR injury. The results are mixed, and the next objectives should be to combine knowledge and experience obtained in the past from animal models and channel future work to translate this into clinical trials in surgical and interventional reperfusion therapy as well as organ transplantation.

A novel TNFR1-triggered apoptosis pathway mediated by class IA PI3Ks in neutrophils

The most common form of neutrophil death is apoptosis. In the present study, we report surprising differences in the molecular mechanisms used for caspase activation between FAS/CD95-stimulated and TNF receptor 1 (TNFR1)-stimulated neutrophils. Whereas FAS-induced apoptosis was followed by caspase-8 activation and required Bid to initiate the mitochondrial amplification loop, TNF-?-induced apoptosis involved class IA PI3Ks, which were activated by MAPK p38. TNF-?-induced PI3K activation resulted in the generation of reactive oxygen species, which activated caspase-3, a mechanism that did not operate in neutrophils without active NADPH oxidase. We conclude that in neutrophils, proapoptotic pathways after TNFR1 stimulation are initiated by p38 and PI3K, but not by caspase-8, a finding that should be considered in anti-inflammatory drug-development strategies.

Inflammation-associated autophagy-related programmed necrotic death of human neutrophils characterized by organelle fusion events

The most common form of neutrophil death, under both physiological and inflammatory conditions, is apoptosis. In this study, we report a novel form of programmed necrotic cell death, associated with cytoplasmic organelle fusion events, that occurs in neutrophils exposed to GM-CSF and other inflammatory cytokines upon ligation of CD44. Strikingly, this type of neutrophil death requires PI3K activation, a signaling event usually involved in cellular survival pathways. In the death pathway reported in this study, PI3K is required for the generation of reactive oxygen species, which somehow trigger the generation of large cytoplasmic vacuoles, generated by the fusion of CD44-containing endosomes with autophagosomes and secondary, but not primary, granules. Neutrophils demonstrating vacuolization undergo rapid cell death that depends on receptor-interacting protein 1 kinase activity and papain family protease(s), but not caspases, that are most likely activated and released, respectively, during or as a consequence of organelle fusion. Vacuolized neutrophils are present in infectious and autoimmune diseases under in vivo conditions. Moreover, isolated neutrophils from such patients are highly sensitive toward CD44-mediated PI3K activation, reactive oxygen species production, and cell death, suggesting that the newly described autophagy-related form of programmed neutrophil necrosis plays an important role in inflammatory responses.

Disruption of the histidine triad nucleotide-binding hint2 gene in mice affects glycemic control and mitochondrial function

The histidine triad nucleotide-binding (Hint2) protein is a mitochondrial adenosine phosphoramidase expressed in liver and pancreas. Its physiological function is unknown. To elucidate the role of Hint2 in liver physiology, the Hint2 gene was deleted. Hint2(-/-) and Hint2(+/+) mice were generated in a mixed C57Bl6/J x 129Sv background. At 20 weeks, the phenotypic changes in Hint2(-/-) relative to Hint2(+/+) mice were an accumulation of hepatic triglycerides, decreased tolerance to glucose, a defective counter-regulatory response to insulin-provoked hypoglycaemia, an increase in plasma interprandial insulin but a decrease in glucose stimulated insulin secretion and defective thermoregulation upon fasting. Leptin mRNA in adipose tissue and plasma leptin were elevated. In mitochondria from Hint2(-/-) hepatocytes, state 3 respiration was decreased, a finding confirmed in HepG2 cells where HINT2 mRNA was silenced. The linked complex II to III electron transfer was decreased in Hint2(-/-) mitochondria, which was accompanied by a lower content of coenzyme Q. HIF-2α expression and the generation of reactive oxygen species were increased. Electron microscopy of mitochondria in Hint2(-/-) mice aged 12 months revealed clustered, fused organelles. The hepatic activities of 3-hydroxyacyl-CoA dehydrogenase short chain and glutamate dehydrogenase (GDH) were decreased by 68% and 60%, respectively, without a change in protein expression. GDH activity was similarly decreased in HINT2-silenced HepG2 cells. When measured in the presence of purified sirtuin 3, latent GDH activity was recovered (126% in Hint2(-/-) vs. 83% in Hint2(+/+) ). This suggests a greater extent of acetylation in Hint2(-/-) than in Hint2(+/+) . Conlusions: Hint2 positively regulates mitochondrial lipid metabolism and respiration, and glucose homeostasis. The absence of Hint2 provokes mitochondrial deformities and a change in the pattern of acetylation of selected proteins. (HEPATOLOGY 2012.).

Sodium nitroprusside induces cell death and cytoskeleton degradation in adult rat cardiomyocytes in vitro: implications for anthracycline-induced cardiotoxicity

Sodium nitroprusside (SNP) is used clinically as a rapid-acting vasodilator and in experimental models as donor of nitric oxide (NO). High concentrations of NO have been reported to induce cardiotoxic effects including apoptosis by the formation of reactive oxygen species. We have therefore investigated effects of SNP on the myofibrillar cytoskeleton, contractility and cell death in long-term cultured adult rat cardiomyocytes at different time points after treatment. Our results show, that SNP treatment at first results in a gradual increase of cytoskeleton degradation marked by the loss of actin labeling and fragmentation of sarcomeric structure, followed by the appearance of TUNEL-positive nuclei. Already lower doses of SNP decreased contractility of cardiomyocytes paced at 2 Hz without changes of intracellular calcium concentration. Ultrastructural analysis of the cultured cells demonstrated mitochondrial changes and disintegration of sarcomeric alignment. These adverse effects of SNP in cardiomyocytes were reminiscent of anthracycline-induced cardiotoxicity, which also involves a dysregulation of NO with the consequence of myofibrillar degradation and ultimately cell death. An inhibition of the pathways leading to the generation of reactive NO products, or their neutralization, may be of significant therapeutic benefit for both SNP and anthracycline-induced cardiotoxicity.