Reactive oxygen species plasmatic levels in ischemic stroke

Oxidative stress is probably one of the mechanisms involved in neuronal damage induced by ischemia-reperfusion, and the antioxidant activity of plasma may be an important factor providing protection from neurological damage caused by stroke-associated oxidative stress. The aim of this study was to investigate the status of oxidative stress, NO and ONOO⁻ levels in patients with atherothrombotic and lacunar acute ischemic stroke and iNOS, eNOS and nitrotyrosine expression in the same patients. Plasma ONOO⁻ levels were significantly higher in patients than in controls while NO decreases in patients in respect to controls. Densitometric analysis of bands indicated that iNOS and N-Tyr protein levels were significantly higher in patients in respect to controls. This study has highlighted a significant NO decrease in our patients compared with controls and this is most probably due to the increased expression of inducible NO synthase by the effect of thrombotic attack. In fact, the constitutive NO isoforms, which produce small amounts of NO, are beneficial, while activation of the inducible isoform of NO, which produces much more NO, causes injury, being its toxicity greatly enhanced by generation of peroxynitrite. The significant ONOO⁻ increase observed in our patients, compared to controls, is most probably due to reaction of NO with O₂^·−. These findings suggest that free radical production and oxidative stress in ischemic stroke might have a major role in the pathogenesis of ischemic brain injury. Peroxynitrite might be the main marker of brain damage and neurological impairment in acute ischemic stroke.     

Evaluation of anticancer effects of cerium oxide nanoparticles on mouse fibrosarcoma cell line

Cerium oxide nanoparticles are associated with anticancer effects. While protecting normal cells, these nanoparticles exert their anticancer effects via oxidative stress and apoptosis in the cancer cells. In this study, the anticancer properties of nanoceria on fibrosarcoma cell line are evaluated. Cerium oxide nanoparticles were synthesized by the coprecipitation method and their anticancer effects on mouse fibrosarcoma tumor cells (WEHI164) were investigated. Viability assay was evaluated by MTT, and the DC-FDA assay performed for the detection of reactive oxygen species. For apoptosis assay, the annexin V/PI test was done as well as measuring the mRNA and protein expression levels of Bax and Bcl2 by real-time PCR and western blot method, respectively. Characterization of nanoceria reveals that synthesized nanoceria has cubic floruit structure with a size of about 30 nm. Toxicity assessment results show that nanoceria increases ROS levels and induced apoptosis in a dose-dependent manner in cancer cells (WEHI164), whereas low levels of toxicity were observed in normal cells (L929), even at the concentrations above 250 µg/ml in MTT assay. Real-time PCR and western blot assays showed that nanoceria could significantly increase the Bax expression in cancer cells. The results showed that nanoceria could act as a potential therapeutic agent for the treatment of fibrosarcoma.     

Cerium Oxide Nanoparticles Protect Endothelial Cells from Apoptosis Induced by Oxidative Stress

Oxidative stress is well documented to cause injury to endothelial cells (ECs), which in turn trigger cardiovascular diseases. Previous studies revealed that cerium oxide nanoparticles (nanoceria) had antioxidant property, but the protective effect of nanoceria on ROS injury to ECs and cardiovascular diseases has not been reported. In the current study, we investigated the protective effect and underlying mechanisms of nanoceria on oxidative injury to ECs. The cell viability, lactate dehydrogenase release, cellular uptake, intracellular localization and reactive oxygen species (ROS) levels, endocytosis mechanism, cell apoptosis, and mitochondrial membrane potential were performed. The results indicated that nanoceria had no cytotoxicity on ECs but had the ability to prevent injury by H₂O₂. Nanoceria could be uptaken into ECs through caveolae- and clathrin-mediated endocytosis and distributed throughout the cytoplasma. The internalized nanoceria effectively attenuated ROS overproduction induced by H₂O₂. Apoptosis was also alleviated greatly by nanoceria pretreatment. These results may be helpful for more rational application of nanoceria in biomedical fields in the future.     

Reflectance structured illumination imaging of internalized cerium oxide nanoparticles modulating dose-dependent reactive oxygen species in breast cancer cells

Cerium oxide nanoparticles have been shown to sensitize cancer cells to radiation damage. Their unique redox properties confer excellent therapeutic potential by augmenting radiation dose with reactive oxygen species mediating bystander effects. Owing to its metallic properties, cerium oxide nanoparticles can be visualized inside cells using reflected light and optical sectioning. This can be advantageous in settings requiring none or minimal sample preparation and modification. We investigated the use of reflectance imaging for the detection of unmodified nanoceria in MDA MB231 breast cancer cells along with differential interference contrast imaging and fluorescent nuclear labeling. We also performed studies to evaluate the uptake capability, cellular toxicity and redox properties of nanocaria in these cells. Our results demonstrate that reflectance structured illumination imaging can effectively localize cerium oxide nanoparticles in breast cancer cells, and when combining with differential interference contrast and fluorescent cell label imaging, effective compartmental localization of the nanoparticles can be achieved. The total number of cells taking up the nanoparticles and the amount of nanoparticle uptake increased significantly in proportion to the dose, with no adverse effects on cell survival. Moreover, significant reduction in reactive oxygen species was also observed in proportion to increasing nanoceria concentrations attesting to its ability to modulate oxidative stress. In conclusion, this work serves as a pre-clinical scientific evaluation of the effective use of reflectance structured illumination imaging of cerium oxide nanoparticles in breast cancer cells and the safe use of these nanoparticles in MDA MB231 cells for further therapeutic applications.     

Inhibition of Peroxynitrite-Induced Mitophagy Activation Attenuates Cerebral Ischemia-Reperfusion Injury

Activated autophagy/mitophagy has been intensively observed in ischemic brain, but its roles remain controversial. Peroxynitrite (ONOO^?), as a representative of reactive nitrogen species, is considered as a critical neurotoxic factor in mediating cerebral ischemia-reperfusion (I/R) injury, but its roles in autophagy/mitophagy activation remain unclear. Herein, we hypothesized that ONOO^? could induce PINK1/Parkin-mediated mitophagy activation via triggering dynamin-related protein 1 (Drp1) recruitment to damaged mitochondria, contributing to cerebral I/R injury. Firstly, we found PINK1/Parkin-mediated mitophagy activation was predominant among general autophagy, leading to rat brain injury at the reperfusion phase after cerebral ischemia. Subsequently, increased nitrotyrosine was found in the plasma of ischemic stroke patients and ischemia-reperfused rat brains, indicating the generation of ONOO^? in ischemic stroke. Moreover, in vivo animal experiments illustrated that ONOO^? was dramatically increased, accompanied with mitochondrial recruitment of Drp1, PINK1/Parkin-mediated mitophagy activation, and progressive infarct size in rat ischemic brains at the reperfusion phase. FeTMPyP, a peroxynitrite decomposition catalyst, remarkably reversed mitochondrial recruitment of Drp1, mitophagy activation, and brain injury. Intriguingly, further study revealed that ONOO^? induced tyrosine nitration of Drp1 peptide, which might contribute to mitochondrial recruitment of Drp1 for mitophagy activation. In vitro cell experiments yielded consistent results with in vivo animal experiments. Taken together, all above findings support the hypothesis that ONOO^?-induced mitophagy activation aggravates cerebral I/R injury via recruiting Drp1 to damaged mitochondria.     

Rare earth cerium oxide nanoparticles attenuated liver fibrosis in bile duct ligation mice model

Liver fibrosis is one of the major liver complications which eventually progresses to liver cirrhosis and liver failure. Cerium oxide nanoparticles, also known as nanoceria (NC) are nanoparticles with potential antioxidant and anti-inflammatory activities. Herein, we evaluated the hepatoprotective and anti-fibrotic effects of nanoceria (NC) against bile duct ligation (BDL) induced liver injury. NC were administered i.p. for 12 days (0.5 and 2 mg/kg) to C57BL/6J mice. The biochemical markers of liver injury, oxidative and nitrosative stress markers, inflammatory cytokines were evaluated. Fibrosis assessment and mechanistic studies were conducted to assess the hepatoprotective effects of NC. Administration of NC proved to significantly ameliorate liver injury as evident by reduction in SGOT, SGPT, ALP and bilirubin levels in the treated animals. NC treatment significantly reduced the hydroxyproline levels and expression of fibrotic markers. In summary, our findings establish the hepatoprotective and anti-fibrotic effects of NC against BDL induced liver injury and liver fibrosis. These protective effects were majorly ascribed to their potential ROS inhibition and antioxidant activities through catalase, superoxide dismutase (SOD)-mimetic properties and auto-regenerating capabilities.     

Catalytic Nanoceria Are Preferentially Retained in the Rat Retina and Are Not Cytotoxic after Intravitreal Injection

Cerium oxide nanoparticles (nanoceria) possess catalytic and regenerative radical scavenging activities. The ability of nanoceria to maintain cellular redox balance makes them ideal candidates for treatment of retinal diseases whose development is tightly associated with oxidative damage. We have demonstrated that our stable water-dispersed nanoceria delay photoreceptor cell degeneration in rodent models and prevent pathological retinal neovascularization in vldlr mutant mice. The objectives of the current study were to determine the temporal and spatial distributions of nanoceria after a single intravitreal injection, and to determine if nanoceria had any toxic effects in healthy rat retinas. Using inductively-coupled plasma mass spectrometry (ICP-MS), we discovered that nanoceria were rapidly taken up by the retina and were preferentially retained in this tissue even after 120 days. We also did not observe any acute or long-term negative effects of nanoceria on retinal function or cytoarchitecture even after this long-term exposure. Because nanoceria are effective at low dosages, nontoxic and are retained in the retina for extended periods, we conclude that nanoceria are promising ophthalmic therapeutics for treating retinal diseases known to involve oxidative stress in their pathogeneses.     

Effects of long term nitrite therapy on functional recovery in experimental ischemia model

Our data have shown that nitrite therapy can rescue the ischemic brain when injected <3 h after cerebral ischemic-reperfusion (I/R) injury and its effects can be prolonged to 4.5 h in combination with memantine. We investigated whether or not long-term nitrite therapy is beneficial in ischemic brains. Sodium nitrite (1-100 μg/kg ip) or saline were administered to rats subjected to focal I/R injury for 7 days beginning 24 h after I/R. Behavioral tests for 5 weeks revealed better functional recovery in the high-dose nitrite group than the control group. Other nitrite groups with relatively low doses showed no functional benefits. Hemispheric atrophy was attenuated by approximately 30% in the high-dose nitrite group. High-dose nitrite therapy also reduced inflammatory cytokine levels and caspase activity in the subacute period, and increased BrdU⁺MAP2⁺ and BrdU⁺laminin⁺ cells, and vascular density in the 5-week ischemic brain. Long-term nitrite therapy, when initiated 24 h after I/R, corrected the subacute hostile environment, induced tissue and vascular regeneration, and improved functional recovery. Early and subsequent long term nitrite therapy may be effective in the management for ischemic stroke patients.     

Nanoceria inhibit the development and promote the regression of pathologic retinal neovascularization in the Vldlr knockout mouse

Many neurodegenerative diseases are known to occur and progress because of oxidative stress, the presence of reactive oxygen species (ROS) in excess of the cellular defensive capabilities. Age related macular degeneration (AMD), diabetic retinopathy (DR) and inherited retinal degeneration share oxidative stress as a common node upstream of the blinding effects of these diseases. Knockout of the Vldlr gene results in a mouse that develops intraretinal and subretinal neovascular lesions within the first month of age and is an excellent model for a form of AMD called retinal angiomatous proliferation (RAP). Cerium oxide nanoparticles (nanoceria) catalytically scavenge ROS by mimicking the activities of superoxide dismutase and catalase. A single intravitreal injection of nanoceria into the Vldlr-/- eye was shown to inhibit: the rise in ROS in the Vldlr-/- retina, increases in vascular endothelial growth factor (VEGF) in the photoreceptor layer, and the formation of intraretinal and subretinal neovascular lesions. Of more therapeutic interest, injection of nanoceria into older mice (postnatal day 28) resulted in the regression of existing vascular lesions indicating that the pathologic neovessels require the continual production of excessive ROS. Our data demonstrate the unique ability of nanoceria to prevent downstream effects of oxidative stress in vivo and support their therapeutic potential for treatment of neurodegenerative diseases such as AMD and DR.     

Peroxynitrite Is a Key Mediator of the Cardioprotection Afforded by Ischemic Postconditioning In Vivo

Myocardial ischemic postconditioning (PosC) describes an acquired resistance to lethal ischemia-reperfusion (I/R) injury afforded by brief episodes of I/R applied immediately after the ischemic insult. Cardioprotection is conveyed by parallel signaling pathways converging to prevent mitochondria permeability transition. Recent observations indicated that PostC is associated with free radicals generation, including nitric oxide (NO^.) and superoxide (O₂ ^.-), and that cardioprotection is abrogated by antioxidants. Since NO. And O₂ ^{. -} react to form peroxynitrite, we hypothesized that postC might trigger the formation of peroxyntrite to promote cardioprotection in vivo. Rats were exposed to 45 min of myocardial ischemia followed by 3h reperfusion. PostC (3 cycles of 30 seconds ischemia/30 seconds reperfusion) was applied at the end of index ischemia. In a subgroup of rats, the peroxynitrite decomposition catalyst 5,10,15,20-tetrakis(4-sulphonatophenyl) porphyrinato iron (FeTPPS) was given intravenously (10 mg/kg^-1) 5 minutes before PostC. Myocardial nitrotyrosine was determined as an index of peroxynitrite formation. Infarct size (colorimetric technique and plasma creatine kinase-CK-levels) and left ventricle (LV) function (micro-tip pressure transducer), were determined. A significant generation of 3-nitrotyrosine was detected just after the PostC manoeuvre. PostC resulted in a marked reduction of infarct size, CK release and LV systolic dysfunction. Treatment with FeTPPS before PostC abrogated the beneficial effects of PostC on myocardial infarct size and LV function. Thus, peroxynitrite formed in the myocardium during PostC induces cardioprotective mechanisms improving both structural and functional integrity of the left ventricle exposed to ischemia and reperfusion in vivo.     

Role of Superoxide Dismutase in Ischemic Brain Injury: A Study Using SOD-1 Transgenic Mice

1. Nitric oxide radicals (NO) play an important role in the pathophysiology of focal cerebral ischemia.2. Vascular NO can reduce ischemic brain injury by increasing CBF, whereas neuronal NO may mediate neurotoxicity following brain ischemia, mainly by its reaction with superoxide to generate peroxynitrite.3. These findings could contribute to a strategy for the treatment of cerebral ischemia.     

Insulin renders diabetic rats resistant to acute ischemic stroke by arresting nitric oxide reaction with superoxide to form peroxynitrite

Background

The functions of free radicals on the effects of insulin that result in protection against cerebral ischemic insult in diabetes remain undefined. This present study aims to explain the contradiction among nitric oxide (NO)/superoxide/peroxynitrite of insulin in amelioration of focal cerebral ischemia–reperfusion (FC I/R) injury in streptozotocin (STZ)-diabetic rats and to delineate the underlying mechanisms. Long-Evans male rats were divided into three groups (age-matched controls, diabetic, and diabetic treated with insulin) with or without being subjected to FC I/R injury.

Results

Hyperglycemia exacerbated microvascular functions, increased cerebral NO production, and aggravated FC I/R-induced cerebral infarction and neurological deficits. Parallel with hypoglycemic effects, insulin improved microvascular functions and attenuated FC I/R injury in STZ-diabetic rats. Diabetes decreased the efficacy of NO and superoxide production, but NO and superoxide easily formed peroxynitrite in diabetic rats after FC I/R injury. Insulin treatment significantly rescued the phenomenon.

Conclusions

These results suggest that insulin renders diabetic rats resistant to acute ischemic stroke by arresting NO reaction with superoxide to form peroxynitrite.     

Caffeic acid phenethyl ester reduces neurovascular inflammation and protects rat brain following transient focal cerebral ischemia

Ischemic stroke is a neurovascular disease treatable by thrombolytic therapy, but the therapy has to be initiated within 3 h of the incident. This therapeutic limitation stems from the secondary injury which results mainly from oxidative stress and inflammation. A potent antioxidant/anti-inflammatory agent, caffeic acid phenethyl ester (CAPE) has potential to mitigate stroke's secondary injury, and thereby widening the therapeutic window. We observed that CAPE protected the brain in a dose-dependent manner (1-10 mg/kg body weight) and showed a wide therapeutic window (about 18 h) in a rat model of transient focal cerebral ischemia and reperfusion. The treatment also increased nitric oxide and glutathione levels, decreased lipid peroxidation and nitrotyrosine levels, and enhanced cerebral blood flow. CAPE down-regulated inflammation by blocking nuclear factor kappa B activity. The affected mediators included adhesion molecules (intercellular adhesion molecule-1 and E-selectin), cytokines (tumor necrosis factor-alpha and interleukin-1beta) and inducible nitric oxide synthase. Anti-inflammatory action of CAPE was further documented through reduction of ED1 (marker of activated macrophage/microglia) expression. The treatment inhibited apoptotic cell death by down-regulating caspase 3 and up-regulating anti-apoptotic protein Bcl-xL. Conclusively, CAPE is a promising drug candidate for ischemic stroke treatment due to its inhibition of oxidative stress and inflammation, and its clinically relevant wide therapeutic window.     

Role of different nitric oxide synthase isoforms in a murine model of acute lung injury and sepsis

Excessive production of nitric oxide (NO) by NO synthase (NOS) with subsequent formation of peroxynitrite and poly(adenosine diphosphate ribose) is critically implemented in the pathophysiology of acute lung injury and sepsis. To elucidate the roles of different isoforms of NOS, we tested the effects of non-selective NOS inhibition and neuronal NOS (nNOS)- and inducible NOS (iNOS)-gene deficiency on the pulmonary oxidative and nitrosative stress reaction in a murine sepsis model. The injury was induced by four sets of cotton smoke using an inhalation chamber and subsequent intranasal administration of live Pseudomonas aeruginosa (3.2 × 10⁷ colony-forming units). In wild type mice, the injury was associated with excessive releases of pro-inflammatory cytokines in the plasma, enhanced neutrophil accumulation, increased lipid peroxidation, and excessive formation of reactive nitrogen species and vascular endothelial growth factor in the lung. Both nNOS- and iNOS-gene deficiency led to significantly reduced oxidative and nitrosative stress markers in the lung, but failed to significantly improve survival. Treatment with a non-selective NOS inhibitor failed to reduce the oxidative and nitrosative stress reaction to the same extent and even tended to increase mortality. In conclusion, the current study demonstrates that both nNOS and iNOS are partially responsible for the pulmonary oxidative and nitrosative stress reaction in this model. Future studies should investigate the effects of specific pharmacological inhibition of nNOS and iNOS at different time points during the disease process.     

Edaravone attenuates cerebral ischemic injury by suppressing aquaporin-4

Aquaporin-4 (AQP4) plays a role in the generation of post-ischemic edema. Pharmacological modulation of AQP4 function may thus provide a novel therapeutic strategy for the treatment of stroke, tumor-associated edema, epilepsy, traumatic brain injury, and other disorders of the central nervous system (CNS) associated with altered brain water balance. Edaravone, a free radical scavenger, is used for the treatment of acute ischemic stroke (AIS) in Japan. In this study, edaravone significantly reduced the infarct area and improved the neurological deficit scores at 24 h after reperfusion in a rat transient focal ischemia model. Furthermore, edaravone markedly reduced AQP4 immunoreactivity and protein levels in the cerebral infarct area. In light of observations that edaravone specifically inhibited AQP4 in a rat transient focal ischemia model, we propose that edaravone might reduce cerebral edema through the inhibition of AQP4 expression following cerebral infarction.     

KatG from Salmonella Typhimurium is a peroxynitritase

Pathogenic bacteria elicit protective responses to oxidative and nitrosative stresses. Although such responses are generally distinct, it was recently reported in Mycobacterium tuberculosis that catalase-peroxidase (KatG), a classical defence against peroxides, also exhibits peroxynitritase activity. Here, the katG gene from Salmonella Typhimurium was cloned and protein purified and characterised. An increase in the rate of decomposition of peroxynitrite was observed for KatG from the enterobacterium with a second-order rate constant of 4.2 × 10⁴ M⁻¹ s⁻¹ at pH 7.4, 25 °C. This enzyme was able to reduce dihydrorhodamine oxidation by peroxynitrite to ∼83%. Given the peroxynitritase activity demonstrated here it is likely that KatG may play a wider role in the detoxification of oxidative stresses than previously thought.     

Kinetic and mechanistic considerations to assess the biological fate of peroxynitrite

Background

Peroxynitrite, the product of the reaction between superoxide radicals and nitric oxide, is an elusive oxidant with a short half-life and a low steady-state concentration in biological systems; it promotes nitroxidative damage.

Scope of review

We will consider kinetic and mechanistic aspects that allow rationalizing the biological fate of peroxynitrite from data obtained by a combination of methods that include fast kinetic techniques, electron paramagnetic resonance and kinetic simulations. In addition, we provide a quantitative analysis of peroxynitrite production rates and conceivable steady–state levels in living systems.

Major conclusions

The preferential reactions of peroxynitrite in vivo include those with carbon dioxide, thiols and metalloproteins; its homolysis represents only < 1% of its fate. To note, carbon dioxide accounts for a significant fraction of peroxynitrite consumption leading to the formation of strong one-electron oxidants, carbonate radicals and nitrogen dioxide. On the other hand, peroxynitrite is rapidly reduced by peroxiredoxins, which represent efficient thiol-based peroxynitrite detoxification systems. Glutathione, present at mM concentration in cells and frequently considered a direct scavenger of peroxynitrite, does not react sufficiently fast with it in vivo; glutathione mainly inhibits peroxynitrite-dependent processes by reactions with secondary radicals. The detection of protein 3-nitrotyrosine, a molecular footprint, can demonstrate peroxynitrite formation in vivo. Basal peroxynitrite formation rates in cells can be estimated in the order of 0.1 to 0.5 μM s^− 1 and its steady-state concentration at ~ 1 nM.

General significance

The analysis provides a handle to predict the preferential fate and steady-state levels of peroxynitrite in living systems. This is useful to understand pathophysiological aspects and pharmacological prospects connected to peroxynitrite. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Oxidizing substrate specificity of Mycobacterium tuberculosis alkyl hydroperoxide reductase E: kinetics and mechanisms of oxidation and overoxidation

Alkyl hydroperoxide reductase E (AhpE), a novel subgroup of the peroxiredoxin family, comprises Mycobacterium tuberculosis AhpE (MtAhpE) and AhpE-like proteins present in many bacteria and archaea, for which functional characterization is scarce. We previously reported that MtAhpE reacted ~ 10³ times faster with peroxynitrite than with hydrogen peroxide, but the molecular reasons for that remained unknown. Herein, we investigated the oxidizing substrate specificity and the oxidative inactivation of the enzyme. In most cases, both peroxidatic thiol oxidation and sulfenic acid overoxidation followed a trend in which those peroxides with the lower leaving-group pK_a reacted faster than others. These data are in agreement with the accepted mechanisms of thiol oxidation and support that overoxidation occurs through sulfenate anion reaction with the protonated peroxide. However, MtAhpE oxidation and overoxidation by fatty acid-derived hydroperoxides (~ 10⁸ and 10⁵ M^− 1 s^− 1, respectively, at pH 7.4 and 25 °C) were much faster than expected according to the Brønsted relationship with leaving-group pK_a. A stoichiometric reduction of the arachidonic acid hydroperoxide 15-HpETE to its corresponding alcohol was confirmed. Interactions of fatty acid hydroperoxides with a hydrophobic groove present on the reduced MtAhpE surface could be the basis of their surprisingly fast reactivity.