Increased Superoxide Generation Research Articles

Mechanisms of Primaquine Induced Hemolysis in a Novel Humanized Murine Model of Mediterranean G6PD Deficiency

Background: Approximately half a billion individuals are genetically deficient in glucose 6-phosphate dehydrogenase (G6PD) due to amino acid variants generally thought to decrease enzyme half-life. Decreased G6PD activity limits the antioxidant capacity of red blood cells (RBCs). As such, certain drugs that induce oxidative stress can cause life-threatening hemolysis in G6PD deficient (G6DPd) individuals. Standard regiments of the only approved drugs that can cure liver phase Plasmodium vivax ( p. vivax) (i.e. primaquine and tafenoquine) are contraindicated in G6PDd patients due to risks of hemolysis. Effective treatment of G6PDd individuals infected with p. vivax requires a prolonged course of primaquine (8 weeks), leading to non-adherence, and subsequent reduced efficacy and increased risk of transmission. Despite decades of research and large drug screening programs, no non-hemolytic compound has been identified that can eradicate p. vivax. Lack of progress in this area has likely been hampered by an incomplete mechanistic understanding of primaquine induced hemolysis, in part due to the lack of an animal model that recapitulates G6PD instability. Methods: Recombinant non-deficient human hG6PD(ND) or the deficient Mediterranean variant (hG6PD(MED)) were expressed, purified, and subjected to thermal proteome profiling in combination with TMT10-assisted quantitative cross-linking proteomics. Mice were generated in which murine genomic G6PD is replaced with either genomic human hG6PD(ND) or hG6PD(MED). Hemolysis in humans is caused by primaquine metabolites (e.g. primaquine-5,6-orthoquinone (5,6-POQ)) and not primaquine itself. As such, RBCs from both strains were exposed to 5,6-POQ and then analyzed by 1) high resolution metabolomics and proteomics, 2) electron paramagnetic resonance (EPR) to measure superoxide, 3) methemoglobin (MetHb) formation, 4) light and electron microscopy, 5) in vitro hemolysis, and 6) in vivo hemolysis (clearance) after infusion into animals. In vivo pulse chase biotinylation allowed visualization and identification of younger (1-6 days) RBCs vs. older (7-55 day) RBCs. Results: Recombinant hG6PD(MED) enzyme had a 2.6-fold lower specific activity compared to hG6PD(ND). Thermal gradient crosslinking proteomics identified specific amino acid spacing (less than 26 angstroms) that was higher in hG6PD(ND) [82-95 and 91-432] vs. higher in hG6PD(MED) [89-205, 97-429, and 429-497]. Analysis of mice demonstrated that hG6PD(MED) RBCs had 5% of G6PD activity as compared to hG6PD(ND) RBCs, with an RBC age dependent decrease in G6PD activity in hG6PD(MED) RBCs. Exposure to 5,6-POQ caused increased superoxide generation, increased MetHb formation, alterations to morphology, in vitro hemolysis, and in vivo hemolysis of hG6PD(MED) RBCs compared to hG6PD(ND) RBCs. However, younger hG6PD(MED) RBCs with higher G6PD activity were highly resistant to 5,6-POQ induced hemolysis (both in vivo and in vitro). All experiments were carried out a minimum of 3 times and all stated findings were statistically significant (p<0.05). Proteomic and metabolomic analysis (with combined samples from multiple experiments) demonstrated oxidation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), resulting in a block in glycolysis that increases dihydroxyacetone phosphate (DHAP) leading to cytotoxic methylglyoxal that broadly oxidizes protein lysines to carboxymethyllysine (CML) - the first step in advanced glycation end products (AGEs) known to facilitate RBC clearance. Discussion: We identify specific conformational changes caused by the hG6PD(MED) variant resulting in decreased specific activity and half-life, and confirm that G6PD activity decreases with RBC age, in a humanized mouse model. The resistance of young hG6PD(MED) RBCs to 5,6-POQ has immediate practical ramifications, in that it supports the rationale of clinical studies currently proposing new dose escalation primaquine regimens to induce low level early hemolysis, leading to reticulocytosis, and decreasing the mean age of circulating RBCs such that increased parricidal doses of primaquine can then be tolerated in G6PDd patients. The elucidation of the metabolic and proteomic lesion (under conditions confirmed to cause 5,6-POQ induced hemolysis of hG6PD(MED) but not hG6PD(ND) RBCs) provides a mechanistic landscape to guide development of non-hemolytic drugs to treat p. vivax.

Abstract 14877: Spike Protein of SARS-CoV-2 Virus Induces Mitochondrial Dysfunction in Swine Heart via Redox Impairment of Heme Proteins and Increasing Superoxide Generation

The cellular entry of the SARS-CoV-2 virus depends on the binding of spike (S) protein to its biological ligand, ACE2. Although the virus commonly causes respiratory distress, cardiac injury can occur in COVID-19 patients, which is consistent with the expression of ACE2 in myocytes. Previous reports indicate SARS-CoV-2 proteins can target cardiac mitochondria and suppress mitochondrial function via enhancing MPTP pore opening and perturbing cardiac bioenergetics. To explore the underlying mechanism of the effect of SARS-CoV-2 on cardiac mitochondria, we measured the interactions of SARS-CoV-2 proteins with swine heart mitochondria in vitro. Incubation of recombinant S protein with isolated mitochondria significantly decreased state-3 oxygen consumption rate (OCR, 95.10 vs 65.68 nmol/min/mg) and FCCP uncoupling OCR (82.94 vs 66.95 nmol/min/mg). We further detected that S protein impaired the enzymatic activities of electron transport chain (ETC) (by 15.62% to 34.44%). However, S protein had no effect on TCA cycle enzymes, indicating the involvement of mitochondrial membrane components in decreased OCR by S proteins. Recombinant nucleocapsid (N) protein of SAR-CoV-2 had no effect on the OCR and ETC activities of swine mitochondria. S proteins decreased the intensity of mitochondrial heme spectrum determined by dithionite reduction with UV/VIS spectroscopy (by 17.52% heme a , 15.82% heme c 1 ). The results were further assessed using isolated complex III (Cx3) and complex IV (Cx4). Treatment of isolated Cx3 and Cx4 with S protein decreased the spectral intensities of heme a and heme c 1 . The spectra of both Cx3 and Cx4 were not affected by N protein. The results suggested S protein downregulates redox potentials of ETC in swine mitochondria. Treatment of swine mitochondria with S proteins enhanced superoxide ( . O 2 – ) generation by Cx1 (by 43.7%) and by Cx3 (by 10.9-fold) assessed by EPR and cytochrome c reduction assays. However, we detected S proteins modestly decreased . O 2 – generation by swine mitochondria under state-2 condition (by 9.52%), indicating impairing pH gradient by S protein. In conclusion, the spike protein of SARS-CoV-2 virus mediates mitochondrial dysfunction of swine heart via impairing the redox function and increasing . O 2 – generation.

Diabetes mellitus and anti-oxidative stress

The development of diabetes complications, both microvascular and cardiovascular, is significantly influenced by oxidative stress. Diabetes-related metabolic problems lead to an overproduction of mitochondrial superoxide in the heart, small and large vascular endothelial cells, and both. Five key pathways implicated in the pathophysiology of problems are activated as a result of the increased superoxide generation. The purpose of this review is to highlight new insights into the processes through which hyperglycemia produces oxygen free radicals.

AGE RAGE Pathways: Cardiovascular Disease and Oxidative Stress.

It is well established that Advanced Glycation End Products (AGEs) and their receptor (RAGE) are primarily responsible for the development of cardiovascular disease. As a result, diabetic therapy is very interested in therapeutic strategies that can target the AGE-RAGE axis. The majority of the AGE-RAGE inhibitors showed encouraging outcomes in animal experiments, but more information is needed to completely understand their clinical effects. The main mechanism implicated in the aetiology of cardiovascular disease in people with diabetes is oxidative stress and inflammation mediated by AGE-RAGE interaction. Numerous PPAR-agonists have demonstrated favourable outcomes in the treatment of cardio-metabolic illness situations by inhibiting the AGE-RAGE axis. The body's ubiquitous phenomena of inflammation occur in reaction to environmental stressors such tissue damage, infection by pathogens, or exposure to toxic substances. Rubor (redness), calor (heat), tumour (swelling), colour (pain), and in severe cases, loss of function, are its cardinal symptoms. When exposed, the lungs develop silicotic granulomas with the synthesis of collagen and reticulin fibres. A natural flavonoid called chyrsin has been found to have PPAR-agonist activity as well as antioxidant and anti-inflammatory properties. The RPE insod2+/animals underwent mononuclear phagocyte-induced apoptosis, which was accompanied with decreased superoxide dismutase 2 (SOD2) and increased superoxide generation. Injections of the serine proteinase inhibitor SERPINA3K decreased proinflammatory factor expression in mice with oxygen-induced retinopathy, decreased ROS production, and increased levels of SOD and GSH.

Curcumin-ZnO nanocomposite mediated inhibition of Pseudomonas aeruginosa biofilm and its mechanism of action

Multidrug-resistance and strong biofilm formation potential of P. aeruginosa makes it a severe risk to public health and nanoparticles/nanocomposites find potential applicability to address this crisis. Curcumin is a potential anti-biofilm/antibacterial molecule that is limited due to large sized particle, reduced water solubility and decreased bioavailability. In this study, curcumin-ZnO nanocomposite was synthesized and its anti-biofilm efficacy as well as mechanism of action against P. aeruginosa biofilm was investigated. Here, using the FTIR peaks, the novel structure of curcumin-ZnO nanocomposites was established and using that as ligand the potential anti-biofilm drug targets were identified followed by superoxide anion, lipid peroxidation, cell viability and TEM analysis to reveal its mechanism of anti-biofilm action. Here, curcumin-ZnO nanocomposites at 300 μg/mL dosage having suitable particle size (110.51 nm), improved zeta potential (−22.3 mV), reduced hydrodynamic size (253.2 nm) were more efficiently able to penetrate through biofilm matrix and bind to P. aeruginosa's cell membrane through the OprM-MexAB receptor and inhibit its growth and biofilm (efficacy = 49.36% compared to control) than ZnO nanoparticle (42.38% compared to control). Curcumin-ZnO nanocomposites could efficiently inhibit P. aeruginosa biofilm by mechanisms involving increased superoxide anion generation by 48.94% possibly due to catalase and rubredoxin-rubredoxin reductase inhibition that mediated increased bacterial membrane lipid peroxidation by 183.75% thus causing severe disintegration of cell membrane and increased cell death by 25.18% compared to control. Curcumin-ZnO nanocomposites can thus be considered as a capable anti-biofilm drug candidate against P. aeruginosa infections.

Transplantation of Astrocytic Mitochondria Modulates Neuronal Antioxidant Defense and Neuroplasticity and Promotes Functional Recovery after Intracerebral Hemorrhage.

Astrocytes release functional mitochondria (Mt) that play regulatory and prosurvival functions on entering adjacent cells. We recently demonstrated that these released Mts could enter microglia to promote their reparative/prophagocytic phenotype that assists in hematoma cleanup and neurological recovery after intracerebral hemorrhage (ICH). However, the relevance of astrocytic Mt transfer into neurons in protecting brain after ICH is unclear. Here, we found that ICH causes a robust increase in superoxide generation and elevated oxidative damage that coincides with loss of the mitochondrial enzyme manganese superoxide dismutase (Mn-SOD). The damaging effect of ICH was reversed by intravenous transplantation of astrocytic Mt, which on entering the brain (and neurons), restored Mn-SOD levels and reduced neurological deficits in male mice subjected to ICH. Using an in vitro ICH-like injury model in cultured neurons, we established that astrocytic Mt on entering neurons prevented reactive oxygen species-induced oxidative stress and neuronal death by restoring neuronal Mn-SOD levels while at the same time promoted neurite extension and upregulation of synaptogenesis-related gene expression. Furthermore, we found that Mt genome-encoded small peptide humanin, which is normally abundant in Mt, could simulate Mt-transfer effect on neuronal Mn-SOD expression, oxidative stress, and neuroplasticity under ICH-like injury. This study demonstrates that adoptive astrocytic Mt transfer enhances neuronal Mn-SOD-mediated antioxidative defense and neuroplasticity in the brain, which potentiate functional recovery following ICH.SIGNIFICANCE STATEMENT Mitochondrial dysfunction and antioxidant defense play essential roles in brain damage after ICH. Astrocytes release functional Mt that enters adjacent cells to help brain homeostatic function. Here, we show that systemic transplantation of astrocytic Mt restores ICH-impaired neuronal antioxidative defense, enhances neurite outgrowth, and improves stroke recovery after ICH. Our study suggests that systemic transplantation of astrocytic Mt could be considered as a novel and potentially promising strategy for ICH treatment.

Alisporivir Normalizes Mitochondrial Function of Primary Mouse Lung Endothelial Cells Under Conditions of Hyperglycemia

Effect of alisporivir (a mitochondrial permeability transition pore inhibitor) on the development of mitochondrial dysfunction under hyperglycemic conditions in the primary culture of mouse lung endothelial cells was investigated in this work. We demonstrated that hyperglycemia (30 mM glucose for 24 h) leads to the decrease in viability of the pulmonary endotheliocytes, causes mitochondrial dysfunction manifested by the drop in membrane potential and increase in superoxide anion generation as well as facilitates opening of the mitochondrial permeability transition pore (MPT pore). Incubation of endothelial cells with 5 µM alisporivir under hyperglycemic conditions leads to the increase in cell viability, restoration of the membrane potential level and of the MPT pore opening activity to control values. Hyperglycemia causes increased mitophagy in the lung endothelial cells: we observed increase in the degree of colocalization of mitochondria and lysosomes and upregulation of the Parkin gene expression. Alisporivir restores these parameters back to the levels observed in the control cells. Hyperglycemia results in the increase in the expression of the Drp1 gene in endotheliocytes responsible for synthesis of the protein involved in the process of mitochondria fission. Alisporivir does not significantly alter expression of the genes. The paper discusses mechanisms of the effect of alisporivir on mitochondrial dysfunction in murine pulmonary endotheliocytes under conditions of hyperglycemia.Electronic supplementary materialThe online version contains supplementary material available at 10.1134/S0006297922070033.

Chronic Exposure to Electronic Cigarettes Induces Lung Oxidative Stress, Inflammation, Fibrosis, and Impaired DNA Repair

BackgroundElectronic cigarettes (e‐cig) have been marketed as a safer alternative to tobacco cigarette smoking (TCS). While there are far less toxic compounds in e‐cig vape (ECV) than in tobacco cigarette smoke, detectable levels of toxicants are still present. Multiple in vitro and short‐term in vivo studies on e‐cig‐induced lung cell damage have been reported; however, questions remain regarding the health effects of chronic e‐cig use on the lungs. Therefore, studies were performed in a chronic mouse exposure model to investigate the deleterious effects of long‐term ECV exposure on the lungs.MethodsC57/BL6 male mice were exposed for 2 hours/day, 5 days/week to either air (control group; 25 mice) or ECV generated from e‐cig liquid containing 24 mg/ml nicotine (e‐cig group; 25 mice) for 16 weeks. At the end of the exposure, animals were euthanized, lungs were lavaged, then the recovered bronchoalveolar lavage fluid (BALF) was prepared to assess the percentages of inflammatory cells in BALF. Fixed lung sections were also prepared and stained with hematoxylin and eosin (H & E) or Masson’s trichrome stain to assess the degree of lung inflammation and fibrosis; respectively. Superoxide radical generation was measured in frozen lung sections using DHE. Western blotting (WB) and immunofluorescence (IF) in lung tissue were used to detect the levels of nitrotyrosine, and the expression of the DNA repair enzymes XPC and OGG1.ResultsThe number of inflammatory cells present in the BALF, including neutrophils, macrophages, and eosinophils, was significantly higher in ECV‐exposed mice compared to that with air exposure. In the ECV‐exposed mice, H & E stained lung sections showed clear inflammatory infiltrates with lymphoid aggregates of lymphocytes, plasma cells and macrophages that stretch from the perivascular and peribronchiolar areas into adjacent septal adipose tissue. Masson’s trichrome stained slides showed increase in connective tissue around airways and bronchioles in e‐cig exposed mice, indicating the presence of pulmonary fibrosis. Using the fluorescence‐based superoxide probe DHE, significantly increased superoxide generation was observed in the lungs of ECV exposed mice, compared to control. This DHE‐derived fluorescence was largely quenched by preincubation with the SOD mimetic MnTBAP, confirming that the observed fluorescence was largely derived from superoxide. WB and IF showed elevated levels of nitrotyrosine. Reduced expression of the DNA damage repair proteins XPC and OGG1 were seen in lung tissue of the ECV exposed mice compared to the controls.ConclusionChronic ECV exposure induced lung inflammation, pulmonary fibrosis, and increased superoxide generation. ECV exposure increased oxidative stress and inflammation in the lungs while decreasing the levels of DNA damage repair proteins. These findings suggest that chronic e‐cig use can trigger oxidative stress with inflammatory lung damage and impaired DNA repair that together may predispose to neoplastic transformation and cancer onset.

Electronic Cigarette Exposure Induces Vascular Endothelial Dysfunction with NADPH Oxidase Activation and eNOS Uncoupling

BackgroundWe recently reported a mouse model of chronic electronic cigarette (e‐cig) exposure‐induced cardiovascular pathology, where long‐term exposure to e‐cig vape (ECV) induces cardiac abnormalities, impairment of endothelial function, and systemic hypertension. Using this model, we performed studies to delineate the underlying mechanisms of ECV‐induced vascular endothelial dysfunction (VED), which is a central trigger of cardiovascular disease.MethodsC57/BL6 male mice were exposed 2 hours/day, 5 days/week to ECV generated from e‐cig liquid containing 0, 6, or 24 mg/ml nicotine for 16 and 60 weeks. Blood pressure was measured by tail cuff and endothelial‐dependent and independent relaxation was measured on aortic segments using wire myography. Western blotting, immunofluorescence and ELISA were used to analyze the pathways involved in the mechanism of ECV‐induced VED.ResultsTime‐dependent elevation in blood pressure and systemic vascular resistance were observed, along with an impairment of acetylcholine‐induced aortic relaxation in ECV‐exposed mice, compared to air‐exposed control. Decreased intravascular nitric oxide (NO) levels and increased superoxide generation with elevated 3‐nitrotyrosine levels in the aorta of ECV‐exposed mice were observed, indicating that ECV‐induced superoxide reacts with NO to generate cytotoxic peroxynitrite. Exposure increased NADPH oxidase expression, supporting its role in ECV‐induced superoxide generation. Downregulation of endothelial nitric oxide synthase (eNOS) expression and Akt‐dependent eNOS phosphorylation occurred in the aorta of ECV‐exposed mice, indicating that exposure inhibited de novo NO synthesis. Following ECV exposure, the critical NOS cofactor tetrahydrobiopterin was decreased, with a concomitant loss of its salvage enzyme, dihydrofolate reductase. NADPH oxidase and NOS inhibitors abrogated ECV‐induced superoxide generation in the aorta of ECV exposed mice.ConclusionOur data demonstrate that ECV exposure activates NADPH oxidase and uncouples eNOS, causing a vicious cycle of superoxide generation and vascular oxidant stress that triggers VED and hypertension with predisposition to other cardiovascular disease.

Natriuretic Peptide Oligomers Cause Proarrhythmic Metabolic and Electrophysiological Effects in Atrial Myocytes.

With aging, the human atrium invariably develops amyloid composed of ANP (atrial natriuretic peptide) and BNP (B-type natriuretic peptide). Preamyloid oligomers are the primary cytotoxic species in amyloidosis, and they accumulate in the atrium during human hypertension and a murine hypertensive model of atrial fibrillation susceptibility. We tested the hypothesis that preamyloid oligomers derived from natriuretic peptides cause cytotoxic and electrophysiological effects in atrial cells that promote arrhythmia susceptibility and that oligomer formation is enhanced for a mutant form of ANP linked to familial atrial fibrillation. Oligomerization was assessed by Western blot analysis. Bioenergic profiling was performed using the Seahorse platform. Mitochondrial dynamics were investigated with immunostaining and gene expression quantitated using quantitative reverse transcription polymerase chain reaction. Action potentials and ionic currents were recorded using patch-clamp methods and intracellular calcium measured using Fura-2. Oligomer formation was markedly accelerated for mutant ANP (mutANP) compared with WT (wild type) ANP. Oligomers derived from ANP, BNP, and mutANP suppressed mitochondrial function in atrial HL-1 cardiomyocytes, associated with increased superoxide generation and reduced biogenesis, while monomers had no effects. In hypertensive mice, atrial cardiomyocytes displayed reduced action potential duration and maximal dV/dT of phase 0, with an elevated resting membrane potential, compared with normotensive mice. Similar changes were observed when atrial cells were exposed to oligomers. mutANP monomers produced similar electrophysiological effects as mutANP oligomers, likely due to accelerated oligomer formation, while ANP and BNP monomers did not. Oligomers decreased Na+ current, inward rectifier K+ current, and L-type Ca++ current, while increasing sustained and transient outward K+ currents, to account for these effects. These findings provide compelling evidence that natriuretic peptide oligomers are novel mediators of atrial arrhythmia susceptibility. Moreover, the accelerated oligomerization by mutANP supports a role for these mediators in the pathophysiology of this mutation in atrial fibrillation.

TREM-1 is required for enhanced OpZ-induced superoxide generation following priming.

Inflammatory agents, microbial products, or stromal factors pre-activate or prime neutrophils to respond to activating stimuli in a rapid and aggressive manner. Primed neutrophils exhibit enhanced chemotaxis, phagocytosis, and respiratory burst when stimulated by secondary activating stimuli. We previously reported that Triggering Receptor Expressed on Myeloid cells-1 (TREM-1) mediates neutrophil effector functions such as increased superoxide generation, transepithelial migration, and chemotaxis. However, it is unclear whether TREM-1 is required for the process of priming itself or for primed responses to subsequent stimulation. To investigate this, we utilized in vitro and in vivo differentiated neutrophils that were primed with TNF-α and then stimulated with the particulate agonist, opsonized zymosan (OpZ). Bone marrow progenitors isolated from WT and Trem-1-/- mice were transduced with estrogen regulated Homeobox8 (ER-Hoxb8) fusion transcription factor and differentiated in vitro into neutrophils following estrogen depletion. The resulting neutrophils expressed high levels of TREM-1 and resembled mature in vivo differentiated neutrophils. The effects of priming on phagocytosis and oxidative burst were determined. Phagocytosis did not require TREM-1 and was not altered by priming. In contrast, priming significantly enhanced OpZ-induced oxygen consumption and superoxide production in WT but not Trem-1-/- neutrophils indicating that TREM-1 is required for primed oxidative burst. TREM-1-dependent effects were not mediated during the process of priming itself as priming enhanced degranulation, ICAM-1 shedding, and IL-1ß release to the same extent in WT and Trem-1-/- neutrophils. Thus, TREM-1 plays a critical role in primed phagocytic respiratory burst and mediates its effects following priming.

Albumin used in human IVF contain different levels of lipids and modify embryo and fetal growth in a mouse model.

Different commercial human embryo culture mediums can alter embryo quality and change birthweight. One component that could be contributing to variations but is not widely investigated is human serum albumin (HSA). HSA plays a multitude of roles during embryo culture and is a carrier for molecules including lipids. It remains unclear if lipid composition of HSA varies among commercial products and its effects on embryo quality, implantation, and fetal outcomes are relatively unknown. Utilizing a mouse model of embryo culture, we cultured zygotes until the blastocyst stage (72-h culture) in G1/G2 containing either Vitrolife HSA, Sage HSA, or Recombinant HSA at 10%. Blastocyst quality (development, total cell number, superoxide generation), blastocyst lipid content (neutral lipids, non-esterified fatty acids, phospholipids, and triglycerides), implantation, and fetal lengths and weights were assessed. Fatty acid quantification of HSA source was assessed by standard thin-layer chromatography. Sage HSA had the greatest fatty acid composition, with an eightfold increase in saturated fatty acids. This coincided with reduced blastocyst development, increased superoxide generation, neutral lipids and triglycerides levels of blastocysts, and decreased implantation rates (p < 0.05). Unexpectedly, while Recombinant HSA had the lowest overall lipids it had 70-fold increase in palmitoleic acid and the lowest fetal weights (p < 0.05). Indicates the importance of a balance between different types/amount of lipids, and an "optimal ratio" required for embryo and fetal development. Therefore, the lipid content of HSA should be considered when choosing a suitable HSA source for use in clinical IVF.

Lutein attenuates angiotensin II- induced cardiac remodeling by inhibiting AP-1/IL-11 signaling

RationaleOxidative stress plays a critical role in the development of cardiac remodeling and heart failure. Lutein, the predominant nonvitamin A carotenoid, has been shown to have profound effects on oxidative stress. However, the effect of lutein on angiotensin II (Ang II)-induced cardiac remodeling and heart failure remains unknown. ObjectiveThe aim of this study was to determine whether lutein is involved in cardiac remodeling and to elucidate the underlying molecular mechanisms. Methods and resultsIn vitro experiments with isolated neonatal rat cardiomyocytes (NRCMs) and cardiac fibroblasts (CFs) revealed that lutein significantly attenuated Ang II-induced collagen expression in CFs, and cardiomyocyte hypertrophy. The Ang II-induced increases in superoxide generation, inflammation and apoptosis in cultured CFs were strikingly prevented by lutein. In vivo, fibrosis, hypertrophic cardiomyocyte and superoxide generation were analyzed, and lutein was demonstrated to confer resistance to Ang II-induced cardiac remodeling in mice. Mechanistically, RNA sequencing revealed that interleukin-11 (IL-11) expression was significantly upregulated in mouse hearts in response to Ang II infusion and was significantly suppressed in the hearts of lutein-treated mice. Furthermore, IL-11 overexpression blocked the effects of lutein on fibrosis and oxidative stress in CFs and impaired the protective effect of lutein on cardiac remodeling. Notably, we discovered that lutein could reduce Ang II-induced IL-11 expression, at least partly through the regulation of activator protein (AP)-1 expression and activity. ConclusionsLutein has potential as a treatment for cardiac remodeling and heart failure via the suppression of IL-11 expression.

FXYD1 Is Protective Against Vascular Dysfunction.

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Neuroprotective Effects of Salicin in a Gerbil Model of Transient Forebrain Ischemia by Attenuating Oxidative Stress and Activating PI3K/Akt/GSK3β Pathway.

Salicin is a major natural compound of willow bark and displays diverse beneficial biological properties, such as antioxidant activity. However, little information available for the neuroprotective potential of salicin against ischemic brain injury has been reported. Thus, this study was performed to investigate the neuroprotective potential of salicin against ischemia and reperfusion (IR) injury and its mechanisms in the hippocampus using a gerbil model of 5-min transient ischemia (TI) in the forebrain, in which a massive loss (death) of pyramidal neurons cells occurred in the subfield Cornu Ammonis 1 (CA1) among the hippocampal subregions (CA1-3) at 5 days after TI. To examine neuroprotection by salicin, gerbils were pretreated with salicin alone or together with LY294002, which is a phosphatidylinositol 3-kinase (PI3K) inhibitor, once daily for 3 days before TI. Treatment with 20 mg/kg of salicin significantly protected CA1 pyramidal neurons against the ischemic injury. Treatment with 20 mg/kg of salicin significantly reduced the TI-induced increase in superoxide anion generation and lipid peroxidation in the CA1 pyramidal neurons after TI. The treatment also reinstated the TI-induced decrease in superoxide dismutases (SOD1 and SOD2), catalase, and glutathione peroxidase in the CA1 pyramidal cells after TI. Moreover, salicin treatment significantly elevated the levels of phosphorylation of Akt and glycogen synthase kinase-3β (GSK3β), which is a major downstream target of PI3K, in the ischemic CA1. Notably, the neuroprotective effect of salicin was abolished by LY294002. Taken together, these findings clearly indicate that salicin protects against ischemic brain injury by attenuating oxidative stress and activating the PI3K/Akt/GSK3β pathway.

Heat shock protein 90α increases superoxide generation from neuronal nitric oxide synthases

Neuronal nitric oxide synthase (nNOS) generates superoxide, particularly at sub-optimal l-arginine (l-Arg) substrate concentrations. Heat shock protein 90 (Hsp90) was reported to inhibit superoxide generation from nNOS protein. However, commercially available Hsp90 product from bovine brain tissues with unspecified Hsp90α and Hsp90β contents and an undefined Hsp90 protein oligomeric state was utilized. These two Hsp90s can have opposite effect on superoxide production by NOS. Importantly, emerging evidence indicates that nNOS splice variants are involved in different biological functions by functioning distinctly in redox signaling. In the present work, purified recombinant human Hsp90α, in its native dimeric state, was used in electron paramagnetic resonance (EPR) spin trapping experiments to study the effects of Hsp90α on superoxide generation from nNOS splice variants nNOSμ and nNOSα. Human Hsp90α was found to significantly increase superoxide generation from nNOSμ and nNOSα proteins under l-Arg-depleted conditions and Hsp90α influenced superoxide production by nNOSμ and nNOSα at varying degrees. Imidazole suppressed the spin adduct signal, indicating that superoxide was produced at the heme site of nNOS in the presence of Hsp90α, whereas l-Arg repletion diminished superoxide production by the nNOS-Hsp90α. Moreover, NADPH consumption rate values exhibited a similar trend/difference as a function of Hsp90α and l-Arg. Together, these EPR spin trapping and NADPH oxidation kinetics results demonstrated noticeable Hsp90α-induced increases in superoxide production by nNOS and a distinguishable effect of Hsp90α on nNOSμ and nNOSα proteins.

Coordination between Calcium/Calmodulin-Dependent Protein Kinase II and Neuronal Nitric Oxide Synthase in Neurons.

Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) is highly abundant in the brain and exhibits broad substrate specificity, thereby it is thought to participate in the regulation of neuronal death and survival. Nitric oxide (NO), produced by neuronal NO synthase (nNOS), is an important neurotransmitter and plays a role in neuronal activity including learning and memory processes. However, high levels of NO can contribute to excitotoxicity following a stroke and neurodegenerative disease. Aside from NO, nNOS also generates superoxide which is involved in both cell injury and signaling. CaMKII is known to activate and translocate from the cytoplasm to the post-synaptic density in response to neuronal activation where nNOS is predominantly located. Phosphorylation of nNOS at Ser847 by CaMKII decreases NO generation and increases superoxide generation. Conversely, NO-induced S-nitrosylation of CaMKII at Cys6 is a prominent determinant of the CaMKII inhibition in ATP competitive fashion. Thus, the “cross-talk” between CaMKII and NO/superoxide may represent important signal transduction pathways in brain. In this review, we introduce the molecular mechanism of and pathophysiological role of mutual regulation between CaMKII and nNOS in neurons.

Divergent effects of genetic and pharmacological inhibition of Nox2 NADPH oxidase on insulin resistance-related vascular damage

Insulin resistance leads to excessive endothelial cell (EC) superoxide generation and accelerated atherosclerosis. The principal source of superoxide from the insulin-resistant endothelium is the Nox2 isoform of NADPH oxidase. Here we examine the therapeutic potential of Nox2 inhibition on superoxide generation in saphenous vein ECs (SVECs) from patients with advanced atherosclerosis and type 2 diabetes and on vascular function, vascular damage, and lipid deposition in apolipoprotein E-deficient (ApoE−/−) mice with EC-specific insulin resistance (ESMIRO). To examine the effect of genetic inhibition of Nox2, ESMIRO mice deficient in ApoE−/− and Nox2 (ESMIRO/ApoE−/−/Nox2−/y) were generated and compared with ESMIRO/ApoE−/−/Nox2+/y littermates. To examine the effect of pharmacological inhibition of Nox2, we administered gp91dstat or scrambled peptide to ESMIRO/ApoE−/− mice. SVECs from diabetic patients had increased expression of Nox2 protein with concomitant increase in superoxide generation, which could be reduced by the Nox2 inhibitor gp91dstat. After 12 wk Western diet, ESMIRO/ApoE−/−/Nox2−/y mice had reduced EC superoxide generation and greater aortic relaxation to acetylcholine. ESMIRO/ApoE−/−/Nox2−/y mice developed more lipid deposition in the thoraco-abdominal aorta with multiple foci of elastin fragmentation at the level of the aortic sinus and greater expression of intercellular adhesion molecule-1 (ICAM-1). Gp91dstat reduced EC superoxide and lipid deposition in the thoraco-abdominal aorta of ESMIRO/ApoE−/− mice without causing elastin fragmentation or increased ICAM-1 expression. These results demonstrate that insulin resistance is characterized by increased Nox2-derived vascular superoxide. Complete deletion of Nox2 in mice with EC insulin resistance exacerbates, whereas partial pharmacological Nox2 inhibition protects against, insulin resistance-induced vascular damage.

Local endothelial DNA repair deficiency causes aging-resembling endothelial-specific dysfunction.

We previously identified genomic instability as a causative factor for vascular aging. In the present study, we determined which vascular aging outcomes are due to local endothelial DNA damage, which was accomplished by genetic removal of ERCC1 (excision repair cross-complementation group 1) DNA repair in mice (EC-knockout (EC-KO) mice). EC-KO showed a progressive decrease in microvascular dilation of the skin, increased microvascular leakage in the kidney, decreased lung perfusion, and increased aortic stiffness compared with wild-type (WT). EC-KO showed expression of DNA damage and potential senescence marker p21 exclusively in the endothelium, as demonstrated in aorta. Also the kidney showed p21-positive cells. Vasodilator responses measured in organ baths were decreased in aorta, iliac and coronary artery EC-KO compared with WT, of which coronary artery was the earliest to be affected. Nitric oxide-mediated endothelium-dependent vasodilation was abolished in aorta and coronary artery, whereas endothelium-derived hyperpolarization and responses to exogenous nitric oxide (NO) were intact. EC-KO showed increased superoxide production compared with WT, as measured in lung tissue, rich in endothelial cells (ECs). Arterial systolic blood pressure (BP) was increased at 3 months, but normal at 5 months, at which age cardiac output (CO) was decreased. Since no further signs of cardiac dysfunction were detected, this decrease might be an adaptation to prevent an increase in BP. In summary, a selective DNA repair defect in the endothelium produces features of age-related endothelial dysfunction, largely attributed to loss of endothelium-derived NO. Increased superoxide generation might contribute to the observed changes affecting end organ perfusion, as demonstrated in kidney and lung.

The inhibitor-evoked shortage of tocopherol and plastoquinol is compensated by other antioxidant mechanisms in Chlamydomonas reinhardtii exposed to toxic concentrations of cadmium and chromium ions.

One of the major mechanisms of heavy metal toxicity is the induction of oxidative stress. Redox-active heavy metals, like chromium, can induce it directly, whereas redox-inactive metals, like cadmium, play an indirect role in the generation of reactive oxygen species (ROS). Living organisms defend themselves against oxidative stress taking advantage of low-molecular-weight antioxidants and ROS-detoxifying enzymes. Tocopherols and plastoquinol are important plastid prenyllipid antioxidants, playing a role during acclimation of Chlamydomonas reinhardtii to heavy metal-induced stress. However, partial inhibition of synthesis of these prenyllipids by pyrazolate did not decrease the tolerance of C. reinhardtii to Cr- and Cd-induced stress, suggesting redundancy between antioxidant mechanisms. To verify this hypothesis we have performed comparative analyses of growth, photosynthetic pigments, low-molecular-weight antioxidants (tocopherols, plastoquinol, plastochromanol, ascorbate, soluble thiols, proline), activities of the ascorbate peroxidase (APX), catalase and superoxide dismutase (SOD) and cumulative superoxide production in C. reinhardtii exposed to Cd2+ and Cr2O72- ions in the presence or absence of pyrazolate. The decreased α-tocopherol and plastoquinol content resulted in the increase in superoxide generation and APX activity in pyrazolate-treated algae. The application of heavy metal ions and pyrazolate had a pronounced impact on Asc and total thiol content, as well as SOD and APX activities (the latter only in Cd-exposed cultures), when compared with algae grown in the presence of heavy metal ions or pyrazolate alone. The superoxide production in cultures exposed to heavy metal ions and pyrazolate decreased when compared to the cultures exposed to either heavy metal ions or an inhibitor alone.