485 results on '"Zweier JL"'
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2. Cardiac myocyte-specific expression of inducible nitric oxide synthase protects against ischemia/reperfusion injury by preventing mitochondrial permeability transition.
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West MB, Rokosh G, Obal D, Velayutham M, Xuan YT, Hill BG, Keith RJ, Schrader J, Guo Y, Conklin DJ, Prabhu SD, Zweier JL, Bolli R, Bhatnagar A, West, Matthew B, Rokosh, Gregg, Obal, Detlef, Velayutham, Murugesan, Xuan, Yu-Ting, and Hill, Bradford G
- Published
- 2008
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3. Endothelium-derived nitric oxide regulates postischemic myocardial oxygenation and oxygen consumption by modulation of mitochondrial electron transport.
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Zhao X, He G, Chen Y, Pandian RP, Kuppusamy P, Zweier JL, Zhao, Xue, He, Guanglong, Chen, Yeong-Renn, Pandian, Ramasamy P, Kuppusamy, Periannan, and Zweier, Jay L
- Published
- 2005
4. Mechanisms of free radical production in the vascular wall.
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Souza HP, Cardounel AJ, Zweier JL, Souza, Heraldo P, Cardounel, Arturo J, and Zweier, Jay L
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- 2003
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5. Targeting dimethylarginine dimethylaminohydrolases in pulmonary arterial hypertension: a new approach to improve vascular dysfunction?
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Zweier JL, Talukder MA, Zweier, Jay L, and Talukder, M A Hassan
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- 2011
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6. Evidence that mitochondrial respiration Is a source of potentially toxic oxygen free radicals in intact rabbit hearts subjected to ischemia and reflow
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Ambrosio, G., Zweier, J. L., Duilio, C., Kuppusamy, P., Santoro, G., Elia, P. P., Tritto, I., Plinio CIRILLO, Condorelli, M., Chiariello, M., Flaherty, J. T., Ambrosio, G, Zweier, Jl, Duilio, C, Kuppusamy, P, Santoro, G, Elia, Pp, Tritto, I, Cirillo, Plinio, Condorelli, Mario, Chiariello, Massimo, and Flaherty, J. T.
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medicine.medical_specialty ,Magnetic Resonance Spectroscopy ,Free Radicals ,Cellular respiration ,Ischemia ,Myocardial Ischemia ,chemistry.chemical_element ,Myocardial Reperfusion ,ischemia ,Mitochondrion ,Biochemistry ,Oxygen ,Mitochondria, Heart ,Phosphocreatine ,Lipid peroxidation ,chemistry.chemical_compound ,Adenosine Triphosphate ,Oxygen Consumption ,Internal medicine ,medicine ,Cytochrome c oxidase ,Animals ,Potassium Cyanide ,Molecular Biology ,Heart metabolism ,free radical ,biology ,Chemistry ,Electron Spin Resonance Spectroscopy ,Cell Biology ,medicine.disease ,reperfusion ,Endocrinology ,biology.protein ,Amobarbital ,Female ,Lipid Peroxidation ,Rabbits - Abstract
Previous in vitro studies have shown that isolated mitochondria can generate oxygen radicals. However, whether a similar phenomenon can also occur in intact organs is unknown. In the present studwy,e tested the hypothesis that resumption of mitochondrial respiration upon reperfusion might be a mechanism of oxygen radical formation in postischemic hearts, and that treatment with inhibitorosf mitochondrial respiration might prevent this phenomenon. Three groups of Langendorff- perfused rabbit hearts were subjected to 30 min of global ischemia at 37 “C, followed by reflow. Throughout ischemia and early reperfusion the hearts received, respectively: (a) 6 mM KC1 (controls), (b) 6 mM sodium amobarbital (Amytal“, which blocks mitochondrial respiration at Site I, at the level of NADH dehydrogenase), and (c) 6 mM potassium cyanide (to block mitochondrial respiration distallya, t the level of cytochrome c oxidase). The hearts were thperonc essed to directly evaluatoex ygen radical generationb y electron paramagnetic resonances pectroscopy, or to measure oxygen radical-induced membrane lipid peroxidation by malonyl dialdehyde (MDA) content of subcellular fractions. Severity of ischemia, as assessed by “P-nuclear magnetic resonance measurements of cardiac ATP, phosphocreatine, and pH, was similar in all groups. Oxygen-centered free radical concentration averaged 3.84 f 0.64 PM in reperfused control hearts, and it was significantly reduced by Amytal treatment (1.98 2 0.26; p < 0.06), but not by KCN (2.68 f 0.96 PM; p = not significant (NS)), consistent with oxygen radicals being formed in them itochondrial respiratory chain at Site I. Membrane lipid peroxidation of reperfused hearts was also reduced by treatment with Amytal, but not with KCN. MDA content of the mitochondrial fraction averaged 0.76 f 0.06 nM/mg protein in controls, 0.72 f 0.06 in KCN-treated hearts, and0 .64groups). Similarly, MDA content of lysosomal membrane fraction was0 .64 f 0.09 nM/mg protein in controls, 0.79 C 0.16 in KCN-treated hearts, and 0.43 2 0.06 in Amytal-treated hearts ( p 0.06 versus both groups). Since the effects of Amytal are known to be reversible, in a second series of experiments we investigated whether transient mitochondrial inhibition during the initial1 0 min of reperfusion wasa lso associated with beneficial effects on subsequent recovery of cardiac function after wash-out of the drug. At the end of the experiment, recoveroyf left ventriculaer nddiastolic and of developed pressure was significantly greater in those hearts that had been treated with Amytal during ischemia and earlyre flow, as compared to untreated hearts. In conclusion, our data demonstrate that in intact hearts electron flow through the respiratory chain may be an important source of oxygen radicals, which may form at the sites of interactions between Fe-S clusters and ubiquinone, and that resumption of mitochondrial respiration upon reoxygenation might contribute to reperfusion injury.
7. Tobacco cigarette smoking induces cerebrovascular dysfunction followed by oxidative neuronal injury with the onset of cognitive impairment.
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Ewees MG, El-Mahdy MA, Hannawi Y, and Zweier JL
- Abstract
While chronic smoking triggers cardiovascular disease, controversy remains regarding its effects on the brain and cognition. We investigated the effects of long-term cigarette smoke (CS) exposure (CSE) on cerebrovascular function, neuronal injury, and cognition in a novel mouse exposure model. Longitudinal studies were performed in CS or air-exposed mice, 2 hours/day, for up to 60 weeks. Hypertension and carotid vascular endothelial dysfunction (VED) occurred by 16 weeks of CSE, followed by reduced carotid artery blood flow, with oxidative stress detected in the carotid artery, and subsequently in the brain of CS-exposed mice with generation of reactive oxygen species (ROS) and secondary protein and DNA oxidation, microglial activation and astrocytosis. Brain small vessels exhibited decreased levels of endothelial NO synthase (eNOS), enlarged perivascular spaces with blood brain barrier (BBB) leak and decreased levels of tight-junction proteins. In the brain, amyloid-β deposition and phosphorylated-tau were detected with increases out to 60 weeks, at which time mice exhibited impaired spatial learning and memory. Thus, long-term CSE initiates a cascade of ROS generation and oxidative damage, eNOS dysfunction with cerebral hypoperfusion, as well as cerebrovascular and BBB damage with intracerebral inflammation, and neuronal degeneration, followed by the onset of impaired cognition and memory., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
- Published
- 2024
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8. Nicotine inhalation and metabolism triggers AOX-mediated superoxide generation with oxidative lung injury.
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Zweier JL, Kundu T, Eid MS, Hemann C, Leimkühler S, and El-Mahdy MA
- Abstract
With the increasing use of vaping devices that deliver high levels of nicotine (NIC) to the lungs, sporadic lung injury has been observed. Commercial vaping solutions can contain high NIC concentrations of 150 mM or more. With high NIC levels, its metabolic products may induce toxicity. NIC is primarily metabolized to form NIC iminium (NICI) which is further metabolized by aldehyde oxidase (AOX) to cotinine. We determine that NICI in the presence of AOX is a potent trigger of superoxide generation. NICI stimulated superoxide generation from AOX with K
m = 2.7 μM and Vmax = 794 nmol/min/mg measured by cytochrome-c reduction. EPR spin-trapping confirmed that NICI in the presence of AOX is a potent source of superoxide. AOX is expressed in the lungs and chronic e-cigarette exposure in mice greatly increased AOX expression. NICI or NIC stimulated superoxide production in the lungs of control mice with an even greater increase after chronic e-cigarette exposure. This superoxide production was quenched by AOX inhibition. Furthermore, e-cigarette-mediated NIC delivery triggered oxidative lung damage that was blocked by AOX inhibition. Thus, NIC metabolism triggers AOX-mediated superoxide generation that can cause lung injury. Therefore, high uncontrolled levels of NIC inhalation, as occur with e-cigarette use, can induce oxidative lung damage., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2024
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9. Improving combination therapies: targeting A2B-adenosine receptor to modulate metabolic tumor microenvironment and immunosuppression.
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Evans JV, Suman S, Goruganthu MUL, Tchekneva EE, Guan S, Arasada RR, Antonucci A, Piao L, Ilgisonis I, Bobko AA, Driesschaert B, Uzhachenko RV, Hoyd R, Samouilov A, Amann J, Wu R, Wei L, Pallerla A, Ryzhov SV, Feoktistov I, Park KP, Kikuchi T, Castro J, Ivanova AV, Kanagasabai T, Owen DH, Spakowicz DJ, Zweier JL, Carbone DP, Novitskiy SV, Khramtsov VV, Shanker A, and Dikov MM
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- Humans, Animals, Mice, Receptor, Adenosine A2B metabolism, Tumor Microenvironment, Immunosuppression Therapy, Adenosine metabolism, Phosphates, Cell Line, Tumor, Carcinoma, Non-Small-Cell Lung drug therapy, Lung Neoplasms drug therapy, Lung Neoplasms pathology
- Abstract
Background: We investigated the role of A2B-adenosine receptor in regulating immunosuppressive metabolic stress in the tumor microenvironment. Novel A2B-adenosine receptor antagonist PBF-1129 was tested for antitumor activity in mice and evaluated for safety and immunologic efficacy in a phase I clinical trial of patients with non-small cell lung cancer., Methods: The antitumor efficacy of A2B-adenosine receptor antagonists and their impact on the metabolic and immune tumor microenvironment were evaluated in lung, melanoma, colon, breast, and epidermal growth factor receptor-inducible transgenic cancer models. Employing electron paramagnetic resonance, we assessed changes in tumor microenvironment metabolic parameters, including pO2, pH, and inorganic phosphate, during tumor growth and evaluated the immunologic effects of PBF-1129, including its pharmacokinetics, safety, and toxicity, in patients with non-small cell lung cancer., Results: Levels of metabolic stress correlated with tumor growth, metastasis, and immunosuppression. Tumor interstitial inorganic phosphate emerged as a correlative and cumulative measure of tumor microenvironment stress and immunosuppression. A2B-adenosine receptor inhibition alleviated metabolic stress, downregulated expression of adenosine-generating ectonucleotidases, increased expression of adenosine deaminase, decreased tumor growth and metastasis, increased interferon γ production, and enhanced the efficacy of antitumor therapies following combination regimens in animal models (anti-programmed cell death 1 protein vs anti-programmed cell death 1 protein plus PBF-1129 treatment hazard ratio = 11.74 [95% confidence interval = 3.35 to 41.13], n = 10, P < .001, 2-sided F test). In patients with non-small cell lung cancer, PBF-1129 was well tolerated, with no dose-limiting toxicities; demonstrated pharmacologic efficacy; modulated the adenosine generation system; and improved antitumor immunity., Conclusions: Data identify A2B-adenosine receptor as a valuable therapeutic target to modify metabolic and immune tumor microenvironment to reduce immunosuppression, enhance the efficacy of immunotherapies, and support clinical application of PBF-1129 in combination therapies., (© The Author(s) 2023. Published by Oxford University Press.)
- Published
- 2023
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10. Bacterial Pyocyanin Inducible Keratin 6A Accelerates Closure of Epithelial Defect under Conditions of Mitochondrial Dysfunction.
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Ghatak S, Hemann C, Boslett J, Singh K, Sharma A, El Masry MS, Abouhashem AS, Ghosh N, Mathew-Steiner SS, Roy S, Zweier JL, and Sen CK
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- Humans, Skin metabolism, Mitochondria metabolism, Pyocyanine chemistry, Pyocyanine metabolism, Keratin-6 metabolism
- Abstract
Repair of epithelial defect is complicated by infection and related metabolites. Pyocyanin (PYO) is one such metabolite that is secreted during Pseudomonas aeruginosa infection. Keratinocyte (KC) migration is required for the closure of skin epithelial defects. This work sought to understand PYO-KC interaction and its significance in tissue repair. Stable Isotope Labeling by Amino acids in Cell culture proteomics identified mitochondrial dysfunction as the top pathway responsive to PYO exposure in human KCs. Consistently, functional studies showed mitochondrial stress, depletion of reducing equivalents, and adenosine triphosphate. Strikingly, despite all stated earlier, PYO markedly accelerated KC migration. Investigation of underlying mechanisms revealed, to our knowledge, a previously unreported function of keratin 6A in KCs. Keratin 6A was PYO inducible and accelerated closure of epithelial defect. Acceleration of closure was associated with poor quality healing, including compromised expression of apical junction proteins. This work recognizes keratin 6A for its role in enhancing KC migration under conditions of threat posed by PYO. Qualitatively deficient junctional proteins under conditions of defensive acceleration of KC migration explain why an infected wound close with deficient skin barrier function as previously reported., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
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11. Trityl Quinodimethane Derivatives as Unimolecular Triple-Function Extracellular EPR Probes for Redox, pH, and Oxygen.
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Feng Y, Tan X, Shi Z, Villamena FA, Zweier JL, Song Y, and Liu Y
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- Electron Spin Resonance Spectroscopy methods, Oxidation-Reduction, Hydrogen-Ion Concentration, Oxygen chemistry, Reducing Agents
- Abstract
Electron paramagnetic resonance (EPR) spectroscopy and imaging coupled with the use of suitable probes is a promising tool for assessment of the tumor microenvironment (TME). Measurement of multiple TME parameters by EPR is very desirable but challenging. Herein, we designed and synthesized a class of negative-charged trityl quinodimethane MTPs as unimolecular triple-function extracellular probes for redox, pH, and oxygen (O
2 ) levels. Using the deuterated analogue, dMTP5, which has an optimal p Ka as well as high sensitivity to bioreduction and O2 , we reasonably evaluated pH effects on efflux of reducing agents from HepG2 cells and cellular O2 consumption.- Published
- 2023
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12. Characterizing CD38 Expression and Enzymatic Activity in the Brain of Spontaneously Hypertensive Stroke-Prone Rats.
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Hannawi Y, Ewees MG, Moore JT, and Zweier JL
- Abstract
Background: CD38 is a transmembrane glycoprotein that catabolizes nicotinamide adenine dinucleotide (NAD
+ ) and is the main source for the age-dependent decrease in NAD+ levels. Increased CD38 enzymatic activity has been implicated in several neurological diseases. However, its role in the pathogenesis of cerebral small vessel disease (CSVD) remains unknown. We aimed to characterize CD38 expression and enzymatic activity in the brain of spontaneously hypertensive stroke-prone rats (SHRSP), a genetic model for hypertension and human CSVD, in comparison to age-matched normotensive Wistar Kyoto rats (WKY). Materials and Methods: Age-matched male 7- and 24-week-old WKY and SHRSP were studied. CD38 enzymatic activity was determined in the brain homogenate. Immunohistochemistry and Western Blotting (WB) were used to characterize CD38 expression and localize it in the different cell types within the brain. In addition, expression of nitric oxide synthase (NOS) isoforms and the levels of nitric oxide (NO), superoxide, nicotinamide dinucleotide (phosphate) NAD(P)H were measured the brain of in WKY and SHRSP. Results: CD38 expression and enzymatic activity were increased in SHRSP brains compared to age matched WKY starting at 7 weeks of age. CD38 expression was localized to the endothelial cells, astrocytes, and microglia. We also identified increased CD38 expression using WB with age in SHRSP and WKY. CD38 enzymatic activity was also increased in 24-week SHRSP compared to 7-week SHRSP. In association, we identified evidence of oxidative stress, reduced NO level, reduced NAD(P)H level and endothelial NOS expression in SHRSP compared to age matched WKY. NAD(P)H also decreased with age in WKY and SHRSP. Additionally, activation of astrocytes and microglia were present in SHRSP compared to WKY. Conclusions: CD38 is overexpressed, and its enzymatic activity is increased in SHRSP, a genetic model for marked hypertension and human CSVD. Our results suggest a potential role for CD38 enzymatic activation in the pathogenesis of CSVD and points to the need for future mechanistic and pharmacological studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hannawi, Ewees, Moore and Zweier.)- Published
- 2022
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13. Electronic cigarette exposure causes vascular endothelial dysfunction due to NADPH oxidase activation and eNOS uncoupling.
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El-Mahdy MA, Ewees MG, Eid MS, Mahgoup EM, Khaleel SA, and Zweier JL
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- Animals, Endothelium, Vascular metabolism, Female, Male, Mice, NADPH Oxidases metabolism, Nicotine, Nitric Oxide metabolism, Nitric Oxide Synthase Type III metabolism, Peroxynitrous Acid metabolism, Superoxides metabolism, Cardiovascular Diseases, Electronic Nicotine Delivery Systems, Hypertension
- Abstract
We 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. Here, we delineate the underlying mechanisms of ECV-induced vascular endothelial dysfunction (VED), a central trigger of cardiovascular disease. C57/BL6 male mice were exposed to ECV generated from e-cig liquid containing 0, 6, or 24 mg/mL nicotine for 16 and 60 wk. Time-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 with 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. Together, our 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. NEW & NOTEWORTHY Underlying mechanisms of e-cig-induced vascular endothelial dysfunction are delineated. e-cig exposure activates and increases expression of NADPH oxidase and disrupts activation and coupling of eNOS, leading to a vicious cycle of superoxide generation and peroxynitrite formation, with tetrahydrobiopterin depletion, causing loss of NO that triggers vascular endothelial dysfunction. This process is progressive, increasing with the duration of e-cig exposure, and is more severe in the presence of nicotine, but observed even with nicotine-free vaping.
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- 2022
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14. In Situ EPR Spin Trapping and Competition Kinetics Demonstrate Temperature-Dependent Mechanisms of Synergistic Radical Production by Ultrasonically Activated Persulfate.
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Fagan WP, Villamena FA, Zweier JL, and Weavers LK
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- Electron Spin Resonance Spectroscopy methods, Free Radicals, Kinetics, Spin Labels, Spin Trapping methods, Temperature, Cyclic N-Oxides
- Abstract
Ultrasound coupled with activated persulfate can synergistically degrade aqueous organic contaminants. Here, in situ electron paramagnetic resonance spin trapping was used to compare radicals produced by ultrasonically activated persulfate (US-PS) and its individual technologies, ultrasound alone (US) and heat-activated persulfate (PS), with respect to temperature. Radicals were trapped using 5,5-dimethyl-1-pyrroline- N -oxide, DMPO, to form detectable nitroxide adducts. Using initial rates of radical adduct formation, and compared to US and PS, US-PS at 40 and 50 °C resulted in the largest synergistic production of radicals. Radicals generated from US were reasonably consistent from 40 to 70 °C, indicating that temperature had little effect on cavitational bubble collapse over this range. However, synergy indexes calculated from initial rates showed that ultrasonic activation of persulfate at the bubble interface changes with temperature. From these results, we speculate that higher temperatures enhance persulfate uptake into cavitation bubbles via nanodroplet injection. DMPO-OH was the predominant adduct detected for all conditions. However, competition modeling and spin trapping in the presence of nitrobenzene and atrazine probes showed that SO
4 •- predominated. Therefore, the DMPO-OH signal is derived from SO4 •- trapping with subsequent DMPO-SO4 - hydrolysis to DMPO-OH. Spin trapping is effective in quantifying total radical adduct formation but limited in measuring primary radical speciation in this case.- Published
- 2022
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15. Role of cytoglobin in cigarette smoke constituent-induced loss of nitric oxide bioavailability in vascular smooth muscle cells.
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Mahgoup EM, Khaleel SA, El-Mahdy MA, Abd-Allah AR, and Zweier JL
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- Animals, Aorta cytology, Cell Survival drug effects, Cytochrome-B(5) Reductase metabolism, Cytochromes b5 metabolism, Cytoglobin genetics, Gene Knockdown Techniques, Mice, Muscle, Smooth, Vascular cytology, Superoxides metabolism, Up-Regulation drug effects, Cytoglobin metabolism, Myocytes, Smooth Muscle metabolism, Nitric Oxide metabolism, Tobacco Smoke Pollution adverse effects
- Abstract
Cytoglobin (Cygb) has been identified as the major nitric oxide (NO) metabolizing protein in vascular smooth muscle cells (VSMCs) and is crucial for the regulation of vascular tone. In the presence of its requisite cytochrome B5a (B5)/B5 reductase-isoform-3 (B5R) reducing system, Cygb controls NO metabolism through the oxygen-dependent process of NO dioxygenation. Tobacco cigarette smoking (TCS) induces vascular dysfunction; however, the role of Cygb in the pathophysiology of TCS-induced cardiovascular disease has not been previously investigated. While TCS impairs NO biosynthesis, its effect on NO metabolism remains unclear. Therefore, we performed studies in aortic VSMCs with tobacco smoke extract (TSE) exposure to investigate the effects of cigarette smoke constituents on the rates of NO decay, with focus on the alterations that occur in the process of Cygb-mediated NO metabolism. TSE greatly enhanced the rates of NO metabolism by VSMCs. An initial increase in superoxide-mediated NO degradation was seen at 4 h of exposure. This was followed by much larger progressive increases at 24 and 48 h, accompanied by parallel increases in the expression of Cygb and B5/B5R. siRNA-mediated Cygb knockdown greatly decreased these TSE-induced elevations in NO decay rates. Therefore, upregulation of the levels of Cygb and its reducing system accounted for the large increase in NO metabolism rate seen after 24 h of TSE exposure. Thus, increased Cygb-mediated NO degradation would contribute to TCS-induced vascular dysfunction and partial inhibition of Cygb expression or its NO dioxygenase function could be a promising therapeutic target to prevent secondary cardiovascular disease., (Copyright © 2021 Elsevier Inc. All rights reserved.)
- Published
- 2022
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16. Cytoglobin has potent superoxide dismutase function.
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Zweier JL, Hemann C, Kundu T, Ewees MG, Khaleel SA, Samouilov A, Ilangovan G, and El-Mahdy MA
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- Animals, Cell Line, Electron Spin Resonance Spectroscopy, Male, Mice, Mice, Knockout, Reactive Oxygen Species metabolism, Cytoglobin chemistry, Cytoglobin genetics, Cytoglobin metabolism, Superoxide Dismutase chemistry, Superoxide Dismutase genetics, Superoxide Dismutase metabolism
- Abstract
Cytoglobin (Cygb) was discovered as a novel type of globin that is expressed in mammals; however, its functions remain uncertain. While Cygb protects against oxidant stress, the basis for this is unclear, and the effect of Cygb on superoxide metabolism is unknown. From dose-dependent studies of the effect of Cygb on superoxide catabolism, we identify that Cygb has potent superoxide dismutase (SOD) function. Initial assays using cytochrome c showed that Cygb exhibits a high rate of superoxide dismutation on the order of 10
8 M-1 ⋅ s-1 Spin-trapping studies also demonstrated that the rate of Cygb-mediated superoxide dismutation (1.6 × 108 M-1 ⋅ s-1 ) was only ∼10-fold less than Cu,Zn-SOD. Stopped-flow experiments confirmed that Cygb rapidly dismutates superoxide with rates within an order of magnitude of Cu,Zn-SOD or Mn-SOD. The SOD function of Cygb was inhibited by cyanide and CO that coordinate to Fe3+ -Cygb and Fe2+ -Cygb, respectively, suggesting that dismutation involves iron redox cycling, and this was confirmed by spectrophotometric titrations. In control smooth-muscle cells and cells with siRNA-mediated Cygb knockdown subjected to extracellular superoxide stress from xanthine/xanthine oxidase or intracellular superoxide stress triggered by the uncoupler, menadione, Cygb had a prominent role in superoxide metabolism and protected against superoxide-mediated death. Similar experiments in vessels showed higher levels of superoxide in Cygb-/- mice than wild type. Thus, Cygb has potent SOD function and can rapidly dismutate superoxide in cells, conferring protection against oxidant injury. In view of its ubiquitous cellular expression at micromolar concentrations in smooth-muscle and other cells, Cygb can play an important role in cellular superoxide metabolism., Competing Interests: The authors declare no competing interest.- Published
- 2021
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17. Reperfusion mediates heme impairment with increased protein cysteine sulfonation of mitochondrial complex III in the post-ischemic heart.
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Chen CL, Kang PT, Zhang L, Xiao K, Zweier JL, Chilian WM, and Chen YR
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- Animals, Benzene Derivatives chemistry, Cattle, Cysteine chemistry, Cytochromes c1 chemistry, Cytochromes c1 metabolism, Electron Transport Complex III chemistry, Heme chemistry, Male, Mice, Transgenic, Mitochondria, Heart metabolism, Mitochondria, Heart pathology, Myocardial Ischemia metabolism, Peroxynitrous Acid chemistry, Rats, Sprague-Dawley, Superoxide Dismutase genetics, Rats, Cysteine metabolism, Electron Transport Complex III metabolism, Heme metabolism, Myocardial Ischemia pathology, Myocardial Reperfusion Injury metabolism
- Abstract
A serious consequence of myocardial ischemia-reperfusion injury (I/R) is oxidative damage, which causes mitochondrial dysfunction. The cascading ROS can propagate and potentially induce heme bleaching and protein cysteine sulfonation (PrSO
3 H) of the mitochondrial electron transport chain. Herein we studied the mechanism of I/R-mediated irreversible oxidative injury of complex III in mitochondria from rat hearts subjected to 30-min of ischemia and 24-h of reperfusion in vivo. In the I/R region, the catalytic activity of complex III was significantly impaired. Spectroscopic analysis indicated that I/R mediated the destruction of hemes b and c + c1 in the mitochondria, supporting I/R-mediated complex III impairment. However, no significant impairment of complex III activity and heme damage were observed in mitochondria from the risk region of rat hearts subjected only to 30-min ischemia, despite a decreased state 3 respiration. In the I/R mitochondria, carbamidomethylated C122 /C125 of cytochrome c1 via alkylating complex III with a down regulation of HCCS was exclusively detected, supporting I/R-mediated thioether defect of heme c1 . LC-MS/MS analysis showed that I/R mitochondria had intensely increased complex III PrSO3 H levels at the C236 ligand of the [2Fe2S] cluster of the Rieske iron‑sulfur protein (uqcrfs1), thus impairing the electron transport activity. MS analysis also indicated increased PrSO3 H of the hinge protein at C65 and of cytochrome c1 at C140 and C220 , which are confined in the intermembrane space. MS analysis also showed that I/R extensively enhanced the PrSO3 H of the core 1 (uqcrc1) and core 2 (uqcrc2) subunits in the matrix compartment, thus supporting the conclusion that complex III releases ROS to both sides of the inner membrane during reperfusion. Analysis of ischemic mitochondria indicated a modest reduction from the basal level of complex III PrSO3 H detected in the mitochondria of sham control hearts, suggesting that the physiologic hyperoxygenation and ROS overproduction during reperfusion mediated the enhancement of complex III PrSO3 H. In conclusion, reperfusion-mediated heme damage with increased PrSO3 H controls oxidative injury to complex III and aggravates mitochondrial dysfunction in the post-ischemic heart., (Copyright © 2021 Elsevier Ltd. All rights reserved.)- Published
- 2021
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18. Characterizing the Neuroimaging and Histopathological Correlates of Cerebral Small Vessel Disease in Spontaneously Hypertensive Stroke-Prone Rats.
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Hannawi Y, Caceres E, Ewees MG, Powell KA, Bratasz A, Schwab JM, Rink CL, and Zweier JL
- Abstract
Introduction: Spontaneously hypertensive stroke-prone rats (SHRSP) are used to model clinically relevant aspects of human cerebral small vessel disease (CSVD). To decipher and understand the underlying disease dynamics, assessment of the temporal progression of CSVD histopathological and neuroimaging correlates is essential. Materials and Methods: Eighty age-matched male SHRSP and control Wistar Kyoto rats (WKY) were randomly divided into four groups that were aged until 7, 16, 24 and 32 weeks. Sensorimotor testing was performed weekly. Brain MRI was acquired at each study time point followed by histological analyses of the brain. Results: Compared to WKY controls, the SHRSP showed significantly higher prevalence of small subcortical hyperintensities on T2w imaging that progressed in size and frequency with aging. Volumetric analysis revealed smaller intracranial and white matter volumes on brain MRI in SHRSP compared to age-matched WKY. Diffusion tensor imaging (DTI) showed significantly higher mean diffusivity in the corpus callosum and external capsule in WKY compared to SHRSP. The SHRSP displayed signs of motor restlessness compared to WKY represented by hyperactivity in sensorimotor testing at the beginning of the experiment which decreased with age. Distinct pathological hallmarks of CSVD, such as enlarged perivascular spaces, microbleeds/red blood cell extravasation, hemosiderin deposits, and lipohyalinosis/vascular wall thickening progressively accumulated with age in SHRSP. Conclusions: Four stages of CSVD severity in SHRSP are described at the study time points. In addition, we find that quantitative analyses of brain MRI enable identification of in vivo markers of CSVD that can serve as endpoints for interventional testing in therapeutic studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hannawi, Caceres, Ewees, Powell, Bratasz, Schwab, Rink and Zweier.)
- Published
- 2021
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19. Serine mutations in overexpressed Hsp27 abrogate the protection against doxorubicin-induced p53-dependent cardiac apoptosis in mice.
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Kanagasabai R, Karthikeyan K, Zweier JL, and Ilangovan G
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- Animals, Cardiomyopathy, Dilated chemically induced, Cardiomyopathy, Dilated genetics, Cardiomyopathy, Dilated pathology, Cardiotoxicity, Cells, Cultured, Disease Models, Animal, Female, Heat-Shock Proteins metabolism, Male, Mice, Transgenic, Molecular Chaperones metabolism, Myocardium pathology, Myosin Heavy Chains genetics, Phosphorylation, Serine, Signal Transduction, Apoptosis, Cardiomyopathy, Dilated metabolism, Doxorubicin, Heat-Shock Proteins genetics, Molecular Chaperones genetics, Mutation, Myocardium metabolism, Tumor Suppressor Protein p53 metabolism
- Abstract
Small heat shock proteins (sHsps) protect the heart from chemotherapeutics-induced heart failure by inhibiting p53-dependent apoptosis. However, mechanism of such protection has not been elucidated yet. Here we test a hypothesis that serine phosphorylation of sHsps is essential to inhibit the doxorubicin-induced and p53-dependent apoptotic pathway. Three transgenic mice (TG) lines with cardiomyocyte-specific overexpression of human heat shock protein 27 (hHsp27), namely, wild-type [myosin heavy chain (MHC)-hHsp27], S82A single mutant [MHC-mut-hHsp27( S82A )], and trimutant [MHC-mut-hHsp27( S15A/S78A/S82A )] were generated. TG mice were treated with Dox (6 mg/kg body wt; once in a week; 4 wk) along with age-matched nontransgenic (non-TG) controls. The Dox-treated MHC-hHsp27 mice showed improved survival and cardiac function (both MRI and echocardiography) in terms of contractility [ejection fraction (%EF)] and left ventricular inner diameter (LVID) compared with the Dox-treated non-TG mice. However, both MHC-mut-hHsp27( S82A ) and MHC-mut-hHsp27( S15A/S78A/S82A ) mutants overexpressing TG mice did not show such a cardioprotection. Furthermore, transactivation of p53 was found to be attenuated only in Dox-treated MHC-hHsp27 mice-derived cardiomyocytes in vitro, as low p53 was detected in the nuclei, not in mutant hHsp27 overexpressing cardiomyocytes. Similarly, only in MHC-hHsp27 overexpressing cardiomyocytes, low Bax, higher mechanistic target of rapamycin (mTOR) phosphorylation, and low apoptotic poly(ADP-ribose) polymerase-1 (PARP-1) cleavage (89 kDa fragment) were detected. Pharmacological inhibition of p53 was more effective in mutant TG mice compared with MHC-hHsp27 mice. We conclude that phosphorylation of overexpressed Hsp27 at S82 and its association with p53 are essential for the cardioprotective effect of overexpressed Hsp27 against Dox-induced dilated cardiomyopathy. Only phosphorylated Hsp27 protects the heart by inhibiting p53 transactivation. NEW & NOTEWORTHY Requirement of serine phosphorylation in Hsp27 for cardioprotective effect against Dox is tested in various mutants overexpressing mice. Cardioprotective effect was found to be compromised in Hsp27 serine mutants overexpressed mice compared with wild-type overexpressing mice. These results indicate that cancer patients, who carry these mutations, may have higher risk of aggravated cardiomyopathy on treated with cardiotoxic chemotherapeutics such as doxorubicin.
- Published
- 2021
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20. Development of an L-band resonator optimized for fast scan EPR imaging of the mouse head.
- Author
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Samouilov A, Komarov D, Petryakov S, Iosilevich A, and Zweier JL
- Subjects
- Animals, Electron Spin Resonance Spectroscopy, Mice, Phantoms, Imaging, Radionuclide Imaging, Magnetic Resonance Imaging
- Abstract
Purpose: To develop a novel resonator for high-quality fast scan electron paramagnetic resonance (EPR) and EPR/NMR co-imaging of the head and brain of mice at 1.25 GHz., Methods: Resonator dimensions were scaled to fit the mouse head with maximum filling factor. A single-loop 6-gap resonator of 20 mm diameter and 20 mm length was constructed. High resonator stability was achieved utilizing a fixed position double coupling loop. Symmetrical mutually inverted connections rendered it insensitive to field modulation and fast scan. Coupling adjustment was provided by a parallel-connected variable capacitor located at the feeding line at λ/4 distance. To minimize radiation loss, the shield around the resonator was supplemented with a planar conductive disc that focuses return magnetic flux., Results: Coupling of the resonator loaded with the mouse head was efficient and easy. This resonator enabled high-quality in vivo 3D EPR imaging of the mouse head following intravenous infusion of nitroxide probes. With this resonator and rapid scan EPR system, 4 ms scans were acquired in forward and reverse directions so that images with 2-scan 3,136 projections were acquired in 25 s. Head images were achieved with resolutions of 0.4 mm, enabling visualization of probe localization and uptake across the blood-brain barrier., Conclusions: This resonator design provides good sensitivity, high stability, and B
1 field homogeneity for in vivo fast scan EPR of the mouse head and brain, enabling faster measurements and higher resolution imaging of probe uptake, localization, and metabolism than previously possible., (© 2021 International Society for Magnetic Resonance in Medicine.)- Published
- 2021
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21. High fidelity triangular sweep of the magnetic field for millisecond scan EPR imaging.
- Author
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Komarov DA, Samouilov A, Hirata H, and Zweier JL
- Subjects
- Electron Spin Resonance Spectroscopy, Diagnostic Imaging, Magnetic Fields
- Abstract
Linearity of the magnetic field sweep is important for high resolution continuous wave EPR imaging. Driving the field with triangular wave function is the most efficient way to scan EPR projections. However, the magnetic field sweep profile can be significantly distorted during fast millisecond projection scan. In this work, we introduce a method to generate highly linear and properly symmetrical triangular sweeps of the magnetic field using calibrated harmonics of the triangular wave function. First, the frequency response function of the EPR magnet and its power circuitry was obtained. For this, the field sweeping coil was driven with sinusoidal signals of different frequencies and the actual magnetic field inside the magnet was recorded. To cover wide range of frequencies, the measurements were carried out independently using gaussmeter, Hall-effect linear sensor integrated circuit, and an inductance coil. For each frequency, the system gain and the phase delay were determined. These data were used to adjust the amplitudes and the phases of individual harmonics of the triangular wave function. After the calibration, the maximum deviation of the magnetic field from the linear function was 0.05% of sweep width for 4 ms scan. The maximum discrepancy between the forward and the reverse scan was less than 0.04%. Sweep overhead time for changing the scan direction was 5%. The proposed approach allows generation of high fidelity triangular magnetic field sweeps with accuracy better than 0.1% for the range of the magnetic field sweep widths up to 48 G and scan duration from 10 s down to 1 ms., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)
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- 2021
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22. Long-term electronic cigarette exposure induces cardiovascular dysfunction similar to tobacco cigarettes: role of nicotine and exposure duration.
- Author
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El-Mahdy MA, Mahgoup EM, Ewees MG, Eid MS, Abdelghany TM, and Zweier JL
- Subjects
- Adrenergic alpha-1 Receptor Agonists pharmacology, Animals, Aorta physiopathology, Blood Pressure drug effects, Blood Pressure physiology, Cardiovascular Diseases physiopathology, Electronic Nicotine Delivery Systems, Male, Mice, Phenylephrine pharmacology, Time Factors, Vasodilation drug effects, Vasodilation physiology, Aorta drug effects, Cardiovascular Diseases chemically induced, Nicotine administration & dosage, Vaping adverse effects
- Abstract
Electronic cigarette (e-cig) vaping (ECV) has been proposed as a safer alternative to tobacco cigarette smoking (TCS); however, this remains controversial due to a lack of long-term comparative studies. Therefore, we developed a chronic mouse exposure model that mimics human vaping and allows comparison with TCS. Longitudinal studies were performed to evaluate alterations in cardiovascular function with TCS and ECV exposure durations of up to 60 wk. For ECV, e-cig liquid with box-mod were used and for TCS, 3R4F-cigarettes. C57/BL6 male mice were exposed 2 h/day, 5 days/wk to TCS, ECV, or air control. The role of vape nicotine levels was evaluated using e-cig-liquids with 0, 6, or 24 mg/mL nicotine. Following 16-wk exposure, increased constriction to phenylephrine and impaired endothelium-dependent and endothelium-independent vasodilation were observed in aortic segents, paralleling the onset of systemic hypertension, with elevations in systemic vascular resistance. Following 32 wk, TCS and ECV induced cardiac hypertrophy. All of these abnormalities further increased out to 60 wk of exposure, with elevated heart weight and aortic thickness along with increased superoxide production in vessels and cardiac tissues of both ECV and TCS mice. While ECV-induced abnormalities were seen in the absence of nicotine, these occurred earlier and were more severe with higher nicotine exposure. Thus, long-term vaping of e-cig can induce cardiovascular disease similar to TCS, and the severity of this toxicity increases with exposure duration and vape nicotine content. NEW & NOTEWORTHY A chronic mouse exposure model that mimics human e-cigarette vaping and allows comparison with tobacco cigarette smoking was developed and utilized to perform longitudinal studies of alterations in cardiovascular function. E-cigarette exposure led to the onset of cardiovascular disease similar to that with tobacco cigarette smoking. Impaired endothelium-dependent and endothelium-independent vasodilation with increased adrenergic vasoconstriction were observed, paralleling the onset of systemic hypertension and subsequent cardiac hypertrophy. This cardiovascular toxicity was dependent on exposure duration and nicotine dose.
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- 2021
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23. Defining the reducing system of the NO dioxygenase cytoglobin in vascular smooth muscle cells and its critical role in regulating cellular NO decay.
- Author
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Ilangovan G, Khaleel SA, Kundu T, Hemann C, El-Mahdy MA, and Zweier JL
- Subjects
- Animals, Biochemical Phenomena, Cells, Cultured, Humans, Kinetics, Mice, Cytochromes b5 metabolism, Cytoglobin metabolism, Muscle, Smooth, Vascular metabolism, Nitric Oxide metabolism, Oxygenases metabolism
- Abstract
In smooth muscle, cytoglobin (Cygb) functions as a potent nitric oxide (NO) dioxygenase and regulates NO metabolism and vascular tone. Major questions remain regarding which cellular reducing systems regulate Cygb-mediated NO metabolism. To better define the Cygb-mediated NO dioxygenation process in vascular smooth muscle cells (SMCs), and the requisite reducing systems that regulate cellular NO decay, we assessed the intracellular concentrations of Cygb and its putative reducing systems and examined their roles in the process of NO decay. Cygb and the reducing systems, cytochrome b5 (B5)/cytochrome b5 reductase (B5R) and cytochrome P450 reductase (CPR) were measured in aortic SMCs. Intracellular Cygb concentration was estimated as 3.5 μM, while B5R, B5, and CPR were 0.88, 0.38, and 0.15 μM, respectively. NO decay in SMCs was measured following bolus addition of NO to air-equilibrated cells. siRNA-mediated knockdown experiments indicated that ∼78% of NO metabolism in SMCs is Cygb-dependent. Of this, ∼87% was B5R- and B5-dependent. CPR knockdown resulted in a small decrease in the NO dioxygenation rate (V
NO ), while depletion of ascorbate had no effect. Kinetic analysis of VNO for the B5/B5R/Cygb system with variation of B5 or B5R concentrations from their SMC levels showed that VNO exhibits apparent Michaelis-Menten behavior for B5 and B5R. In contrast, linear variation was seen with change in Cygb concentration. Overall, B5/B5R was demonstrated to be the major reducing system supporting Cygb-mediated NO metabolism in SMCs with changes in cellular B5/B5R levels modulating the process of NO decay., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2021
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24. The novel SOD mimetic GC4419 increases cancer cell killing with sensitization to ionizing radiation while protecting normal cells.
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El-Mahdy MA, Alzarie YA, Hemann C, Badary OA, Nofal S, and Zweier JL
- Subjects
- Apoptosis, Cell Death, Humans, Radiation, Ionizing, Superoxide Dismutase, Neoplasms, Organometallic Compounds
- Abstract
While radiotherapy is a widely used treatment for many types of human cancer, problems of radio-resistance and side effects remain. Side effects induced by ionizing radiation (IR) arise primarily from its propensity to trigger inflammation and oxidative stress with damage of normal cells and tissues near the treatment area. The highly potent superoxide dismutase mimetic, GC4419 (Galera Therapeutics), rapidly enters cells and is highly effective in dismutating superoxide (O
2 •- ). We performed studies to assess the potency of GC4419 in cancer killing and radio-sensitization in human lung cancer cells and normal immortalized lung cells. Treatment with GC4419 did not alter the radical generation during IR, primarily hydroxyl radical (. OH); however, it quenched the increased levels of O2 •- detected in the cancer cells before and following IR. GC4419 triggered cancer cell death and inhibited cancer cell proliferation with no adverse effect on normal cells. Combination of GC4419 with IR augmented the cytotoxic effects of IR on cancer cells compared to monotherapy, while protecting normal cells from IR-induced cell death. DNA fragmentation and caspase-3 activity assays showed that combination of GC4419 with IR enhances cancer cell apoptosis. Moreover, GC4419 increased IR-induced Bax levels with decreased Bcl-2 and elevated Bax/Bcl-2 ratio following treatment. GC4419 increased TrxR activity in the normal cells but decreased activity in cancer cells, conferring increased cancer cell sensitivity to oxidative stress. In conclusion, GC4419 increases the cytotoxic and pro-apoptotic activity of IR in lung cancer cells while decreasing injury in normal cells., (Copyright © 2020 Elsevier Inc. All rights reserved.)- Published
- 2020
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25. Whole body electronic cigarette exposure system for efficient evaluation of diverse inhalation conditions and products.
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Zweier JL, Shalaan MT, Samouilov A, Saleh IG, and El-Mahdy MA
- Subjects
- Administration, Inhalation, Animals, Carbon Dioxide analysis, Carbon Monoxide analysis, Cotinine blood, Equipment Design, Humidity, Male, Mice, Inbred C57BL, Oxygen analysis, Particulate Matter analysis, Particulate Matter toxicity, Temperature, Electronic Nicotine Delivery Systems, Toxicity Tests instrumentation
- Abstract
Objectives: To develop and test a new system for whole body exposure of small animals to support investigation of the biological effects of aerosol generated by electronic cigarette (e-cig) products under diverse inhalation conditions with improved control and monitoring of the e-cig vape exposure and nicotine delivered to the animal's systemic circulation. Methods: A computer-controlled design, with built-in sensors for real time monitoring of O
2 , CO2 , relative humidity, and temperature within the exposure chambers and port for measuring total particulate matter (TPM) was developed, constructed and tested. This design accommodates a variety of commercial vaping devices, offers software flexibility to adjust exposure protocols to mimic different users' puffing patterns, enables variable nicotine delivery to the animal's systemic circulation; minimizes travel time and alterations of aerosol quality or quantity by delivering aerosol directly to the exposure chamber, offers local or remote operation of up to six distinct exposure chambers from a single control unit, and can simultaneously test different exposure conditions or products in diverse animal groups, which reduces inter-run variability, saves time, and increases productivity. Results: The time course pattern of TPM concentration during different phases of the exposure cycle was measured. With increased puffing duration or number of exposure cycles, higher TPM exposure and plasma cotinine levels were observed with plasma cotinine levels in the range reported in light or heavy smokers. Conclusion: Overall, this novel, versatile, and durable exposure system facilitates high-throughput evaluation of the relative safety and potential toxicity of a variety of e-cig devices and liquids.- Published
- 2020
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26. Synergistic, aqueous PAH degradation by ultrasonically-activated persulfate depends on bulk temperature and physicochemical parameters.
- Author
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Fagan WP, Zhao J, Villamena FA, Zweier JL, and Weavers LK
- Abstract
Coupling ultrasound with other remediation technologies has potential to result in synergistic degradation of contaminants. In this work, we evaluated synergisms from adding high-power ultrasound (20 kHz; 250 W) to activated persulfate over a range of bulk temperatures (20-60 °C). We studied the aqueous degradation kinetics of three polycyclic aromatic hydrocarbons (PAHs: naphthalene, phenanthrene, and fluoranthene) treated by ultrasound-alone, heat-activated persulfate, and combined ultrasonically-activated persulfate (US-PS). At 20 °C, observed US-PS rate constants strongly correlated with Wilke-Chang diffusion coefficients. This correlation indicates PAH molecules diffuse to the bubble-water interface prior to reaction with sulfate radicals (SO
4 - ) generated at the interface. At higher temperatures, observed US-PS rate constants appear to be a more complicated function of temperature and diffusion coefficients. Synergy indexes for PAHs with fast diffusion coefficients were greatest at 20 °C. Fluoranthene, the largest and most hydrophobic PAH, had a maximum synergy index at 30 °C; it benefited from additional thermal persulfate activation in bulk solution. Fluoranthene synergy indexes, however, decreased above 30 °C and became antagonistic at 60 °C. Electron paramagnetic resonance (EPR) spin trapping was used to quantify hydroxyl radical (OH) produced from acoustic cavitation in the absence of persulfate. These data showed consistent OH production from 20 to 60 °C, indicating PAH antagonisms at 60 °C were not due to lower bubble collapse temperatures. Instead, the results suggest that PAH antagonisms are caused by increased radical-radical recombination as bulk temperature increases. In effort to develop an efficient, combined remediation technology, this work suggests bulk temperatures between 20 and 40 °C maximize US-PS synergisms., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)- Published
- 2020
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27. Algebraic reconstruction of 3D spatial EPR images from high numbers of noisy projections: An improved image reconstruction technique for high resolution fast scan EPR imaging.
- Author
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Komarov DA, Samouilov A, Ahmad R, and Zweier JL
- Subjects
- Algorithms, Phantoms, Imaging, Electron Spin Resonance Spectroscopy, Image Processing, Computer-Assisted methods, Imaging, Three-Dimensional
- Abstract
A novel method for reconstructing 3D spatial EPR images from large numbers of noisy projections was developed that minimizes mean square error between the experimental projections and those from the reconstructed image. The method utilizes raw projection data and zero gradient spectrum to account for EPR line shape and hyperfine structure of the paramagnetic probe without the need for deconvolution techniques that are poorly suited for processing of high noise projections. A numerical phantom was reconstructed for method validation. Reconstruction time for the matrix of 128
3 voxels and 16,384 noiseless projections was 4.6 min for a single iteration. The algorithm converged quickly, reaching R2 ~ 0.99975 after the very first iteration. An experimental phantom sample with nitroxyl radical was measured. With 16,384 projections and a field gradient of 8 G/cm, resolutions of 0.4 mm were achieved for a cubical area of 25 × 25 × 25 mm3 . Reconstruction was sufficiently fast and memory efficient making it suitable for applications with large 3D matrices and fully determined system of equations. The developed algorithm can be used with any gradient distribution and does not require adjustable filter parameters that makes for simple application. A thorough analysis of the strengths and limitations of this method for 3D spatial EPR imaging is provided., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)- Published
- 2020
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28. Chronic cigarette smoke exposure triggers a vicious cycle of leukocyte and endothelial-mediated oxidant stress that results in vascular dysfunction.
- Author
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El-Mahdy MA, Abdelghany TM, Hemann C, Ewees MG, Mahgoup EM, Eid MS, Shalaan MT, Alzarie YA, and Zweier JL
- Subjects
- Animals, Aorta metabolism, Aorta physiopathology, Blood Pressure, Endothelium, Vascular physiopathology, Male, Mesenteric Arteries metabolism, Mesenteric Arteries physiopathology, Mice, Mice, Inbred C57BL, NADPH Oxidases metabolism, Nitric Oxide Synthase Type III metabolism, Proto-Oncogene Proteins c-akt metabolism, Smoke Inhalation Injury etiology, Smoke Inhalation Injury physiopathology, Superoxides metabolism, Endothelium, Vascular metabolism, Leukocytes metabolism, Oxidative Stress, Smoke Inhalation Injury metabolism, Tobacco Smoke Pollution adverse effects, Vasodilation
- Abstract
Although there is a strong association between cigarette smoking exposure (CSE) and vascular endothelial dysfunction (VED), the underlying mechanisms by which CSE triggers VED remain unclear. Therefore, studies were performed to define these mechanisms using a chronic mouse model of cigarette smoking (CS)-induced cardiovascular disease mirroring that in humans. C57BL/6 male mice were subjected to CSE for up to 48 wk. CSE impaired acetylcholine (ACh)-induced relaxation of aortic and mesenteric segments and triggered hypertension, with mean arterial blood pressure at 32 and 48 wk of exposure of 122 ± 6 and 135 ± 5 mmHg compared with 99 ± 4 and 102 ± 6 mmHg, respectively, in air-exposed mice. CSE led to monocyte activation with superoxide generation in blood exiting the pulmonary circulation. Macrophage infiltration with concomitant increase in NADPH oxidase subunits p22
phox and gp91phox was seen in aortas of CS-exposed mice at 16 wk, with further increase out to 48 wk. Associated with this, increased superoxide production was detected that decreased with Nox inhibition. Tetrahydrobiopterin was progressively depleted in CS-exposed mice but not in air-exposed controls, resulting in endothelial nitric oxide synthase (eNOS) uncoupling and secondary superoxide generation. CSE led to a time-dependent decrease in eNOS and Akt expression and phosphorylation. Overall, CSE induces vascular monocyte infiltration with increased NADPH oxidase-mediated reactive oxygen species generation and depletes the eNOS cofactor tetrahydrobiopterin, uncoupling eNOS and triggering a vicious cycle of oxidative stress with VED and hypertension. Our study provides important insights toward understanding the process by which smoking contributes to the genesis of cardiovascular disease and identifies biomarkers predictive of disease. NEW & NOTEWORTHY In a chronic model of smoking-induced cardiovascular disease, we define underlying mechanisms of smoking-induced vascular endothelial dysfunction (VED). Smoking exposure triggered VED and hypertension and led to vascular macrophage infiltration with concomitant increase in superoxide and NADPH oxidase levels as early as 16 wk of exposure. This oxidative stress was accompanied by tetrahydrobiopterin depletion, resulting in endothelial nitric oxide synthase uncoupling with further superoxide generation triggering a vicious cycle of oxidative stress and VED.- Published
- 2020
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29. Imbalance in zinc homeostasis enhances lung Tissue Loss following cigarette smoke exposure.
- Author
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Knoell DL, Smith D, Bao S, Sapkota M, Wyatt TA, Zweier JL, Flury J, Borchers MT, and Knutson M
- Subjects
- Animals, Cation Transport Proteins genetics, Cation Transport Proteins metabolism, Diet, Disease Models, Animal, Lung pathology, Male, Mice, Mice, Inbred C57BL, Pulmonary Disease, Chronic Obstructive pathology, Zinc administration & dosage, Zinc deficiency, Homeostasis, Lung metabolism, Pulmonary Disease, Chronic Obstructive metabolism, Smoking adverse effects, Tobacco Products adverse effects, Zinc metabolism
- Abstract
Cigarette smoke exposure is a major cause of chronic obstructive pulmonary disease. Cadmium is a leading toxic component of cigarette smoke. Cadmium and zinc are highly related metals. Whereas, zinc is an essential metal required for normal health, cadmium is highly toxic. Zrt- and Irt-like protein 8 (ZIP8) is an avid transporter of both zinc and cadmium into cells and is abundantly expressed in the lung of smokers compared to nonsmokers. Our objective was to determine whether disturbed zinc homeostasis through diet or the zinc transporter ZIP8 increase susceptibility to lung damage following prolonged cigarette smoke exposure., Methods: Cigarette smoke exposure was evaluated in the lungs of mice subject to insufficient and sufficient zinc intakes, in transgenic ZIP8 overexpressing mice, and a novel myeloid-specific ZIP8 knockout strain., Results: Moderate depletion of zinc intakes in adult mice resulted in a significant increase in lung cadmium burden and permanent lung tissue loss following prolonged smoke exposure. Overexpression of ZIP8 resulted in increased lung cadmium burden and more extensive lung damage, whereas cigarette smoke exposure in ZIP8 knockout mice resulted in increased lung tissue loss without a change in lung cadmium content, but a decrease in zinc., Conclusions: Overall, findings were consistent with past human studies. Imbalance in Zn homeostasis increases susceptibility to permanent lung injury following prolonged cigarette smoke exposure. Based on animal studies, both increased and decreased ZIP8 expression enhanced irreversible tissue damage in response to prolonged tobacco smoke exposure. We believe these findings represent an important advancement in our understanding of how imbalance in zinc homeostasis and cadmium exposure via tobacco smoke may increase susceptibility to smoking-induced lung disease., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest. The authors claim no competing financial or nonfinancial interests., (Copyright © 2020 Elsevier GmbH. All rights reserved.)
- Published
- 2020
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30. Dioxygen Binding and Sensing Proteins.
- Author
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Estrín DA, Luque FJ, Ilangovan G, and Zweier JL
- Subjects
- Binding Sites, Humans, Oxygen metabolism, Proteins metabolism
- Abstract
Oxygen binding proteins (O
2 BIP) have been actively investigated for the past five decades due to their rich redox chemistry and function as O2 carriers in blood cells, as well as their function as gasotransmitters and sensors that modulate cellular signaling. A series of meetings on the periodic advances in the knowledge gained in the field of globin structure and function are conducted typically on a biannual basis. In the fall of 2018, the XXth International Conference was conducted, and very important articles with breakthrough discoveries were presented and very enthusiastically discussed. This was yet another highly successful meeting in the series. Select articles from this meeting were recently reviewed, updated, and published over several issues of Antioxidants and Redox Signaling , as Forum articles communicating the latest advances in this important area of redox biology. This Forum editorial introduces these articles and highlights their scientific significance in advancing the field. Each of these articles grew out of lectures presented in the meeting, and appears either as an original contribution or a comprehensive review in the journal. Overall, the articles published in the Forum provide in-depth details on the recent developments in the field as well as point the way to future directions. These Forum articles thus serve as an important summary of progress and the ongoing direction of this field, and serve to highlight recent advances in our understanding of O2 BIP.- Published
- 2020
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31. Regulation of Nitric Oxide Metabolism and Vascular Tone by Cytoglobin.
- Author
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Zweier JL and Ilangovan G
- Subjects
- Animals, Cytoglobin deficiency, Humans, Oxygenases metabolism, Cytoglobin metabolism, Nitric Oxide metabolism
- Abstract
Significance: Cytoglobin (Cygb) was discovered as a new addition to the globin superfamily and subsequently identified to have potent nitric oxide (NO) dioxygenase function. Cygb plays a critical role in the oxygen-dependent regulation of NO levels and vascular tone. Recent Advances: In recent years, the mechanism of the Cygb-mediated NO dioxygenation has been studied in isolated protein, smooth muscle cell, isolated blood vessel, and in vivo animal model systems. Studies in Cygb
-/- mice have demonstrated that Cygb plays a critical role in regulating blood pressure and vascular tone. This review summarizes advances in the knowledge of NO dioxygenation/metabolism regulated by Cygb. Advances in measurement of NO diffusion dynamics across blood vessels and kinetic modeling of Cygb-mediated NO dioxygenation are summarized. The oxygen-dependent regulation of NO degradation by Cygb is also reviewed along with how Cygb paradoxically generates NO from nitrite under anaerobic conditions. The important role of Cygb in the regulation of vascular function and disease is reviewed. Critical Issues: Cygb is a more potent NO dioxygenase (NOD) than previously known globins with structural differences in heme coordination and environment, conferring it with a higher rate of reduction and more rapid process of NO dioxygenation with unique oxygen dependence. Various cellular reducing systems regenerate the catalytic oxyferrous Cygb species, supporting a high rate of NO dioxygenation. Future Directions: There remains a critical need to further characterize the factors and processes that modulate Cygb-mediated NOD function, and to develop pharmacological or other approaches to modulate Cygb function and expression.- Published
- 2020
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32. Discriminative Detection of Biothiols by Electron Paramagnetic Resonance Spectroscopy using a Methanethiosulfonate Trityl Probe.
- Author
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Tan X, Ji K, Wang X, Yao R, Han G, Villamena FA, Zweier JL, Song Y, Rockenbauer A, and Liu Y
- Subjects
- Humans, Biosensing Techniques methods, Electron Spin Resonance Spectroscopy methods, Mesylates chemistry, Sulfhydryl Compounds chemistry
- Abstract
Biothiols, such as glutathione (GSH), homocysteine (Hcy), and cysteine (Cys), coexist in biological systems with diverse biological roles. Thus, analytical techniques that can detect, quantify, and distinguish between multiple biothiols are desirable but challenging. Herein, we demonstrate the simultaneous detection and quantitation of multiple biothiols, including up to three different biothiols in a single sample, using electron paramagnetic resonance (EPR) spectroscopy and a trityl-radical-based probe (MTST). We term this technique EPR thiol-trapping. MTST could trap thiols through its methanethiosulfonate group to form the corresponding disulfide conjugate with an EPR spectrum characteristic of the trapped thiol. MTST was used to investigate effects of l-buthionine sulfoximine (BSO) and pyrrolidine dithiocarbamate (PDTC) on the efflux of GSH and Cys from HepG2 cells., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2020
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33. Membrane-specific spin trap, 5-dodecylcarbamoyl-5-N-dodecylacetamide-1-pyroline-N-oxide (diC 12 PO): theoretical, bioorthogonal fluorescence imaging and EPR studies.
- Author
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Headley CA, Hoffman CN, Freisen JM, Han Y, Macklin JM, Zweier JL, Rockenbauer A, Kuret J, and Villamena FA
- Subjects
- Acetamides chemical synthesis, Animals, Cattle, Cells, Cultured, Electron Spin Resonance Spectroscopy, Molecular Structure, Acetamides chemistry, Density Functional Theory, Fluorescence, Optical Imaging
- Abstract
Membranous organelles are major endogenous sources of reactive oxygen and nitrogen species. When present at high levels, these species can cause macromolecular damage and disease. To better detect and scavenge free radical forms of the reactive species at their sources, we investigated whether nitrone spin traps could be selectively targeted to intracellular membranes using a bioorthogonal imaging approach. Electron paramagnetic resonance imaging demonstrated that the novel cyclic nitrone 5-dodecylcarbamoyl-5-N-dodecylacetamide-1-pyroline-N-oxide (diC
12 PO) could be used to target the nitrone moiety to liposomes composed of phosphatidyl choline. To test localization with authentic membranes in living cells, fluorophores were introduced via strain-promoted alkyne-nitrone cycloaddition (SPANC). Two fluorophore-conjugated alkynes were investigated: hexynamide-fluoresceine (HYA-FL) and dibenzylcyclooctyne-PEG4-5/6-sulforhodamine B (DBCO-Rhod). Computational and mass spectrometry experiments confirmed the cycloadduct formation of DBCO-Rhod (but not HYA-FL) with diC12 PO in cell-free solution. Confocal microscopy of bovine aortic endothelial cells treated sequentially with diC12 PO and DBCO-Rhod demonstrated clear localization of fluorescence with intracellular membranes. These results indicate that targeting of nitrone spin traps to cellular membranes is feasible, and that a bioorthogonal approach can aid the interrogation of their intracellular compartmentalization properties.- Published
- 2019
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34. [D-Ala 2 , D-Leu 5 ] Enkephalin Improves Liver Preservation During Normothermic Ex Vivo Perfusion.
- Author
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Beal EW, Kim JL, Reader BF, Akateh C, Maynard K, Washburn WK, Zweier JL, Whitson BA, and Black SM
- Subjects
- Allografts metabolism, Allografts pathology, Animals, Disease Models, Animal, Hepatocytes, Humans, Liver metabolism, Liver pathology, Male, Mitochondria drug effects, Mitochondria metabolism, Oxidative Stress drug effects, Perfusion adverse effects, Perfusion methods, Primary Cell Culture, Rats, Receptors, Opioid, delta metabolism, Reperfusion Injury etiology, Reperfusion Injury pathology, Tissue and Organ Harvesting adverse effects, Tissue and Organ Harvesting methods, Allografts drug effects, Enkephalin, Leucine-2-Alanine pharmacology, Liver drug effects, Organ Preservation Solutions pharmacology, Reperfusion Injury prevention & control
- Abstract
Background: Meeting the metabolic demands of donor livers using normothermic ex vivo liver perfusion (NEVLP) preservation technology is challenging. The delta opioid agonist [D-Ala2, D-Leu5] enkephalin (DADLE) has been reported to decrease the metabolic demand in models of ischemia and cold preservation. We evaluated the therapeutic potential of DADLE by investigating its ability to protect against oxidative stress and hepatic injury during normothermic perfusion., Materials and Methods: Primary rat hepatocytes were used in an in vitro model of oxidative stress to determine the minimum dose of DADLE needed to induce protection and the mechanisms associated with protection. NEVLP was then used to induce injury in rat livers and determine the effectiveness of DADLE in preventing liver injury., Results: In hepatocytes, DADLE was protective against oxidative stress and led to a decrease in phosphorylation of JNK and p38. Naltrindole, a δ-opioid receptor antagonist, blocked this effect. DADLE also activated the PI3K/Akt signaling pathway, and PI3K/Akt inhibition decreased the protective effects of DADLE treatment. In addition, DADLE treatment during NEVLP resulted in lower perfusate alanine aminotransferase and tissue malondialdehyde and better tissue adenosine triphosphate and glutathione. Furthermore, perfusion with DADLE compared with perfusate alone preserved tissue architecture., Conclusions: DADLE confers protection against oxidative stress in hepatocytes and during NEVLP. These data suggest that the mechanism of protection involved the prevention of mitochondrial dysfunction by opioid receptor signaling and subsequent increased expression of prosurvival/antiapoptotic signaling pathways. Altogether, data suggest that opioid receptor agonism may serve as therapeutic target for improved liver protection during NEVLP., (Copyright © 2019 Elsevier Inc. All rights reserved.)
- Published
- 2019
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35. Development of a fast-scan EPR imaging system for highly accelerated free radical imaging.
- Author
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Samouilov A, Ahmad R, Boslett J, Liu X, Petryakov S, and Zweier JL
- Subjects
- Animals, Free Radicals, Heart diagnostic imaging, Phantoms, Imaging, Rats, Electron Spin Resonance Spectroscopy methods, Imaging, Three-Dimensional methods, Magnetic Resonance Imaging methods
- Abstract
Purpose: In continuous wave EPR imaging, the acquisition of high-quality images was previously limited by the requisite long acquisition times of each image projection that was typically greater than 1 second. To accelerate the process of image acquisition facilitating greater numbers of projections and higher image resolution, instrumentation was developed to greatly accelerate the magnetic field scan that is used to obtain each EPR image projection., Methods: A low-inductance solenoidal coil for field scanning was used along with a spherical solenoid air core magnet, and scans were driven by triangular symmetric waves, allowing forward and reverse spectrum acquisition as rapid as 3.8 ms. The uniform distribution of projections was used to optimize the contribution of projections for 3D image reconstruction., Results: Using this fast-scan EPR system, high-quality EPR images of phantoms and perfused rat hearts were performed using trityl or nanoparticulate LiNcBuO (lithium octa-n-butoxy-substituted naphthalocyanine) probes with fast-scan EPR imaging at L-band, achieving spatial resolutions of up to 250 micrometers in 1 minute., Conclusion: Fast-scan EPR imaging can greatly facilitate the efficient and precise mapping of the spatial distribution of free radical and other paramagnetic probes in living systems., (© 2019 International Society for Magnetic Resonance in Medicine.)
- Published
- 2019
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36. Preclinical Development of a vWF Aptamer to Limit Thrombosis and Engender Arterial Recanalization of Occluded Vessels.
- Author
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Nimjee SM, Dornbos D 3rd, Pitoc GA, Wheeler DG, Layzer JM, Venetos N, Huttinger A, Talentino SE, Musgrave NJ, Moody H, Rempel RE, Jones C, Carlisle K, Wilson J, Bratton C, Joseph ME, Khan S, Hoffman MR, Sommerville L, Becker RC, Zweier JL, and Sullenger BA
- Subjects
- Animals, Antidotes pharmacology, Aptamers, Nucleotide chemical synthesis, Aptamers, Nucleotide metabolism, Blood Platelets drug effects, Blood Platelets metabolism, Carotid Artery Injuries drug therapy, Dogs, Dose-Response Relationship, Drug, Female, Healthy Volunteers, Humans, Male, Mice, Mice, Inbred C57BL, Oligonucleotides pharmacology, Platelet Adhesiveness drug effects, Platelet Aggregation drug effects, von Willebrand Factor metabolism, Aptamers, Nucleotide pharmacology, Arterial Occlusive Diseases drug therapy, Drug Evaluation, Preclinical methods, Fibrinolytic Agents pharmacology, Thrombosis drug therapy, Thrombosis prevention & control, von Willebrand Factor antagonists & inhibitors
- Abstract
Endothelial surface and circulating glycoprotein von Willebrand factor (vWF) regulates platelet adhesion and is associated with thrombotic diseases, including ischemic stroke, myocardial infarction, and peripheral vascular disease. Thrombosis, as manifested in these diseases, is the leading cause of disability and death in the western world. Current parenteral antithrombotic and thrombolytic agents used to treat these conditions are limited by a short therapeutic window, irreversibility, and major risk of hemorrhage. To overcome these limitations, we developed a novel anti-vWF aptamer, called DTRI-031, that selectively binds and inhibits vWF-mediated platelet adhesion and arterial thrombosis while enabling rapid reversal of this antiplatelet activity by an antidote oligonucleotide (AO). Aptamer DTRI-031 exerts dose-dependent inhibition of platelet aggregation and thrombosis in whole blood and mice, respectively. Moreover, DTRI-031 can achieve potent vascular recanalization of platelet-rich thrombotic occlusions in murine and canine carotid arteries. Finally, DTRI-031 activity is rapidly (<5 min) and completely reversed by AO administration in a murine saphenous vein hemorrhage model, and murine toxicology studies indicate the aptamer is well tolerated. These findings suggest that targeting vWF with an antidote-controllable aptamer potentially represents an effective and safer treatment for thrombosis patients having platelet-rich arterial occlusions in the brain, heart, or periphery., (Copyright © 2019. Published by Elsevier Inc.)
- Published
- 2019
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37. Intrahepatic Delivery of Pegylated Catalase Is Protective in a Rat Ischemia/Reperfusion Injury Model.
- Author
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Akateh C, Beal EW, Kim JL, Reader BF, Maynard K, Zweier JL, Whitson BA, and Black SM
- Subjects
- Alanine Transaminase blood, Animals, Aspartate Aminotransferases blood, Cell Survival drug effects, Disease Models, Animal, Humans, Hydrogen Peroxide pharmacology, Injections, Intralesional, Liver blood supply, Liver cytology, Male, Oxidative Stress drug effects, Primary Cell Culture, Rats, Reperfusion Injury blood, Reperfusion Injury etiology, Treatment Outcome, Warm Ischemia adverse effects, Catalase administration & dosage, Liver surgery, Polyethylene Glycols administration & dosage, Reperfusion Injury drug therapy
- Abstract
Background: Ischemia/reperfusion injury (IRI) can occur during liver surgery. Endogenous catalase is important to cellular antioxidant defenses and is critical to IRI prevention. Pegylation of catalase (PEG-CAT) improves its therapeutic potential by extending plasma half-life, but systemic administration of exogenous PEG-CAT has been only mildly therapeutic for hepatic IRI. Here, we investigated the protective effects of direct intrahepatic delivery of PEG-CAT during IRI using a rat hilar clamp model., Materials and Methods: PEG-CAT was tested in vitro and in vivo. In vitro, enriched rat liver cell populations were subjected to oxidative stress injury (H
2 O2 ), and measures of cell health and viability were assessed. In vivo, rats underwent segmental (70%) hepatic warm ischemia for 1 h, followed by 6 h of reperfusion, and plasma alanine aminotransferase and aspartate aminotransferase, tissue malondialdehyde, adenosine triphosphate, and GSH, and histology were assessed., Results: In vitro, PEG-CAT pretreatment of liver cells showed substantial uptake and protection against oxidative stress injury. In vivo, direct intrahepatic, but not systemic, delivery of PEG-CAT during IRI significantly reduced alanine aminotransferase and aspartate aminotransferase in a time-dependent manner (P < 0.01, P < 0.0001, respectively, for all time points) compared to control. Similarly, tissue malondialdehyde (P = 0.0048), adenosine triphosphate (P = 0.019), and GSH (P = 0.0015), and the degree of centrilobular necrosis, were improved by intrahepatic compared to systemic PEG-CAT delivery., Conclusions: Direct intrahepatic administration of PEG-CAT achieved significant protection against IRI by reducing the volume distribution and taking advantage of the substantial hepatic first-pass uptake of this molecule. The mode of delivery was an important factor for protection against hepatic IRI by PEG-CAT., (Copyright © 2019 Elsevier Inc. All rights reserved.)- Published
- 2019
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38. Inhibition of CD38 with the Thiazoloquin(az)olin(on)e 78c Protects the Heart against Postischemic Injury.
- Author
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Boslett J, Reddy N, Alzarie YA, and Zweier JL
- Subjects
- Animals, Biological Transport, Biopterins analogs & derivatives, Biopterins metabolism, Endothelium drug effects, Endothelium metabolism, Glycoside Hydrolase Inhibitors metabolism, Heart physiopathology, Mice, Mice, Inbred C57BL, Myocardial Contraction drug effects, Myocardial Reperfusion Injury metabolism, Myocardial Reperfusion Injury physiopathology, NADP metabolism, Nitric Oxide Synthase metabolism, Quinazolines metabolism, ADP-ribosyl Cyclase 1 antagonists & inhibitors, Glycoside Hydrolase Inhibitors chemistry, Glycoside Hydrolase Inhibitors pharmacology, Heart drug effects, Myocardial Reperfusion Injury prevention & control, Quinazolines chemistry, Quinazolines pharmacology
- Abstract
Inhibition of and genetic deletion of the NAD(P)
+ hydrolase [NAD(P)ase] CD38 have been shown to protect against ischemia/reperfusion (I/R) injury in rat and mouse hearts. CD38 has been shown to enhance salvage of NADP(H), which in turn prevents impairment of endothelial nitric oxide synthase function, a hallmark of endothelial dysfunction. Despite growing evidence for a role of CD38 in postischemic injury, until recently there had been a lack of potent CD38 inhibitors. Recently, a new class of thiazoloquin(az)olin(on)e compounds were identified as highly potent and specific CD38 inhibitors. Herein, we investigate the ability of one of these compounds, 78c, to inhibit CD38 and protect the heart in an ex vivo model of myocardial I/R injury. The potency and mechanism of CD38 inhibition by 78c was assessed in vitro using recombinant CD38. The dose-dependent tissue uptake of 78c in isolated mouse hearts was determined, and high tissue permeability of 78c was observed when delivered in perfusate. Treatment of hearts with 78c was protective against both postischemic endothelial and cardiac myocyte injury, with preserved nitric oxide synthase-dependent vasodilatory and contractile function, respectively. Myocardial infarction was also significantly decreased in 78c-treated hearts, with preserved levels of high-energy phosphates. Protective effects peaked at 10 μ M treatment, and similar protection without toxicity was seen at 5-fold higher doses. Overall, 78c was shown to be a potent and biologically active CD38 inhibitor with favorable tissue uptake and marked protective effects against I/R injury with enhanced preservation of contractile function, coronary flow, and decreased infarction., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)- Published
- 2019
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39. Mitochondrial complex I in the post-ischemic heart: reperfusion-mediated oxidative injury and protein cysteine sulfonation.
- Author
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Kang PT, Chen CL, Lin P, Zhang L, Zweier JL, and Chen YR
- Subjects
- Animals, Cysteine analogs & derivatives, Cysteine metabolism, Humans, Mice, Mitochondria genetics, Mitochondria metabolism, Mitochondria pathology, Mitochondria, Heart chemistry, Mitochondria, Heart pathology, Myocardial Infarction genetics, Myocardial Infarction physiopathology, Myocardial Reperfusion Injury physiopathology, Rats, Tandem Mass Spectrometry, Electron Transport Complex I genetics, Mitochondria, Heart genetics, Myocardial Reperfusion Injury genetics, Oxidative Stress genetics
- Abstract
A serious consequence of ischemia-reperfusion injury (I/R) is oxidative damage leading to mitochondrial dysfunction. Such I/R-induced mitochondrial dysfunction is observed as impaired state 3 respiration and overproduction of O
2 - . The cascading ROS can propagate cysteine oxidation on mitochondrial complex I and add insult to injury. Herein we employed LC-MS/MS to identify protein sulfonation of complex I in mitochondria from the infarct region of rat hearts subjected to 30-min of coronary ligation and 24-h of reperfusion in vivo as well as the mitochondria of sham controls. Mitochondrial preparations from the I/R regions had enhanced sulfonation levels on the cysteine ligands of iron‑sulfur clusters, including N3 (C425 ), N1b (C92 ), N4 (C226 ), N2 (C158 /C188 ), and N1a (C134 /C139 ). The 4Fe-4S centers of N3, N1b, N4, and N2 are key redox-active components of complex I, thus sulfonation of metal-binding sites impaired the main electron transfer pathway. The binuclear N1a has a very low redox potential and an antioxidative function. Increased C134 /C139 sulfonation by I/R impaired the N1a cluster, potentially contributing to overall O2 - generation by the FMN moiety of complex I. MS analysis also revealed I/R-mediated increased sulfonation at the core subunits of 51 kDa (C125 , C187 , C206 , C238 , C255 , C286 ), 75 kDa (C367 , C554 , C564 , C727 ), 49 kDa (C146 , C326 , C347 ), and PSST (C188 ). These results were consistent with the consensus indicating that 51 kDa and 75 kDa are two of major subunits hosting regulatory thiols, and their enhanced sulfonation by I/R predisposed the myocardium to further oxidant stress with impaired ubiquinone reduction. MS analysis further showed I/R-mediated enhanced sulfonation at the supernumerary subunits of 42 kDa (C67 , C112 , C183 , C253 ), 15 kDa (C43 ), and 13 kDa (C79 ). The 42 kDa protein is metazoan-specific, which was reported to stabilize mammalian complex I. C43 of the 15 kDa subunit forms an intramolecular disulfide bond with C56 , which was reported to stabilize complex I structure. C79 of the 13 kDa subunit is involved in Zn2+ -binding, which was reported functionally important for complex I assembly. C79 sulfonation by I/R was found to impair Zn2+ -binding. No significant enhancement of protein sulfonation was observed in mitochondrial complex I from the rat heart subjected to 30-min ischemia alone in vivo despite a decreased state 3 respiration, suggesting that the physiologic conditions of hyperoxygenation during reperfusion mediated an increase in complex I sulfonation and oxidative injury. In conclusion, sulfonation of specific cysteines of complex I mediates I/R-induced mitochondrial dysfunction via impaired ETC activity, increasing• O2 - production, and mediating redox dysfunction of complex I., (Copyright © 2018 Elsevier Ltd. All rights reserved.)- Published
- 2018
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40. A Small Animal Model of Ex Vivo Normothermic Liver Perfusion.
- Author
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Beal EW, Dumond C, Kim JL, Akateh C, Eren E, Maynard K, Sen CK, Zweier JL, Washburn K, Whitson BA, and Black SM
- Subjects
- Animals, Disease Models, Animal, Male, Organ Preservation methods, Rats, Liver pathology, Liver Transplantation methods, Perfusion methods
- Abstract
There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, normothermic ex vivo liver perfusion (NEVLP) has been introduced as a method to evaluate and modify organ function. NEVLP has many advantages in comparison to hypothermic and subnormothermic perfusion including reduced preservation injury, restoration of normal organ function under physiologic conditions, assessment of organ performance, and as a platform for organ repair, remodeling, and modification. Both murine and porcine NEVLP models have been described. We demonstrate a rat model of NEVLP and use this model to show one of its important applications - the use of a therapeutic molecule added to liver perfusate. Catalase is an endogenous reactive oxygen species (ROS) scavenger and has been demonstrated to decrease ischemia-reperfusion in the eye, brain, and lung. Pegylation has been shown to target catalase to the endothelium. Here, we added pegylated-catalase (PEG-CAT) to the base perfusate and demonstrated its ability to mitigate liver preservation injury. An advantage of our rodent NEVLP model is that it is inexpensive in comparison to larger animal models. A limitation of this study is that it does not currently include post-perfusion liver transplantation. Therefore, prediction of the function of the organ post-transplantation cannot be made with certainty. However, the rat liver transplant model is well established and certainly could be used in conjunction with this model. In conclusion, we have demonstrated an inexpensive, simple, easily replicable NEVLP model using rats. Applications of this model can include testing novel perfusates and perfusate additives, testing software designed for organ evaluation, and experiments designed to repair organs.
- Published
- 2018
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41. Cigarette smoke constituents cause endothelial nitric oxide synthase dysfunction and uncoupling due to depletion of tetrahydrobiopterin with degradation of GTP cyclohydrolase.
- Author
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Abdelghany TM, Ismail RS, Mansoor FA, Zweier JR, Lowe F, and Zweier JL
- Subjects
- Animals, Aorta drug effects, Aorta enzymology, Biopterins antagonists & inhibitors, Biopterins metabolism, Cattle, Cells, Cultured, Endothelial Cells enzymology, GTP Cyclohydrolase metabolism, Nitric Oxide, Nitric Oxide Synthase Type III metabolism, Biopterins analogs & derivatives, Cigarette Smoking, Endothelial Cells drug effects, GTP Cyclohydrolase antagonists & inhibitors, Nitric Oxide Synthase Type III antagonists & inhibitors
- Abstract
Cigarette smoking (CS) is a well-established risk factor for cardiovascular disease (CVD). Endothelial dysfunction (ED) with loss of nitric oxide (NO) production is a central mechanism leading to the advent of CVD. Despite many prior studies of this major health problem, the exact mechanism by which CS induces ED is not well understood. This study examines the mechanism by which CS induces ED with altered endothelial NO synthase (eNOS) function in aortic endothelial cells (AECs). Exposure of AECs to cigarette smoke extract (CSE) resulted in a marked decrease in NO production with concomitant increase in superoxide (O
2 .- ) generation and accumulation of 4-hydroxy-2-nonenal protein adducts. CSE exposure led to depletion of the essential eNOS cofactor tetrahydrobiopterin (BH4 ) as well as total biopterin levels and decreased the expression level of guanosine triphosphate cyclohydrolase (GTPCH), the rate limiting enzyme in BH4 biosynthesis. Moreover, exposure of AECs to CSE increased the level of ubiquitinated proteins and increased 26 S proteasomal activity in a concentration-dependent manner. Pre-treatment with MG132, a 26 S proteasome inhibitor, partially prevented CSE-induced loss of BH4, total biopterin, GTPCH, and increased NO production following CSE exposure, indicating a role of the ubiquitin-proteasome system in CSE-induced eNOS dysfunction. In conclusion, CSE-induced eNOS dysfunction and uncoupling occurs due to BH4 depletion with BH4 de novo synthesis limited by diminished GTPCH expression., (Copyright © 2018. Published by Elsevier Inc.)- Published
- 2018
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42. Synthesis and Characterization of the Perthiatriarylmethyl Radical and Its Dendritic Derivatives with High Sensitivity and Selectivity to Superoxide Radical.
- Author
-
Tan X, Tao S, Liu W, Rockenbauer A, Villamena FA, Zweier JL, Song Y, and Liu Y
- Abstract
EPR spectroscopy, coupled with the use of tetrathiatriarylmethyl (TAM) radicals, has been a reliable method to detect the superoxide radical (O
2 .- ). However, the specificity and biocompatibility of TAM radicals need to be further improved. Although derivatization may overcome the drawbacks of current TAM radicals, esterification or amidation through the carboxylic groups greatly changes their redox properties and makes them inert to O2 .- . Herein, the synthesis of a perthiatriarylmethyl (PST) radical and its dendritic derivatives, PST-TA and PST-NA, in which PST is covalently linked with dendrons containing three (TA) and nine (NA) carboxylic acids, respectively. The results show that PST rapidly reacts with O2 .- to yield a unique quinone methide product. Dendritic modification of PST slightly decreases the reactivities of PST-TA and PST-NA, but notably increases their biostability toward various oxidoreductants. The detection limit of PST-NA to O2 .- was estimated to be 2.1 nm min-1 over 60 min of detection. Importantly, PST-NA shows threefold higher sensitivity to O2 .- in the presence and absence of ascorbic acid than that of the classic spin-trapping technique. In addition, the application of PST-NA to detect extracellular O2 .- generation in stimulated RAW 264.7 macrophages was also explored. This study demonstrates that PST-NA has great potential for specific detection and quantitation of O2 .- in extracellular sites., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)- Published
- 2018
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43. A Potent and Specific CD38 Inhibitor Ameliorates Age-Related Metabolic Dysfunction by Reversing Tissue NAD + Decline.
- Author
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Tarragó MG, Chini CCS, Kanamori KS, Warner GM, Caride A, de Oliveira GC, Rud M, Samani A, Hein KZ, Huang R, Jurk D, Cho DS, Boslett JJ, Miller JD, Zweier JL, Passos JF, Doles JD, Becherer DJ, and Chini EN
- Subjects
- AMP-Activated Protein Kinase Kinases, Aging metabolism, Animals, DNA Damage drug effects, Enzyme Inhibitors chemistry, Glucose Intolerance blood, Glucose Intolerance drug therapy, Humans, MAP Kinase Signaling System drug effects, Mice, Physical Functional Performance, Poly(ADP-ribose) Polymerases metabolism, Protein Kinases metabolism, Quinolines chemistry, Sirtuins metabolism, TOR Serine-Threonine Kinases metabolism, Triazoles chemistry, ADP-ribosyl Cyclase 1 antagonists & inhibitors, Aging drug effects, Cellular Senescence drug effects, Enzyme Inhibitors pharmacology, NAD metabolism, Quinolines pharmacology, Triazoles pharmacology
- Abstract
Aging is characterized by the development of metabolic dysfunction and frailty. Recent studies show that a reduction in nicotinamide adenine dinucleotide (NAD
+ ) is a key factor for the development of age-associated metabolic decline. We recently demonstrated that the NADase CD38 has a central role in age-related NAD+ decline. Here we show that a highly potent and specific thiazoloquin(az)olin(on)e CD38 inhibitor, 78c, reverses age-related NAD+ decline and improves several physiological and metabolic parameters of aging, including glucose tolerance, muscle function, exercise capacity, and cardiac function in mouse models of natural and accelerated aging. The physiological effects of 78c depend on tissue NAD+ levels and were reversed by inhibition of NAD+ synthesis. 78c increased NAD+ levels, resulting in activation of pro-longevity and health span-related factors, including sirtuins, AMPK, and PARPs. Furthermore, in animals treated with 78c we observed inhibition of pathways that negatively affect health span, such as mTOR-S6K and ERK, and attenuation of telomere-associated DNA damage, a marker of cellular aging. Together, our results detail a novel pharmacological strategy for prevention and/or reversal of age-related NAD+ decline and subsequent metabolic dysfunction., (Copyright © 2018 Elsevier Inc. All rights reserved.)- Published
- 2018
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44. Genetic deletion of CD38 confers post-ischemic myocardial protection through preserved pyridine nucleotides.
- Author
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Boslett J, Helal M, Chini E, and Zweier JL
- Subjects
- ADP-ribosyl Cyclase 1 metabolism, Animals, Biopterins analogs & derivatives, Biopterins metabolism, Cyclic GMP metabolism, Endothelial Cells metabolism, Glutathione metabolism, Hemodynamics, Male, Mice, Inbred C57BL, Myocardial Contraction, Myocardial Infarction complications, Myocardial Infarction metabolism, Myocardial Infarction pathology, Myocardial Infarction physiopathology, Myocardial Ischemia complications, Myocardial Ischemia pathology, Myocardial Ischemia physiopathology, Myocardial Reperfusion Injury complications, Myocardial Reperfusion Injury metabolism, Myocardial Reperfusion Injury pathology, Myocardial Reperfusion Injury physiopathology, Myocardium metabolism, Myocardium pathology, Myocytes, Cardiac metabolism, NAD metabolism, NADP metabolism, Nitric Oxide metabolism, Nitric Oxide Synthase Type III metabolism, Nucleotidases metabolism, Protein Multimerization, Signal Transduction, Superoxides metabolism, ADP-ribosyl Cyclase 1 genetics, Gene Deletion, Myocardial Ischemia genetics, Nucleotides metabolism, Pyridines metabolism
- Abstract
Following the onset of ischemia/reperfusion (I/R), CD38 activation occurs and is associated with depletion of NAD(P)(H) in the heart as well as myocardial injury and endothelial dysfunction. Studies with pharmacological inhibitors suggest that the NADP
+ -hydrolyzing ability of CD38 can deplete the NAD(P)(H) pools. However, there is a need for more specific studies on the importance of CD38 and its role in the process of endothelial dysfunction and myocardial injury in the post-ischemic heart. Therefore, experiments were performed in hearts of mice with global gene knockout of CD38. Isolated perfused CD38-/- and wild type (WT) mouse hearts were studied to determine the link between CD38 activation, the levels of NADP(H), endothelial dysfunction, and myocardial injury after I/R. Genetic deletion of CD38 preserves the myocardial and endothelial NADP(H) pools compared to WT. Whole heart BH4 levels in CD38-/- hearts were also preserved. Post-ischemic levels of cGMP were greatly depleted in WT hearts, but preserved to near baseline levels in CD38-/- hearts. The preservation of these metabolite pools in CD38-/- hearts was accompanied by near full recovery of NOS-dependent coronary flow, while in WT hearts, severe impairment of endothelial function and NOS uncoupling occurred with decreased NO and enhanced superoxide generation. CD38-/- hearts also exhibited marked protection against I/R with preserved glutathione levels, increased recovery of left ventricular contractile function, decreased myocyte enzyme release, and decreased infarct size. Thus, CD38 activation causes post-ischemic depletion of NADP(H) within the heart, with severe depletion from the endothelium, resulting in endothelial dysfunction and myocardial injury., (Copyright © 2018 Elsevier Ltd. All rights reserved.)- Published
- 2018
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45. Characterization of CD38 in the major cell types of the heart: endothelial cells highly express CD38 with activation by hypoxia-reoxygenation triggering NAD(P)H depletion.
- Author
-
Boslett J, Hemann C, Christofi FL, and Zweier JL
- Subjects
- ADP-ribosyl Cyclase 1 deficiency, ADP-ribosyl Cyclase 1 genetics, Animals, Cell Hypoxia, Coronary Vessels pathology, Endothelial Cells pathology, Enzyme Activation, Fibroblasts metabolism, Membrane Glycoproteins deficiency, Membrane Glycoproteins genetics, Mice, Inbred C57BL, Mice, Knockout, Myocardial Reperfusion Injury genetics, Myocytes, Cardiac enzymology, Nitric Oxide metabolism, Nitric Oxide Synthase Type III metabolism, Rats, Sprague-Dawley, Signal Transduction, Superoxides metabolism, Time Factors, ADP-ribosyl Cyclase metabolism, ADP-ribosyl Cyclase 1 metabolism, Coronary Vessels enzymology, Endothelial Cells enzymology, Membrane Glycoproteins metabolism, Myocardial Reperfusion adverse effects, Myocardial Reperfusion Injury enzymology, NADP metabolism
- Abstract
The NAD(P)
+ -hydrolyzing enzyme CD38 is activated in the heart during the process of ischemia and reperfusion, triggering NAD(P)(H) depletion. However, the presence and role of CD38 in the major cell types of the heart are unknown. Therefore, we characterize the presence and function of CD38 in cardiac myocytes, endothelial cells, and fibroblasts. To comprehensively evaluate CD38 in these cells, we measured gene transcription via mRNA, as well as protein expression and enzymatic activity. Endothelial cells strongly expressed CD38, while only low expression was present in cardiac myocytes with intermediate levels in fibroblasts. In view of this high level expression in endothelial cells and the proposed role of CD38 in the pathogenesis of endothelial dysfunction, endothelial cells were subjected to hypoxia-reoxygenation to characterize the effect of this stress on CD38 expression and activity. An activity-based CD38 imaging method and CD38 activity assays were used to characterize CD38 activity in normoxic and hypoxic-reoxygenated endothelial cells, with marked CD38 activation seen following hypoxia-reoxygenation. To test the impact of hypoxia-reoxygenation-induced CD38 activation on endothelial cells, NAD(P)(H) levels and endothelial nitric oxide synthase (eNOS)-derived NO production were measured. Marked NADP(H) depletion with loss of NO and increase in superoxide production occurred following hypoxia-reoxygenation that was prevented by CD38 inhibition or knockdown. Thus, endothelial cells have high expression of CD38 which is activated by hypoxia-reoxygenation triggering CD38-mediated NADP(H) depletion with loss of eNOS-mediated NO generation and increased eNOS uncoupling. This demonstrates the importance of CD38 in the endothelium and explains the basis by which CD38 triggers post-ischemic endothelial dysfunction.- Published
- 2018
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46. Angiotensin Receptor Expression and Vascular Endothelial Dysfunction in Obstructive Sleep Apnea.
- Author
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Khayat RN, Varadharaj S, Porter K, Sow A, Jarjoura D, Gavrilin MA, and Zweier JL
- Subjects
- Adult, Female, Humans, Male, Middle Aged, Nitric Oxide analysis, Polysomnography methods, Renin-Angiotensin System physiology, Subcutaneous Tissue blood supply, Subcutaneous Tissue pathology, Treatment Outcome, Up-Regulation, Continuous Positive Airway Pressure methods, Endothelium, Vascular metabolism, Endothelium, Vascular physiopathology, Hypertension metabolism, Hypertension physiopathology, Microvessels metabolism, Microvessels pathology, Microvessels physiopathology, Oxidative Stress, Receptor, Angiotensin, Type 1 metabolism, Sleep Apnea, Obstructive metabolism, Sleep Apnea, Obstructive physiopathology, Sleep Apnea, Obstructive therapy
- Abstract
Background: Obstructive sleep apnea (OSA) is associated with vascular endothelial dysfunction (VED) in otherwise healthy patients. The role of renin-angiotensin system (RAS) in the OSA induced VED is not well understood., Methods: Recently diagnosed OSA patients with very low cardiovascular disease (CVD) risk (Framingham score <5%) were studied at diagnosis and after 12 weeks of verified continuous positive airway pressure (CPAP) therapy. Participants underwent biopsy of gluteal subcutaneous tissue at baseline and after CPAP. Microcirculatory endothelial expression of angiotensin receptors type-1 (AT-1) and type-2 (AT-2) was measured in the subcutaneous tissue using quantitative confocal microscopy techniques. The ex-vivo effect of AT-1 receptor blockade (ARB) on endothelial superoxide production was also measured before and after CPAP treatment., Results: In OSA patients (n = 11), microcirculatory endothelial AT1 expression decreased from 873 (200) (fluorescence units) at baseline to 393 (59) units after 12 weeks of CPAP (P = 0.02). AT2 expression did not decrease significantly in these patients (479 (75) to 329 (58) post CPAP (P = 0.08)). The ex-vivo addition of the losartan to the microcirculatory endothelium resulted in decreased superoxide expression in the vascular walls from 14.2 (2.2) units to 4.2 (0.8) P < 0.001; while it had no effect on post-CPAP patient tissue (P = 0.64)., Conclusions: In OSA patients with no to minimal CVD risk, VED is associated with upregulation of AT-1 expression that is reversible with CPAP. Endothelial oxidative stress was reversible with ARB. RAS activation may play an important role in the development of early CVD risk in OSA patients., (© American Journal of Hypertension, Ltd 2017. All rights reserved. For Permissions, please email: journals.permissions@oup.com)
- Published
- 2018
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47. Role of Dietary Antioxidants in the Preservation of Vascular Function and the Modulation of Health and Disease.
- Author
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Varadharaj S, Kelly OJ, Khayat RN, Kumar PS, Ahmed N, and Zweier JL
- Abstract
In vascular diseases, including hypertension and atherosclerosis, vascular endothelial dysfunction (VED) occurs secondary to altered function of endothelial nitric oxide synthase (eNOS). A novel redox regulated pathway was identified through which eNOS is uncoupled due to S -glutathionylation of critical cysteine residues, resulting in superoxide free radical formation instead of the vasodilator molecule, nitric oxide. In addition, the redox sensitive cofactor tetrahydrobiopterin, BH
4 , is also essential for eNOS coupling. Antioxidants, either individually or combined, can modulate eNOS uncoupling by scavenging free radicals or impairing specific radical generating pathways, thus preventing oxidative stress and ameliorating VED. Epidemiological evidence and dietary guidelines suggest that diets high in antioxidants, or antioxidant supplementation, could preserve vascular health and prevent cardiovascular diseases (CVDs). Therefore, the purpose of this review is to highlight the possible role of dietary antioxidants in regulating eNOS function and uncoupling which is critical for maintenance of vascular health with normal blood flow/circulation and prevention of VED. We hypothesize that a conditioned dietary approach with suitable antioxidants may limit systemic oxidation, maintain a beneficial ratio of reduced to oxidized glutathione, and other redox markers, and minimize eNOS uncoupling serving to prevent CVD and possibly other chronic diseases.- Published
- 2017
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48. Thiol-Dependent Reduction of the Triester and Triamide Derivatives of Finland Trityl Radical Triggers O 2 -Dependent Superoxide Production.
- Author
-
Tan X, Chen L, Song Y, Rockenbauer A, Villamena FA, Zweier JL, and Liu Y
- Subjects
- Ascorbic Acid chemistry, Electron Spin Resonance Spectroscopy, Free Radicals chemistry, Glutathione chemistry, Kinetics, Oxidation-Reduction, Spectrophotometry, Ultraviolet, Trityl Compounds chemical synthesis, Oxygen chemistry, Sulfhydryl Compounds chemistry, Superoxides chemistry, Trityl Compounds chemistry
- Abstract
Tetrathiatriaylmethyl (trityl) radicals have found wide biomedical applications as magnetic resonance probes. Trityl radicals and their derivatives are generally stable toward biological reducing agents such as glutathione (GSH) and ascorbate. We demonstrate that the triester (ET-03) and triamide (AT-03) derivatives of the Finland trityl radical exhibit unique reduction by thiols such as GSH and cysteine (Cys) to generate the corresponding trityl carbanions as evidenced by the loss of EPR signal and appearance of characteristic UV-vis absorbance at 644 nm under anaerobic conditions. The trityl carbanions can be quickly converted back to the original trityl radicals by oxygen (O
2 ) in air, thus rendering the reaction between the trityl derivative and biothiol undetectable under aerobic conditions. The reduction product of O2 by the trityl carbanions was shown to be superoxide radical (O2 •- ) by EPR spin-trapping. Kinetic studies showed that the reaction rate constants (k) depend on the types of both trityl radicals and thiols with the order of kET-03/Cys (0.336 M-1 s-1 ) > kET-03/GSH (0.070 M-1 s-1 ) > kAT-03/Cys (0.032 M-1 s-1 ) > kAT-03/GSH (0.027 M-1 s-1 ). The reactivity of trityl radicals with thiols is closely related to the para-substituents of trityl radicals as well as the pKa of the thiols and is further reflected by the rate of O2 •- production and consumptions of O2 and thiols. This novel reaction represents a new metabolic process of trityl derivatives and should be considered in the design and application of new trityl radical probes.- Published
- 2017
- Full Text
- View/download PDF
49. Nitro-Triarylmethyl Radical as Dual Oxygen and Superoxide Probe.
- Author
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Driesschaert B, Bobko AA, Khramtsov VV, and Zweier JL
- Subjects
- Electrochemical Techniques, Electron Spin Resonance Spectroscopy, Hydrogen-Ion Concentration, Molecular Probes chemical synthesis, Oxygen chemistry, Solutions, Superoxides chemistry, Trityl Compounds chemical synthesis, Water chemistry, Xanthine Oxidase chemistry, Molecular Probes chemistry, Oxygen analysis, Superoxides analysis, Trityl Compounds chemistry
- Abstract
Superoxide radical is involved in numerous physiological and pathophysiological processes. Tetrathiatriarylmethyl (TAM) radicals are known to react with superoxide allowing measurement of superoxide production in biological media. We report the synthesis of a Nitro conjugated TAM radical showing a rate constant of 7 × 10
5 M-1 s-1 which is two order of magnitude higher than other TAMs, allowing high sensitivity measurement of superoxide.- Published
- 2017
- Full Text
- View/download PDF
50. Oxygen binding and nitric oxide dioxygenase activity of cytoglobin are altered to different extents by cysteine modification.
- Author
-
Zhou D, Hemann C, Boslett J, Luo A, Zweier JL, and Liu X
- Abstract
Cytoglobin (Cygb), like other members of the globin family, is a nitric oxide (NO) dioxygenase, metabolizing NO in an oxygen (O
2 )-dependent manner. We examined the effect of modification of cysteine sulfhydryl groups of Cygb on its O2 binding and NO dioxygenase activity. The two cysteine sulfhydryls of Cygb were modified to form either an intramolecular disulfide bond (Cygb_SS), thioether bonds to N -ethylmaleimide (NEM; Cygb_SC), or were maintained as free SH groups (Cygb_SH). It was observed that the NO dioxygenase activity of Cygb only slightly changed (~ 25%) while the P50 of O2 binding to Cygb changed over four-fold with these modifications. Our results suggest that it is possible to separately regulate one Cygb function (such as O2 binding) without largely affecting the other Cygb functions (such as its NO dioxygenase activity).- Published
- 2017
- Full Text
- View/download PDF
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