RESUMO
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) that 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 Km=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 lungs of control mice with 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.
RESUMO
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 108 M-1 â s-1 Spin-trapping studies also demonstrated that the rate of Cygb-mediated superoxide dismutation (1.6 × 108 M-1 â s-1) was only â¼10-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.
Assuntos
Citoglobina , Superóxido Dismutase , Animais , Linhagem Celular , Citoglobina/química , Citoglobina/genética , Citoglobina/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Masculino , Camundongos , Camundongos Knockout , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/química , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismoRESUMO
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 (O2) levels. Using the deuterated analogue, dMTP5, which has an optimal pKa 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.
Assuntos
Oxigênio , Substâncias Redutoras , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Oxigênio/química , Oxirredução , Concentração de Íons de HidrogênioRESUMO
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 â¼78% of NO metabolism in SMCs is Cygb-dependent. Of this, â¼87% was B5R- and B5-dependent. CPR knockdown resulted in a small decrease in the NO dioxygenation rate (VNO), 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.
Assuntos
Citocromos b5/metabolismo , Citoglobina/metabolismo , Músculo Liso Vascular/metabolismo , Óxido Nítrico/metabolismo , Oxigenases/metabolismo , Animais , Fenômenos Bioquímicos , Células Cultivadas , Humanos , Cinética , CamundongosRESUMO
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.
Assuntos
Doenças Cardiovasculares , Sistemas Eletrônicos de Liberação de Nicotina , Hipertensão , Animais , Endotélio Vascular/metabolismo , Feminino , Masculino , Camundongos , NADPH Oxidases/metabolismo , Nicotina , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Ácido Peroxinitroso/metabolismo , Superóxidos/metabolismoRESUMO
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.
Assuntos
Citoglobina/metabolismo , Miócitos de Músculo Liso/metabolismo , Óxido Nítrico/metabolismo , Poluição por Fumaça de Tabaco/efeitos adversos , Animais , Aorta/citologia , Sobrevivência Celular/efeitos dos fármacos , Citocromo-B(5) Redutase/metabolismo , Citocromos b5/metabolismo , Citoglobina/genética , Técnicas de Silenciamento de Genes , Camundongos , Músculo Liso Vascular/citologia , Superóxidos/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
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 SO4â¢- predominated. Therefore, the DMPO-OH signal is derived from SO4â¢- 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.
Assuntos
Óxidos N-Cíclicos , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Radicais Livres , Cinética , Marcadores de Spin , Detecção de Spin/métodos , TemperaturaRESUMO
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 (PrSO3H) 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 PrSO3H levels at the C236 ligand of the [2Fe2S] cluster of the Rieske ironsulfur protein (uqcrfs1), thus impairing the electron transport activity. MS analysis also indicated increased PrSO3H 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 PrSO3H 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 PrSO3H detected in the mitochondria of sham control hearts, suggesting that the physiologic hyperoxygenation and ROS overproduction during reperfusion mediated the enhancement of complex III PrSO3H. In conclusion, reperfusion-mediated heme damage with increased PrSO3H controls oxidative injury to complex III and aggravates mitochondrial dysfunction in the post-ischemic heart.
Assuntos
Cisteína/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Heme/metabolismo , Isquemia Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Animais , Derivados de Benzeno/química , Bovinos , Cisteína/química , Citocromos c1/química , Citocromos c1/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/química , Heme/química , Masculino , Camundongos Transgênicos , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Isquemia Miocárdica/metabolismo , Ácido Peroxinitroso/química , Ratos Sprague-Dawley , Superóxido Dismutase/genéticaRESUMO
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.
Assuntos
Apoptose , Cardiomiopatia Dilatada/metabolismo , Doxorrubicina , Proteínas de Choque Térmico/genética , Chaperonas Moleculares/genética , Mutação , Miocárdio/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Cardiomiopatia Dilatada/induzido quimicamente , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/patologia , Cardiotoxicidade , Células Cultivadas , Modelos Animais de Doenças , Feminino , Proteínas de Choque Térmico/metabolismo , Masculino , Camundongos Transgênicos , Chaperonas Moleculares/metabolismo , Miocárdio/patologia , Cadeias Pesadas de Miosina/genética , Fosforilação , Serina , Transdução de SinaisRESUMO
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.
Assuntos
Aorta/efeitos dos fármacos , Doenças Cardiovasculares/induzido quimicamente , Nicotina/administração & dosagem , Vaping/efeitos adversos , Agonistas de Receptores Adrenérgicos alfa 1/farmacologia , Animais , Aorta/fisiopatologia , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Doenças Cardiovasculares/fisiopatologia , Sistemas Eletrônicos de Liberação de Nicotina , Masculino , Camundongos , Fenilefrina/farmacologia , Fatores de Tempo , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologiaRESUMO
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 B1 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.
Assuntos
Imageamento por Ressonância Magnética , Animais , Espectroscopia de Ressonância de Spin Eletrônica , Camundongos , Imagens de Fantasmas , CintilografiaRESUMO
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 p22phox 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.
Assuntos
Endotélio Vascular/metabolismo , Leucócitos/metabolismo , Estresse Oxidativo , Lesão por Inalação de Fumaça/metabolismo , Poluição por Fumaça de Tabaco/efeitos adversos , Vasodilatação , Animais , Aorta/metabolismo , Aorta/fisiopatologia , Pressão Sanguínea , Endotélio Vascular/fisiopatologia , Masculino , Artérias Mesentéricas/metabolismo , Artérias Mesentéricas/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , NADPH Oxidases/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Lesão por Inalação de Fumaça/etiologia , Lesão por Inalação de Fumaça/fisiopatologia , Superóxidos/metabolismoRESUMO
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.
Assuntos
Aptâmeros de Nucleotídeos/farmacologia , Arteriopatias Oclusivas/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos/métodos , Fibrinolíticos/farmacologia , Trombose/tratamento farmacológico , Trombose/prevenção & controle , Fator de von Willebrand/antagonistas & inibidores , Animais , Antídotos/farmacologia , Aptâmeros de Nucleotídeos/síntese química , Aptâmeros de Nucleotídeos/metabolismo , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Lesões das Artérias Carótidas/tratamento farmacológico , Cães , Relação Dose-Resposta a Droga , Feminino , Voluntários Saudáveis , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oligonucleotídeos/farmacologia , Adesividade Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Fator de von Willebrand/metabolismoRESUMO
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 O2, 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.
Assuntos
Sistemas Eletrônicos de Liberação de Nicotina , Testes de Toxicidade/instrumentação , Administração por Inalação , Animais , Dióxido de Carbono/análise , Monóxido de Carbono/análise , Cotinina/sangue , Desenho de Equipamento , Umidade , Masculino , Camundongos Endogâmicos C57BL , Oxigênio/análise , Material Particulado/análise , Material Particulado/toxicidade , TemperaturaRESUMO
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.
Assuntos
Técnicas Biossensoriais/métodos , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Mesilatos/química , Compostos de Sulfidrila/química , HumanosRESUMO
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 µM 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.
Assuntos
ADP-Ribosil Ciclase 1/antagonistas & inibidores , Inibidores de Glicosídeo Hidrolases/química , Inibidores de Glicosídeo Hidrolases/farmacologia , Coração/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Quinazolinas/química , Quinazolinas/farmacologia , Animais , Transporte Biológico , Biopterinas/análogos & derivados , Biopterinas/metabolismo , Endotélio/efeitos dos fármacos , Endotélio/metabolismo , Inibidores de Glicosídeo Hidrolases/metabolismo , Coração/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Contração Miocárdica/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/fisiopatologia , NADP/metabolismo , Óxido Nítrico Sintase/metabolismo , Quinazolinas/metabolismoRESUMO
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.
Assuntos
Espectroscopia de Ressonância de Spin Eletrônica/métodos , Imageamento Tridimensional/métodos , Imageamento por Ressonância Magnética/métodos , Animais , Radicais Livres , Coração/diagnóstico por imagem , Imagens de Fantasmas , RatosRESUMO
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 (H2O2), 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.
Assuntos
Catalase/administração & dosagem , Fígado/cirurgia , Polietilenoglicóis/administração & dosagem , Traumatismo por Reperfusão/tratamento farmacológico , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Peróxido de Hidrogênio/farmacologia , Injeções Intralesionais , Fígado/irrigação sanguínea , Fígado/citologia , Masculino , Estresse Oxidativo/efeitos dos fármacos , Cultura Primária de Células , Ratos , Traumatismo por Reperfusão/sangue , Traumatismo por Reperfusão/etiologia , Resultado do Tratamento , Isquemia Quente/efeitos adversosRESUMO
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.
Assuntos
Aloenxertos/efeitos dos fármacos , Leucina Encefalina-2-Alanina/farmacologia , Fígado/efeitos dos fármacos , Soluções para Preservação de Órgãos/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Aloenxertos/metabolismo , Aloenxertos/patologia , Animais , Modelos Animais de Doenças , Hepatócitos , Humanos , Fígado/metabolismo , Fígado/patologia , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Perfusão/efeitos adversos , Perfusão/métodos , Cultura Primária de Células , Ratos , Receptores Opioides delta/metabolismo , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/patologia , Coleta de Tecidos e Órgãos/efeitos adversos , Coleta de Tecidos e Órgãos/métodosRESUMO
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 (diC12PO) 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 diC12PO in cell-free solution. Confocal microscopy of bovine aortic endothelial cells treated sequentially with diC12PO 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.