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ABSTRACT: Acute hyperhemolysis is a severe life-threatening complication in patients with sickle cell disease (SCD) that may occur during delayed hemolytic transfusion reaction (DHTR), or vaso-occlusive crises associated with multiorgan failure. Here, we developed in vitro and in vivo animal models to mimic endothelial damage during the early phase of hyperhemolysis in SCD. We then used the carbon monoxide (CO)-releasing molecule CORM-401 and examined its effects against endothelial activation, damage, and inflammation inflicted by hemolysates containing red blood cell membrane-derived particles. The in vitro results revealed that CORM-401: (1) prevented the upregulation of relevant proinflammatory and proadhesion markers controlled by the NF-κB enhancer of activated B cells, and (2) abolished the expression of the nuclear factor erythroid-2-related factor 2 (Nrf2) that regulates the inducible antioxidant cell machinery. We also show in SCD mice that CORM-401 protects against hemolysate-induced acute damage of target organs such as the lung, liver, and kidney through modulation of NF-κB proinflammatory and Nrf2 antioxidant pathways. Our data demonstrate the efficacy of CORM-401 as a novel therapeutic agent to counteract hemolysate-induced organ damage during hyperhemolysis in SCD. This approach might be considered as possible preventive treatment in high-risk situations such as patients with SCD with history of DHTR.
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Anemia de Células Falciformes , Monóxido de Carbono , Hemólisis , Factor 2 Relacionado con NF-E2 , Anemia de Células Falciformes/tratamiento farmacológico , Anemia de Células Falciformes/complicaciones , Animales , Ratones , Monóxido de Carbono/farmacología , Humanos , Hemólisis/efectos de los fármacos , Factor 2 Relacionado con NF-E2/metabolismo , Administración Oral , Modelos Animales de Enfermedad , Masculino , Ratones Endogámicos C57BLRESUMEN
Simultaneous poisoning by carbon monoxide (CO) and hydrogen cyanide is the major cause of mortality in fire gas accidents. Here, we report on the invention of an injectable antidote against CO and cyanide (CN-) mixed poisoning. The solution contains four compounds: iron(III)porphyrin (FeIIITPPS, F), two methyl-ß-cyclodextrin (CD) dimers linked by pyridine (Py3CD, P) and imidazole (Im3CD, I), and a reducing agent (Na2S2O4, S). When these compounds are dissolved in saline, the solution contains two synthetic heme models including a complex of F with P (hemoCD-P) and another one of F with I (hemoCD-I), both in their iron(II) state. hemoCD-P is stable in its iron(II) state and captures CO more strongly than native hemoproteins, while hemoCD-I is readily autoxidized to its iron(III) state to scavenge CN- once injected into blood circulation. The mixed solution (hemoCD-Twins) exhibited remarkable protective effects against acute CO and CN- mixed poisoning in mice (~85% survival vs. 0% controls). In a model using rats, exposure to CO and CN- resulted in a significant decrease in heart rate and blood pressure, which were restored by hemoCD-Twins in association with decreased CO and CN- levels in blood. Pharmacokinetic data revealed a fast urinary excretion of hemoCD-Twins with an elimination half-life of 47 min. Finally, to simulate a fire accident and translate our findings to a real-life scenario, we confirmed that combustion gas from acrylic cloth caused severe toxicity to mice and that injection of hemoCD-Twins significantly improved the survival rate, leading to a rapid recovery from the physical incapacitation.
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Monóxido de Carbono , Porfirinas , Ratas , Ratones , Animales , Antídotos/farmacología , Oxígeno , Compuestos Férricos , Cianuros/toxicidad , Hierro , Compuestos FerrososRESUMEN
Bioenergetic metabolism is a key regulator of cellular function and signaling, but how it can instruct the behavior of cells and their fate during embryonic development remains largely unknown. Here, we investigated the role of glucose metabolism in the development of avian trunk neural crest cells (NCCs), a migratory stem cell population of the vertebrate embryo. We uncovered that trunk NCCs display glucose oxidation as a prominent metabolic phenotype, in contrast to what is seen for cranial NCCs, which instead rely on aerobic glycolysis. In addition, only one pathway downstream of glucose uptake is not sufficient for trunk NCC development. Indeed, glycolysis, mitochondrial respiration and the pentose phosphate pathway are all mobilized and integrated for the coordinated execution of diverse cellular programs, epithelial-to-mesenchymal transition, adhesion, locomotion, proliferation and differentiation, through regulation of specific gene expression. In the absence of glucose, the OXPHOS pathway fueled by pyruvate failed to promote trunk NCC adaptation to environmental stiffness, stemness maintenance and fate-decision making. These findings highlight the need for trunk NCCs to make the most of the glucose pathway potential to meet the high metabolic demands appropriate for their development.
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Glucosa , Cresta Neural , Codorniz , Codorniz/crecimiento & desarrollo , Codorniz/metabolismo , Animales , Cresta Neural/crecimiento & desarrollo , Cresta Neural/metabolismo , Glucosa/metabolismo , Tubo Neural/citología , Células Cultivadas , Técnicas In Vitro , Fosforilación Oxidativa , Redes y Vías Metabólicas , Adhesión CelularRESUMEN
Metal carbonyls have been developed as carbon monoxide-releasing molecules (CO-RMs) to deliver CO for therapeutic purposes. The manganese-based CORM-401 has been recently reported to exert beneficial effects in obese animals by reducing body weight gain, improving glucose metabolism and reprogramming adipose tissue towards a healthy phenotype. Here, we report on the synthesis and characterization of glyco-CORMs, obtained by grafting manganese carbonyls on dextrans (70 and 40 kDa), based on the fact that polysaccharides facilitate the targeting of drugs to adipose tissue. We found that glyco-CORMs efficiently deliver CO to cells in vitro with higher CO accumulation in adipocytes compared to other cell types. Oral administration of two selected glyco-CORMs (5b and 6b) resulted in CO accumulation in various organs, including adipose tissue. In addition, glyco-CORM 6b administered for eight weeks elicited anti-obesity and positive metabolic effects in mice fed a high fat diet. Our study highlights the feasibility of creating carriers with multiple functionalized CO-RMs.
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Monóxido de Carbono , Compuestos Organometálicos , Ratones , Animales , Monóxido de Carbono/metabolismo , Manganeso , Obesidad/tratamiento farmacológico , Obesidad/metabolismo , Aumento de Peso , Polisacáridos , Compuestos Organometálicos/farmacologíaRESUMEN
Understanding the processes governing the ability of the heart to repair and regenerate after injury is crucial for developing translational medical solutions. New avenues of exploration include cardiac cell therapy and cellular reprogramming targeting cell death and regeneration. An attractive possibility is the exploitation of cytoprotective genes that exist solely for self-preservation processes and serve to promote and support cell survival. Although the antioxidant and heat-shock proteins are included in this category, one enzyme that has received a great deal of attention as a master protective sentinel is heme oxygenase-1 (HO-1), the rate-limiting step in the catabolism of heme into the bioactive signaling molecules carbon monoxide, biliverdin, and iron. The remarkable cardioprotective effects ascribed to heme oxygenase-1 are best evidenced by its ability to regulate inflammatory processes, cellular signaling, and mitochondrial function ultimately mitigating myocardial tissue injury and the progression of vascular-proliferative disease. We discuss here new insights into the role of heme oxygenase-1 and heme on cardiovascular health, and importantly, how they might be leveraged to promote heart repair after injury.
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Monóxido de Carbono/metabolismo , Hemo-Oxigenasa 1/metabolismo , Isquemia Miocárdica/metabolismo , Miocardio/metabolismo , Animales , Hemo-Oxigenasa 1/genética , Humanos , Mitocondrias Cardíacas/metabolismoRESUMEN
Carbon monoxide (CO) is continuously produced in mammalian cells during the degradation of heme. It is a stable gaseous molecule that reacts selectively with transition metals in a specific redox state, and these characteristics restrict the interaction of CO with defined biological targets that transduce its signaling activity. Because of the high affinity of CO for ferrous heme, these targets can be grouped into heme-containing proteins, representing a large variety of sensors and enzymes with a series of diverse function in the cell and the organism. Despite this notion, progress in identifying which of these targets are selective for CO has been slow and even the significance of elevated carbonmonoxy hemoglobin, a classical marker used to diagnose CO poisoning, is not well understood. This is also due to the lack of technologies capable of assessing in a comprehensive fashion the distribution and local levels of CO between the blood circulation, the tissue, and the mitochondria, one of the cellular compartments where CO exerts its signaling or detrimental effects. Nevertheless, the use of CO gas and CO-releasing molecules as pharmacological approaches in models of disease has provided new important information about the signaling properties of CO. In this review we will analyze the most salient effects of CO in biology and discuss how the binding of CO with key ferrous hemoproteins serves as a posttranslational modification that regulates important processes as diverse as aerobic metabolism, oxidative stress, and mitochondrial bioenergetics.
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Velocidad del Flujo Sanguíneo/fisiología , Vasos Sanguíneos/fisiología , Monóxido de Carbono/metabolismo , Hemo/metabolismo , Modelos Biológicos , Transducción de Señal/fisiología , Animales , HumanosRESUMEN
Carbon monoxide (CO) is produced in mammalian cells during heme metabolism and serves as an important signaling messenger. Here we report the bioactive properties of selective CO scavengers, hemoCD1 and its derivative R8-hemoCD1, which have the ability to detect and remove endogenous CO in cells. HemoCD1 is a supramolecular hemoprotein-model complex composed of 5,10,15,20-tetrakis(4-sulfonatophenyl)porphinatoiron(II) and a per-O-methylated ß-cyclodextrin dimer having an pyridine linker. We demonstrate that hemoCD1 can be used effectively to quantify endogenous CO in cell lysates by a simple spectrophotometric method. The hemoCD1 assay detected ca. 260 pmol of CO in 106 hepatocytes, which was well-correlated with the amount of intracellular bilirubin, the final breakdown product of heme metabolism. We then covalently attached an octaarginine peptide to a maleimide-appended hemoCD1 to synthesize R8-hemoCD1, a cell-permeable CO scavenger. Indeed, R8-hemoCD1 was taken up by intact cells and captured intracellular CO with high efficiency. Moreover, we revealed that removal of endogenous CO by R8-hemoCD1 in cultured macrophages led to a significant increase (ca. 2.5-fold) in reactive oxygen species production and exacerbation of inflammation after challenge with lipopolysaccharide. Thus, R8-hemoCD1 represents a powerful expedient for exploring specific and still unidentified biological functions of CO in cells.
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Monóxido de Carbono/análisis , Hemoproteínas/química , Modelos Biológicos , Animales , Monóxido de Carbono/metabolismo , Células Cultivadas , Hemoproteínas/metabolismo , Células Hep G2 , Humanos , Ratones , Microscopía Confocal , Estructura Molecular , Células RAW 264.7RESUMEN
Carbon monoxide (CO), a product of heme degradation by heme oxygenases, plays an important role in vascular homeostasis. Recent evidence indicates that mitochondria are among a number of molecular targets that mediate the cellular actions of CO. In the present study we characterized the effects of CO released from CORM-401 on mitochondrial respiration and glycolysis in intact human endothelial cells using electron paramagnetic resonance (EPR) oximetry and the Seahorse XF technology. We found that CORM-401 (10-100µM) induced a persistent increase in the oxygen consumption rate (OCR) that was accompanied by inhibition of glycolysis (extracellular acidification rate, ECAR) and a decrease in ATP-turnover. Furthermore, CORM-401 increased proton leak, diminished mitochondrial reserve capacity and enhanced non-mitochondrial respiration. Inactive CORM-401 (iCORM-401) neither induced mitochondrial uncoupling nor inhibited glycolysis, supporting a direct role of CO in the endothelial metabolic response induced by CORM-401. Interestingly, blockade of mitochondrial large-conductance calcium-regulated potassium ion channels (mitoBKCa) with paxilline abolished the increase in OCR promoted by CORM-401 without affecting ECAR; patch-clamp experiments confirmed that CO derived from CORM-401 activated mitoBKCa channels present in mitochondria. Conversely, stabilization of glycolysis by MG132 prevented CORM-401-mediated decrease in ECAR but did not modify the OCR response. In summary, we demonstrated in intact endothelial cells that CO induces a two-component metabolic response: uncoupling of mitochondrial respiration dependent on the activation of mitoBKCa channels and inhibition of glycolysis independent of mitoBKCa channels.
RESUMEN
We hypothesized that O2 tension influences the redox state and the immunomodulatory responses of inflammatory cells to dimethyl fumarate (DMF), an activator of the nuclear factor Nrf2 that controls antioxidant genes expression. This concept was investigated in macrophages permanently cultured at either physiological (5% O2) or atmospheric (20% O2) oxygen levels and then treated with DMF or challenged with lipopolysaccharide (LPS) to induce inflammation. RAW 264.7 macrophages cultured at 20% O2 exhibited a pro-oxidant phenotype, reflected by a lower content of reduced glutathione, higher oxidized glutathione and increased production of reactive oxygen species when compared to macrophages continuously grown at 5% O2. At 20% O2, DMF induced a stronger antioxidant response compared to 5% O2 as evidenced by a higher expression of heme oxygenase-1, NAD(P)H:quinone oxydoreductase-1 and superoxide dismutase-2. After challenge of macrophages with LPS, several pro-inflammatory (iNOS, TNF-α, MMP-2, MMP-9), anti-inflammatory (arginase-1, IL-10) and pro-angiogenic (VEGF-A) mediators were evaluated in the presence or absence of DMF. All markers, with few interesting exceptions, were significantly reduced at 5% O2. This study brings new insights on the effects of O2 in the cellular adaptation to oxidative and inflammatory stimuli and highlights the importance of characterizing the effects of chemicals and drugs at physiologically relevant O2 tension. Our results demonstrate that the common practice of culturing cells at atmospheric O2 drives the endogenous cellular environment towards an oxidative stress phenotype, affecting inflammation and the expression of antioxidant pathways by exogenous modulators.
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Antioxidantes/farmacología , Técnicas de Cultivo de Célula/métodos , Fumaratos/farmacología , Factores Inmunológicos/farmacología , Macrófagos/citología , Oxígeno/farmacología , Animales , Antioxidantes/metabolismo , Células Cultivadas , Dimetilfumarato , Regulación de la Expresión Génica/efectos de los fármacos , Hemo-Oxigenasa 1/metabolismo , Inflamación/patología , Mediadores de Inflamación/metabolismo , Lipopolisacáridos , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Ratones , Óxido Nítrico Sintasa de Tipo II/metabolismo , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Consumo de Oxígeno/efectos de los fármacos , Consumo de Oxígeno/genética , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Retinal pigment epithelial cells exert an important supporting role in the eye and develop adaptive responses to oxidative stress or high glucose levels, as observed during diabetes. Endogenous antioxidant defences are mainly regulated by Nrf2, a transcription factor that is activated by naturally-derived and electrophilic compounds. Here we investigated the effect of the Nrf2 activators dimethylfumarate (DMF) and carnosol on antioxidant pathways, oxygen consumption rate and wound healing in human retinal pigment epithelial cells (ARPE-19) cultured in medium containing normal (NG, 5mM) or high (HG, 25 mM) glucose levels. We also assessed wound healing using an in vivo corneal epithelial injury model. We found that Nrf2 nuclear translocation and heme oxygenase activity increased in ARPE cells treated with 10 µM DMF or carnosol irrespective of glucose culture conditions. However, HG rendered retinal cells more sensitive to regulators of glutathione synthesis or inhibition and caused a decrease of both cellular and mitochondrial reactive oxygen species. Culture in HG also reduced ATP production and mitochondrial function as measured with the Seahorse XF analyzer and electron microscopy analysis revealed morphologically damaged mitochondria. Acute treatment with DMF or carnosol did not restore mitochondrial function in HG cells; conversely, the compounds reduced cellular maximal respiratory and reserve capacity, which were completely prevented by N-acetylcysteine thus suggesting the involvement of thiols in this effect. Interestingly, the scratch assay showed that wound closure was faster in cells cultured in HG than NG and was accelerated by carnosol. This effect was reversed by an inhibitor of heme oxygenase activity. Moreover, topical application of carnosol to the cornea of diabetic rats significantly accelerated wound healing. In summary, these data indicate that culture of retinal epithelial cells in HG does not affect the activation of the Nrf2/heme oxygenase axis but influences other crucial oxidative and mitochondrial-dependent cellular functions. The additional effect on wound closure suggests that results obtained in in vitro experimental settings need to be carefully evaluated in the context of the glucose concentrations used in cell culture.
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Metabolismo Energético/fisiología , Células Epiteliales/metabolismo , Glucosa/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/fisiología , Retina/metabolismo , Abietanos/metabolismo , Acetilcisteína/metabolismo , Animales , Antioxidantes/metabolismo , Línea Celular , Hemo Oxigenasa (Desciclizante)/metabolismo , Humanos , Masculino , Mitocondrias/metabolismo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The transcription factor Nrf2 and its downstream target heme oxygenase-1 (HO-1) are essential protective systems against oxidative stress and inflammation. The products of HO-1 enzymatic activity, biliverdin and carbon monoxide (CO), actively contribute to this protection, suggesting that exploitation of these cellular systems may offer new therapeutic avenues in a variety of diseases. Starting from a CO-releasing compound and a chemical scaffold exhibiting electrophilic characteristics (esters of fumaric acid), we report the synthesis of hybrid molecules that simultaneously activate Nrf2 and liberate CO. These hybrid compounds, which we termed "HYCOs", release CO to myoglobin and activate the CO-sensitive fluorescent probe COP-1, while also potently inducing nuclear accumulation of Nrf2 and HO-1 expression and activity in different cell types. Thus, we provide here the first example of a new class of pharmacologically active molecules that target the HO-1 pathway by combining an Nrf2 activator coordinated to a CO-releasing group.
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Monóxido de Carbono/química , Monóxido de Carbono/farmacología , Complejos de Coordinación/química , Complejos de Coordinación/farmacología , Hemo-Oxigenasa 1/química , Factor 2 Relacionado con NF-E2/metabolismo , Alquinos/química , Animales , Cobalto/química , Complejos de Coordinación/síntesis química , Diseño de Fármacos , Ésteres/química , Hemo-Oxigenasa 1/metabolismo , Macrófagos/efectos de los fármacos , Ratones , Estrés Oxidativo/efectos de los fármacosRESUMEN
Heme oxygenase-1 (HO-1) is a redox sensitive inducible enzyme endowed with important antioxidant and cytoprotective activities. Here we report that two water-soluble isothiocyanate-cysteine conjugates, S-[N-benzyl(thiocarbamoyl)]-l-cysteine (BTTC) and S-[N-(3-phenylpropyl)(thiocarbamoyl)]-l-cysteine (PTTC), potently increase HO-1 protein expression and heme oxygenase activity in renal tubular epithelial cells at 5 and 10µM, while higher concentrations are themselves cytotoxic and pro-apoptotic. Inhibitors of the pro-survival pathways ERK, MAPK and PI3K almost completely abolished the increase in HO-1 induction and heme oxygenase activity, while the JNK pathway appeared to be mainly involved in the apoptosis triggered by the isothiocyanates. We also found that renal cells exposed to 50µM cisplatin (CDDP), a chemotherapeutic agent known for its nephrotoxic actions, displayed a marked increase in caspase-3 activity and the number of apoptotic cells. These effects were abolished by pre-incubation of cells with concentrations of BTCC or PTCC that maximize HO-1 induction and were reversed by the inhibitor of heme oxygenase activity tin protoporphyrin IX (SnPPIX). Moreover, in a model of CDDP-induced nephrotoxicity in vivo, pre-treatment of rats with a daily dose of BTCC or PTCC (25mg/kg, i.p.) completely abolished the increase in serum creatinine and urea levels and markedly reduced the severity of renal tissue apoptosis caused by CDDP. The renoprotective effects of BTCC and PTCC in vivo were markedly attenuated by administration of rats with SnPPIX. These findings indicate that water-soluble isothiocyanates counteract renal dysfunction and apoptosis by up-regulating the HO-1 system and could be used as a supplementary treatment to mitigate CDDP-induced nephrotoxic effects.
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Cisteína/análogos & derivados , Riñón/efectos de los fármacos , Sustancias Protectoras/farmacología , Tiocarbamatos/farmacología , Animales , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Supervivencia Celular/efectos de los fármacos , Cisplatino , Creatinina/sangre , Cisteína/farmacología , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Riñón/metabolismo , Riñón/patología , Células LLC-PK1 , Masculino , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , ARN Mensajero/metabolismo , Ratas Wistar , Porcinos , Urea/sangreRESUMEN
Carbon monoxide (CO), a gaseous signaling molecule, has shown promise in preventing body weight gain and metabolic dysfunction induced by high fat diet (HFD), but the mechanisms underlying these effects are largely unknown. An essential component in response to HFD is the gut microbiome, which is significantly altered during obesity and represents a target for developing new therapeutic interventions to fight metabolic diseases. Here, we show that CO delivered to the gut by oral administration with a CO-releasing molecule (CORM-401) accumulates in faeces and enriches a variety of microbial species that were perturbed by a HFD regimen. Notably, Akkermansia muciniphila, which exerts salutary metabolic effects in mice and humans, was strongly depleted by HFD but was the most abundant gut species detected after CORM-401 treatment. Analysis of bacterial transcripts revealed a restoration of microbial functional activity, with partial or full recovery of the Krebs cycle, ß-oxidation, respiratory chain and glycolysis. Mice treated with CORM-401 exhibited normalization of several plasma and fecal metabolites that were disrupted by HFD and are dependent on Akkermansia muciniphila's metabolic activity, including indoles and tryptophan derivatives. Finally, CORM-401 treatment led to an improvement in gut morphology as well as reduction of inflammatory markers in colon and cecum and restoration of metabolic profiles in these tissues. Our findings provide therapeutic insights on the efficacy of CO as a potential prebiotic to combat obesity, identifying the gut microbiota as a crucial target for CO-mediated pharmacological activities against metabolic disorders.
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Monóxido de Carbono , Dieta Alta en Grasa , Microbioma Gastrointestinal , Obesidad , Animales , Microbioma Gastrointestinal/efectos de los fármacos , Ratones , Obesidad/metabolismo , Obesidad/tratamiento farmacológico , Obesidad/microbiología , Monóxido de Carbono/metabolismo , Dieta Alta en Grasa/efectos adversos , Administración Oral , Akkermansia/efectos de los fármacos , Masculino , Heces/microbiología , Modelos Animales de Enfermedad , Ratones Endogámicos C57BLRESUMEN
Activation of inflammation is tightly associated with metabolic reprogramming in macrophages. The iron-containing tetrapyrrole heme can induce pro-oxidant and pro-inflammatory effects in murine macrophages, but has been associated with polarization towards an anti-inflammatory phenotype in human macrophages. In the current study, we compared the regulatory responses to heme and the prototypical Toll-like receptor (TLR)4 ligand lipopolysaccharide (LPS) in human and mouse macrophages with a particular focus on alterations of cellular bioenergetics. In human macrophages, bulk RNA-sequencing analysis indicated that heme led to an anti-inflammatory transcriptional profile, whereas LPS induced a classical pro-inflammatory gene response. Co-stimulation of heme with LPS caused opposing regulatory patterns of inflammatory activation and cellular bioenergetics in human and mouse macrophages. Specifically, in LPS-stimulated murine, but not human macrophages, heme led to a marked suppression of oxidative phosphorylation and an up-regulation of glycolysis. The species-specific alterations in cellular bioenergetics and inflammatory responses to heme were critically dependent on the availability of nitric oxide (NO) that is generated in inflammatory mouse, but not human macrophages. Accordingly, studies with an inducible nitric oxide synthase (iNOS) inhibitor in mouse, and a pharmacological NO donor in human macrophages, reveal that NO is responsible for the opposing effects of heme in these cells. Taken together, the current findings indicate that NO is critical for the immunomodulatory role of heme in macrophages.
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Hemo , Inflamación , Lipopolisacáridos , Macrófagos , Óxido Nítrico , Humanos , Hemo/metabolismo , Animales , Óxido Nítrico/metabolismo , Ratones , Macrófagos/metabolismo , Macrófagos/efectos de los fármacos , Lipopolisacáridos/farmacología , Inflamación/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Fosforilación Oxidativa/efectos de los fármacos , Metabolismo Energético/efectos de los fármacos , Glucólisis/efectos de los fármacosRESUMEN
The cytoprotective transcription factor NRF2 regulates the expression of several hundred genes in mammalian cells and is a promising therapeutic target in a number of diseases associated with oxidative stress and inflammation. Hence, an ability to monitor basal and inducible NRF2 signalling is vital for mechanistic understanding in translational studies. Due to some caveats related to the direct measurement of NRF2 levels, the modulation of NRF2 activity is typically determined by measuring changes in the expression of one or more of its target genes and/or the associated protein products. However, there is a lack of consensus regarding the most relevant set of these genes/proteins that best represents NRF2 activity across cell types and species. We present the findings of a comprehensive literature search that according to stringent criteria identifies GCLC, GCLM, HMOX1, NQO1, SRXN1 and TXNRD1 as a robust panel of markers that are directly regulated by NRF2 in multiple cell and tissue types. We assess the relevance of these markers in clinically accessible biofluids and highlight future challenges in the development and use of NRF2 biomarkers in humans.
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Biomarcadores , Factor 2 Relacionado con NF-E2 , Estrés Oxidativo , Transducción de Señal , Factor 2 Relacionado con NF-E2/metabolismo , Factor 2 Relacionado con NF-E2/genética , Humanos , Animales , Regulación de la Expresión GénicaRESUMEN
Carbon monoxide-releasing molecules (CO-RMs) are a class of organometallo carbonyl complexes capable of delivering controlled quantities of CO gas to cells and tissues thus exerting a broad spectrum of pharmacological effects. Here we report on the chemical synthesis, CO releasing properties, cytotoxicity profile and pharmacological activities of four novel structurally related iron-allyl carbonyls. The major difference among the new CO-RMs tested was that three compounds (CORM-307, CORM-308 and CORM-314) were soluble in dimethylsulfoxide (DMSO), whereas a fourth one (CORM-319) was rendered water-soluble by reacting the iron-carbonyl with hydrogen tetrafluoroborate. We found that despite the fact all compounds liberated CO, CO-RMs soluble in DMSO caused a more pronounced toxic effect both in vascular and inflammatory cells as well as in isolated vessels. More specifically, iron carbonyls soluble in DMSO released CO with a fast kinetic and displayed a marked cytotoxic effect in smooth muscle cells and RAW 247.6 macrophages despite exerting a rapid and pronounced vasorelaxation ex vivo. In contrast, CORM-319 that is soluble in water and liberated CO with a slower rate, preserved smooth muscle cell viability, relaxed aortic tissue and exerted a significant anti-inflammatory effect in macrophages challenged with endotoxin. These data suggest that iron carbonyls can be used as scaffolds for the design and synthesis of pharmacologically active CO-RMs and indicate that increasing water solubility and controlling the rate of CO release are important parameters for limiting their potential toxic effects.
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Monóxido de Carbono/metabolismo , Óxido Nítrico/antagonistas & inhibidores , Compuestos Organometálicos/farmacología , Vasodilatadores/farmacología , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/fisiología , Monóxido de Carbono/química , Línea Celular , Técnicas In Vitro , Hierro/química , Macrófagos , Masculino , Ratones , Compuestos Organometálicos/química , Ratas , Ratas Sprague-Dawley , Vasodilatación/efectos de los fármacos , Vasodilatadores/químicaRESUMEN
The nuclear factor erythroid derived 2-related factor 2 (Nrf2) and the antioxidant protein heme oxygenase-1 (HO-1) are crucial components of the cellular stress response. These two systems work together to combat oxidative stress and inflammation and are attractive drug targets for counteracting different pathologies, including neuroinflammation. We aimed to identify the most effective Nrf2/HO-1 activators that modulate the inflammatory response in microglia cells. In the present study, we searched the literature and selected 56 compounds reported to activate Nrf2 or HO-1 and analyzed them for HO-1 induction at 6 and 24h and cytotoxicity in BV2 microglial cells in vitro. Approximately 20 compounds up-regulated HO-1 at the concentrations tested (5-20 µM) with carnosol, supercurcumin, cobalt protoporphyrin-IX and dimethyl fumarate exhibiting the best induction/low cytotoxicity profile. Up-regulation of HO-1 by some compounds resulted in increased cellular bilirubin levels but did not augment the expression of proteins involved in heme synthesis (ALAS 1) or biliverdin reductase. Bilirubin production by HO-1 inducers correlated with their potency in inhibiting nitrite production after challenge with interferon-γ (INF-γ) or lipopolysaccharide (LPS). The compounds down-regulated the inflammatory response (TNF-α, PGE2 and nitrite) more strongly in cells challenged with INF-γ than LPS, and silencing HO-1 or Nrf2 with shRNA differentially affected the levels of inflammatory markers. These findings indicate that some small activators of Nrf2/HO-1 are effective modulators of microglia inflammation and highlight the chemical scaffolds that can serve for the synthesis of potent new derivatives to counteract neuroinflammation and neurodegeneration.
Asunto(s)
Hemo-Oxigenasa 1/metabolismo , Hemo/metabolismo , Microglía/efectos de los fármacos , Factor 2 Relacionado con NF-E2/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Antioxidantes/metabolismo , Bilirrubina/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/inmunología , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Interferón gamma/inmunología , Lipopolisacáridos/inmunología , Ratones , Microglía/citología , Microglía/inmunología , Microglía/metabolismo , Factor 2 Relacionado con NF-E2/inmunologíaRESUMEN
RATIONALE: Lung fibroblast senescence is involved in the pathophysiology of chronic obstructive pulmonary disease (COPD). However, the mechanisms underlining this phenomenon are still poorly understood. Secreted phospholipases (sPLA2, a subclass of phospholipases) are secreted by senescent cells and can in turn induce senescence. However, their role in fibroblasts senescence in COPD is unknown. OBJECTIVES: The aim of this study was to analyze the role of sPLA2 in pulmonary fibroblast senescence. METHODS: Fibroblasts were isolated from patients with COPD and control subjects, and senescence markers and inflammatory profile was analyzed. sPLA2 levels were quantified in serum of COPD and controls. MAIN RESULTS: In comparison with non-smokers and smoker controls, senescent lung COPD fibroblasts exhibited a higher mRNA and protein expression of the sPLA2 isoform XIIA and of syndecan 4 (one of its receptors). sPLA2 XIIA induced in turn senescence of non-senescent pulmonary fibroblasts via a pathway involving consecutively syndecan 4, activation of MAPK and p-serine 727 STAT-3, increased mitochondrial ROS production, and activation of AMPK/p53. This pathway was associated with a specific inflammatory secretome (IL-10, IL-12 and TNFα), globally suggesting occurrence of a mitochondrial damage-induced senescence. COPD fibroblasts were more susceptible to this sPLA2 XIIA effect than cells from controls subjects. sPLA2 XIIA levels were significantly higher in serum from COPD patients as compared to controls. CONCLUSION: sPLA2 XIIA is involved in senescence in COPD and could be a potential target to dampen this process.
Asunto(s)
Fosfolipasas A2 Secretoras , Enfermedad Pulmonar Obstructiva Crónica , Humanos , Sindecano-4/metabolismo , Sindecano-4/farmacología , Senescencia Celular , Enfermedad Pulmonar Obstructiva Crónica/genética , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Pulmón/metabolismo , Fibroblastos/metabolismo , Fosfolipasas A2 Secretoras/metabolismo , Fosfolipasas A2 Secretoras/farmacologíaRESUMEN
OBJECTIVE: Intracerebral hemorrhage is accompanied by a pronounced inflammatory response that mediates brain damage but is also essential for the tissue reparative process. We assessed the effect of CORM-3, a water-soluble carbon monoxide-releasing molecule possessing anti-inflammatory properties, on inflammation and brain injury after intracerebral hemorrhage. DESIGN: In vivo and in vitro laboratory study. SETTING: Research laboratory. SUBJECTS: Male Sprague-Dawley rats, 250-350 g. INTERVENTIONS: A model of collagenase injection (2 µL) in the brain was established to induce intracerebral hemorrhage. CORM-3 (4 or 8 mg/kg) was administered intravenously at different times as follows: 1) 5 mins before collagenase; 2) 3 hrs after collagenase; and 3) 3 days after collagenase challenge. MEASUREMENTS AND MAIN RESULTS: Saline was used as a negative control. Brain damage, brain water content, and behavioral assessment were evaluated. The inflammatory response was determined at set intervals after intracerebral hemorrhage by counting peripheral neutrophils and lymphocytes, neutrophils, and activated microglia/macrophages in the intracerebral hemorrhage area and measuring plasma tumor necrosis factor-á levels. BV2 microglia and DI-TNC1 astrocytes were exposed to triton (1%) or CORM-3 (10-100 ìM) and cytotoxicity (lactic dehydrogenase assay) measured at 24 hrs. A challenge with collagenase to induce intracerebral hemorrhage caused marked brain damage and modified the levels of inflammatory markers. Pretreatment with CORM-3 significantly prevented injury, modulated inflammation, and reduced plasma tumor necrosis factor-α. CORM-3 given 3 hrs after collagenase significantly increased brain injury and tumor necrosis factor-α production. In contrast, CORM-3 given 3 days after collagenase afforded partial protection, modulated inflammation, and decreased tumor necrosis factor-α starting from the day of application. No dose-dependent effects were observed. CONCLUSIONS: CORM-3 promotes neuroprotection or neurotoxicity after intracerebral hemorrhage depending on the time of administration. Beneficial effects are achieved when CORM-3 is given either before or 3 days after intracerebral hemorrhage, namely, as a prophylactic agent or during the postacute inflammatory phase.
Asunto(s)
Lesiones Encefálicas/tratamiento farmacológico , Hemorragia Cerebral/tratamiento farmacológico , Fármacos Neuroprotectores/farmacología , Compuestos Organometálicos/farmacología , Animales , Lesiones Encefálicas/etiología , Lesiones Encefálicas/mortalidad , Lesiones Encefálicas/patología , Hemorragia Cerebral/complicaciones , Hemorragia Cerebral/mortalidad , Hemorragia Cerebral/patología , Modelos Animales de Enfermedad , Inmunohistoquímica , Inflamación/tratamiento farmacológico , Inflamación/etiología , Mediadores de Inflamación/análisis , Mediadores de Inflamación/metabolismo , Masculino , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Valores de Referencia , Medición de Riesgo , Índice de Severidad de la Enfermedad , Tasa de Supervivencia , Resultado del Tratamiento , Factor de Necrosis Tumoral alfa/análisis , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Thermoregulation is critical in health and disease and is tightly controlled to maintain body temperature homeostasis. Carbon monoxide (CO), an endogenous gasotransmitter produced during heme degradation by heme oxygenases, has been suggested to play a role in body core temperature (Tb) regulation. However, a direct involvement of CO in thermoregulation has not been confirmed and its mechanism(s) of action remain largely unknown. In the present study we characterized the effects of systemic delivery of CO by administration of an orally active CO-releasing molecule (CORM-401) on Tb regulation in conscious freely moving rats. Specifically, we evaluated the main thermo effectors in rats treated with CORM-401 by assessing: (i) non-shivering thermogenesis, i.e. the increased metabolism of brown fat measured through oxygen consumption and (ii) the rate of heat loss from the tail through calculations of heat loss index. We found that oral administration of CORM-401 (30 mg/kg) resulted in augmented CO delivery into the blood circulation as evidenced a by significant increase in carbon monoxy hemoglobin levels(COHb). In addition, treatment with CORM-401 increased Tb, which was caused by an elevated non-shivering thermogenesis indicated by increased oxygen consumption without significant changes in the tail heat loss. On the other hand, CORM-401 did not affect blood pressure, but significantly decreased heart rate. In summary, the findings of the present study reveal that increased circulating CO levels lead to a rise in Tb, which could have important implications in the emerging role of CO in the modulation of energetic metabolism.