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Cardiovascular disease (CVD) stands as one of the leading causes of morbidity and mortality worldwide, and the continued search for novel therapeutics is vital for addressing this global health challenge. Over the past decade, hydrogen sulfide (H2S) has garnered significant attention in the field of medical research, as it has been proven to be a cardioprotective gaseous signaling molecule. It joins nitric oxide and carbon monoxide as endogenously produced gasotransmitters. As for its mechanism, H2S functions through the posttranslational addition of a sulfur group to cysteine residues on target proteins in a process called sulfhydration. As a result, the observed physiological effects of H2S can include vasodilation, anti-apoptosis, anti-inflammation, antioxidant effects, and regulation of ion channels. Various studies have observed the cardioprotective benefits of H2S in diseases such as myocardial infarction, ischemia-reperfusion injury, cardiac remodeling, heart failure, arrhythmia, and atherosclerosis. In this review, we discuss the mechanisms and therapeutic potential of H2S in various CVDs.
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PURPOSE OF REVIEW: Diabetic neuropathy is a common complication of diabetes mellitus (DM) and can affect up to 50% of DM patients during their lifetime. Patients typically present with numbness, tingling, pain, and loss of sensation in the extremities. Since there is no treatment targeting the underlying mechanism of neuropathy, strategies focus on preventative care and pain management. RECENT FINDINGS: Up to 69% of patients with diabetic neuropathy receive pharmacological treatment for neuropathic pain. The United States Food and Drug Administration (FDA) confirmed four drugs for painful diabetic neuropathy (PDN): pregabalin, duloxetine, tapentadol, and the 8% capsaicin patch. Nonpharmacological treatments such as spinal cord stimulation (SCS) and transcutaneous electrical nerve stimulation (TENS) both show promise in reducing pain in DM patients. Despite the high burden associated with PDN, effective management remains challenging. This update covers the background and management of diabetic neuropathy, including its epidemiology, pathogenesis, preventative care, and current therapeutic strategies.
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Peroxidative damage to human spermatozoa has been shown to be the primary cause of male infertility. The possible role of nitric oxide (NO) in affecting sperm motility, capacitation, and acrosome reaction has been reported, too. The overproduction of NO by the enzyme inducible nitric oxide synthase (iNOS) could be responsible as it has been implicated in the pathogenesis of many diseases. There have been many studies on regulating iNOS function in various tissues, especially by protein-protein interaction; however, no study has looked for iNOS-interacting proteins in the human testis. Here, we have reported the identification of two proteins that interact with iNOS. We initially undertook a popular yeast two-hybrid assay to screen a human testis cDNA library in yeast using an iNOS-peptide fragment (amino acids 181-335) as bait. We verified our data using the mammalian chemiluminescent co-IP method; first, employing the same peptide and, then, a full-length protein co-expressed in HEK293 cells in addition to the candidate protein. In both cases, these two protein partners of iNOS were revealed: (a) sperm acrosome-associated 7 protein and (b) retinoblastoma tumor-suppressor binding protein.
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Óxido Nítrico Sintasa de Tipo II , Testículo , Técnicas del Sistema de Dos Híbridos , Humanos , Masculino , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Testículo/metabolismo , Células HEK293 , Unión ProteicaRESUMEN
Endogenously produced hydrogen sulfide (H2S) is critical for cardiovascular homeostasis. Therapeutic strategies aimed at increasing H2S levels have proven cardioprotective in models of acute myocardial infarction (MI) and heart failure (HF). The present study was undertaken to investigate the effects of a novel H2S prodrug, SG-1002, on stress induced hypertrophic signaling in murine HL-1 cardiac muscle cells. Treatment of HL-1 cells with SG-1002 under serum starvation without or with H2O2 increased the levels of H2S, H2S producing enzyme, and cystathionine ß-synthase (CBS), as well as antioxidant protein levels, such as super oxide dismutase1 (SOD1) and catalase, and additionally decreased oxidative stress. SG-1002 also decreased the expression of hypertrophic/HF protein markers such as atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), galectin-3, TIMP1, collagen type III, and TGF-ß1 in stressed HL-1 cells. Treatment with SG-1002 caused a significant induction of cell viability and a marked reduction of cellular cytotoxicity in HL-1 cells under serum starvation incubated without or with H2O2. Experimental results of this study suggest that SG-1002 attenuates myocardial cellular oxidative damage and/or hypertrophic signaling via increasing H2S levels or H2S producing enzymes, CBS, and antioxidant proteins.
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BACKGROUND: Currently there are no standard of care treatment strategies for IH prevention (IHP). Dehydrated human amnion-chorion (dHACM) is a healing adjunct that elutes growth factors including several that have reduced IH in animal models. We therefore performed a double-blinded, prospective randomized controlled trial (RCT) to test the hypothesis that dHACM significantly reduces IH formation in a well-studied animal model of acute IH. MATERIAL AND METHODS: Forty 16-week-old male Sprague-Dawley rats were randomized to one of four groups: No Treatment vs. dHACM Sheet (Group A), and Saline vs. dHACM Injection (Group B). Each animal underwent a 5-cm midline laparotomy which was incompletely closed with 5-0 plain gut sutures; this was performed by a surgeon blinded to treatment group (first blind). After 28 days, the primary endpoints of IH formation and hernia size were determined by study staff blinded to treatment (second blind). Secondary endpoints included healed fascia tensile strength as determined by tensiometry, systemic and local inflammatory markers as measured by ELISA, and fascial scar collagen I/III ratios per Western blotting. RESULTS: In Group A, No Treatment developed IH at 87.5% vs. 62.5% for Sheet (P = 0.28). Hernias that formed in the Sheet group were significantly smaller (P = 0.036). In Group B, Injection and Saline yielded identical IH rates of 77.8%. Molecular characterization of fascial scar demonstrated non-inferior tensile strength, collagen I/III ratios, and inflammatory markers in dHACM-treated animals. CONCLUSIONS: dHACM sheets significantly reduced the size of IH following laparotomy when compared to no treatment.
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Hernia Incisional , Amnios , Animales , Corion , Modelos Animales de Enfermedad , Humanos , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Human immunodeficiency virus (HIV) attacks the immune system and weakens the ability to fight infections/disease. Furthermore, HIV infection confers approximately two-fold higher risk of cardiac events compared with the general population. The pathological mechanisms responsible for the increased incidence of cardiovascular disease in HIV patients are largely unknown. We hypothesized that increased oxidative stress and attenuated circulating levels of the cardioprotective gaseous signaling molecules, nitric oxide (NO), and hydrogen sulfide (H2S) were involved in the cardiovascular pathobiology observed in HIV patients. Plasma samples from both HIV patients and age-matched normal subjects were used for all assays. Oxidative stress was determined by analyzing the levels of advanced oxidation protein products (AOPP) and H2O2. Antioxidant levels were determined by measuring the levels of trolox equivalent capacity. ADMA, hs-CRP, and IL-6 were determined by using ELISA. The levels of H2S (free H2S and sulfane sulfur) and NO2 (nitrite) were determined in the plasma samples by using gas chromatography and HPLC, respectively. In the present study we observed a marked induction in the levels of oxidative stress and decreased antioxidant status in the plasma of HIV patients as compared with the controls. Circulating levels of the cardiovascular disease biomarkers: ADMA, hs-CRP (high-sensitivity C-reactive protein), and IL-6 were significantly increased in the circulatory system of HIV patients. The levels of both nitrite and H2S/sulfane sulfur were significantly reduced in the plasma of HIV patients as compared with normal subjects. Our data demonstrate significant increases in circulating biomarkers of oxidative stress and cardiovascular (CV) in conjunction with decreased bioavailability of H2S and NO in HIV patients. Diminished levels of these two cardioprotective gaseous signaling molecules may be involved in the pathogenesis of CV disease in the setting of HIV.
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White adipose tissue (WAT) is a critical organ in both health and disease. However, physiologically faithful tissue culture models of primary human WAT remain limited, at best. In this study we describe a novel WAT culture system in which primary human WAT is sandwiched between tissue-engineered sheets of adipose-derived stromal cells. This construct, called "sandwiched white adipose tissue" (SWAT), can be defined as a microphysiological system (MPS) since it is a tissue-engineered, multicellular, three-dimensional organ construct produced using human cells. We validated SWAT against the National Institutes of Health MPS standards and found that SWAT is viable in culture for 8 weeks, retains physiologic responses to exogenous signaling, secretes adipokines, and engrafts into animal models. These attributes position SWAT as a powerful tool for the study of WAT physiology, pathophysiology, personalized medicine, and pharmaceutical development.
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Adipocitos/citología , Tejido Adiposo Blanco/citología , Células del Estroma/citología , Técnicas de Cultivo de Tejidos/métodos , Ingeniería de Tejidos/métodos , Adipocitos/metabolismo , Adipoquinas/metabolismo , Tejido Adiposo Blanco/metabolismo , Tejido Adiposo Blanco/trasplante , Adulto , Animales , Diferenciación Celular , Femenino , Perfilación de la Expresión Génica , Humanos , Lipólisis , Masculino , Ratones , Persona de Mediana Edad , Células del Estroma/metabolismoRESUMEN
Oxidative stress results in mtDNA damage and contributes to myocardial cell death. mtDNA repair enzymes are crucial for mtDNA repair and cell survival. We investigated a novel, mitochondria-targeted fusion protein (Exscien1-III) containing endonuclease III in myocardial ischemia-reperfusion injury and transverse aortic constriction (TAC)-induced heart failure. Male C57/BL6J mice (10-12 wk) were subjected to 45 min of myocardial ischemia and either 24 h or 4 wk of reperfusion. Exscien1-III (4 mg/kg ip) or vehicle was administered at the time of reperfusion. Male C57/BL6J mice were subjected to TAC, and Exscien1-III (4 mg/kg i.p) or vehicle was administered daily starting at 3 wk post-TAC and continued for 12 wk. Echocardiography was performed to assess left ventricular (LV) structure and function. Exscien1-III reduced myocardial infarct size ( P < 0.01) at 24 h of reperfusion and preserved LV ejection fraction at 4 wk postmyocardial ischemia. Exscien1-III attenuated TAC-induced LV dilation and dysfunction at 6-12 wk post-TAC ( P < 0.05). Exscien1-III reduced ( P < 0.05) cardiac hypertrophy and maladaptive remodeling after TAC. Assessment of cardiac mitochondria showed that Exscien1-III localized to mitochondria and increased mitochondrial antioxidant and reduced apoptotic markers. In conclusion, our results indicate that administration of Exscien1-III provides significant protection against myocardial ischemia and preserves myocardial structure and LV performance in the setting of heart failure. NEW & NOTEWORTHY Oxidative stress-induced mitochondrial DNA damage is a prominent feature in the pathogenesis of cardiovascular diseases. In the present study, we demonstrate the efficacy of a novel, mitochondria-targeted fusion protein that traffics endonuclease III specifically for mitochondrial DNA repair in two well-characterized murine models of cardiac injury and failure.
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Fármacos Cardiovasculares/farmacología , Daño del ADN/efectos de los fármacos , ADN Mitocondrial/efectos de los fármacos , Insuficiencia Cardíaca/tratamiento farmacológico , Hipertrofia Ventricular Izquierda/tratamiento farmacológico , Disfunción Ventricular Izquierda/tratamiento farmacológico , Función Ventricular Izquierda/efectos de los fármacos , Remodelación Ventricular/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/metabolismo , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Modelos Animales de Enfermedad , Fibrosis , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/fisiopatología , Hipertrofia Ventricular Izquierda/metabolismo , Hipertrofia Ventricular Izquierda/patología , Hipertrofia Ventricular Izquierda/fisiopatología , Masculino , Ratones Endogámicos C57BL , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/patología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Estrés Oxidativo/efectos de los fármacos , Proteínas Recombinantes de Fusión/farmacología , Transducción de Señal/efectos de los fármacos , Volumen Sistólico/efectos de los fármacos , Disfunción Ventricular Izquierda/metabolismo , Disfunción Ventricular Izquierda/patología , Disfunción Ventricular Izquierda/fisiopatologíaRESUMEN
BACKGROUND: Zofenopril, a sulfhydrylated angiotensin-converting enzyme inhibitor (ACEI), reduces mortality and morbidity in infarcted patients to a greater extent than do other ACEIs. Zofenopril is a unique ACEI that has been shown to increase hydrogen sulfide (H2S) bioavailability and nitric oxide (NO) levels via bradykinin-dependent signaling. Both H2S and NO exert cytoprotective and antioxidant effects. We examined zofenopril effects on H2S and NO bioavailability and cardiac damage in murine and swine models of myocardial ischemia/reperfusion (I/R) injury. METHODS AND RESULTS: Zofenopril (10 mg/kg PO) was administered for 1, 8, and 24 hours to establish optimal dosing in mice. Myocardial and plasma H2S and NO levels were measured along with the levels of H2S and NO enzymes (cystathionine ß-synthase, cystathionine γ-lyase, 3-mercaptopyruvate sulfur transferase, and endothelial nitric oxide synthase). Mice received 8 hours of zofenopril or vehicle pretreatment followed by 45 minutes of ischemia and 24 hours of reperfusion. Pigs received placebo or zofenopril (30 mg/daily orally) 7 days before 75 minutes of ischemia and 48 hours of reperfusion. Zofenopril significantly augmented both plasma and myocardial H2S and NO levels in mice and plasma H2S (sulfane sulfur) in pigs. Cystathionine ß-synthase, cystathionine γ-lyase, 3-mercaptopyruvate sulfur transferase, and total endothelial nitric oxide synthase levels were unaltered, while phospho-endothelial nitric oxide synthase(1177) was significantly increased in mice. Pretreatment with zofenopril significantly reduced myocardial infarct size and cardiac troponin I levels after I/R injury in both mice and swine. Zofenopril also significantly preserved ischemic zone endocardial blood flow at reperfusion in pigs after I/R. CONCLUSIONS: Zofenopril-mediated cardioprotection during I/R is associated with an increase in H2S and NO signaling.
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Antihipertensivos/farmacología , Captopril/análogos & derivados , Corazón/efectos de los fármacos , Sulfuro de Hidrógeno/metabolismo , Daño por Reperfusión Miocárdica/prevención & control , Miocardio/metabolismo , Óxido Nítrico/metabolismo , Animales , Disponibilidad Biológica , Western Blotting , Captopril/farmacología , Cistationina betasintasa/efectos de los fármacos , Cistationina betasintasa/genética , Cistationina betasintasa/metabolismo , Cistationina gamma-Liasa/efectos de los fármacos , Cistationina gamma-Liasa/genética , Cistationina gamma-Liasa/metabolismo , Ratones , Infarto del Miocardio/patología , Miocardio/patología , Óxido Nítrico Sintasa de Tipo III/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Ramipril/farmacología , Distribución Aleatoria , Flujo Sanguíneo Regional , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sulfurtransferasas/efectos de los fármacos , Sulfurtransferasas/genética , Sulfurtransferasas/metabolismo , Porcinos , Porcinos Enanos , Troponina I/efectos de los fármacos , Troponina I/metabolismoRESUMEN
BACKGROUND: Bioavailability of nitric oxide (NO) and hydrogen sulfide (H2S) is reduced in heart failure (HF). Recent studies suggest cross-talk between NO and H2S signaling. We previously reported that sodium nitrite (NaNO2) ameliorates myocardial ischemia-reperfusion injury and HF. Nuclear factor-erythroid-2-related factor 2 (Nrf2) regulates the antioxidant proteins expression and is upregulated by H2S. We examined the NaNO2 effects on endogenous H2S bioavailability and Nrf2 activation in mice subjected to ischemia-induced chronic heart failure (CHF). METHODS AND RESULTS: Mice underwent 60 minutes of left coronary artery occlusion and 4 weeks of reperfusion. NaNO2 (165 µg/kgic) or vehicle was administered at reperfusion and then in drinking water (100 mg/L) for 4 weeks. Left ventricular (LV), ejection fraction (EF), LV end diastolic (LVEDD) and systolic dimensions (LVESD) were determined at baseline and at 4 weeks of reperfusion. Myocardial tissue was analyzed for oxidative stress and respective gene/protein-related assays. We found that NaNO2 therapy preserved LVEF, LVEDD and LVSD at 4 weeks during ischemia-induced HF. Myocardial malondialdehyde and protein carbonyl content were significantly reduced in NaNO2-treated mice as compared to vehicle, suggesting a reduction in oxidative stress. NaNO2 therapy markedly increased expression of Cu,Zn-superoxide dismutase, catalase, and glutathione peroxidase during 4 weeks of reperfusion. Furthermore, NaNO2 upregulated the activity of Nrf2, as well as H2S-producing enzymes, and ultimately increased H2S bioavailability in ischemia-induced CHF in mice as compared with vehicle. CONCLUSIONS: Our results demonstrate that NaNO2 therapy significantly improves LV function via increasing H2S bioavailability, Nrf2 activation, and antioxidant defenses.
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Antioxidantes/farmacología , Cardiotónicos/farmacología , Insuficiencia Cardíaca/metabolismo , Sulfuro de Hidrógeno/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Nitrito de Sodio/farmacología , Animales , Oclusión Coronaria/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Isquemia Miocárdica/fisiopatología , Daño por Reperfusión Miocárdica/fisiopatología , Miocardio/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Estrés Oxidativo/fisiología , Transducción de Señal/efectos de los fármacos , Volumen Sistólico/fisiologíaRESUMEN
BACKGROUND AND PURPOSE: Published data on nebivolol reveal selective ß1 adrenergic selectively along with novel nitric oxide (NO)-dependent vasodilatory properties. However, the exact molecular mechanism is unknown. Protein S-nitrosylation constitutes a large part of the ubiquitous influence of NO on cellular signal transduction and is involved in a number of human diseases. More recently, protein denitrosylation has been shown to play a major role in controlling cellular S-nitrosylation (SNO). Several enzymes have been reported to catalyze the reduction of SNOs and are viewed as candidate denitrosylases. One of the first described is known as S-nitrosoglutathione reductase (GSNOR). Importantly, GSNOR has been shown to play a role in regulating SNO signaling downstream of the ß-adrenergic receptor and is therefore operative in cellular signal transduction. Pharmacological inhibition or genetic deletion of GSNOR leads to enhanced vasodilation and characteristic of known effects of nebivolol. Structurally, nebivolol is similar to known inhibitors of GSNOR. Therefore, we hypothesize that some of the known effects of nebivolol may occur through this mechanism. EXPERIMENTAL APPROACH: Using cell culture systems, tissue organ bath, and intact animal models, we report that nebivolol treatment leads to a dose-dependent accumulation of nitrosothiols in cells, and this is associated with an enhanced vasodilation by S-nitrosoglutathione. KEY RESULTS: These data suggest a new mechanism of action of nebivolol that may explain in part the reported NO activity. CONCLUSIONS AND IMPLICATIONS: Because exogenous mediators of protein SNO or denitrosylation can substantially affect the development or progression of disease, this may call for new utility of nebivolol.
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Aldehído Oxidorreductasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Nebivolol/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , S-Nitrosoglutatión/metabolismo , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacología , Aldehído Oxidorreductasas/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/enzimología , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/deficiencia , Óxido Nítrico Sintasa de Tipo III/genética , Oxidación-Reducción , Células RAW 264.7 , Ratas Sprague-DawleyRESUMEN
Metabolic syndrome (MetS) reduces endothelial nitric oxide (NO) bioavailability and exacerbates vascular dysfunction in patients with preexisting vascular diseases. Nitrite, a storage form of NO, can mediate vascular function during pathological conditions when endogenous NO is reduced. The aims of the present study were to characterize the effects of severe MetS and obesity on dyslipidemia, myocardial oxidative stress, and endothelial NO synthase (eNOS) regulation in the obese Ossabaw swine (OS) model and to examine the effects of a novel, sustained-release formulation of sodium nitrite (SR-nitrite) on coronary vascular reactivity and myocardial redox status in obese OS subjected to critical limb ischemia (CLI). After 6 mo of an atherogenic diet, obese OS displayed a MetS phenotype. Obese OS had decreased eNOS functionality and NO bioavailability. In addition, obese OS exhibited increased oxidative stress and a significant reduction in antioxidant enzymes. The efficacy of SR-nitrite therapy was examined in obese OS subjected to CLI. After 3 wk of treatment, SR-nitrite (80 mg · kg(-1) · day(-1) bid po) increased myocardial nitrite levels and eNOS function. Treatment with SR-nitrite reduced myocardial oxidative stress while increasing myocardial antioxidant capacity. Ex vivo assessment of vascular reactivity of left anterior descending coronary artery segments demonstrated marked improvement in vasoreactivity to sodium nitroprusside but not to substance P and bradykinin in SR-nitrite-treated animals compared with placebo-treated animals. In conclusion, in a clinically relevant, large-animal model of MetS and CLI, treatment with SR-nitrite enhanced myocardial NO bioavailability, attenuated oxidative stress, and improved ex vivo coronary artery vasorelaxation.
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Vasos Coronarios/efectos de los fármacos , Cardiopatías/prevención & control , Síndrome Metabólico/tratamiento farmacológico , Miocardio/metabolismo , Estrés Oxidativo/efectos de los fármacos , Enfermedades Vasculares Periféricas/tratamiento farmacológico , Nitrito de Sodio/farmacología , Vasodilatación/efectos de los fármacos , Animales , Vasos Coronarios/metabolismo , Vasos Coronarios/fisiopatología , Preparaciones de Acción Retardada , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Dislipidemias/tratamiento farmacológico , Dislipidemias/metabolismo , Dislipidemias/fisiopatología , Femenino , Cardiopatías/metabolismo , Cardiopatías/fisiopatología , Masculino , Síndrome Metabólico/metabolismo , Síndrome Metabólico/fisiopatología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Obesidad/tratamiento farmacológico , Obesidad/metabolismo , Obesidad/fisiopatología , Oxidación-Reducción , Enfermedades Vasculares Periféricas/metabolismo , Enfermedades Vasculares Periféricas/fisiopatología , Índice de Severidad de la Enfermedad , PorcinosRESUMEN
BACKGROUND: Cystathionine γ-lyase, cystathionine ß-synthase, and 3-mercaptopyruvate sulfurtransferase are endogenous enzymatic sources of hydrogen sulfide (H2S). Functions of H2S are mediated by several targets including ion channels and signaling proteins. Nuclear factor-erythroid 2-related factor 2 is responsible for the expression of antioxidant response element-regulated genes and is known to be upregulated by H2S. We examined the levels of H2S, H2S-producing enzymes, and nuclear factor-erythroid 2-related factor 2 activation status in skeletal muscle obtained from critical limb ischemia (CLI) patients. METHODS AND RESULTS: Gastrocnemius tissues were attained postamputation from human CLI and healthy control patients. We found mRNA and protein levels of cystathionine γ-lyase, cystathionine ß-synthase, and 3-mercaptopyruvate sulfurtransferase were significantly decreased in skeletal muscle of CLI patients as compared to control. H2S and sulfane sulfur levels were significantly decreased in skeletal muscle of CLI patients. We also observed significant reductions in nuclear factor-erythroid 2-related factor 2 activation as well as antioxidant proteins, such as Cu, Zn-superoxide dismutase, catalase, and glutathione peroxidase in skeletal muscle of CLI patients. Biomarkers of oxidative stress, such as malondialdehyde and protein carbonyl formation, were significantly increased in skeletal muscle of CLI patients as compared to healthy controls. CONCLUSIONS: The data demonstrate that H2S bioavailability and nuclear factor-erythroid 2-related factor 2 activation are both attenuated in CLI tissues concomitant with significantly increased oxidative stress. Reductions in the activity of H2S-producing enzymes may contribute to the pathogenesis of CLI.
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Amputación Quirúrgica , Sulfuro de Hidrógeno/análisis , Isquemia/cirugía , Pierna/irrigación sanguínea , Músculo Esquelético/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Anciano , Biomarcadores/análisis , Cistationina betasintasa/genética , Cistationina betasintasa/metabolismo , Cistationina gamma-Liasa/genética , Cistationina gamma-Liasa/metabolismo , Femenino , Glutatión Peroxidasa/metabolismo , Humanos , Pierna/cirugía , Masculino , Estrés Oxidativo/genética , ARN Mensajero , Transducción de Señal/genética , Sulfuros/análisis , Sulfurtransferasas/genética , Sulfurtransferasas/metabolismo , Superóxido Dismutasa/metabolismoRESUMEN
Nitrite is a storage reservoir of nitric oxide that is readily reduced to nitric oxide under pathological conditions. Previous studies have demonstrated that nitrite levels are significantly reduced in cardiovascular disease states, including peripheral vascular disease. We investigated the cytoprotective and proangiogenic actions of a novel, sustained-release formulation of nitrite (SR-nitrite) in a clinically relevant in vivo swine model of critical limb ischemia (CLI) involving central obesity and metabolic syndrome. CLI was induced in obese Ossabaw swine (n = 18) by unilateral external iliac artery deployment of a full cross-sectional vessel occlusion device positioned within an endovascular expanded polytetrafluoroethylene-lined nitinol stent-graft. At post-CLI day 14, pigs were randomized to placebo (n = 9) or SR-nitrite (80 mg, n = 9) twice daily by mouth for 21 days. SR-nitrite therapy increased nitrite, nitrate, and S-nitrosothiol in plasma and ischemic skeletal muscle. Oxidative stress was reduced in ischemic limb tissue of SR-nitrite- compared with placebo-treated pigs. Ischemic limb tissue levels of proangiogenic growth factors were increased following SR-nitrite therapy compared with placebo. Despite the increases in cytoprotective and angiogenic signals with SR-nitrite therapy, new arterial vessel formation and enhancement of blood flow to the ischemic limb were not different from placebo. Our data clearly demonstrate cytoprotective and proangiogenic signaling in ischemic tissues following SR-nitrite therapy in a very severe model of CLI. Further studies evaluating longer-duration nitrite therapy and/or additional nitrite dosing strategies are warranted to more fully evaluate the therapeutic potential of nitrite therapy in peripheral vascular disease.
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Inductores de la Angiogénesis/farmacología , Arteria Ilíaca/cirugía , Isquemia , Síndrome Metabólico , Músculo Esquelético/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Enfermedad Arterial Periférica , Nitrito de Sodio/farmacología , Animales , Preparaciones de Acción Retardada , Modelos Animales de Enfermedad , Miembro Posterior/irrigación sanguínea , Miembro Posterior/efectos de los fármacos , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/metabolismo , Nitratos/metabolismo , Nitritos/metabolismo , S-Nitrosotioles/metabolismo , PorcinosRESUMEN
Previous studies have demonstrated that hydrogen sulfide (H2S) protects against multiple cardiovascular disease states in a similar manner as nitric oxide (NO). H2S therapy also has been shown to augment NO bioavailability and signaling. The purpose of this study was to investigate the impact of H2S deficiency on endothelial NO synthase (eNOS) function, NO production, and ischemia/reperfusion (I/R) injury. We found that mice lacking the H2S-producing enzyme cystathionine γ-lyase (CSE) exhibit elevated oxidative stress, dysfunctional eNOS, diminished NO levels, and exacerbated myocardial and hepatic I/R injury. In CSE KO mice, acute H2S therapy restored eNOS function and NO bioavailability and attenuated I/R injury. In addition, we found that H2S therapy fails to protect against I/R in eNOS phosphomutant mice (S1179A). Our results suggest that H2S-mediated cytoprotective signaling in the setting of I/R injury is dependent in large part on eNOS activation and NO generation.
Asunto(s)
Citoprotección/fisiología , Sulfuro de Hidrógeno/metabolismo , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico/metabolismo , Transducción de Señal/fisiología , Alanina Transaminasa/sangre , Análisis de Varianza , Animales , Aspartato Aminotransferasas/sangre , Western Blotting , Cromatografía Líquida de Alta Presión , Cistationina gamma-Liasa/genética , Citoprotección/efectos de los fármacos , Sulfuro de Hidrógeno/farmacología , Inmunohistoquímica , Ratones , Ratones Noqueados , Mitocondrias/fisiología , Daño por Reperfusión Miocárdica/metabolismo , Estrés Oxidativo/fisiología , Consumo de Oxígeno/fisiología , Troponina I/metabolismoRESUMEN
G protein-coupled receptor kinase 5 GRK5 plays a key role in regulating cardiac signaling and its expression is increased in heart failure. GRK5 activity in the nucleus of myocytes has been shown to be detrimental in the setting of pressure-overload hypertrophy. The ubiquitous nuclear transcription factor κB (NF-κB) is involved in the regulation of numerous genes in various tissues, and activation of NF-κB has been shown to be associated with heart disease. Herein, we investigated whether GRK5 can specifically regulate the NF-κB signaling pathway in myocytes. We found that overexpression of GRK5 increased the levels of NF-κB -p50 and p65 in vitro and in vivo, whereas loss of GRK5 resulted in lower cardiac NF-κB levels. Furthermore, increased GRK5 expression induced the phosphorylation status of p65, increased the activity of a NF-κB reporter, and increased NF-κB DNA binding activity in cultured neonatal rat ventricular myocytes. Importantly, siRNA against GRK5 presented with the opposite results in neonatal rat ventricular myocytes as p65 and p50 were decreased, and there was a loss of NF-κB DNA binding activity. The influence of GRK5 on NF-κB expression and activity was dependent on its nuclear localization as overexpression of a mutant GRK5 that cannot enter the nucleus was devoid of NF-κB activation or DNA binding. Our study demonstrates that a novel pathological consequence of GRK5 up-regulation in the injured and failing heart is the induction of NF-κB expression and activity.
Asunto(s)
Núcleo Celular/metabolismo , Quinasa 5 del Receptor Acoplado a Proteína-G/metabolismo , Miocitos Cardíacos/citología , Subunidad p50 de NF-kappa B/metabolismo , Factor de Transcripción ReIA/metabolismo , Animales , Animales Recién Nacidos , Aorta/metabolismo , Aorta/fisiología , Quinasa 5 del Receptor Acoplado a Proteína-G/deficiencia , Quinasa 5 del Receptor Acoplado a Proteína-G/genética , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Ventrículos Cardíacos/citología , Luciferasas/metabolismo , Ratones , Ratones Transgénicos , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/metabolismo , Subunidad p50 de NF-kappa B/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Factor de Transcripción ReIA/genética , VasoconstricciónRESUMEN
Oxidative stress has been implicated in the pathogenesis of various cardiovascular diseases, including ischemic heart disease and heart failure. The peroxisome proliferator-activated receptor gamma (PPARγ) agonist improves insulin sensitivity and limits tissue inflammation and cellular apoptosis, but there are few data on the relationship between the PPARγ agonist, rosiglitazone (RSG), and the thioredoxin (TRx) system in oxidatively stressed cardiomyocytes (CMCs). Here we provide evidence that the PPARγ agonist RSG protects rat CMCs from hydrogen peroxide (H2O2)-induced apoptosis by TRx overexpression. The expression levels of pAkt/Akt, pErk/Erk, survivin, Bcl-2/Bax-α, and manganese-superoxide dismutase were increased by RSG pretreatment in H2O2-injured rat CMCs. On the contrary, the expression levels of caspase-3 and p53 were decreased by RSG pretreatment. These effects of RSG were reversed by chemical inhibitors of TRx and the PPARγ antagonist. This suggests that RSG protects rCMCs from H2O2-induced oxidative stress through TRx overexpression and a PPARγ-dependent mechanism.
Asunto(s)
Apoptosis/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , PPAR gamma/agonistas , Tiazolidinedionas/farmacología , Tiorredoxinas/genética , Regulación hacia Arriba/efectos de los fármacos , Animales , Citoprotección/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Ratas , Ratas Sprague-Dawley , RosiglitazonaRESUMEN
G protein-coupled receptor kinase 5 (GRK5) plays a key role in cardiac signaling regulation, and its expression is increased in heart failure. Recently, increased expression of GRK5 in the myocardium of mice has been shown to be detrimental in the setting of pressure-overload hypertrophy. The ubiquitous nuclear transcription factor κB (NF-κB) is involved in the regulation of numerous genes in various tissues, and activation of NF-κB has been shown to be associated with heart disease. Here, we investigated the role of NF-κB signaling in the regulation of the GRK5 gene and expression of this kinase in cardiomyocytes. First, in analyzing the 5'-flanking DNA of GRK5, the presence of a potential NF-κB binding site was observed in the promoter region. Phorbol myristate acetate, a known stimulator of NF-κB, increased the levels of GRK5 in myocytes whereas treatment of cells with N-acetyl cysteine, a known inhibitor of NF-κB, or with SC 514, an inhibitor of IκB kinase 2 decreased GRK5. Utilizing EMSA or ChIP assays, we found that both p50 and p65 NF-κB could interact with the promoter of GRK5 following myocytes NF-κB activation. Importantly, short interfering RNA (siRNA)-mediated loss of p65 in myocytes decreased the stimulated increased levels of GRK5 mRNA and protein. Finally, adenovirus-mediated overexpression of a dominant-negative IκBα in myocytes inhibited the levels of GRK5. Taken together, our study demonstrates that NF-κB plays a critical role in the regulation of GRK5 transcription in myocytes and that this may translate to the significant expression changes seen in heart disease.
Asunto(s)
Quinasa 5 del Receptor Acoplado a Proteína-G/genética , Regulación Enzimológica de la Expresión Génica/fisiología , Miocitos Cardíacos/enzimología , Subunidad p50 de NF-kappa B/metabolismo , Transducción de Señal/fisiología , Factor de Transcripción ReIA/metabolismo , Acetilcisteína/farmacología , Animales , Quinasa 5 del Receptor Acoplado a Proteína-G/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Cardiopatías/enzimología , Cardiopatías/genética , Subunidad p50 de NF-kappa B/genética , Cultivo Primario de Células , Regiones Promotoras Genéticas/fisiología , ARN Interferente Pequeño/genética , Ratas , Especies Reactivas de Oxígeno/metabolismo , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacología , Tiofenos/farmacología , Factor de Transcripción ReIA/genéticaRESUMEN
Induction of surfactant protein-A (SP-A) gene expression in fetal lung type II cells by cAMP and IL-1 is mediated by increased binding of thyroid transcription factor-1 (TTF-1) and NF-B proteins p50 and p65 to the TTF-1-binding element (TBE) at -183 bp. In type II cell transfections, dexamethasone (Dex) markedly inhibits cAMP-induced expression of rabbit SP-A:human growth hormone (hGH) fusion genes containing as little as 300 bp of the SP-A 5'-flanking sequence. Dex inhibition is blocked by RU-486, suggesting a role of the glucocorticoid receptor (GR). The present study was undertaken to define the mechanisms for GR inhibition of SP-A expression. Cotransfection of primary cultures of type II cells with a GR expression vector abrogated cAMP induction of SP-A promoter activity while, at the same time, causing a 60-fold induction of cotransfected mouse mammary tumor virus (MMTV) promoter. In lung cells transfected with a fusion gene containing three TBEs fused to the basal SP-A promoter, Dex prevented the stimulatory effect of IL-1 on TTF-1 induction of SP-A promoter activity, suggesting that the GR inhibits SP-A promoter activity through the TBE. In gel shift assays using nuclear extracts from human fetal type II cells cultured in the absence or presence of cAMP, Dex markedly reduced binding of nuclear proteins to the TBE and blocked the stimulatory effect of cAMP on TBE-binding activity. Our finding that Dex increased expression of the NF-kappaB inhibitory partner IkappaB-alpha suggests that the decrease in TBE-binding activity may be caused, in part, by GR inhibition of NF-kappaB interaction with this site.