RESUMEN
The maintenance of cellular function relies on the close regulation of adenosine triphosphate (ATP) synthesis and hydrolysis. ATP hydrolysis by mitochondrial ATP Synthase (CV) is induced by loss of proton motive force and inhibited by the mitochondrial protein ATPase inhibitor (ATPIF1). The extent of CV hydrolytic activity and its impact on cellular energetics remains unknown due to the lack of selective hydrolysis inhibitors of CV. We find that CV hydrolytic activity takes place in coupled intact mitochondria and is increased by respiratory chain defects. We identified (+)-Epicatechin as a selective inhibitor of ATP hydrolysis that binds CV while preventing the binding of ATPIF1. In cells with Complex-III deficiency, we show that inhibition of CV hydrolytic activity by (+)-Epichatechin is sufficient to restore ATP content without restoring respiratory function. Inhibition of CV-ATP hydrolysis in a mouse model of Duchenne Muscular Dystrophy is sufficient to improve muscle force without any increase in mitochondrial content. We conclude that the impact of compromised mitochondrial respiration can be lessened using hydrolysis-selective inhibitors of CV.
Asunto(s)
Adenosina Trifosfato , Mitocondrias , Ratones , Animales , Adenosina Trifosfato/metabolismo , Mitocondrias/metabolismo , ATPasas de Translocación de Protón/metabolismo , Proteínas/metabolismo , Homeostasis , HidrólisisRESUMEN
Consumption of (-)-epicatechin (Epi), a cacao flavanol improves cognition. The aim was to compare the effects of (-)-Epi or its stereoisomer (+)-Epi on mouse frontal cortex-dependent short-term working memory and modulators of neurogenesis. Three-month-old male mice (n = 7 per group) were provided by gavage either water (vehicle; Veh), (-)-Epi, at 1 mg kg-1 or (+)-Epi at 0.1 mg per kg of body weight for 15 days. After treatment, spontaneous alternation was evaluated by Y-maze. Brain frontal cortex was isolated for nitrate/nitrite measurements, Western blotting for nerve growth factor (NGF), microtubule associated protein 2 (MAP2), endothelial and neuronal nitric oxide synthase (eNOS and nNOS) and immunohistochemistry for neuronal specific protein (NeuN), doublecortin (DCX), capillary (CD31) and neurofilaments (NF200). Results demonstrate the stimulatory capacity of (-)-Epi and (+)-Epi on markers of neuronal proliferation as per increases in immunoreactive cells for NeuN (74 and 120% respectively), DCX (70 and 124%) as well as in NGF (34.4, 63.6%) and MAP2 (41.8, 63.8%). Capillary density yielded significant increases with (-)-Epi (â¼80%) vs. (+)-Epi (â¼160%). CD31 protein levels increased with (-)-Epi (â¼70%) and (+)-Epi (â¼140%). Effects correlated with nitrate/nitrite stimulation by (-)-Epi and (+)-Epi (110.2, 246.5%) and enhanced eNOS phosphorylation (Ser1177) with (-)-Epi and (+)-Epi (21.4, 41.2%) while nNOS phosphorylation only increased with (+)-Epi (18%). Neurofilament staining was increased in (-)-Epi by 135.6 and 84% with (+)-Epi. NF200 increased with (-)-Epi (116%) vs. (+)-Epi (84.5%). Frontal cortex-dependent short-term spatial working improved with (-)-Epi and (+)-Epi (15, 13%). In conclusion, results suggest that both enantiomers, but more effectively (+)-Epi, upregulate neurogenesis markers likely through stimulation of capillary formation and NO triggering, improvements in memory.
Asunto(s)
Catequina/farmacología , Lóbulo Frontal/fisiología , Memoria a Corto Plazo/efectos de los fármacos , Neurogénesis/efectos de los fármacos , Animales , Biomarcadores/análisis , Química Encefálica , Cacao/química , Catequina/análisis , Proliferación Celular/efectos de los fármacos , Proteína Doblecortina , Lóbulo Frontal/irrigación sanguínea , Lóbulo Frontal/efectos de los fármacos , Masculino , Aprendizaje por Laberinto , Ratones , Ratones Endogámicos C57BL , Neuronas/fisiología , Óxido Nítrico/metabolismo , EstereoisomerismoRESUMEN
BACKGROUND: (+)-Epicatechin (EPI) induces mitochondrial biogenesis and antioxidant metabolism in muscle fibers and neurons. We aimed to evaluate safety and efficacy of (+)-EPI in pediatric subjects with Friedreich's ataxia (FRDA). METHODS: This was a phase II, open-label, baseline-controlled single-center trial including 10 participants ages 10 to 22 with confirmed FA diagnosis. (+)-EPI was administered orally at 75 mg/d for 24 weeks, with escalation to 150 mg/d at 12 weeks for subjects not showing improvement of neuromuscular, neurological or cardiac endpoints. Neurological endpoints were change from baseline in Friedreich's Ataxia Rating Scale (FARS) and 8-m timed walk. Cardiac endpoints were changes from baseline in left ventricular (LV) structure and function by cardiac magnetic resonance imaging (MRI) and echocardiogram, changes in cardiac electrophysiology, and changes in biomarkers for heart failure and hypertrophy. RESULTS: Mean FARS/modified (m)FARS scores showed nonstatistically significant improvement by both group and individual analysis. FARS/mFARS scores improved in 5/9 subjects (56%), 8-m walk in 3/9 (33%), 9-peg hole test in 6/10 (60%). LV mass index by cardiac MRI was significantly reduced at 12 weeks (P = .045), and was improved in 7/10 (70%) subjects at 24 weeks. Mean LV ejection fraction was increased at 24 weeks (P = .008) compared to baseline. Mean maximal septal thickness by echocardiography was increased at 24 weeks (P = .031). There were no serious adverse events. CONCLUSION: (+)-EPI was well tolerated over 24 weeks at up to 150 mg/d. Improvement was observed in cardiac structure and function in subset of subjects with FRDA without statistically significant improvement in primary neurological outcomes. SYNOPSIS: A (+)-epicatechin showed improvement of cardiac function, nonsignificant reduction of FARS/mFARS scores, and sustained significant upregulation of muscle-regeneration biomarker follistatin.
Asunto(s)
Antioxidantes/administración & dosificación , Catequina/administración & dosificación , Ataxia de Friedreich/tratamiento farmacológico , Corazón/diagnóstico por imagen , Adolescente , Niño , Ecocardiografía , Femenino , Ataxia de Friedreich/fisiopatología , Humanos , Imagen por Resonancia Magnética , Masculino , Estudios Prospectivos , Índice de Severidad de la Enfermedad , Resultado del Tratamiento , CaminataRESUMEN
INTRODUCTION: We conducted an open-label study to examine the effects of the flavonoid (-)-epicatechin in seven ambulatory adult patients with Becker muscular dystrophy (BMD). METHODS: Seven participants received (-)-epicatechin 50 mg twice per day for 8 weeks. Pre- and postprocedures included biceps brachii biopsy to assess muscle structure and growth-relevant endpoints by western blotting, mitochondria volume measurement, and cristae abundance by electron microscopy, graded exercise testing, and muscle strength and function tests. RESULTS: Western blotting showed significantly increased levels of enzymes modulating cellular bioenergetics (liver kinase B1 and 5'-adenosine monophosphate-activated protein kinase). Peroxisome proliferator-activated receptor gamma coactivator-1alpha, a transcriptional coactivator of genes involved in mitochondrial biogenesis and cristae-associated mitofilin levels, increased as did cristae abundance. Muscle and plasma follistatin increased significantly while myostatin decreased. Markers of skeletal muscle regeneration myogenin, myogenic regulatory factor-5, myoblast determination protein 1, myocyte enhancer factor-2, and structure-associated proteins, including dysferlin, utrophin, and intracellular creatine kinase, also increased. Exercise testing demonstrated decreased heart rate, maximal oxygen consumption per kilogram, and plasma lactate levels at defined workloads. Tissue saturation index improved in resting and postexercise states. DISCUSSION: (-)-Epicatechin, an exercise mimetic, appears to have short-term positive effects on tissue biomarkers indicative of mitochondrial biogenesis and muscle regeneration, and produced improvements in graded exercise testing parameters in patients with BMD.
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Catequina/uso terapéutico , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/tratamiento farmacológico , Adulto , Biopsia , Western Blotting , Creatina Quinasa/metabolismo , Disferlina/metabolismo , Prueba de Esfuerzo , Folistatina/metabolismo , Frecuencia Cardíaca , Humanos , Ácido Láctico/sangre , Factores de Transcripción MEF2/metabolismo , Masculino , Microscopía Electrónica , Persona de Mediana Edad , Mitocondrias/ultraestructura , Proteínas Mitocondriales/metabolismo , Tamaño Mitocondrial , Proteínas Musculares/metabolismo , Fuerza Muscular , Músculo Esquelético/fisiopatología , Músculo Esquelético/ultraestructura , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patología , Distrofia Muscular de Duchenne/fisiopatología , Proteína MioD/metabolismo , Factor 5 Regulador Miogénico/metabolismo , Miogenina/metabolismo , Miostatina/metabolismo , Biogénesis de Organelos , Consumo de Oxígeno , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Regeneración , Utrofina/metabolismoRESUMEN
Currently, there is great interest in identifying endogenous (i.e. physiological) stimulators of mitochondrial biogenesis (MB), in particular, those that may mediate the effects of exercise. The molecular size of the cacao flavanols (epicatechin and catechin) highly resembles that of sterols and epicatechin has been reported to activate cells surface receptors leading to the stimulation of MB in endothelial and skeletal muscle cells translating into enhanced exercise capacity. We therefore hypothesize, that epicatechin may be acting as a structural mimic of an as yet unknown sterol capable of stimulating MB. We developed a new synthetic process for obtaining enantiomerically pure preparations of (-)-epicatechin and (+)-epicatechin. Applying spatial analytics and molecular modeling, we found that the two isoforms of epicatechin, (-) and (+), have a structural resemblance to 11-ß-hydroxypregnenolone, a sterol with no previously described biological activity. As reported in this proof-of-concept study performed in primary cultures of endothelial and muscle cells, 11-ß-hydroxypregnenolone is one of the most potent inducers of MB as significant activity can be detected at femtomolar levels. The relative potency of (-)/(+)-epicatechin isoforms and on inducing MB correlates with their degree of spatial homology towards the 11-ß-hydroxypregnenolone. On the basis of these results, the detailed in vivo characterization of the potential for these sterols to act as endogenous modulators of MB is warranted.
Asunto(s)
Catequina/química , Catequina/farmacología , Imitación Molecular , Biogénesis de Organelos , Esteroles/química , Esteroles/farmacología , Animales , Bovinos , Línea Celular , Células Cultivadas , Ratones , Modelos Moleculares , EstereoisomerismoRESUMEN
We reported that (-)-epicatechin can stimulate mitochondria biogenesis and improve metabolism. However, preliminary studies indicate that the (+) stereoisomer form may be more potent. We evaluated in a preliminary manner, the pharmacokinetics (PK) and initial safety analysis of (+)-epicatechin ((+)-Epi) in healthy and pre-diabetic subjects. Using a mouse model of diet-induced obesity and insulin resistance, we also evaluated the metabolic effects of (+)-Epi vs. (+)-catechin (Cat) to determine class effects. In the Phase I PK study, subjects were provided a single incremental oral dose of (+)-Epi (10, 30 or 100 mg). For the PD study, subjects were provided a single 30 mg dose per day for 7 days. Blood samples were collected and safety measures were performed. Incremental doses of (+)-Epi increase the half-life of blood metabolites from 1.2-4.9 h. The compound was well tolerated and no adverse effects were reported. Seven day dosing of pre-diabetic subjects led to tendencies for reductions in circulating levels of tumor necrosis factor-α and monocyte chemoattractant protein-1, which returned to baseline by 7 days after treatment. In animals, 2 weeks of oral dosing (0.003, 0.01, 0.03, 0.1 and 0.3 mg kg-1 day-1) dose dependently improved metabolism-related endpoints (weight gain, glucose, cholesterol, triglyceride, with thresholds as low as 0.01 mg kg-1 day-1). Cat yielded no effects at 0.1 mg kg-1 day-1. Results indicate that (+)-Epi evidences a favorable PK and safety profile. Using a pre-clinical model, the compound positively modulates metabolism, which may link to mitochondrial effects. Effects are not due to general antioxidant actions, as Cat yielded no effects.
Asunto(s)
Catequina/farmacocinética , Estado Prediabético/tratamiento farmacológico , Adulto , Anciano , Animales , Glucemia , Catequina/administración & dosificación , Catequina/sangre , Colesterol , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Proyectos Piloto , Estado Prediabético/sangre , Estado Prediabético/metabolismo , Triglicéridos , Adulto JovenRESUMEN
BACKGROUND: Cardiometabolic disruptions such as insulin resistance, obesity, high blood pressure, hyperglycemia, and dyslipidemias, are known to increase the risk for cardiovascular and metabolic diseases such as type 2 diabetes mellitus and atherosclerosis. Several screening tools for assessing cardiometabolic risk have been developed including the TG/HDLc ratio, which has been, demonstrated to possess a strong association with insulin resistance and coronary disease. Dietary modifications, together with regular moderate exercise have proven to be effective in attenuating cardiometabolic disruptions. However, they often exhibit poor long-term patient compliance. Nutraceutics, including (-)-epicatechin (EPI), have gained increasing interest as coadjuvant effective and safe therapies that are able to attenuate hypertension, hyperglycemia, hyperinsulinemia, hypertriglyceridemia and hypoalphalipoproteinemia. METHODS: The aims of this study were: 1) to compare the in vitro effect of EPI vs. (+)-catechin on fructose induced triglyceride accumulation and mitochondrial function in Hep2 cells in culture, 2) to evaluate the efficacy of EPI treatment in reducing fasting blood triglycerides and improving the TG/HDLc ratio in hypertriglyceridemic patients with a total daily dose of 100mg of EPI. Secondary clinical variables included total cholesterol, LDLc, fructosamine, glucose, insulin, and high sensitivity C-reactive protein blood levels. RESULTS AND CONCLUSION: Our results provide preliminary evidence as to favorable effects of EPI on glycemia homeostasis, lipid profile and systemic inflammation such bioactive actions are not class-effects (i.e. limited to their antioxidant potential) but instead, may result from the specific activation of associated downstream signaling pathways since catechin has no effects.
Asunto(s)
Enfermedades Cardiovasculares/etiología , Catequina/uso terapéutico , HDL-Colesterol/sangre , Hipertrigliceridemia/tratamiento farmacológico , Síndrome Metabólico/etiología , Triglicéridos/sangre , Adolescente , Adulto , Enfermedades Cardiovasculares/sangre , Enfermedades Cardiovasculares/tratamiento farmacológico , Células Cultivadas , Método Doble Ciego , Femenino , Humanos , Hipertrigliceridemia/sangre , Hipertrigliceridemia/etiología , Masculino , Síndrome Metabólico/sangre , Síndrome Metabólico/tratamiento farmacológico , Persona de Mediana Edad , Factores de Riesgo , Adulto JovenRESUMEN
(-)-Epicatechin ((-)-EPI), a naturally occurring flavanol, has emerged as a likely candidate for cocoa-based product reported reductions in cardiometabolic risk. The present study aimed to determine the safety, tolerability, pharmacokinetics and pharmacodynamics of purified (-)-EPI administered to healthy volunteers. In this phase I, open-label, two-part single- and multiple-dose study, subjects received either a single dose (n = 9) of 50, 100 or 200 mg or multiple doses (n = 8) of 50 mg daily (q.d.) or twice daily (b.i.d) for 5 days. Blood was collected at 0, 0.5, 1, 2, 4 and 6 h after (-)-EPI administration in the single and multiple dose groups (blood collection repeated in day 5). Samples were analyzed by HPLC-HR-ESI-MS for EPI and metabolite quantification. In the q.d. and b.i.d. groups, blood samples were analyzed for NO surrogates and follistatin levels as well as, platelet mitochondrial complexes I, V and citrate synthase activity levels. (-)-EPI was well tolerated and readily absorbed with further phase 2 metabolism. On day 5, in the q.d. and b.i.d. groups, there were significant increases in plasma nitrite of 30% and 17%, respectively. In the q.d. group on day 5 vs. day 1, platelet mitochondrial complexes I, IV and citrate synthase activities demonstrated a significant increase of â¼92, 62 and 8%, respectively. Average day 5 follistatin AUC levels were â¼2.5 fold higher vs. day 1 AUC levels in the b.i.d. group. (-)-EPI was safe to use, with no observed adverse effects, and our findings suggest that increases in NO metabolites, mitochondrial enzyme function and plasma follistatin levels may underlie some of the beneficial effects of cocoa products or (-)-EPI as reported in other studies.
Asunto(s)
Antiinflamatorios no Esteroideos/efectos adversos , Antiinflamatorios no Esteroideos/metabolismo , Enfermedades Cardiovasculares/prevención & control , Catequina/efectos adversos , Catequina/metabolismo , Suplementos Dietéticos/efectos adversos , Absorción Intestinal , Adulto , Anciano , Antiinflamatorios no Esteroideos/administración & dosificación , Antiinflamatorios no Esteroideos/sangre , Biomarcadores/sangre , Biomarcadores/metabolismo , Plaquetas/enzimología , Enfermedades Cardiovasculares/inmunología , Catequina/administración & dosificación , Catequina/sangre , Citrato (si)-Sintasa/química , Citrato (si)-Sintasa/metabolismo , Suplementos Dietéticos/análisis , Proteínas del Complejo de Cadena de Transporte de Electrón/agonistas , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Femenino , Folistatina/sangre , Folistatina/metabolismo , Humanos , Cinética , Masculino , Persona de Mediana Edad , Óxido Nítrico/agonistas , Óxido Nítrico/sangre , Óxido Nítrico/metabolismo , Pruebas de Toxicidad Subcrónica , Adulto JovenRESUMEN
AIMS: Chronic heart failure is a costly epidemic that affects up to 2% of people in developed countries. The purpose of this study was to discover novel blood proteomic biomarker signatures of recovered heart function that could lead to more effective heart failure patient management by both primary care and specialty physicians. METHODS AND RESULTS: The discovery cohort included 41 heart transplant patients and 20 healthy individuals. Plasma levels of 138 proteins were detected in at least 75% of these subjects by iTRAQ mass spectrometry. Eighteen proteins were identified that had (i) differential levels between pre-transplant patients with end-stage heart failure and healthy individuals; and (ii) levels that returned to normal by 1 month post-transplant in patients with stable heart function after transplantation. Seventeen of the 18 markers were validated by multiple reaction monitoring mass spectrometry in a cohort of 39 heart failure patients treated with drug therapy, of which 30 had recovered heart function and 9 had not. This 17-protein biomarker panel had 93% sensitivity and 89% specificity, while the RAMP® NT-proBNP assay had the same specificity but 80% sensitivity. Performance further improved when the panel was combined with NT-proBNP, yielding a net reclassification index relative to NT-proBNP of 0.28. CONCLUSIONS: We have identified potential blood biomarkers of recovered heart function by harnessing data from transplant patients. These biomarkers can lead to the development of an inexpensive protein-based blood test that could be used by physicians to monitor response to therapy in heart failure, resulting in more personalized, front-line heart failure patient management.
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Proteínas Sanguíneas , Fármacos Cardiovasculares/uso terapéutico , Insuficiencia Cardíaca , Trasplante de Corazón/métodos , Adulto , Anciano , Biomarcadores/análisis , Biomarcadores/sangre , Proteínas Sanguíneas/análisis , Proteínas Sanguíneas/clasificación , Interpretación Estadística de Datos , Monitoreo de Drogas/métodos , Femenino , Insuficiencia Cardíaca/sangre , Insuficiencia Cardíaca/diagnóstico , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/cirugía , Humanos , Masculino , Persona de Mediana Edad , Péptido Natriurético Encefálico/sangre , Evaluación de Resultado en la Atención de Salud , Fragmentos de Péptidos/sangre , Atención Perioperativa/métodos , Recuperación de la Función/fisiología , Proyectos de Investigación , Sensibilidad y EspecificidadRESUMEN
Excessive dietary fructose intake may have an important role in the current epidemics of fatty liver, obesity and diabetes as its intake parallels the development of these syndromes and because it can induce features of metabolic syndrome. The effects of fructose to induce fatty liver, hypertriglyceridemia and insulin resistance, however, vary dramatically among individuals. The first step in fructose metabolism is mediated by fructokinase (KHK), which phosphorylates fructose to fructose-1-phosphate; intracellular uric acid is also generated as a consequence of the transient ATP depletion that occurs during this reaction. Here we show in human hepatocytes that uric acid up-regulates KHK expression thus leading to the amplification of the lipogenic effects of fructose. Inhibition of uric acid production markedly blocked fructose-induced triglyceride accumulation in hepatocytes in vitro and in vivo. The mechanism whereby uric acid stimulates KHK expression involves the activation of the transcription factor ChREBP, which, in turn, results in the transcriptional activation of KHK by binding to a specific sequence within its promoter. Since subjects sensitive to fructose often develop phenotypes associated with hyperuricemia, uric acid may be an underlying factor in sensitizing hepatocytes to fructose metabolism during the development of fatty liver.
Asunto(s)
Hígado Graso/metabolismo , Fructoquinasas/metabolismo , Fructosa/metabolismo , Hepatocitos/metabolismo , Ácido Úrico/metabolismo , Alopurinol/farmacología , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Inhibidores Enzimáticos/farmacología , Hígado Graso/genética , Hígado Graso/patología , Fructoquinasas/genética , Células Hep G2 , Hepatocitos/efectos de los fármacos , Hepatocitos/patología , Humanos , Hígado/metabolismo , Hígado/patología , Masculino , Ratas , Ratas Sprague-Dawley , Activación Transcripcional , Regulación hacia Arriba/efectos de los fármacos , Ácido Úrico/antagonistas & inhibidoresRESUMEN
BACKGROUND: Uric acid is an independent risk factor in fructose-induced fatty liver, but whether it is a marker or a cause remains unknown. RESULTS: Hepatocytes exposed to uric acid developed mitochondrial dysfunction and increased de novo lipogenesis, and its blockade prevented fructose-induced lipogenesis. CONCLUSION: Rather than a consequence, uric acid induces fatty liver SIGNIFICANCE: Hyperuricemic people are more prone to develop fructose-induced fatty liver. Metabolic syndrome represents a collection of abnormalities that includes fatty liver, and it currently affects one-third of the United States population and has become a major health concern worldwide. Fructose intake, primarily from added sugars in soft drinks, can induce fatty liver in animals and is epidemiologically associated with nonalcoholic fatty liver disease in humans. Fructose is considered lipogenic due to its ability to generate triglycerides as a direct consequence of the metabolism of the fructose molecule. Here, we show that fructose also stimulates triglyceride synthesis via a purine-degrading pathway that is triggered from the rapid phosphorylation of fructose by fructokinase. Generated AMP enters into the purine degradation pathway through the activation of AMP deaminase resulting in uric acid production and the generation of mitochondrial oxidants. Mitochondrial oxidative stress results in the inhibition of aconitase in the Krebs cycle, resulting in the accumulation of citrate and the stimulation of ATP citrate lyase and fatty-acid synthase leading to de novo lipogeneis. These studies provide new insights into the pathogenesis of hepatic fat accumulation under normal and diseased states.
Asunto(s)
Hígado Graso/metabolismo , Mitocondrias/metabolismo , Estrés Oxidativo , Ácido Úrico/metabolismo , Fructosa/metabolismo , Células Hep G2 , Humanos , Lipogénesis , Triglicéridos/metabolismo , Ácido Úrico/efectos adversosRESUMEN
Cisplatin nephropathy can be regarded as a mitochondrial disease. Intervention to halt such deleterious injury is under investigation. Recently, the flavanol (-)-epicatechin emerges as a novel compound to protect the cardiovascular system, owing in part to mitochondrial protection. Here, we have hypothesized that epicatechin prevents the progression of cisplatin-induced kidney injury by protecting mitochondria. Epicatechin was administered 8 h after cisplatin injury was induced in the mouse kidney. Cisplatin significantly induced renal dysfunction and tubular injury along with an increase in oxidative stress. Mitochondrial damages were also evident as a decrease in loss of mitochondrial mass with a reduction in the oxidative phosphorylation complexes and low levels of MnSOD. The renal damages and mitochondrial injuries were significantly prevented by epicatechin treatment. Consistent with these observations, an in vitro study using cultured mouse proximal tubular cells demonstrated that cisplatin-induced mitochondrial injury, as revealed by a decrease in mitochondrial succinate dehydrogenase activity, an induction of cytochrome c release, mitochondrial fragmentation, and a reduction in complex IV protein, was prevented by epicatechin. Such a protective effect of epicatechin might be attributed to decreased oxidative stress and reduced ERK activity. Finally, we confirmed that epicatechin did not perturb the anticancer effect of cisplatin in HeLa cells. In conclusion, epicatechin exhibits protective effects due in part to its ability to prevent the progression of mitochondrial injury in mouse cisplatin nephropathy. Epicatechin may be a novel option to treat renal disorders associated with mitochondrial dysfunction.
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Catequina/uso terapéutico , Cisplatino/efectos adversos , Enfermedades Renales/inducido químicamente , Enfermedades Renales/prevención & control , Mitocondrias/fisiología , Enfermedades Mitocondriales/inducido químicamente , Enfermedades Mitocondriales/prevención & control , Animales , Antioxidantes/farmacología , Antioxidantes/uso terapéutico , Catequina/farmacología , Células Cultivadas , Citocromos c/metabolismo , Modelos Animales de Enfermedad , Células HeLa/efectos de los fármacos , Células HeLa/patología , Humanos , Técnicas In Vitro , Enfermedades Renales/fisiopatología , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/patología , Túbulos Renales Proximales/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Enfermedades Mitocondriales/fisiopatología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Especies Reactivas de Oxígeno/metabolismo , Succinato Deshidrogenasa/metabolismoRESUMEN
Nicorandil exhibits a protective effect in the vascular system, which is thought to be due to vasodilatation from opening ATP-dependent potassium channels and donation of nitric oxide. Recently, nicorandil was shown to be renoprotective in models of acute kidney injury and glomerulonephritis. However, the specific mechanisms of renoprotection are unclear. We evaluated the effect of nicorandil on the rat remnant kidney model of chronic kidney disease. Blood pressure was unchanged by a 10-wk course of nicorandil, while albuminuria was significantly reduced. Glomerular injury and tubulointerstitial injury were also ameliorated by nicorandil. Oxidative stress, as noted by renal nitrotyrosine level and urine 8-hydroxy-2'-deoxyguanosine, were elevated in this model and was significantly reduced by nicorandil treatment. Treatment was associated with maintenance of the mitochondrial antioxidant, manganese SOD, in podocytes and with suppression of xanthine oxidase expression in infiltrating macrophages. Interestingly, these two cell types express sulfonylurea receptor 2 (SUR2), a binding site of nicorandil in the ATP-dependent K channel. Consistently, we found that stimulating SUR2 with nicorandil prevented angiotensin II-mediated upregulation of xanthine oxidase in the cultured macrophage, while xanthine oxidase expression was rather induced by blocking SUR2 with glibenclamide. In conclusion, nicorandil reduces albuminuria and ameliorates renal injury by blocking oxidative stress in chronic kidney disease.
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Canales KATP/agonistas , Fallo Renal Crónico/tratamiento farmacológico , Riñón/metabolismo , Nicorandil/farmacología , Transportadoras de Casetes de Unión a ATP/metabolismo , Albuminuria/prevención & control , Animales , Western Blotting , Células Cultivadas , Progresión de la Enfermedad , Inmunohistoquímica , Riñón/efectos de los fármacos , Riñón/patología , Fallo Renal Crónico/patología , Glomérulos Renales/patología , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Macrófagos/metabolismo , Masculino , Ratones , Nefritis Intersticial/patología , Nefritis Intersticial/prevención & control , Óxido Nítrico/metabolismo , Estrés Oxidativo/efectos de los fármacos , Adhesión en Parafina , Canales de Potasio de Rectificación Interna/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Droga/metabolismo , Receptores de Sulfonilureas , Xantina Oxidasa/biosíntesisRESUMEN
Nicorandil is an orally available drug that can act as a nitric oxide donor, an antioxidant, and an ATP-dependent K channel activator. We hypothesized that it may have a beneficial role in treating diabetic nephropathy. We administered nicorandil to a model of advanced diabetic nephropathy (the streptozotocin-induced diabetes in mice lacking endothelial nitric oxide synthase, eNOSKO); controls included diabetic eNOS KO mice without nicorandil and nondiabetic eNOS KO mice treated with either nicorandil or vehicle. Mice were treated for 8 wk. Histology, blood pressure, and renal function were determined. Additional studies involved examining the effects of nicorandil on cultured human podocytes. Here, we found that nicorandil did not affect blood glucose levels, blood pressure, or systemic endothelial function, but significantly reduced proteinuria and glomerular injury (mesangiolysis and glomerulosclerosis). Nicorandil protected against podocyte loss and podocyte oxidative stress. Studies in cultured podocytes showed that nicorandil likely protects against glucose-mediated oxidant stress via the ATP-dependent K channel as opposed to its NO-stimulating effects. In conclusion, nicorandil may be beneficial in diabetic nephropathy by preserving podocyte function. We recommend clinical trials to determine whether nicorandil may benefit diabetic nephropathy or other conditions associated with podocyte dysfunction.
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Antioxidantes/uso terapéutico , Nefropatías Diabéticas/tratamiento farmacológico , Nicorandil/uso terapéutico , Donantes de Óxido Nítrico/uso terapéutico , Óxido Nítrico Sintasa de Tipo III/deficiencia , Índice de Severidad de la Enfermedad , Animales , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/fisiología , Células Cultivadas , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/complicaciones , Nefropatías Diabéticas/etiología , Nefropatías Diabéticas/fisiopatología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Nicorandil/farmacología , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa de Tipo III/genética , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Podocitos/citología , Podocitos/efectos de los fármacos , Podocitos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Estreptozocina/efectos adversosRESUMEN
Inhibitors of angiogenic factors are known to be upregulated, and their levels increase in the maternal circulation before the onset of preeclampsia. We reproduced a previously characterized model of preeclampsia by adenoviral overexpression of the soluble vascular endothelial growth factor (VEGF) receptor sFlt-1 (also referred to as sVEGFR-1) in pregnant and nonpregnant Sprague-Dawley rats. Animals were treated with VEGF121 at 0, 100, 200, or 400 microg/kg once or twice daily (n=8 per group; 64 total) and compared with normal control animals (n=4 per group) by examination of systolic blood pressure, urinary albumin and creatinine, renal histopathology, and glomerular gene expression profiling. sFlt-1 expression induced hypertension with proteinuria and glomerular endotheliosis and significant changes in gene expression. VEGF121 treatment alleviated these symptoms and reversed 125 of 268 sFlt-1-induced changes in gene expression. VEGF121 had beneficial effects in this rat model of preeclampsia without apparent harm to the fetus. Further study of VEGF121 as a potential therapeutic agent for preeclampsia is warranted.
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Hipertensión/tratamiento farmacológico , Riñón/efectos de los fármacos , Riñón/patología , Preeclampsia/tratamiento farmacológico , Proteínas Recombinantes/uso terapéutico , Factor A de Crecimiento Endotelial Vascular/uso terapéutico , Animales , Presión Sanguínea/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Hipertensión/fisiopatología , Riñón/metabolismo , Preeclampsia/fisiopatología , Embarazo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/farmacología , Factor A de Crecimiento Endotelial Vascular/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
OBJECTIVES: These studies describe molecular forms of circulating B-type natriuretic peptide (BNP) as well as their biological activity. BACKGROUND: Increased circulating levels of immunoreactive BNP correlate with the severity of heart failure and are considered a sensitive biomarker. However, little is known about the molecular forms of circulating BNP and their biological activity. METHODS: Western blot analysis was used to characterize immunoreactive BNP species in heart failure plasma. Recombinant proBNP was assessed for reactivity in commercially available BNP assays and cell activity by cyclic guanosine monophosphate production in vascular cells. RESULTS: Heart failure plasma contained both low- (LMW-BNP) and high-molecular-weight (HMW-BNP) forms. The LMW-BNP migrated similarly to a 32-amino acid BNP standard, whereas HMW-BNP, when deglycosylated, was similar to deglycosylated recombinant proBNP. Recombinant proBNP and BNP were equally recognized by the Triage BNP assay (Biosite, San Diego, California). Furthermore, recombinant proBNP and BNP were both recognized by the Advia Centaur BNP test (Bayer Diagnostics, Tarrytown, New York), but only recombinant proBNP was recognized by the Elecsys NTproBNP assay (Roche Diagnostics, Indianapolis, Indiana). Recombinant proBNP exerted significantly less biological activity than BNP on human endothelial and vascular smooth muscle cells. Comparison of effective concentration (50%) values indicates that proBNP is 6- to 8-fold less potent than BNP in these human cells. CONCLUSIONS: This study demonstrates that proBNP, constituting a substantial portion of immunoreactive BNP in heart failure plasma, possesses significantly lower biological activity than the processed 32-amino acid hormone. These results implicate a discordance in heart failure between the high circulating levels of immunoreactive BNP and hormone activity, suggesting that some patients may be in a state of natriuretic peptide deficiency.
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Insuficiencia Cardíaca/sangre , Péptido Natriurético Encefálico/metabolismo , Fragmentos de Péptidos/sangre , Biomarcadores/sangre , Western Blotting , Células Cultivadas , ADN Complementario/análisis , Progresión de la Enfermedad , Regulación hacia Abajo , Endotelio Vascular/citología , Regulación de la Expresión Génica , Humanos , Inmunoprecipitación , Biología Molecular , Péptido Natriurético Encefálico/sangre , Péptido Natriurético Encefálico/genética , Fragmentos de Péptidos/genética , Pronóstico , Sensibilidad y EspecificidadRESUMEN
Pancreatic cancer is one of the deadliest forms of cancer and effective treatment remains a clinical challenge. Transforming growth factor-beta (TGF-beta) has important roles in primary tumor progression and in promoting metastasis, and has become an attractive target for therapy. Previously, we reported that treatment of pancreatic cancer cells in vitro with SD-208, a small molecule inhibitor of the TGF-beta receptor I kinase (TGF-betaRI), inhibited expression of genes associated with tumor progression and inhibited invasiveness in a cell-based assay. In a demonstration of efficacy of TGF-beta signaling inhibition in an in vivo model of pancreatic cancer, we showed significantly reduced primary tumor weight and decreased incidence of metastasis in the Panc-1 orthotopic xenograft model of established pancreatic cancer. In this report, we extend these in vivo findings to examine the mechanistic consequences of TGF-betaRI inhibition on Panc-1 primary tumors and their microenvironment in situ. In a longitudinal study of TGF-betaRI inhibition in the Panc-1 orthotopic model, we show that SD-208 treatment significantly reduced tumor growth measured as bioluminescence intensity throughout the study. Histological evaluation revealed that SD-208 treatment reduced proliferation and induced apoptosis in the primary tumors, and reduced fibrosis in the tumor microenvironment. An immune contribution (greater B-cell infiltration in SD-208-treated tumors) was also suggested by the histological analyses. SD-208 not only blocked direct TGF-beta signaling in Panc-1 primary tumors (reduced phospho SMAD2/3), but also down-regulated the expression of TGF-beta-regulated genes (PAI-1 and COL7A1). Taken together, our results indicate that a TGF-betaRI kinase inhibitor has a potential therapeutic benefit for pancreatic cancer patients.
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Adenocarcinoma/tratamiento farmacológico , Neoplasias Pancreáticas/tratamiento farmacológico , Pteridinas/farmacología , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Adenocarcinoma/inmunología , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Linfocitos B/inmunología , Procesos de Crecimiento Celular/efectos de los fármacos , Procesos de Crecimiento Celular/fisiología , Femenino , Humanos , Ratones , Ratones Desnudos , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Receptores de Factores de Crecimiento Transformadores beta/genética , Transducción de Señal , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Transforming growth factor-beta (TGF-beta) suppresses tumor development by inhibiting cellular proliferation, inducing differentiation and apoptosis, and maintaining genomic integrity. However, once tumor cells escape from the tumor-suppressive effects of TGF-beta, they often constitutively overexpress and activate TGF-beta, which may promote tumor progression by enhancing invasion, metastasis, and angiogenesis and by suppressing antitumor immunity. The purpose of this study was to test this hypothesis using TGF-beta pathway antagonists. EXPERIMENTAL DESIGN: We examined the effects of selective TGF-beta type I receptor kinase inhibitors, SD-093 and SD-208, on two murine mammary carcinoma cell lines (R3T and 4T1) in vitro and in vivo. RESULTS: Both agents blocked TGF-beta-induced phosphorylation of the receptor-associated Smads, Smad2 and Smad3, in a dose-dependent manner, with IC50 between 20 and 80 nmol/L. TGF-beta failed to inhibit growth of these cell lines but stimulated epithelial-to-mesenchymal transdifferentiation, migration, and invasiveness into Matrigel in vitro. These effects were inhibited by SD-093, indicating that these processes are partly driven by TGF-beta. Treatment of syngeneic R3T or 4T1 tumor-bearing mice with orally given SD-208 inhibited primary tumor growth as well as the number and size of metastases. In contrast, SD-208 failed to inhibit R3T tumor growth or metastasis in athymic nude mice. Moreover, in vitro anti-4T1 cell cytotoxic T-cell responses of splenocytes from drug-treated animals were enhanced compared with cells from control animals. In addition, SD-208 treatment resulted in a decrease in tumor angiogenesis. CONCLUSION: TGF-beta type I receptor kinase inhibitors hold promise as novel therapeutic agents for metastatic breast cancer.