RESUMEN
Supporting cells of the ovary, termed granulosa cells, are essential for ovarian differentiation and oogenesis by providing a nurturing environment for oocyte maintenance and maturation. Granulosa cells are specified in the fetal and perinatal ovary, and sufficient numbers of granulosa cells are critical for the establishment of follicles and the oocyte reserve. Identifying the cellular source from which granulosa cells and their progenitors are derived is an integral part of efforts to understand basic ovarian biology and the etiology of female infertility. In particular, the contribution of mesenchymal cells, especially perivascular cells, to ovarian development is poorly understood but is likely to be a source of new information regarding ovarian function. Here we have identified a cell population in the fetal ovary, which is a Nestin-expressing perivascular cell type. Using lineage tracing and ex vivo organ culture methods, we determined that perivascular cells are multipotent progenitors that contribute to granulosa, thecal, and pericyte cell lineages in the ovary. Maintenance of these progenitors is dependent on ovarian vasculature, likely reliant on endothelial-mesenchymal Notch signaling interactions. Depletion of Nestin+ progenitors resulted in a disruption of granulosa cell specification and in an increased number of germ cell cysts that fail to break down, leading to polyovular ovarian follicles. These findings highlight a cell population in the ovary and uncover a key role for vasculature in ovarian differentiation, which may lead to insights into the origins of female gonad dysgenesis and infertility.
Asunto(s)
Ovario , Pericitos , Animales , Femenino , Células de la Granulosa/metabolismo , Nestina/genética , Nestina/metabolismo , Oogénesis/fisiología , Folículo Ovárico , Ovario/metabolismoRESUMEN
Loss-of-function mutations in the secreted enzyme ADAMTS7 (a disintegrin and metalloproteinase with thrombospondin motifs 7) are associated with protection for coronary artery disease. ADAMTS7 catalytic inhibition has been proposed as a therapeutic strategy for treating coronary artery disease; however, the lack of an endogenous substrate has hindered the development of activity-based biomarkers. To identify ADAMTS7 extracellular substrates and their cleavage sites relevant to vascular disease, we used TAILS (terminal amine isotopic labeling of substrates), a method for identifying protease-generated neo-N termini. We compared the secreted proteome of vascular smooth muscle and endothelial cells expressing either full-length mouse ADAMTS7 WT, catalytic mutant ADAMTS7 E373Q, or a control luciferase adenovirus. Significantly enriched N-terminal cleavage sites in ADAMTS7 WT samples were compared to the negative control conditions and filtered for stringency, resulting in catalogs of high confidence candidate ADAMTS7 cleavage sites from our three independent TAILS experiments. Within the overlap of these discovery sets, we identified 24 unique cleavage sites from 16 protein substrates, including cleavage sites in EFEMP1 (EGF-containing fibulin-like extracellular matrix protein 1/Fibulin-3). The ADAMTS7 TAILS preference for EFEMP1 cleavage at the amino acids 123.124 over the adjacent 124.125 site was validated using both endogenous EFEMP1 and purified EFEMP1 in a binary in vitro cleavage assay. Collectively, our TAILS discovery experiments have uncovered hundreds of potential substrates and cleavage sites to explore disease-related biological substrates and facilitate activity-based ADAMTS7 biomarker development.
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Enfermedad de la Arteria Coronaria , Péptido Hidrolasas , Proteína ADAMTS7 , Animales , Biomarcadores , Endopeptidasas , Células Endoteliales/metabolismo , Ratones , Péptido Hidrolasas/metabolismo , Proteoma/química , Cola (estructura animal)/metabolismoRESUMEN
[Figure: see text].
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Enfermedad de la Arteria Coronaria/metabolismo , Proteína ADAMTS7/química , Proteína ADAMTS7/genética , Proteína ADAMTS7/metabolismo , Animales , Dominio Catalítico , Células Cultivadas , Enfermedad de la Arteria Coronaria/genética , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Músculo Liso Vascular/metabolismo , Mutación , Polimorfismo de Nucleótido SimpleRESUMEN
RATIONALE: Hypertrophic cardiomyopathy occurs with a frequency of about 1 in 500 people. Approximately 30% of those affected carry mutations within the gene encoding cMyBP-C (cardiac myosin binding protein C). Cardiac stress, as well as cMyBP-C mutations, can trigger production of a 40kDa truncated fragment derived from the amino terminus of cMyBP-C (Mybpc340kDa). Expression of the 40kDa fragment in mouse cardiomyocytes leads to hypertrophy, fibrosis, and heart failure. Here we use genetic approaches to establish a causal role for excessive myofibroblast activation in a slow, progressive genetic cardiomyopathy-one that is driven by a cardiomyocyte-intrinsic genetic perturbation that models an important human disease. OBJECTIVE: TGFß (transforming growth factor-ß) signaling is implicated in a variety of fibrotic processes, and the goal of this study was to define the role of myofibroblast TGFß signaling during chronic Mybpc340kDa expression. METHODS AND RESULTS: To specifically block TGFß signaling only in the activated myofibroblasts in Mybpc340kDa transgenic mice and quadruple compound mutant mice were generated, in which the TGFß receptor II (TßRII) alleles ( Tgfbr2) were ablated using the periostin ( Postn) allele, myofibroblast-specific, tamoxifen-inducible Cre ( Postnmcm) gene-targeted line. Tgfbr2 was ablated either early or late during pathological fibrosis. Early myofibroblast-specific Tgfbr2 ablation during the fibrotic response reduced cardiac fibrosis, alleviated cardiac hypertrophy, preserved cardiac function, and increased lifespan of the Mybpc340kDa transgenic mice. Tgfbr2 ablation late in the pathological process reduced cardiac fibrosis, preserved cardiac function, and prolonged Mybpc340kDa mouse survival but failed to reverse cardiac hypertrophy. CONCLUSIONS: Fibrosis and cardiac dysfunction induced by cardiomyocyte-specific expression of Mybpc340kDa were significantly decreased by Tgfbr2 ablation in the myofibroblast. Surprisingly, preexisting fibrosis was partially reversed if the gene was ablated subsequent to fibrotic deposition, suggesting that continued TGFß signaling through the myofibroblasts was needed to maintain the heart fibrotic response to a chronic, disease-causing cardiomyocyte-only stimulus.
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Cardiomiopatía Hipertrófica/metabolismo , Proteínas Portadoras/genética , Miocitos Cardíacos/metabolismo , Miofibroblastos/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta/metabolismo , Transducción de Señal , Animales , Cardiomiopatía Hipertrófica/genética , Proteínas Portadoras/metabolismo , Células Cultivadas , Ratones , Mutación , Receptor Tipo II de Factor de Crecimiento Transformador beta/genéticaRESUMEN
Cardiac myosin-binding protein C (cMyBP-C) is an integral part of the sarcomeric machinery in cardiac muscle that enables normal function. cMyBP-C regulates normal cardiac contraction by functioning as a brake through interactions with the sarcomere's thick, thin, and titin filaments. cMyBP-C's precise effects as it binds to the different filament systems remain obscure, particularly as it impacts on the myosin heavy chain's head domain, contained within the subfragment 2 (S2) region. This portion of the myosin heavy chain also contains the ATPase activity critical for myosin's function. Mutations in myosin's head, as well as in cMyBP-C, are a frequent cause of familial hypertrophic cardiomyopathy (FHC). We generated transgenic lines in which endogenous cMyBP-C was replaced by protein lacking the residues necessary for binding to S2 (cMyBP-C(S2-)). We found, surprisingly, that cMyBP-C lacking the S2 binding site is incorporated normally into the sarcomere, although systolic function is compromised. We show for the first time the acute and chronic in vivo consequences of ablating a filament-specific interaction of cMyBP-C. This work probes the functional consequences, in the whole animal, of modifying a critical structure-function relationship, the protein's ability to bind to a region of the critical enzyme responsible for muscle contraction, the subfragment 2 domain of the myosin heavy chain. We show that the binding is not critical for the protein's correct insertion into the sarcomere's architecture, but is essential for long-term, normal function in the physiological context of the heart.
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Proteínas Portadoras/metabolismo , Miocardio/metabolismo , Miosinas/metabolismo , Animales , Sitios de Unión , Proteínas Portadoras/genética , Ratones , Contracción Muscular , Mutación , Unión Proteica , Sarcómeros/metabolismoRESUMEN
Heart Failure (HF) continues to be a complex public health issue with increasing world population prevalence. Although overall mortality has decreased for HF and hypertrophic cardiomyopathy (HCM), a precursor for HF, their prevalence continues to increase annually. Because the etiology of HF and HCM is heterogeneous, it has been difficult to identify novel therapies to combat these diseases. Isoproterenol (ISP), a non-selective ß-adrenoreceptor agonist, is commonly used to induce cardiotoxicity and cause acute and chronic HCM and HF in mice. However, the variability in dose and duration of ISP treatment used in studies has made it difficult to determine the optimal combination of ISP dose and delivery method to develop a reliable ISP-induced mouse model for disease. Here we examined cardiac effects induced by ISP via subcutaneous (SQ) and SQ-minipump (SMP) infusions across 3 doses (2, 4, and 10mg/kg/day) over 2 weeks to determine whether SQ and SMP ISP delivery induced comparable disease severity in C57BL/6J mice. To assess disease, we measured body and heart weight, surface electrocardiogram (ECG), and echocardiography recordings. We found all 3 ISP doses comparably increase heart weight, but these increases are more pronounced when ISP was administered via SMP. We also found that the combination of ISP treatment and delivery method induces contrasting heart rate, RR interval, and R and S amplitudes that may place SMP treated mice at higher risk for sustained disease burden. Mice treated via SMP also had increased heart wall thickness and LV Mass, but mice treated via SQ showed greater increase in gene markers for hypertrophy and fibrosis. Overall, these data suggest that at 2 weeks, mice treated with 2, 4, or 10mg/kg/day ISP via SQ and SMP routes cause similar pathological heart phenotypes but highlight the importance of drug delivery method to induce differing disease pathways.
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Cardiomegalia , Isoproterenol , Ratones Endogámicos C57BL , Animales , Isoproterenol/administración & dosificación , Cardiomegalia/inducido químicamente , Cardiomegalia/patología , Ratones , Masculino , Modelos Animales de Enfermedad , Ecocardiografía , Relación Dosis-Respuesta a Droga , ElectrocardiografíaRESUMEN
Mitochondria, also known as "Power House of cell," are crucial organelles, regulating energy metabolism. Recently, an involvement of mitochondria in cancer occurrence and metastasis has been proposed. The roles of mitochondria in cancer progression/metastasis include alteration of glycolysis, regulation of ROS and suppression of intrinsic apoptosis. This mini-review explains the specific mitochondrial characteristics during cancer metastasis with past and recent findings. It may contribute to understanding mitochondria-related mechanisms of cancer metastasis.
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Apoptosis , Metabolismo Energético , Mitocondrias/metabolismo , Neoplasias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Humanos , Mitocondrias/patología , Metástasis de la Neoplasia , Neoplasias/patologíaRESUMEN
Hyperlipidemia is a major contributor for atherosclerosis and hypolipidemic drugs such as statin are highly prescribed to treat elevated lipid level in plasma. Rubus coreanus, which is widely cultivated in south eastern Asia, have been reported to show significant cholesterol lowering action in hyperlipidemic subjects. Our objective was to determine the cellular effect of Rubus coreanus extract (RCE) on cholesterol biosynthesis in human hepatic cells (HepG2) and to elucidate the molecular mechanism by which it causes change in cholesterol metabolism. RCE treatment lowered cholesterol biosynthesis as well as secretion from HepG2 cells. This effect was associated with lowering the release of apolipoproteins from hepatic cells. RCE treatment also showed an increase in phosphorylation of foxhead box protein 01 (FoXo-1) and 5-adenosine monophosphate-activated protein kinase (AMPK), thus lowering expression of phosphoenolpyruvate carboxykinase (PEPCK) and G6Pase, which might be a major pathway for cholesterol biosynthesis inhibition. Apart from this; RCE also lowered sterol regulatory element-binding protein-1 (SREBP-1) expression in HepG2 cells, showing a long term regulation of cholesterol biosynthesis activity. These results indicate that one of the anti-hyperlipidemic actions of RCE is due to inhibition of cholesterol biosynthesis in hepatic cells and provides first documentation of a hypolipidemic bio-molecular action of Rubus coreanus.
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Colesterol/metabolismo , Ácidos Grasos/metabolismo , Hipolipemiantes/farmacología , Extractos Vegetales/farmacología , Rosaceae , Proteínas Quinasas Activadas por AMP/metabolismo , Apolipoproteína A-I/metabolismo , Apolipoproteínas B/metabolismo , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/metabolismo , Glucosa-6-Fosfatasa/metabolismo , Células Hep G2 , Humanos , Hipolipemiantes/análisis , Hígado/citología , Hígado/metabolismo , Extractos Vegetales/análisis , Proteínas Serina-Treonina Quinasas/metabolismo , Solventes/química , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Agua/químicaRESUMEN
OBJECTIVES: Gastrodia elata (GE) Blume (Orchidaceae) has been previously known for its therapeutic benefits against neurodegenerative diseases. Microglial activation and death have been implicated in the pathogenesis of a variety of neurodegenerative diseases, including Alzheimer's disease. In this study, GE and its pure components, gastrodin and 4-hydroxybenzyl alcohol (4HBA), were applied to ß-amyloid-induced BV2 mouse microglial cells. MATERIALS AND METHODS: Cell viability was assessed by the MTT assay and Western blotting was also performed. RESULTS: ß-amyloid-induced cell death was shown to be induced time- and dose-dependently. To examine the cell death mechanism, we confirmed the involvement of ER stress signaling. C/EBP homologous protein (CHOP), a pro-apoptotic ER stress protein, was expressed at high levels but glucose-regulated protein 78 (GRP78), an anti-apoptotic ER stress protein with chaperone activity, was only slightly affected by treatment with ß-amyloid. However, pretreatment with GE and its components inhibited the expression of CHOP but increased that of GRP78 in ß-amyloid-treated cells. This study also showed that a single treatment with GE extracts, gastrodin, or 4HBA induced the expression of GRP78, a marker for enhanced protein folding machinery, suggesting a protective mechanism for GE against ß-amyloid. CONCLUSIONS: This study reveals the protective effects of GE against ß-amyloid-induced cell death, possibly through the enhancement of protein folding machinery of a representative protein, GRP78, and the regulation of CHOP in BV2 mouse microglial cells.
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Amiloide/farmacología , Alcoholes Bencílicos/farmacología , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Gastrodia/química , Glucósidos/farmacología , Microglía/efectos de los fármacos , Animales , Alcoholes Bencílicos/aislamiento & purificación , Chaperón BiP del Retículo Endoplásmico , Glucósidos/aislamiento & purificación , RatonesRESUMEN
The endoplasmic reticulum (ER) is the major site of calcium storage and protein folding. It has a unique oxidizing-folding environment due to the predominant disulfide bond formation during the process of protein folding. Alterations in the oxidative environment of the ER and also intra-ER Ca2+ cause the production of ER stress-induced reactive oxygen species (ROS). Protein disulfide isomerases, endoplasmic reticulum oxidoreductin-1, reduced glutathione and mitochondrial electron transport chain proteins also play crucial roles in ER stress-induced production of ROS. In this article, we discuss ER stress-associated ROS and related diseases, and the current understanding of the signaling transduction involved in ER stress.
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Enfermedad , Estrés del Retículo Endoplásmico , Animales , Humanos , Modelos Biológicos , Especies Reactivas de Oxígeno/metabolismo , Respuesta de Proteína DesplegadaRESUMEN
BACKGROUND: Salvia miltiorrhiza (SM) has long been used as a traditional oriental medicine for cardiovascular disease. Accumulating evidence also indicates that SM has anti-osteoporotic effects. This study was conducted to examine the SM-induced anti-osteoporotic effect and its possible mechanisms with various doses of SM. METHODS: We studied Sprague-Dawley female rats aged 12 weeks, divided into six groups: sham-operated control (SHAM), OVX rats supplemented with SM (1, 3, 10 and 30 mg/kg) orally for 8 weeks. At the end of the experiment, blood samples were collected and biochemistry analysis was performed. Specimens from both tibia and liver were processed for light microscopic examination. DEXA and µ-CT analyses of the tibia were also performed. RESULTS: SM treatment significantly ameliorated the decrease in BMD and trabecular bone mass according to DEXA and trabecular bone architecture analysis of trabecular bone structural parameters by µ-CT scanning. In serum biochemical analysis, SM decreased the released TRAP-5b, an osteoclast activation marker and oxidative stress parameters including MDA and NO induced by OVX. CONCLUSIONS: The preventive effect of SM was presumably due to its anti-oxidative stress partly via modulation of osteoclast maturation and number. In current study, SM appears to be a promising osteoporosis therapeutic natural product.
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Conservadores de la Densidad Ósea/uso terapéutico , Densidad Ósea/efectos de los fármacos , Osteoporosis Posmenopáusica/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Fitoterapia , Extractos Vegetales/uso terapéutico , Salvia miltiorrhiza , Animales , Antioxidantes/farmacología , Antioxidantes/uso terapéutico , Conservadores de la Densidad Ósea/farmacología , Femenino , Humanos , Malondialdehído/sangre , Óxido Nítrico/sangre , Osteoclastos/efectos de los fármacos , Osteoporosis Posmenopáusica/diagnóstico por imagen , Osteoporosis Posmenopáusica/prevención & control , Ovariectomía , Extractos Vegetales/farmacología , Radiografía , Ratas , Ratas Sprague-Dawley , Tibia/anatomía & histología , Tibia/efectos de los fármacos , Tibia/metabolismoRESUMEN
Sertoli cells are highly polarized testicular supporting cells that simultaneously nurture multiple stages of germ cells during spermatogenesis. Proper localization of polarity protein complexes within Sertoli cells, including those responsible for blood-testis barrier formation, is vital for spermatogenesis. However, the mechanisms and developmental timing that underlie Sertoli cell polarity are poorly understood. We investigate this aspect of testicular function by conditionally deleting Cdc42, encoding a Rho GTPase involved in regulating cell polarity, specifically in Sertoli cells. Sertoli Cdc42 deletion leads to increased apoptosis and disrupted polarity of juvenile and adult testes but does not affect fetal and postnatal testicular development. The onset of the first wave of spermatogenesis occurs normally, but it fails to progress past round spermatid stages, and by young adulthood, conditional knockout males exhibit a complete loss of spermatogenic cells. These findings demonstrate that Cdc42 is essential for Sertoli cell polarity and for maintaining steady-state sperm production.
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Células de Sertoli/enzimología , Espermátides/enzimología , Espermatogénesis , Proteína de Unión al GTP cdc42/metabolismo , Animales , Masculino , Ratones , Proteína de Unión al GTP cdc42/genéticaRESUMEN
Background Transforming growth factor beta ( TGF -ß) is an important cytokine in mediating the cardiac fibrosis that often accompanies pathogenic cardiac remodeling. Cardiomyocyte-specific expression of a mutant αB-crystallin (Cry ABR120G), which causes human desmin-related cardiomyopathy, results in significant cardiac fibrosis. During onset of fibrosis, fibroblasts are activated to the so-called myofibroblast state and TGF -ß binding mediates an essential signaling pathway underlying this process. Here, we test the hypothesis that fibroblast-based TGF -ß signaling can result in significant cardiac fibrosis in a disease model of cardiac proteotoxicity that has an exclusive cardiomyocyte-based etiology. Methods and Results Against the background of cardiomyocyte-restricted expression of Cry ABR120G, we have partially ablated TGF -ß signaling in cardiac myofibroblasts to observe whether cardiac fibrosis is reduced despite the ongoing pathogenic stimulus of Cry ABR120G production. Transgenic Cry ABR120G mice were crossed with mice containing a floxed allele of TGF -ß receptor 2 ( Tgfbr2 f/f). The double transgenic animals were subsequently crossed to another transgenic line in which Cre expression was driven from the periostin locus ( Postn) so that Tgfbr2 would be ablated with myofibroblast conversion. Structural and functional assays were then used to determine whether general fibrosis was affected and cardiac function rescued in Cry ABR120G mice lacking Tgfbr2 in the myofibroblasts. Ablation of myofibroblast specific TGF -ß signaling led to decreased morbidity in a proteotoxic disease resulting from cardiomyocyte autonomous expression of Cry ABR120G. Cardiac fibrosis was decreased and hypertrophy was also significantly attenuated, with a significant improvement in survival probability over time, even though the primary proteotoxic insult continued. Conclusions Myofibroblast-targeted knockdown of Tgfbr2 signaling resulted in reduced fibrosis and improved cardiac function, leading to improved probability of survival.
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Miocardio/patología , Miofibroblastos/fisiología , Factor de Crecimiento Transformador beta/fisiología , Análisis de Varianza , Animales , Cardiomiopatías/patología , Modelos Animales de Enfermedad , Femenino , Fibroblastos/fisiología , Fibrosis/etiología , Cardiopatías/patología , Masculino , Ratones Transgénicos , Distrofias Musculares/patología , Miocitos Cardíacos/fisiología , Receptor Tipo II de Factor de Crecimiento Transformador beta/metabolismo , Transducción de Señal/fisiología , Cadena B de alfa-Cristalina/metabolismoRESUMEN
Hyperactivation of phosphoinositol 3-kinase (PI3K) has been suggested to be a potential mechanism for endoplasmic reticulum (ER) stress-enhanced airway hyperresponsiveness, and PI3K inhibitors have been examined as asthma therapeutics. However, the regulatory mechanism linking PI3K to ER stress and related pathological signals in asthma have not been defined. To elucidate these pathogenic pathways, we investigated the influence of a selective PI3Kδ inhibitor, IC87114, on airway inflammation in an ovalbumin/lipopolysaccharide (OVA/LPS)-induced asthma model. In OVA/LPS-induced asthmatic mice, the activity of PI3K, downstream phosphorylation of AKT and activation of nuclear factor-κB (NF-κB) were all significantly elevated; these effects were reversed by IC87114. IC87114 treatment also reduced the OVA/LPS-induced ER stress response by enhancing the intra-ER oxidative folding status through suppression of protein disulfide isomerase activity, ER-associated reactive oxygen species (ROS) accumulation and NOX4 activity. Furthermore, inositol-requiring enzyme-1α (IRE1α)-dependent degradation (RIDD) of IRE1α was reduced by IC87114, resulting in a decreased release of proinflammatory cytokines from bronchial epithelial cells. These results suggest that PI3Kδ may induce severe airway inflammation and hyperresponsiveness by activating NF-κB signaling through ER-associated ROS and RIDD-RIG-I activation. The PI3Kδ inhibitor IC87114 is a potential therapeutic agent against neutrophil-dominant asthma.
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Asma/metabolismo , Estrés del Retículo Endoplásmico , Oxidación-Reducción , Fosfatidilinositol 3-Quinasas/metabolismo , Adenina/análogos & derivados , Adenina/farmacología , Animales , Asma/etiología , Modelos Animales de Enfermedad , Estrés del Retículo Endoplásmico/efectos de los fármacos , Peroxidación de Lípido , Lipopolisacáridos/inmunología , Proteínas de la Membrana/metabolismo , Ratones , FN-kappa B/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Ovalbúmina/inmunología , Estrés Oxidativo/efectos de los fármacos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Quinazolinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Receptores de Superficie Celular , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Cardiac stress can trigger production of a 40-kDa peptide fragment derived from the amino terminus of the cardiac myosin-binding protein C. Cardiac stress, as well as cMyBP-C mutations, can trigger production of 1 such truncated protein fragment, a 40-kDa peptide fragment derived from the amino terminus of cMyBP-C. Genetic expression of this 40-kDa fragment in mouse cardiomyocytes (cMyBP-C40k) leads to cardiac disease, fibrosis, and death within the first year. Fibrosis can occur in many cardiovascular diseases, and mitogen-activated protein kinase--activated protein kinase-2 signaling has been implicated in a variety of fibrotic processes. Recent studies demonstrated that mitogen-activated protein kinase--activated protein kinase-2 inhibition using the cell-permeant peptide inhibitor MMI-0100 is protective in the setting of acute myocardial infarction. We hypothesized that MMI-0100 might also be protective in a chronic model of fibrosis, produced as a result of cMyBP-C40k cardiomyocyte expression. METHODS AND RESULTS: Nontransgenic and cMyBP-C40k inducible transgenic mice were given MMI-0100 or PBS daily for 30 weeks. In control groups, long-term MMI-0100 was benign, with no measurable effects on cardiac anatomy, function, cell viability, hypertrophy, or probability of survival. In the inducible transgenic group, MMI-0100 treatment reduced cardiac fibrosis, decreased cardiac hypertrophy, and prolonged survival. CONCLUSIONS: Pharmaceutical inhibition of mitogen-activated protein kinase--activated protein kinase-2 signaling via MMI-0100 treatment is beneficial in the context of fibrotic cMyBPC40k disease.
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Cardiomiopatías/prevención & control , Proteínas Portadoras/metabolismo , Hipertrofia Ventricular Izquierda/prevención & control , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Miocitos Cardíacos/efectos de los fármacos , Péptidos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Remodelación Ventricular/efectos de los fármacos , Actinas/metabolismo , Animales , Cardiomiopatías/enzimología , Cardiomiopatías/genética , Cardiomiopatías/fisiopatología , Proteínas Portadoras/genética , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Fibroblastos/patología , Fibrosis , Hipertrofia Ventricular Izquierda/genética , Hipertrofia Ventricular Izquierda/fisiopatología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Regulación hacia ArribaRESUMEN
Endoplasmic reticulum (ER) stress and associated protein aggregation are closely associated with human diseases, including alterations in hepatic lipid metabolism. Inhibition of ER stress can have a significant effect on the prevention of hepatic dyslipidemia. Here, we studied the role of 4-phenylbutyric acid (4-PBA), a chemical chaperone, on ER stress-induced hepatic lipid accumulation. We studied ER stress induction following CCl4 exposure and delineated mechanisms of the CCl4-induced ER stress response in liver tissue from mice. CCl4 affected the formation of disulfide bonds through excessive hyper-oxidation of protein disulfide isomerase (PDI). Increased complex formation between PDI and its client proteins persisted in CCl4-exposed samples. Conversely, 4-PBA inhibited ER stress via secretion of apolipoprotein B and prevention of hepatic lipid accumulation. We also studied the mechanism-based pharmacokinetic and pharmacodynamic profiles and identified the ER stress-related proteins GRP78 and CHOP, along with plasma apolipoprotein B and triglyceride levels, as novel biomarkers of ER stress-induced hepatic lipid accumulation. ER stress and its clinical relevance for therapeutic approaches were well correlated with the activity of the ER stress regulator 4-PBA, which may be a promising drug candidate for the treatment of hepatic lipid accumulation, such as hepatic steatosis.
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Tetracloruro de Carbono/efectos adversos , Dislipidemias/inducido químicamente , Dislipidemias/tratamiento farmacológico , Hígado/efectos de los fármacos , Hígado/metabolismo , Fenilbutiratos/farmacología , Fenilbutiratos/farmacocinética , Animales , Apolipoproteínas B/metabolismo , Citocromo P-450 CYP2E1/metabolismo , Modelos Animales de Enfermedad , Disulfuros/química , Dislipidemias/metabolismo , Dislipidemias/patología , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Metabolismo de los Lípidos/efectos de los fármacos , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Fenilbutiratos/uso terapéutico , Pliegue de Proteína/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
IC87114 is a selective PI3Kδ inhibitor. A simple, sensitive and reliable LC-MS/MS method with rapid sample preparation was developed and validated for the determination of IC87114 in mouse plasma, bronchoalveolar lavage, and lung. Chromatographic separation was achieved using an Agilent Zorbax Eclipse XDB-C18 column (150mm×2.1mm internal diameter, 3.5µm particle size). Mass spectrometric detection was conducted by electrospray ionization in positive ion multiple reaction monitoring modes. The calibration curve was linear over a concentration range of 0.01-1000ng/mL for plasma/BAL and 0.1-250ng/mL for lung tissue. Recoveries were as high as 97.29%, 102.81% and 89.70% for plasma, BAL fluid and lung sample, respectively. The lower limit of quantification was 0.01ng/mL. Intra-day and inter-day accuracy and precision were within the acceptable limits of ±15% at all concentrations. Finally, the method was successfully used in a pharmacokinetic study that measured IC87114 in mouse plasma, BAL fluid and lung tissue after administration of a single 1mg/kg intratracheal dose of IC87114. The percentage change for incurred sample reanalysis (ISR) was within ±15.0% and met the acceptance criteria for ISR.
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Adenina/análogos & derivados , Cromatografía Liquida/métodos , Pulmón/metabolismo , Quinazolinas/farmacocinética , Espectrometría de Masas en Tándem/métodos , Adenina/biosíntesis , Adenina/metabolismo , Adenina/farmacocinética , Animales , Lavado Broncoalveolar/métodos , Ratones , Ratones Endogámicos C57BL , Quinazolinas/metabolismoRESUMEN
The aim of this study was to investigate the effects of soy on the pharmacokinetics and pharmacodynamics of valproic acid (VPA). In a preclinical study, rats were pretreated with two different amounts of soy extract for five days (150 mg/kg and 500 mg/kg), which resulted in decreases of 57% and 65% in the Cmax of VPA, respectively. AUC of VPA decreased to 83% and 70% in the soy pretreatment groups. Interestingly, the excretion rate of VPA glucuronide (VPAG) was higher in the soy-fed groups. Levels of UDP-glucuronosyltransferase (UGT) UGT1A3, UGT1A6, UGT2B7 and UGT2B15 were elevated in the soy-treated group, and GABA concentrations were elevated in the brain after VPA administration. However, this was less pronounced in soy extract pretreated group than for the untreated group. This is the first study to report the effects of soy pretreatment on the pharmacokinetics and pharmacodynamics of VPA in rodents.
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Glycine max/química , Extractos Vegetales/farmacología , Ácido Valproico/análogos & derivados , Ácido Valproico/farmacocinética , Animales , Área Bajo la Curva , Química Encefálica , Evaluación Preclínica de Medicamentos , Interacciones Alimento-Droga , Glucuronosiltransferasa/sangre , Isoenzimas/sangre , Masculino , Extractos Vegetales/química , Ratas , Ratas Sprague-Dawley , Ácido Valproico/sangre , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Endoplasmic reticulum (ER) stress is associated with various human diseases. Phenylbutyric acid (PBA) is a well-known chemical chaperone that regulates ER stress. The main objective of this study was to develop a simple, rapid, and sensitive method for the simultaneous determination of phenylbutyric acid and its metabolite, phenylacetic acid (PAA). A LC-MS/MS analysis using negative electrospray ionization was used. Samples were analyzed by multiple reaction monitoring (MRM) in 15 min of total run time, using d11-PBA and d7-PAA as internal standards. The limit of quantification was 1 µg/g for tissue and 0.8 µg/mL for plasma. Recoveries for plasma and tissues were higher than 81% for both PBA and PAA. The inter-day and intra-day accuracy and precision were within ±15%. We then further successfully validated this method by applying it to determine the tissue distribution of PBA and its metabolite PAA after i.p. injection of PBA at a dose of 500 mg/kg in mice. The maximum concentrations of PBA and PAA in plasma and tissues were seen at 15 min and 45 min, respectively. The PBA plasma concentration was 15-fold higher than the concentration in the kidney, whereas the PAA plasma concentration was 6-fold higher than the concentration in the liver. The area under the curve decreased in the order of plasma > kidney > liver > heart > muscle > lung for PBA and plasma > liver > kidney > heart > muscle > lung for PAA. The tissue to plasma ratio ranged from 0.007 to 0.063 for PBA and 0.016 to 0.109 for PAA. In summary, the LC-ESI-MS method developed in this study is simple, sensitive and reliable.