RESUMEN
Maternal immune activation results in altered levels of cytokines in the maternal-fetal system, which has a negative impact on fetal development, including the gonadotropin-releasing hormone (GnRH) system, which is crucial for the reproduction. Suppression of GnRH-neuron migration may be associated with cytokine imbalances, and primarily with proinflammatory cytokine interleukin (IL)-6. This study aimed to determine the effects of IL-6 and monoclonal antibody to IL-6 or IL-6R or polyclonal IgG on the formation of migration route of GnRH-neurons in ex vivo and in vivo rodent models on day 11.5 of embryonic development. The increased level of IL-6 in mouse nasal explants suppressed peripherin-positive fiber outgrowth, while this led to an increase in the number of GnRH-neurons in the nose and olfactory bulbs and a decrease in their number in the fetal brain. This effect is likely to be realized via IL-6 receptors along the olfactory nerves. The suppressive effect of IL-6 was diminished by monoclonal antibodies to IL-6 or its receptors and by IgG.
Asunto(s)
Citocinas , Hormona Liberadora de Gonadotropina , Animales , Femenino , Ratones , Embarazo , Encéfalo/metabolismo , Movimiento Celular , Citocinas/farmacología , Hormona Liberadora de Gonadotropina/metabolismo , Inmunoglobulina G/farmacología , Interleucina-6/farmacología , Roedores/metabolismoRESUMEN
The development of the neuroendocrine system, including the hypothalamic-pituitary-gonadal (HPG) axis, is sensitive to environmental impacts during critical developmental periods. Maternal immune system activation by bacterial or viral infection may be one of the negative impacts. This study focused on the effect of systemic inflammation induced by lipopolysaccharides (LPS E. coli) on the HPG axis development in male rat offspring, corrected by the anti-inflammatory action of polyclonal IgG and monoclonal anti-interleukin (IL)-6 receptor antibodies (IL-6RmAbs). A single LPS exposure on the 12th embryonic day (ED) led to a decrease in the number of afferent synaptic inputs on gonadotropin-releasing, hormone-producing neurons in adult male offspring. LPS exposure on ED18 did not lead to such disruptions. Moreover, after the LPS injections on ED12, circulating follicle-stimulating hormone and sex steroid levels were reduced, and the gonadal structure was disrupted. A prenatal IL-6R blockade with IL-6RmAbs and polyclonal IgG reduced the negative effects of inflammation on fetal HPG axis development. Overall, the data obtained confirm the morphogenetic effect of inflammation on fetal HPG development and IL-6 involvement in these processes.
Asunto(s)
Eje Hipotálamico-Pituitario-Gonadal , Sistema Hipotálamo-Hipofisario , Embarazo , Femenino , Ratas , Animales , Masculino , Sistema Hipotálamo-Hipofisario/metabolismo , Lipopolisacáridos/toxicidad , Escherichia coli/metabolismo , Hormona Liberadora de Gonadotropina/metabolismo , Inflamación , Inmunoglobulina G/farmacologíaRESUMEN
Sexual performance in adult male rats is highly sensitive to prenatal stress which can affect the functionality of the reproductive system and various brain structures involved in modulating sexual behavior. The immunomodulatory effect of mouse IgG on reproductive maturity in male offspring after LPS exposure in vivo and in vitro was studied. Prenatal IgG injection (20 µg/mouse) had a positive impact on the puberty of male mice whose mothers were exposed to LPS (100 µg/kg) on the 12th day of pregnancy. The numbers of Sertoli cells were increased, whereas the body weight and the number of symplastic spermatids were decreased in offspring as compared to LPS-treated animals. Besides, IgG had a positive effect on altered hormone levels: reduced estradiol level on the 5th and 14th postnatal days and increased testosterone level on the 30th postnatal day in blood that led to an increased number of mounting attempts in sexually mature males. The cAMP-dependent pathway may be involved in the regulation of the LPS-induced inflammation. IgG reduced the increased level of cAMP in mouse peritoneal macrophages activated by LPS in vitro. IgG is able to modulate inflammation processes but its exposure time is important.
Asunto(s)
Inmunoglobulina G/farmacología , Infertilidad Masculina/tratamiento farmacológico , Inflamación/complicaciones , Lipopolisacáridos/toxicidad , Efectos Tardíos de la Exposición Prenatal/patología , Reproducción , Conducta Sexual Animal , Animales , Femenino , Infertilidad Masculina/sangre , Infertilidad Masculina/etiología , Infertilidad Masculina/patología , Inflamación/inducido químicamente , Inflamación/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Embarazo , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Maduración Sexual , Testosterona/sangreRESUMEN
Different aspects of the reciprocal regulatory influence on the development of gonadotropin-releasing hormone (GnRH)-producing- and immune systems in the perinatal ontogenesis and their functioning in adults in normal and pathological conditions are discussed. The influence of GnRH on the development of the immune system, on the one hand, and the influence of proinflammatory cytokines on the development of the hypothalamic-pituitary-gonadal system, on the other hand, and their functioning in adult offspring are analyzed. We have focused on the effects of GnRH on the formation and functional activity of the thymus, as the central organ of the immune system, in the perinatal period. The main mechanisms of reciprocal regulation of these systems are discussed. The reproductive health of an individual is programmed by the establishment and development of physiological systems during critical periods. Regulatory epigenetic mechanisms of development are not strictly genetically controlled. These processes are characterized by a high sensitivity to various regulatory factors, which provides possible corrections for disorders.
Asunto(s)
Citocinas/metabolismo , Regulación de la Expresión Génica , Hormona Liberadora de Gonadotropina/metabolismo , Sistema Inmunológico , Animales , Moléculas de Adhesión Celular/metabolismo , Epigénesis Genética , Femenino , Humanos , Inflamación , Masculino , Ratones , Neuronas/metabolismo , Neurotransmisores/metabolismo , Ratas , Transducción de Señal , Timo/metabolismo , Factores de Transcripción/metabolismoRESUMEN
This review provides information on the structure of estrogen receptors (ERs), their localization and functions in mammalian cells. Additionally, the structure of proteasomes and mechanisms of protein ubiquitination and cleavage are described. According to the modern concept, the ubiquitin proteasome system (UPS) is involved in the regulation of the activity of ERs in several ways. First, UPS performs the ubiquitination of ERs with a change in their functional activity. Second, UPS degrades ERs and their transcriptional regulators. Third, UPS affects the expression of ER genes. In addition, the opportunity of the regulation of proteasome functioning by ERs-in particular, the expression of immune proteasomes-is discussed. Understanding the complex mechanisms underlying the regulation of ERs and proteasomes has great prospects for the development of new therapeutic agents that can make a significant contribution to the treatment of diseases associated with the impaired function of these biomolecules.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/metabolismo , Receptores de Estrógenos/metabolismo , Ubiquitina/metabolismo , Animales , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Regulación de la Expresión Génica , Humanos , Complejo de la Endopetidasa Proteasomal/química , Receptores de Estrógenos/genética , Complejos de Ubiquitina-Proteína Ligasa/metabolismoRESUMEN
Viral and bacterial infections causing systemic inflammation are significant risk factors for developing body. Inflammatory processes can alter physiological levels of regulatory factors and interfere with developmental mechanisms. The brain is the main target for the negative impact of inflammatory products during critical ontogenetic periods. Subsequently, the risks of various neuropsychiatric diseases such as Alzheimer's and Parkinson's diseases, schizophrenia, and depression are increased in the offspring. Inflammation-induced physiological disturbances can cause immune and behavioral disorders, reproductive deficiencies, and infertility. The influence of maternal immune stress is mediated by the regulation of pro-inflammatory cytokines such as interleukin (IL)-1ß, IL-6, monocyte chemotactic protein 1, leukemia-inhibiting factor, and tumor necrosis factor-alpha secretion in the maternal-fetal system. The increasing number of patients with neuronal and reproductive disorders substantiates the identification of biomarkers for these disorders targeted at their therapy.
Asunto(s)
Citocinas/metabolismo , Fenómenos del Sistema Inmunológico/fisiología , Mediadores de Inflamación/metabolismo , Sistemas Neurosecretores/metabolismo , Efectos Tardíos de la Exposición Prenatal/metabolismo , Reproducción/fisiología , Animales , Biomarcadores/metabolismo , Encéfalo/inmunología , Encéfalo/metabolismo , Citocinas/inmunología , Femenino , Humanos , Inflamación/complicaciones , Inflamación/inmunología , Inflamación/metabolismo , Mediadores de Inflamación/inmunología , Sistemas Neurosecretores/inmunología , Embarazo , Síndrome de Respuesta Inflamatoria Sistémica/complicaciones , Síndrome de Respuesta Inflamatoria Sistémica/inmunología , Síndrome de Respuesta Inflamatoria Sistémica/metabolismoRESUMEN
An increasing body of recent experimental data confirms the impact of neurohormones on fetal development and function of different body systems. The synthesis of many neurohormones starts in fetal tissues before the hypothalamic-pituitary-adrenal and hypothalamic-pituitary-gonadal systems are formed, and their high levels are detected in the bloodstream. Here, we studied the role of gonadotropin-releasing hormone (GnRH) in rat thymus development and tried to reveal possible mechanisms underlying the GnRH effects in early development. Western blotting and reverse transcription-polymerase chain reaction allowed us to identify receptor for GnRH in the fetal thymus with peak expression on embryonic days 17-18 (ED17-18). Blocking the receptors in utero on ED17 by a GnRH antagonist suppressed the concanavalin A-induced proliferative response of T cells in adults. GnRH (10-7 M) increased mRNA expression of interleukin (IL)-4, IL-10, IL-1ß, interferon γ (IFNγ), and tumor necrosis factor α (TNFα) in the thymus of 18-day fetuses after an ex vivo culture for 24 h. The increased mRNA levels of the cytokines in the thymus were accompanied by increased numbers of CD4+ T helpers. Overall, the data obtained confirm the regulatory or morphogenetic effect of GnRH on fetal thymus development mediated by synthesis of thymic cytokines.
Asunto(s)
Citocinas/metabolismo , Hormona Liberadora de Gonadotropina/metabolismo , Timo/embriología , Animales , Diferenciación Celular , Femenino , Ratas , Ratas Wistar , Receptores LHRH/metabolismo , Linfocitos T/citología , Linfocitos T/metabolismo , Timo/metabolismoRESUMEN
During prenatal and early postnatal periods of development, multiple environmental factors have profound and long-lasting effects on the immune and reproductive functions. The aim of this study was to investigate the effects of maternal lipopolysaccharide (LPS) exposure (50 mg/kg, i.p.) at day 12 of pregnancy and estradiol antagonist treatment (fulvestrant, 1.5 mg/kg, s.c. in neck) at postnatal days 5-14 (PND5-14) with high estradiol levels on reproductive parameters in adult rat males. Serum steroid concentrations were measured in male offspring at PND80 by ELISA. Body, testis weights and ano-genital distance (AGD) were recorded at different stages of postnatal development. Testis was also processed to cytohistological studies at PND80. Our results demonstrate that body weight was decreased from PND14 to 30 after prenatal LPS treatment and was increased after fulvestrant treatment. AGD was decreased after prenatal LPS treatment and was increased after fulvestrant injections. Testis weight, testosterone level, seminiferous tubule diameter, and number of Sertoli and spermatid cells were also decreased in rats exposed prenatally to LPS and were restored to the normal control level after fulvestrant treatment. According to results, we can conclude that the development of sexual disorders in males after prenatal immune stress is potentiated by estradiol during the pre-pubertal period.
Asunto(s)
Peso Corporal/efectos de los fármacos , Antagonistas del Receptor de Estrógeno/uso terapéutico , Fulvestrant/uso terapéutico , Infertilidad Masculina/tratamiento farmacológico , Efectos Tardíos de la Exposición Prenatal/tratamiento farmacológico , Testículo/efectos de los fármacos , Animales , Antagonistas del Receptor de Estrógeno/farmacología , Femenino , Fulvestrant/farmacología , Lipopolisacáridos , Masculino , Embarazo , Ratas , Células de Sertoli/efectos de los fármacos , Espermátides/efectos de los fármacosRESUMEN
Various stress factors during critical periods of fetal development modulate the epigenetic mechanisms controlling specific genes, which can affect the structure and function of physiological systems. Maternal immune stress by bacterial infection simulated by lipopolysaccharide (LPS) in an experiment is considered to be a powerful programming factor of fetal development. Studies of the molecular mechanisms controlling the formation and functioning of physiological systems are in the pilot stage. LPSs are the most potent natural inflammation factors. LPS-induced increases in fetal levels of pro- and anti-inflammatory cytokines can affect brain development and have long-term effects on behavior and neuroendocrine functions. The degradation of serotonergic neurons induced by LPS in the fetus is attributed to the increased levels of interleukin (IL)-6 and tumor necrosis factor (TNFα) as well as to anxiety and depression in children. Dopamine deficiency causes dysthymia, learning disability, and Parkinson's disease. According to our data, an LPS-induced increase in the levels of IL-6, leukemia inhibitory factor (LIF), and monocyte chemotactic protein (MCP-1) in maternal and fetal rats during early pregnancy disturbs the development and functioning of gonadotropin-releasing hormone production and reproductive systems. It is important to note the high responsiveness of epigenetic developmental mechanisms to many regulatory factors, which offers opportunities to correct the defects.
Asunto(s)
Lipopolisacáridos/farmacología , Sistemas Neurosecretores/embriología , Efectos Tardíos de la Exposición Prenatal/metabolismo , Animales , Citocinas/genética , Citocinas/metabolismo , Femenino , Humanos , Lipopolisacáridos/efectos adversos , Sistemas Neurosecretores/efectos de los fármacos , Sistemas Neurosecretores/metabolismo , Embarazo , Efectos Tardíos de la Exposición Prenatal/etiologíaRESUMEN
UNLABELLED: Cardiac c-Kit+ cells have a modest cardiogenic potential that could limit their efficacy in heart disease treatment. The present study was designed to augment the cardiogenic potential of cardiac c-Kit+ cells through class I histone deacetylase (HDAC) inhibition and evaluate their therapeutic potency in the chronic heart failure (CHF) animal model. Myocardial infarction (MI) was created by coronary artery occlusion in rats. c-Kit+ cells were treated with mocetinostat (MOCE), a specific class I HDAC inhibitor. At 3 weeks after MI, CHF animals were retrogradely infused with untreated (control) or MOCE-treated c-Kit+ cells (MOCE/c-Kit+ cells) and evaluated at 3 weeks after cell infusion. We found that class I HDAC inhibition in c-Kit+ cells elevated the level of acetylated histone H3 (AcH3) and increased AcH3 levels in the promoter regions of pluripotent and cardiac-specific genes. Epigenetic changes were accompanied by increased expression of cardiac-specific markers. Transplantation of CHF rats with either control or MOCE/c-Kit+ cells resulted in an improvement in cardiac function, retardation of CHF remodeling made evident by increased vascularization and scar size, and cardiomyocyte hypertrophy reduction. Compared with CHF infused with control cells, infusion of MOCE/c-Kit+ cells resulted in a further reduction in left ventricle end-diastolic pressure and total collagen and an increase in interleukin-6 expression. The low engraftment of infused cells suggests that paracrine effects might account for the beneficial effects of c-Kit+ cells in CHF. In conclusion, selective inhibition of class I HDACs induced expression of cardiac markers in c-Kit+ cells and partially augmented the efficacy of these cells for CHF repair. SIGNIFICANCE: The study has shown that selective class 1 histone deacetylase inhibition is sufficient to redirect c-Kit+ cells toward a cardiac fate. Epigenetically modified c-Kit+ cells improved contractile function and retarded remodeling of the congestive heart failure heart. This study provides new insights into the efficacy of cardiac c-Kit+ cells in the ischemic heart failure model.
Asunto(s)
Epigénesis Genética , Insuficiencia Cardíaca/terapia , Infarto del Miocardio/terapia , Miocitos Cardíacos/trasplante , Proteínas Proto-Oncogénicas c-kit/genética , Acetilación , Animales , Benzamidas/farmacología , Biomarcadores/metabolismo , Diferenciación Celular , Células Cultivadas , Colágeno/genética , Colágeno/metabolismo , Modelos Animales de Enfermedad , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Inhibidores de Histona Desacetilasas/farmacología , Histonas/genética , Histonas/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , Infarto del Miocardio/genética , Infarto del Miocardio/metabolismo , Infarto del Miocardio/fisiopatología , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Pirimidinas/farmacología , Ratas , Ratas Sprague-Dawley , Recuperación de la Función , Técnicas de Cultivo de Tejidos , Función Ventricular Izquierda , Remodelación VentricularRESUMEN
BACKGROUND: Recent studies have linked histone deacetylases (HDAC) to remodeling of the heart and cardiac fibrosis in heart failure. However, the molecular mechanisms linking chromatin remodeling events with observed anti-fibrotic effects are unknown. Here, we investigated the molecular players involved in anti-fibrotic effects of HDAC inhibition in congestive heart failure (CHF) myocardium and cardiac fibroblasts in vivo. METHODS AND RESULTS: MI was created by coronary artery occlusion. Class I HDACs were inhibited in three-week post MI rats by intraperitoneal injection of Mocetinostat (20 mg/kg/day) for duration of three weeks. Cardiac function and heart tissue were analyzed at six week post-MI. CD90+ cardiac fibroblasts were isolated from ventricles through enzymatic digestion of heart. In vivo treatment of CHF animals with Mocetinostat reduced CHF-dependent up-regulation of HDAC1 and HDAC2 in CHF myocardium, improved cardiac function and decreased scar size and total collagen amount. Moreover, expression of pro-fibrotic markers, collagen-1, fibronectin and Connective Tissue Growth Factor (CTGF) were reduced in the left ventricle (LV) of Mocetinostat-treated CHF hearts. Cardiac fibroblasts isolated from Mocetinostat-treated CHF ventricles showed a decrease in expression of collagen I and III, fibronectin and Timp1. In addition, Mocetinostat attenuated CHF-induced elevation of IL-6 levels in CHF myocardium and cardiac fibroblasts. In parallel, levels of pSTAT3 were reduced via Mocetinostat in CHF myocardium. CONCLUSIONS: Anti-fibrotic effects of Mocetinostat in CHF are associated with the IL-6/STAT3 signaling pathway. In addition, our study demonstrates in vivo regulation of cardiac fibroblasts via HDAC inhibition.
Asunto(s)
Benzamidas/farmacología , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Interleucina-6/metabolismo , Isquemia Miocárdica/complicaciones , Pirimidinas/farmacología , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Cicatriz , Colágeno/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/genética , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Modelos Animales de Enfermedad , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Fibrosis , Expresión Génica , Insuficiencia Cardíaca/patología , Histona Desacetilasas/metabolismo , Ratas , Función Ventricular/efectos de los fármacosRESUMEN
Recent studies provide evidence that prenatal immunological stress may affect the programming of reproductive health and sexual behavior in adult animals. The aim of this study was to investigate the influence of maternal inflammation, induced by an intraperitoneal (i.p.) injection of lipopolysaccharide (LPS, 45 µg/kg) on embryonic day 11.5 (E 11.5), on the development of the gonadotropin-releasing hormone (GnRH) system in mouse fetuses as well as on the proinflammatory cytokine level in pregnant mice and their fetuses. In the fetuses, the GnRH neuron migration from the olfactory pit to the forebrain was estimated on embryonic days 14.5 and 18.5. The levels of the proinflammatory cytokines interleukin (IL)-6, monocyte chemotactic protein (MCP)-1, tumor necrosis factor (TNF)-α and leukemia inhibitory factor (LIF) were measured with the cytometric bead and ELISA array method in the maternal and fetal blood, amniotic fluid and fetal cerebrospinal fluid (CSF). According to our data, activation of the immune system by LPS treatment on embryonic day 11.5 leads to an increased quantity of neurons in the nasal and olfactory bulb areas and a decreased quantity in the forebrain area on embryonic day 14.5. There was a slight decrease in the total number of neurons in the forebrain area on embryonic day 18.5. The levels of proinflammatory cytokines were significantly increased within 3 h after LPS treatment in the maternal and fetal blood, amniotic fluid and fetal CSF. IL-6-receptor immunoreactivity was detected on olfactory/vomeronasal axons. Thus, prenatal immunological stress delays the GnRH neuron migration in the nasal compartment of mouse fetuses, which may be mediated by the regulation of IL-6, MCP-1 and LIF secretion in the maternal-fetal system.
Asunto(s)
Movimiento Celular/fisiología , Citocinas/metabolismo , Hormona Liberadora de Gonadotropina/metabolismo , Inflamación/metabolismo , Lipopolisacáridos/farmacología , Neuronas/fisiología , Animales , Femenino , Desarrollo Fetal , Feto/metabolismo , Inflamación/inducido químicamente , Lipopolisacáridos/administración & dosificación , Ratones , Ratones Endogámicos BALB C , EmbarazoRESUMEN
BACKGROUND: Progenitor cells isolated from cardiac explant-derived cells improve cardiac function after myocardial infarction (MI). To fully realize the therapeutic potential of these cells, it is essential to develop a safe and efficient delivery method. Therefore, the objective of this study was to determine the efficacy of our newly developed approach to retrograde coronary vein (RCV) infusion of cardiac c-Kit(+) cells in a small-animal model of congestive heart failure (CHF). METHODS: Sprague-Dawley rats underwent experimental MI. After 21 days, cardiac explant-derived c-Kit(+) cells were delivered to both sham and CHF animals using RCV delivery. Vehicle-treated (serum-free medium) sham and CHF animals were used as controls. Cardiac function and heart tissues were evaluated 21 days post-transplantation. RESULTS: RCV-delivered cells were retained in infarcted hearts for at least 21 days after transplantation. At 21 days post-RCV infusion, the majority of transplanted c-Kit(+)/GFP(+) cells were localized in the left ventricle. Compared with vehicle-treated CHF animals, RCV-treated rats showed a significant improvement in cardiac function. Furthermore, RCV-treated rats exhibited an increase in capillary density, a decrease in total heart collagen, and a reduction in both infarct size and cardiomyocyte hypertrophy when compared with vehicle-treated CHF rats. CONCLUSIONS: Our study showed that the RCV infusion approach is an efficient technique for targeted cell delivery to the infarcted myocardium. Cardiac c-Kit(+) cells, delivered using RCV infusion ameliorated progression of heart failure, improved cardiac function and retarded myocardial remodeling in heart failure rats.
Asunto(s)
Insuficiencia Cardíaca/terapia , Infarto del Miocardio/complicaciones , Miocitos Cardíacos/trasplante , Proteínas Proto-Oncogénicas c-kit , Trasplante de Células Madre/métodos , Animales , Vasos Coronarios , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/patología , Infusiones Intravenosas , Masculino , Infarto del Miocardio/patología , Infarto del Miocardio/terapia , Ratas Sprague-Dawley , Remodelación VentricularRESUMEN
BACKGROUND: Cardiac c-Kit+ cells isolated from cardiac explant-derived cells modestly improve cardiac functions after myocardial infarction; however, their full potential has not yet been realized. For instance, the majority of potential candidates for cell therapy suffer from chronic heart failure (CHF), and it is unclear how this disease affects the explant-derived progenitor cells. Therefore, the objective of this study was to determine the effect of CHF on the number and phenotype of cardiac explant c-Kit+ progenitors and elucidate mechanisms of their regulation. METHODS AND RESULTS: Myocardial infarction was created by left anterior descending coronary artery occlusion. Sham-operated animals were used as a control group. CHF-developed infarcted animals were selected on the basis of left ventricle end-diastolic pressure ≥ 20 mm Hg and scar size ≥ 30%. Here, we found that CHF atrial explants produced less c-Kit+ cells than sham explants. CHF-derived c-Kit+ cells exhibited upregulated transforming growth factor-ß (TGF-ß) signaling, increased level of epithelial to mesenchymal transition markers, and diminished expression of pluripotency markers compared with shams. We show that intervention with TGF-ß signaling by inhibiting TGF-ß receptor type I or Smad 2/3 using small-molecule inhibitors improved c-Kit+ cell yield, attenuated epithelial to mesenchymal transition markers, stimulated the pluripotency marker Nanog, and improved efficiency of c-Kit+ cell differentiation toward cardiomyocyte-like cells in vitro. CONCLUSIONS: Taken together, our findings suggest that TGF-ß inhibition positively modulates c-Kit+ cell phenotype and function in vitro, and this strategy may be considered in optimizing cardiac progenitor function and cell expansion protocols for clinical application.
Asunto(s)
Atrios Cardíacos/citología , Insuficiencia Cardíaca/patología , Factor de Crecimiento Transformador beta/fisiología , Animales , Células Cultivadas , Enfermedad Crónica , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Progenitor cell therapy is emerging as a novel treatment for heart failure. However the molecular mechanisms regulating the generation of cardiac progenitor cells is not fully understood. We hypothesized that cardiac progenitor cells are generated from cardiac explant via a process similar to epithelial to mesenchymal transition (EMT). METHODS/FINDINGS: Explant-derived cells were generated from partially digested atrial tissue. After 21 days in culture, c-Kit+ cells were isolated from cell outgrowth. The majority of explant-originated c-Kit+ cells expressed the epicardial marker Wt1. Cardiac cell outgrowth exhibits a temporal up-regulation of EMT-markers. Notch stimulation augmented, while Notch inhibition suppressed, mesenchymal transition in both c-Kit+ and c-Kit- cells. In c-Kit+ cells, Notch stimulation reduced, while Notch inhibition up-regulated pluripotency marker expressions such as Nanog and Sox2. Notch induction was associated with degradation of ß-catenin in c-Kit- cells. In contrast, Notch inhibition resulted in ß-catenin accumulation, acquisition of epitheloid morphology, and up-regulation of Wnt target genes in c-Kit- cells. CONCLUSION: Our study suggests that Notch-mediated reversible EMT process is a mechanism that regulates explant-derived c-Kit+ and c-Kit- cells.
Asunto(s)
Transición Epitelial-Mesenquimal , Miocardio/citología , Receptores Notch/metabolismo , Células Madre/metabolismo , Animales , Proteínas de Unión al Calcio/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Miocardio/metabolismo , Fenotipo , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor Notch1/metabolismo , Proteínas Serrate-Jagged , Transducción de Señal , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMEN
Dynamics of the expression of MHC class I, immune proteasomes and proteasome regulators 19S, PA28, total proteasome pool and proteasome chymotrypsin-like activity in Walker 256 tumor after implantation into Brattleboro rats with the hereditary defect of arginine-vasopressin synthesis was studied. The tumor growth and regression in Brattleboro rats were accompanied by changes in the proteasome subunit level unlike the tumor growth in WAG rats with normal expression of arginine-vasopressin gene. In the tumor implanted into Brattleboro rats the immune proteasome level was maximal between days 14 and 17, when the tumor underwent regression. Conversely, the expression of proteasome regulators tended to decrease during this period. Immune proteasomes are known to produce antigen epitopes for MHC class I to be presented to CD8+ T lymphocytes. Enhanced expression of immune proteasomes coincided with the recovery of MHC class I expression, suggesting the efficient presentation of tumor antigens in Brattleboro rats.
Asunto(s)
Arginina Vasopresina/genética , Carcinoma 256 de Walker/genética , Carcinoma 256 de Walker/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Complejo de la Endopetidasa Proteasomal/inmunología , Animales , Presentación de Antígeno , Antígenos de Neoplasias/metabolismo , Arginina Vasopresina/biosíntesis , Carcinoma 256 de Walker/metabolismo , Carcinoma 256 de Walker/patología , Quimotripsina/inmunología , Quimotripsina/metabolismo , Cisteína Endopeptidasas/inmunología , Cisteína Endopeptidasas/metabolismo , Masculino , Regresión Neoplásica Espontánea/genética , Regresión Neoplásica Espontánea/inmunología , Trasplante de Neoplasias , Complejo de la Endopetidasa Proteasomal/metabolismo , Ratas , Ratas BrattleboroRESUMEN
Immune proteasomes in thymus are involved in processing of self-antigens, which are presented by MHC class I molecules for rejection of autoreactive thymocytes in adults and probably in perinatal rats. The distribution of immune proteasome subunits LMP7 and LMP2 in thymic cells have been investigated during rat perinatal ontogenesis. Double immunofluorescent labeling revealed LMP7 and LMP2 in thymic epithelial and dendritic cells, as well as in CD68 positive cells - macrophages, monocytes - at all developmental stages. LMP2 and LMP7 were also detected by flow cytometry in almost all thymic CD90 lymphocytes through pre- and postnatal ontogenesis. Our results demonstrate that the immune proteasomes are expressed in all types of thymic antigen presenting cells during perinatal ontogenesis, suggesting the establishment of the negative selection in the thymus at the end of fetal life. The observation of the immune proteasome expression in T lymphocytes suggests their role in thymocyte differentiation besides antigen processing in thymus.
Asunto(s)
Sistema Inmunológico/embriología , Sistema Inmunológico/crecimiento & desarrollo , Complejo de la Endopetidasa Proteasomal/metabolismo , Timo/enzimología , Timo/crecimiento & desarrollo , Animales , Animales Recién Nacidos , Células Presentadoras de Antígenos/citología , Células Presentadoras de Antígenos/metabolismo , Antígenos CD/metabolismo , Antígenos de Diferenciación/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Cisteína Endopeptidasas/metabolismo , Células Dendríticas/citología , Células Dendríticas/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Queratina-18/metabolismo , Queratina-19/metabolismo , Complejo de la Endopetidasa Proteasomal/inmunología , Subunidades de Proteína/metabolismo , Proteínas/metabolismo , Ratas , Ratas Wistar , Linfocitos T/citología , Linfocitos T/metabolismo , Antígenos Thy-1/metabolismo , Timo/citología , Timo/embriologíaRESUMEN
AIMS: Cell-based therapy for myocardial infarction (MI) holds great promise; however, the ideal cell type and delivery system have not been established. Obstacles in the field are the massive cell death after direct injection and the small percentage of surviving cells differentiating into cardiomyocytes. To overcome these challenges we designed a novel study to deliver cardiac progenitor cells as a cell sheet. METHODS AND RESULTS: Cell sheets composed of rat or human cardiac progenitor cells (cardiospheres), and cardiac stromal cells were transplanted onto the infarcted myocardium after coronary artery ligation in rats. Three weeks later, transplanted cells survived, proliferated, and differentiated into cardiomyocytes (14.6 +/- 4.7%). Cell sheet transplantation suppressed cardiac wall thinning and increased capillary density (194 +/- 20 vs. 97 +/- 24 per mm(2), P < 0.05) compared with the untreated MI. Cell migration from the sheet was observed along the necrotic trails within the infarcted area. The migrated cells were located in the vicinity of stromal-derived factor (SDF-1) released from the injured myocardium, and about 20% of these cells expressed CXCR4, suggesting that the SDF-1/CXCR4 axis plays, at least, a role in cell migration. Transplantation of cell sheets resulted in a preservation of cardiac contractile function after MI, as was shown by a greater ejection fraction and lower left ventricular end diastolic pressure compared with untreated MI. CONCLUSION: The scaffold-free cardiosphere-derived cell sheet approach seeks to efficiently deliver cells and increase cell survival. These transplanted cells effectively rescue myocardium function after infarction by promoting not only neovascularization but also inducing a significant level of cardiomyogenesis.
Asunto(s)
Desarrollo de Músculos , Infarto del Miocardio/cirugía , Miocitos Cardíacos/trasplante , Regeneración , Trasplante de Células Madre , Función Ventricular Izquierda , Animales , Diferenciación Celular , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Quimiocina CXCL12/metabolismo , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Humanos , Masculino , Contracción Miocárdica , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Neovascularización Fisiológica , Ratas , Ratas Sprague-Dawley , Recuperación de la Función , Volumen Sistólico , Células del Estroma/trasplante , Factores de Tiempo , Presión Ventricular , Remodelación VentricularRESUMEN
A jumonji domain containing gene 6 (Jmjd6), previously referred to as phosphatidylserine receptor (PSR) gene, plays an important role in cell differentiation and development of multiple organs, although mechanisms of its action are not known. The Jmjd6 gene product was initially identified as a membrane protein that participates in phagocytosis. However, the later findings that recombinant Jmjd6 in expression systems was targeted to the nucleus challenged the role of Jmjd6 as a membrane receptor. Using immunocytochemistry approach we studied the subcellular distribution of endogenous Jmjd6 protein in THP-1 cells activated with phorbol 12-myristate 13 acetate (PMA). We found that treatment with PMA stimulated Jmjd6 expression in the cytosol of activated cells. Furthermore, Jmjd6 initially appeared at the cell surface of immature phagocytes (1-2 days after activation) but then translocated into the nucleus of differentiated macrophage-like cells (5-9 days after activation). Anti-Jmjd6 antibodies suppressed the engulfment of dead cell corpses by THP-1 cells expressing the Jmjd6 at the cell surface. These data indicate that Jmjd6 serves as a membrane-associated receptor that regulates phagocytosis in immature macrophages but is dispensable for phagocytosis and has other functions when it is expressed in the cytosol and nucleus of mature macrophage-like cells.
Asunto(s)
Diferenciación Celular , Membrana Celular/metabolismo , Monocitos/citología , Monocitos/metabolismo , Fagocitosis , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Anticuerpos/farmacología , Muerte Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular , Membrana Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Citosol/efectos de los fármacos , Citosol/metabolismo , Humanos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Histona Demetilasas con Dominio de Jumonji , Monocitos/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The expression pattern and distribution of proteasome immune subunits LMP7 and LMP2 in the developing rat spleen and liver as well as the periarterial lymphoid sheath formation were investigated. LMP7 and LMP2 were detected by immunoblotting in the spleen on the 21st embryonic day and during the first postnatal days in equal amounts. Their levels increased by the 8th and 18th postnatal days. Double immunofluorescent labeling the spleen cells revealed LMP7 and LMP2 in T and B lymphocytes localized in the red pulp in embryogenesis. Few T lymphocytes were discovered in periarterial zones on the 8th postnatal day. T lymphocytes filled these zones and formed lymphoid sheaths by the 18-19th day. In the liver, LMP7 and LMP2 were revealed by the 17-19th postnatal day. Immunofluorescent analysis showed their presence in hepatocytes at this period. The data suggest that T cell-mediated immune response in relation to hepatocytes is possible beginning from 18th to 19th postnatal day.