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Wnt signaling plays critical roles in development of various organs and pathogenesis of many diseases, and augmented Wnt signaling has recently been implicated in mammalian aging and aging-related phenotypes. We here report that complement C1q activates canonical Wnt signaling and promotes aging-associated decline in tissue regeneration. Serum C1q concentration is increased with aging, and Wnt signaling activity is augmented during aging in the serum and in multiple tissues of wild-type mice, but not in those of C1qa-deficient mice. C1q activates canonical Wnt signaling by binding to Frizzled receptors and subsequently inducing C1s-dependent cleavage of the ectodomain of Wnt coreceptor low-density lipoprotein receptor-related protein 6. Skeletal muscle regeneration in young mice is inhibited by exogenous C1q treatment, whereas aging-associated impairment of muscle regeneration is restored by C1s inhibition or C1qa gene disruption. Our findings therefore suggest the unexpected role of complement C1q in Wnt signal transduction and modulation of mammalian aging.
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Envejecimiento/metabolismo , Complemento C1q/metabolismo , Vía de Señalización Wnt , Animales , Complemento C1s/metabolismo , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Ratones , Suero/metabolismoRESUMEN
BACKGROUND: Adipose tissue is one of the sources of mesenchymal stem cells, which have the potential to differentiate into various types of cells, including myocytes. Whether brown adipose tissue (BAT)-derived cells might differentiate into the cardiac pacemaking-conducting cells, and have the potential to regenerate the cardiac conduction system (CCS), is investigated in this study. METHODSâANDâRESULTS: BAT was isolated from the interscapular area of mice and enzymatically digested before culture. Round or fusiform cells showed spontaneous beating at 4-7 days after culturing of BAT-derived cells. Reverse transcriptase-polymerase chain reaction analysis and immunocytochemical analysis revealed that BAT-derived cells expressed several cardiomyocytes, the CCS and pacemaker (PM) cell marker genes and proteins. Patch-clamp techniques revealed that spontaneous electrical activity and the shape of the action potential showed properties of cardiac PM cells. Next, a complete atrioventricular (AV) block was created in mice and green fluorescent protein-positive (GFP (+)) BAT-derived cells were injected intramyocardially around the AV node. At 1 week after transplantation, 50% of BAT-derived cells injected mice showed a sinus rhythm or a 2:1 AV block. Immunohistochemical analysis revealed that injected GFP (+) cells were engrafted and some GFP (+) cells co-expressed several cardiac PM cell marker proteins. CONCLUSIONS: BAT-derived cells differentiate into the CCS and PM-like cells in vitro and in vivo, and may become a useful cell source for arrhythmia therapy.
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Tejido Adiposo Pardo/metabolismo , Sistema de Conducción Cardíaco/metabolismo , Trasplante de Células Madre , Células Madre/metabolismo , Aloinjertos , Animales , Masculino , RatonesRESUMEN
The orientation map is one of the most well-studied functional maps of the visual cortex. However, results from the literature are of different qualities. Clear boundaries among different orientation domains and blurred uncertain distinctions were shown in different studies. These unclear imaging results will lead to an inaccuracy in depicting cortical structures, and the lack of consideration in experimental design will also lead to biased depictions of the cortical features. How we accurately define orientation domains will impact the entire field of research. In this study, we test how spatial frequency (SF), stimulus size, location, chromatic, and data processing methods affect the orientation functional maps (including a large area of dorsal V4, and parts of dorsal V1) acquired by intrinsic signal optical imaging. Our results indicate that, for large imaging fields, large grating stimuli with mixed SF components should be considered to acquire the orientation map. A diffusion model image enhancement based on the difference map could further improve the map quality. In addition, the similar outcomes of achromatic and chromatic gratings indicate two alternative types of afferents from LGN, pooling in V1 to generate cue-invariant orientation selectivity.
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OBJECTIVE: To evaluate the effects of low molecular weight heparin (LMWH) and heparin-binding epidermal growth factor (HB-EGF) on the biological function of human trophoblast in first trimester. METHODS: From Feb. 2011 to Nov. 2011, the trophoblast isolated from human first trimester chorionic villi was cultured in vitro. Based on variation of LMWH concentration, the trophoblast was classified into 0.025 U/ml group, 0.25 U/ml group, 2.5 U/ml group, 25 U/ml group and 250 U/ml group. In the mean time, based on treatment of heparin, the trophoblast was classified into LMWH group (0.25 U/ml), HB-EGF group (10 µg/L), combination group (LMWH at 0.25 U/ml + HB-EGF at 10 µg/L) and add with DMEM as control group. Cell proliferation was assessed by the methyl thiazolyl tetrazolium (MTT) test, which was showed with the mean absorbance as A value. Cell invasion was measured by transwell, which counted the number of cells migrated to the superficies inferia of filter membrane. Cell differentiation was assessed by the concentration of hCG secretion. RESULTS: Compared with control group, the trophoblast proliferation and invasion treated by LMWH at 0.025 U/ml did not show significant difference (P > 0.05). When treated by LWMH at 0.25 U/ml and 2.5 U/ml, trophoblast proliferation and invasion was increased significantly (P < 0.05). When LMWH at 25 U/ml and 250 U/ml, it could inhibit trophoblast proliferation and invasion (P < 0.05). When compared with A value of 0.44 ± 0.04 in control group, the increased A value were 0.51 ± 0.05 in LMWH group, 0.56 ± 0.04 in HB-EGF group and 0.69 ± 0.06 in combination group (P < 0.05). In the transwell test, the cell number were 511 ± 78 in LMWH group, 669 ± 67 in HB-EGF group and 872 ± 64 in combination group, which were significantly higher than 405 ± 67 in control group (P < 0.05), respectively. And the hCG concentration were (7143 ± 649) U/L in LMWH group, (11 762 ± 1059) U/L in HB-EGF group and (11 015 ± 1084) U/L in combination group, which showed statistical difference with (8182 ± 666) U/L in control group (P < 0.05). CONCLUSION: LMWH could modulate trophoblast proliferation, invasion, and differentiation. HB-EGF is one of important factors involved in effects of LMWH on trophoblast function.
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Proliferación Celular/efectos de los fármacos , Heparina de Bajo-Peso-Molecular/farmacología , Péptidos y Proteínas de Señalización Intercelular/farmacología , Trofoblastos/efectos de los fármacos , Adulto , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Gonadotropina Coriónica/metabolismo , Femenino , Heparina de Bajo-Peso-Molecular/administración & dosificación , Factor de Crecimiento Similar a EGF de Unión a Heparina , Humanos , Péptidos y Proteínas de Señalización Intercelular/administración & dosificación , Embarazo , Primer Trimestre del Embarazo , Trofoblastos/metabolismoRESUMEN
RATIONALE: The number of patients with coronary heart disease, including myocardial infarction, is increasing and novel therapeutic strategy is awaited. Tumor suppressor protein p53 accumulates in the myocardium after myocardial infarction, causes apoptosis of cardiomyocytes, and plays an important role in the progression into heart failure. OBJECTIVES: We investigated the molecular mechanisms of p53 accumulation in the heart after myocardial infarction and tested whether anti-p53 approach would be effective against myocardial infarction. METHODS AND RESULTS: Through expression screening, we found that CHIP (carboxyl terminus of Hsp70-interacting protein) is an endogenous p53 antagonist in the heart. CHIP suppressed p53 level by ubiquitinating and inducing proteasomal degradation. CHIP transcription was downregulated after hypoxic stress and restoration of CHIP protein level prevented p53 accumulation after hypoxic stress. CHIP overexpression in vivo prevented p53 accumulation and cardiomyocyte apoptosis after myocardial infarction. Promotion of CHIP function by heat shock protein (Hsp)90 inhibitor, 17-allylamino-17-demethoxy geldanamycin (17-AAG), also prevented p53 accumulation and cardiomyocyte apoptosis both in vitro and in vivo. CHIP-mediated p53 degradation was at least one of the cardioprotective effects of 17-AAG. CONCLUSIONS: We found that downregulation of CHIP level by hypoxia was responsible for p53 accumulation in the heart after myocardial infarction. Decreasing the amount of p53 prevented myocardial apoptosis and ameliorated ventricular remodeling after myocardial infarction. We conclude that anti-p53 approach would be effective to treat myocardial infarction.
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Infarto del Miocardio/terapia , Miocitos Cardíacos/enzimología , Complejo de la Endopetidasa Proteasomal/metabolismo , Procesamiento Proteico-Postraduccional , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Animales Recién Nacidos , Apoptosis , Secuencia de Bases , Benzoquinonas/farmacología , Células COS , Hipoxia de la Célula , Chlorocebus aethiops , Modelos Animales de Enfermedad , Terapia Genética/métodos , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Lactamas Macrocíclicas/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Mutación , Infarto del Miocardio/enzimología , Infarto del Miocardio/genética , Infarto del Miocardio/fisiopatología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Regiones Promotoras Genéticas , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Procesamiento Proteico-Postraduccional/genética , Interferencia de ARN , Ratas , Ratas Wistar , Activación Transcripcional , Ubiquitina-Proteína Ligasas/deficiencia , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Remodelación VentricularRESUMEN
Implantation of various types of cells into the heart has been reported to be effective for heart failure, however, it is unknown what kinds of cells are most suitable for myocardial repair. To examine which types of cells are most effective, we injected cell-Puramatrix™ (PM) complex into the border area and overlaid the cell-PM patch on the myocardial infarction (MI) area. We compared cardiac morphology and function at 2 weeks after transplantation. Among clonal stem cell antigen-1 positive cardiac progenitors with PM (cSca-1/PM), bone marrow mononuclear cells with PM (BM/PM), skeletal myoblasts with PM (SM/PM), adipose tissue-derived mesenchymal cells with PM (AMC/PM), PM alone (PM), and non-treated MI group (MI), the infarct area of cSca-1/PM was smaller than that of BM/PM, SM/PM, PM and MI. cSca-1/PM and AMC/PM attenuated ventricular enlargement and restored cardiac function in comparison with MI. Capillary density in the infarct area of cSca-1/PM was higher than that of other five groups. The percentage of TUNEL positive cardiomyocytes in the infarct area of cSca-1/PM was lower than that of MI and PM. cSca-1 secreted VEGF and some of them differentiated into cardiomyocytes and vascular smooth muscle cells. These results suggest that transplantation of cSca-1/PM most effectively prevents cardiac remodeling and dysfunction through angiogenesis, inhibition of apoptosis and myocardial regeneration.
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Pruebas de Función Cardíaca/efectos de los fármacos , Infarto del Miocardio/fisiopatología , Infarto del Miocardio/terapia , Miocardio/citología , Péptidos/farmacología , Trasplante de Células Madre , Células Madre/citología , Actinas/metabolismo , Inductores de la Angiogénesis/metabolismo , Animales , Antígenos Ly/metabolismo , Apoptosis/efectos de los fármacos , Capilares/efectos de los fármacos , Capilares/metabolismo , Capilares/patología , Línea Celular , Transdiferenciación Celular/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Ratones , Infarto del Miocardio/diagnóstico por imagen , Infarto del Miocardio/patología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Células Madre/efectos de los fármacos , Sístole/efectos de los fármacos , Ultrasonografía , Remodelación Ventricular/efectos de los fármacos , Factor de von Willebrand/metabolismoRESUMEN
This study examined the associations of overall and domain-specific (i.e., occupational, transport, and leisure-time) sedentary behaviors with cardiovascular disease (CVD) risk factors among high-tech company employees in Taiwan. A total of 363 participants employed at high-tech companies (mean age ± standard deviation: 37.4 ± 7.2 years) completed a questionnaire administered by email regarding their overall, occupational, transport, and leisure-time sedentary behaviors. Self-reported data of height and weight, blood pressure, blood sugar, and total cholesterol levels were also collected in 2018. An adjusted binary logistic regression model was employed in the analysis. After adjusting for sociodemographic variables, high-tech company employees who used a computer (or Internet) for more than 2 h per day during their leisure time were more likely to have CVD risk factors (odds ratio: 1.80; 95% confidence interval: 1.08-3.00). No significant associations with CVD risk factors were detected for total sedentary time, occupational sitting, television viewing time, and transport-related sitting. Despite the nature of cross-sectional design in this study, our findings may have considerable implications for intervention designers and policymakers of Taiwan. Developing effective strategies for limiting leisure-time computer use should be considered for the prevention of CVD among high-tech company employees.
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Enfermedades Cardiovasculares , Actividades Recreativas , Ocupaciones , Conducta Sedentaria , Adulto , Enfermedades Cardiovasculares/epidemiología , Estudios Transversales , Femenino , Humanos , Masculino , Riesgo , Taiwán , TecnologíaRESUMEN
Replication of the Japanese encephalitis virus (JEV) genome depends on host factors for successfully completing their life cycles; to do this, host factors have been recruited and/or relocated to the site of viral replication. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a cellular metabolic protein, was found to colocalize with viral RNA-dependent RNA polymerase (NS5) in JEV-infected cells. Subcellular fractionation further indicated that GAPDH remained relatively constant in the cytosol, while increasing at 12 to 24 hours postinfection (hpi) and decreasing at 36 hpi in the nuclear fraction of infected cells. In contrast, the redistribution patterns of GAPDH were not observed in the uninfected cells. Co-immunoprecipitation of GAPDH and JEV NS5 protein revealed no direct protein-protein interaction; instead, GAPDH binds to the 3' termini of plus- and minus-strand RNAs of JEV by electrophoretic mobility shift assays. Accordingly, GAPDH binds to the minus strand more efficiently than to the plus strand of JEV RNAs. This study highlights the findings that infection of JEV changes subcellular localization of GAPDH suggesting that this metabolic enzyme may play a role in JEV replication.
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Virus de la Encefalitis Japonesa (Especie)/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/análisis , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , ARN Viral/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas no Estructurales Virales/metabolismo , Animales , Sitios de Unión , Células Cultivadas , Cricetinae , Virus de la Encefalitis Japonesa (Especie)/metabolismo , Técnica del Anticuerpo Fluorescente , HumanosRESUMEN
Clear identification among early-stage cancer patients of those at highest risk of having metastatic disease would be of great benefit in treatment planning and management. Considerable additional benefit would accrue to high-risk patients if their responses to specific therapeutic alternatives could be predicted. Molecular biomarkers in the form of gene expression profiles are proving to be more effective tools for both prognostic and predictive patient stratification than more traditional methods such as patient demographics and histopathology indicators. Such biomarkers must be clinically validated before they can be effectively used to manage patients in clinical studies or clinical practice. This can be most efficiently accomplished by analyzing archived clinical samples with well-characterized clinical outcomes. Doing studies of this type requires reoptimization of traditional molecular expression profiling techniques to analyze RNA from fixed paraffin-embedded tissues. We have modified molecular methods for RNA extraction, RNA quantification, reverse transcription, and quantitative PCR to work optimally in archived clinical samples in order to develop a clinically validated assay for breast cancer prognosis and prediction of patient response to hormonal and chemotherapy.
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Biomarcadores de Tumor/análisis , Formaldehído/metabolismo , Perfilación de la Expresión Génica/métodos , Adhesión en Parafina/métodos , ARN/genética , Fijación del Tejido/métodos , Desoxirribonucleasa I/metabolismo , Endopeptidasa K/metabolismo , Humanos , Reacción en Cadena de la Polimerasa , ARN/aislamiento & purificación , ARN/metabolismo , Transcripción Reversa , SolucionesRESUMEN
BACKGROUND: Methionine synthase (MS) catalyzes the folate-dependent remethylation of homocysteine to methionine. We tested the hypothesis that deficiency of MS impairs endothelial function in mice heterozygous for disruption of the Mtr gene, which encodes MS. METHODS AND RESULTS: Plasma total homocysteine was similar in wild-type (Mtr(+/+)) and heterozygous (Mtr(+/-)) mice fed a control diet (4.5+/-0.3 and 5.3+/-0.4 micromol/L, respectively) and mildly elevated in Mtr(+/+) and Mtr(+/-) mice fed a low-folate (LF) diet (7.5+/-0.7 and 9.6+/-1.2 micromol/L, respectively; P<0.001 versus control diet). Dilatation of cerebral arterioles to the endothelium-dependent dilator, acetylcholine (10 micromol/L) was blunted in Mtr(+/-) mice compared with Mtr(+/+) mice fed the control diet (21+/-4 versus 32+/-4%; P<0.05). Both Mtr(+/+) and Mtr(+/-) mice exhibited impaired dilatation of cerebral arterioles to acetylcholine when they were fed the LF diet (12+/-2 and 14+/-2%, respectively; P<0.01 versus Mtr(+/+) mice fed the control diet). Elevated levels of superoxide and hydrogen peroxide were detected by confocal microscopy in cerebral arterioles of Mtr(+/-) mice fed the control diet and in both Mtr(+/+) and Mtr(+/-) mice fed the LF diet. CONCLUSIONS: These findings demonstrate that defective homocysteine remethylation caused by deficiency of either MS or folate produces oxidative stress and endothelial dysfunction in the cerebral microcirculation of mice.
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5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/deficiencia , 5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/genética , Trastornos Cerebrovasculares/genética , Animales , Aorta/efectos de los fármacos , Aorta/fisiología , Trastornos Cerebrovasculares/enzimología , Tamización de Portadores Genéticos , Homocisteína/sangre , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Estrés Oxidativo , Vasodilatación/efectos de los fármacosRESUMEN
PURPOSE: This study, along with two others, was done to develop the 21-gene Recurrence Score assay (Oncotype DX) that was validated in a subsequent independent study and is used to aid decision making about chemotherapy in estrogen receptor (ER)-positive, node-negative breast cancer patients. EXPERIMENTAL DESIGN: Patients with >or=10 nodes diagnosed from 1979 to 1999 were identified. RNA was extracted from paraffin blocks, and expression of 203 candidate genes was quantified using reverse transcription-PCR (RT-PCR). RESULTS: Seventy-eight patients were studied. As of August 2002, 77% of patients had distant recurrence or breast cancer death. Univariate Cox analysis of clinical and immunohistochemistry variables indicated that HER2/immunohistochemistry, number of involved nodes, progesterone receptor (PR)/immunohistochemistry (% cells), and ER/immunohistochemistry (% cells) were significantly associated with distant recurrence-free survival (DRFS). Univariate Cox analysis identified 22 genes associated with DRFS. Higher expression correlated with shorter DRFS for the HER2 adaptor GRB7 and the macrophage marker CD68. Higher expression correlated with longer DRFS for tumor protein p53-binding protein 2 (TP53BP2) and the ER axis genes PR and Bcl2. Multivariate methods, including stepwise variable selection and bootstrap resampling of the Cox proportional hazards regression model, identified several genes, including TP53BP2 and Bcl2, as significant predictors of DRFS. CONCLUSION: Tumor gene expression profiles of archival tissues, some more than 20 years old, provide significant information about risk of distant recurrence even among patients with 10 or more nodes.
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Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Antígeno Ki-67/análisis , Antígeno Ki-67/genética , Ganglios Linfáticos/patología , Metástasis Linfática , Persona de Mediana Edad , Pronóstico , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Receptor ErbB-2/análisis , Receptor ErbB-2/genética , Receptores de Estrógenos/análisis , Receptores de Estrógenos/genética , Receptores de Progesterona/análisis , Receptores de Progesterona/genética , Recurrencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Índice de Severidad de la EnfermedadRESUMEN
Cardiac stem cells or precursor cells regenerate cardiomyocytes; however, the mechanism underlying this effect remains unclear. We generated CreLacZ mice in which more than 99.9% of the cardiomyocytes in the left ventricular field were positive for 5-bromo-4-chloro-3-indolyl-ß-d-galactoside (X-gal) staining immediately after tamoxifen injection. Three months after myocardial infarction (MI), the MI mice had more X-gal-negative (newly generated) cells than the control mice (3.04 ± 0.38/mm2, MI; 0.47 ± 0.16/mm2, sham; p < 0.05). The cardiac side population (CSP) cell fraction contained label-retaining cells, which differentiated into X-gal-negative cardiomyocytes after MI. We injected a leukemia inhibitory factor (LIF)-expression construct at the time of MI and identified a significant functional improvement in the LIF-treated group. At 1 month after MI, in the MI border and scar area, the LIF-injected mice had 31.41 ± 5.83 X-gal-negative cardiomyocytes/mm2, whereas the control mice had 12.34 ± 2.56 X-gal-negative cardiomyocytes/mm2 (p < 0.05). Using 5-ethynyl-2'-deoxyurinide (EdU) administration after MI, the percentages of EdU-positive CSP cells in the LIF-treated and control mice were 29.4 ± 2.7% and 10.6 ± 3.7%, respectively, which suggests that LIF influenced CSP proliferation. Moreover, LIF activated the Janus kinase (JAK)signal transducer and activator of transcription (STAT), mitogen-activated protein kinase/extracellular signal-regulated (MEK)extracellular signal-regulated kinase (ERK), and phosphatidylinositol 3-kinase (PI3K)-AKT pathways in CSPs in vivo and in vitro. The enhanced green fluorescent protein (EGFP)-bone marrow-chimeric CreLacZ mouse results indicated that LIF did not stimulate cardiogenesis via circulating bone marrow-derived cells during the 4 weeks following MI. Thus, LIF stimulates, in part, stem cell-derived cardiomyocyte regeneration by activating cardiac stem or precursor cells. This approach may represent a novel therapeutic strategy for cardiogenesis.
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Factor Inhibidor de Leucemia/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Infarto del Miocardio/tratamiento farmacológico , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Regeneración/efectos de los fármacos , Animales , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Quinasas Janus/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Ratones , Ratones Transgénicos , Infarto del Miocardio/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factores de Transcripción STAT/metabolismo , Células Madre/metabolismoRESUMEN
BACKGROUND: Cardiac cell therapy has been proposed as one of the new strategies against myocardial infarction. Although several reports showed improvement of the function of ischemic heart, the effects of cell therapy vary among the studies and the mechanisms of the beneficial effects are still unknown. Previously, we reported that clonal stem cell antigen-1-positive cardiac progenitor cells exerted a therapeutic effect when transplanted into the ischemic heart. Our aims were to identify the cardiac progenitor-specific paracrine factor and to elucidate the mechanism of its beneficial effect. METHODS AND RESULTS: By using an antibody array, we found that soluble junctional adhesion molecule-A (JAM-A) was abundantly secreted from cardiac progenitor cells. Pretreatment of neutrophils with conditioned medium from cultured cardiac progenitor cells or soluble JAM-A inhibited transendothelial migration and reduced motility of neutrophils. These inhibitory effects were attenuated by anti-JAM-A neutralizing antibody. Injection of cardiac progenitor cells into infarct heart attenuated neutrophil infiltration and expression of inflammatory cytokines. Injection of soluble JAM-A-expressing, but not of JAM-A siRNA-expressing, cardiac progenitor cells into the infarct heart prevented cardiac remodeling and reduced fibrosis area. CONCLUSIONS: Soluble JAM-A secreted from cardiac progenitor cells reduces infiltration of neutrophils after myocardial infarction and ameliorates tissue damage through prevention of excess inflammation. Our finding may lead to a new therapy for cardiovascular disease by using the anti-inflammatory effect of JAM-A.
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Antiinflamatorios/metabolismo , Moléculas de Adhesión Celular/metabolismo , Inflamación/prevención & control , Infarto del Miocardio/cirugía , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/trasplante , Receptores de Superficie Celular/metabolismo , Trasplante de Células Madre/métodos , Células Madre/metabolismo , Animales , Anticuerpos Neutralizantes/farmacología , Moléculas de Adhesión Celular/antagonistas & inhibidores , Moléculas de Adhesión Celular/genética , Células Cultivadas , Técnicas de Cocultivo , Medios de Cultivo Condicionados/metabolismo , Modelos Animales de Enfermedad , Inflamación/metabolismo , Inflamación/patología , Inflamación/fisiopatología , Ratones Endogámicos C57BL , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Miocardio/patología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Infiltración Neutrófila , Neutrófilos/metabolismo , Interferencia de ARN , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/genética , Células Madre/efectos de los fármacos , Migración Transendotelial y Transepitelial , Transfección , Función Ventricular Izquierda , Remodelación VentricularRESUMEN
The identification of gene fusions promises to play an important role in personalized cancer treatment decisions. Many rare gene fusion events have been identified in fresh frozen solid tumors from common cancers employing next-generation sequencing technology. However the ability to detect transcripts from gene fusions in RNA isolated from formalin-fixed paraffin-embedded (FFPE) tumor tissues, which exist in very large sample repositories for which disease outcome is known, is still limited due to the low complexity of FFPE libraries and the lack of appropriate bioinformatics methods. We sought to develop a bioinformatics method, named gFuse, to detect fusion transcripts in FFPE tumor tissues. An integrated, cohort based strategy has been used in gFuse to examine single-end 50 base pair (bp) reads generated from FFPE RNA-Sequencing (RNA-Seq) datasets employing two breast cancer cohorts of 136 and 76 patients. In total, 118 fusion events were detected transcriptome-wide at base-pair resolution across the 212 samples. We selected 77 candidate fusions based on their biological relevance to cancer and supported 61% of these using TaqMan assays. Direct sequencing of 19 of the fusion sequences identified by TaqMan confirmed them. Three unique fused gene pairs were recurrent across the 212 patients with 6, 3, 2 individuals harboring these fusions respectively. We show here that a high frequency of fusion transcripts detected at the whole transcriptome level correlates with poor outcome (P<0.0005) in human breast cancer patients. This study demonstrates the ability to detect fusion transcripts as biomarkers from archival FFPE tissues, and the potential prognostic value of the fusion transcripts detected.
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Biomarcadores de Tumor/análisis , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Formaldehído/química , Proteínas de Fusión Oncogénica/análisis , Adhesión en Parafina/métodos , Femenino , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Técnicas In VitroRESUMEN
NOD2, one of the cytosolic proteins that contain a nuclear oligomerization domain (NOD), is a pattern recognition receptor (PRR) involved in innate immune responses to intracellular pathogens. Little is known, however, about the effect of NOD2 expression on the maternal-fetal relationship. Our aim was to elucidate the functions of NOD2 in normal decidual stromal cells (DSCs) from the first trimester. Tissues and DSCs were isolated from 26 patients with normal pregnancies that required abortion. The expression of NOD2 in deciduas/decidual stromal cells (DSCs) was examined by real-time PCR, immunohistochemistry, and In-cell western. DSCs containing NOD2 were stimulated by its ligand, muramyl dipeptide (MDP). The secretion of various cytokines and chemokines were measured by ELISA and the apoptotic rate was determined by flow cytometry. Treatment with MDP significantly elevated the expression of both NOD2 mRNA and protein levels in DSCs. In addition, MDP activation of NOD2 significantly increased IL-1ß and MCP-1 cytokine expression in a dose dependent manner but had no effect on IL-12 expression. IL-1ß and TNF-α also significantly increased the expression of NOD2 in DSCs, suggesting a positive feedback loop mechanism. Moreover, MDP stimulation augmented DSC apoptosis. In summary, the results suggest that NOD2 expression in DSCs plays an important role in protecting the embryo and preventing infection in the maternal-fetal interface.
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Decidua/inmunología , Transmisión Vertical de Enfermedad Infecciosa/prevención & control , Proteína Adaptadora de Señalización NOD2/fisiología , Primer Trimestre del Embarazo/inmunología , Células del Estroma/metabolismo , Acetilmuramil-Alanil-Isoglutamina/farmacología , Adulto , Apoptosis , Citocinas/metabolismo , Citocinas/farmacología , Decidua/citología , Decidua/metabolismo , Retroalimentación Fisiológica , Femenino , Enfermedades Fetales/prevención & control , Infecciones por Bacterias Gramnegativas/prevención & control , Infecciones por Bacterias Gramnegativas/transmisión , Infecciones por Bacterias Grampositivas/prevención & control , Infecciones por Bacterias Grampositivas/transmisión , Humanos , Lipopolisacáridos/farmacología , Intercambio Materno-Fetal/inmunología , Proteína Adaptadora de Señalización NOD2/agonistas , Proteína Adaptadora de Señalización NOD2/análisis , Embarazo , Células del Estroma/efectos de los fármacosRESUMEN
OBJECTIVE: To evaluate the effects of low molecular weight heparin (LMWH) and heparin-binding epidermal growth factor (HB-EGF) on the biological function of human trophoblast in first trimester. DESIGN: Control experiment. SETTING: Two academic-based reproductive centers. PATIENT(S): The first trimester human placentas (gestational age, 5-10 weeks) from patients who underwent electively induced abortions. INTERVENTION(S): Cultured trophoblast treated with LMWH or LMWH and HB-EGF. MAIN OUTCOME MEASURE(S): The biological function (proliferation, invasion, and differentiation) of trophoblast and the interaction between LMWH and HB-EGF on trophoblast in vitro. RESULT(S): 1) At a dose of 0.25 IU/mL-2.5 IU/mL LMWH promoted trophoblast proliferation, enhanced their invasion, and increased hCG secretion. The LMWH had little effect or the opposite effect at other concentrations. These differences were statistically significant. 2) Combined use of LMWH and HB-EGF significantly promoted proliferation and invasion, but there was no difference in hCG secretion compared with solo LMWH or solo HB-EGF. CONCLUSION(S): The LMWH exerts a cytoprotective effect by regulating trophoblast proliferation, invasion, and differentiation. The HB-EGF is an important factor in the effects of LMWH on trophoblast function.
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Enoxaparina/farmacología , Péptidos y Proteínas de Señalización Intercelular/farmacología , Trofoblastos/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Gonadotropina Coriónica/metabolismo , Citoprotección , Relación Dosis-Respuesta a Droga , Femenino , Factor de Crecimiento Similar a EGF de Unión a Heparina , Humanos , Embarazo , Tercer Trimestre del Embarazo , Trofoblastos/metabolismoRESUMEN
RNA biomarkers discovered by RT-PCR-based gene expression profiling of archival formalin-fixed paraffin-embedded (FFPE) tissue form the basis for widely used clinical diagnostic tests; however, RT-PCR is practically constrained in the number of transcripts that can be interrogated. We have developed and optimized RNA-Seq library chemistry as well as bioinformatics and biostatistical methods for whole transcriptome profiling from FFPE tissue. The chemistry accommodates low RNA inputs and sample multiplexing. These methods both enable rediscovery of RNA biomarkers for disease recurrence risk that were previously identified by RT-PCR analysis of a cohort of 136 patients, and also identify a high percentage of recurrence risk markers that were previously discovered using DNA microarrays in a separate cohort of patients, evidence that this RNA-Seq technology has sufficient precision and sensitivity for biomarker discovery. More than two thousand RNAs are strongly associated with breast cancer recurrence risk in the 136 patient cohort (FDR <10%). Many of these are intronic RNAs for which corresponding exons are not also associated with disease recurrence. A number of the RNAs associated with recurrence risk belong to novel RNA networks. It will be important to test the validity of these novel associations in whole transcriptome RNA-Seq screens of other breast cancer cohorts.
Asunto(s)
Neoplasias de la Mama/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Recurrencia Local de Neoplasia/genética , Adhesión en Parafina , Análisis de Secuencia de ARN , Fijación del Tejido , Secuencia de Bases , Biomarcadores de Tumor/genética , ADN Intergénico/genética , Femenino , Formaldehído , Humanos , Intrones/genética , Modelos de Riesgos Proporcionales , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico , Factores de RiesgoRESUMEN
A molecular test providing clear identification of individuals at highest risk for developing metastatic disease from among early stage breast cancer patients has proven to be of great benefit in breast cancer treatment planning and therapeutic management. Patients with high risk of disease recurrence can also get an estimate of the magnitude of benefit to be gained by adding chemotherapy to surgery and hormonal therapy. Developing this clinical test was made possible by the availability of technologies capable of identifying molecular biomarkers from the gene expression profiles of preserved surgical specimens. Molecular tests such as the Oncotype DX(®) breast cancer test are proving to be more effective tools for individualized patient stratification and treatment planning than traditional methods such as patient demographic variables and histopathology indicators.Molecular biomarkers must be clinically validated before they can be effectively applied toward patient management in clinical practice. The most effective and efficient means of clinical validation is to use archived surgical specimens annotated with well-characterized clinical outcomes. However, carrying out this type of clinical study requires optimization of traditional molecular expression profiling techniques to analyze RNA from fixed, paraffin-embedded (FPE) tissues. In order to develop our clinically validated breast cancer assay, we modified molecular methods for RNA extraction, RNA quantitation, reverse transcription, and quantitative PCR to work optimally in archived clinical samples. Here, we present an updated description of current best practices for isolating both mRNA and microRNA from FPE tissues for RT-PCR-based expression profiling.
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Biomarcadores de Tumor/genética , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Adhesión en Parafina/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Fijación del Tejido/métodos , Humanos , MicroARNs/genética , Reacción en Cadena de la Polimerasa , ARN Neoplásico/aislamiento & purificación , Transcripción Reversa , Estadística como AsuntoRESUMEN
Although RNA is isolated from archival fixed tissues routinely for reverse transcription polymerase chain reaction (RT-PCR) and microarray analyses to identify biomarkers of cancer prognosis and therapeutic response prediction, the sensitivity of these molecular profiling methods to variability in pathology tissue processing has not been described in depth. As increasing numbers of expression analysis studies using fixed archival tumor specimens are reported, it is important to examine how dependent these results are on tissue-processing methods.We carried out a series of studies to systematically evaluate the effects of various tissue-fixation reagents and protocols on RNA quality and RT-PCR gene expression profiles. Human placenta was selected as a model specimen for these studies since it is relatively easily obtained and has proliferative and invasive qualities similar to solid tumors. In addition, each specimen is relatively homogeneous and large enough to provide sufficient tissue to systematically compare a range of fixation conditions and reagents, thereby avoiding the variability inherent in studying collections of tumor tissue specimens. Since anatomical pathology laboratories generally offer hundreds of different tissue-fixation protocols, we focused on fixation reagents and conditions used to process the most common solid tumors for primary cancer diagnosis. Fresh placentas donated under an IRB-approved protocol were collected at delivery and immediately submerged in cold saline for transport to a central pathology laboratory for processing. RNA was extracted from each specimen, quantified, and analyzed for size distribution and analytical performance using a panel of 24 RT-PCR gene expression assays. We found that different tissue-fixation reagents and tissue-processing conditions resulted in widely varying RNA extraction yields and extents of RNA fragmentation. However, the RNA extraction method and RT-PCR assays could be optimized to achieve successful gene expression analysis for nearly all fixation conditions represented in these studies.
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Fijadores/química , Perfilación de la Expresión Génica/métodos , Adhesión en Parafina/métodos , ARN/genética , ARN/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Fijación del Tejido/métodos , Humanos , Indicadores y Reactivos , Estándares de Referencia , Transcripción Reversa , Estadística como AsuntoRESUMEN
Previously, we had identified gene expression patterns that predicted response to neoadjuvant docetaxel. Other studies have validated that a high Recurrence Score (RS) by the 21-gene RT-PCR assay is predictive of worse prognosis but better response to chemotherapy. We investigated whether tumor expression of these 21 genes and other candidate genes can predict response to docetaxel. Core biopsies from 97 patients were obtained before treatment with neoadjuvant docetaxel (4 cycles, 100 mg/m2 q3 weeks). Three 10-microm FFPE sections were submitted for quantitative RT-PCR assays of 192 genes that were selected from our previous work and the literature. Of the 97 patients, 81 (84%) had sufficient invasive cancer, 80 (82%) had sufficient RNA for QRTPCR assay, and 72 (74%) had clinical response data. Mean age was 48.5 years, and the median tumor size was 6 cm. Clinical complete responses (CR) were observed in 12 (17%), partial responses in 41 (57%), stable disease in 17 (24%), and progressive disease in 2 patients (3%). A significant relationship (P<0.05) between gene expression and CR was observed for 14 genes, including CYBA. CR was associated with lower expression of the ER gene group and higher expression of the proliferation gene group from the 21 gene assay. Of note, CR was more likely with a high RS (P=0.008). We have established molecular profiles of sensitivity to docetaxel. RT-PCR technology provides a potential platform for a predictive test of docetaxel chemosensitivity using small amounts of routinely processed material.