RESUMEN

Glaucoma is a multifactorial neurodegenerative disease, characterized by degeneration of the retinal ganglion cells (RGCs). There has been little progress in developing efficient strategies for neuroprotection in glaucoma. We profiled the retina transcriptome of Lister Hooded rats at 2 weeks after optic nerve crush (ONC) and analyzed the data from the genomic fabric paradigm (GFP) to bring additional insights into the molecular mechanisms of the retinal remodeling after induction of RGC degeneration. GFP considers three independent characteristics for the expression of each gene: level, variability, and correlation with each other gene. Thus, the 17,657 quantified genes in our study generated a total of 155,911,310 values to analyze. This represents 8830x more data per condition than a traditional transcriptomic analysis. ONC led to a 57% reduction in RGC numbers as detected by retrograde labeling with 1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarbocyanine perchlorate (DiI). We observed a higher relative expression variability after ONC. Gene expression stability was used as a measure of transcription control and disclosed a robust reduction in the number of very stably expressed genes. Predicted protein-protein interaction (PPI) analysis with STRING revealed axon and neuron projection as mostly decreased processes, consistent with RGC degeneration. Conversely, immune response PPIs were found among upregulated genes. Enrichment analysis showed that complement cascade and Notch signaling pathway, as well as oxidative stress and kit receptor pathway were affected after ONC. To expand our studies of altered molecular pathways, we examined the pairwise coordination of gene expressions within each pathway and within the entire transcriptome using Pearson correlations. ONC increased the number of synergistically coordinated pairs of genes and the number of similar profiles mainly in complement cascade and Notch signaling pathway. This deep bioinformatic study provided novel insights beyond the regulation of individual gene expression and disclosed changes in the control of expression of complement cascade and Notch signaling functional pathways that may be relevant for both RGC degeneration and remodeling of the retinal tissue after ONC.

Asunto(s)

Glaucoma , Traumatismos del Nervio Óptico , Nervio Óptico , Células Ganglionares de la Retina , Transcriptoma , Animales , Femenino , Glaucoma/genética , Glaucoma/metabolismo , Glaucoma/patología , Nervio Óptico/metabolismo , Nervio Óptico/patología , Traumatismos del Nervio Óptico/genética , Traumatismos del Nervio Óptico/metabolismo , Traumatismos del Nervio Óptico/patología , Ratas , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/patología

RESUMEN

Chagas disease is responsible for more than 10,000 deaths per year and about 6 to 7 million infected people worldwide. In its chronic stage, patients can develop mega-colon, mega-esophagus, and cardiomyopathy. Differences in clinical outcomes may be determined, in part, by the genetic background of the parasite that causes Chagas disease. Trypanosoma cruzi has a high genetic diversity, and each group of strains may elicit specific pathological responses in the host. Conflicting results have been reported in studies using various combinations of mammalian host-T. cruzi strains. We previously profiled the transcriptomic signatures resulting from infection of L6E9 rat myoblasts with four reference strains of T. cruzi (Brazil, CL, Y, and Tulahuen). The four strains induced similar overall gene expression alterations in the myoblasts, although only 21 genes were equally affected by all strains. Cardiotrophin-like cytokine factor 1 (Clcf1) was one of the genes found to be consistently upregulated by the infection with all four strains of T. cruzi. This cytokine is a member of the interleukin-6 family that binds to glycoprotein 130 receptor and activates the JAK/STAT signaling pathway, which may lead to muscle cell hypertrophy. Another commonly upregulated gene was tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein theta (Ywhaq, 14-3-3 protein Θ), present in the Cell Cycle Pathway. In the present work, we reanalyzed our previous microarray dataset, aiming at understanding in more details the transcriptomic impact that each strain has on JAK/STAT signaling and Cell Cycle pathways. Using Pearson correlation analysis between the expression levels of gene pairs in biological replicas from each pathway, we determined the coordination between such pairs in each experimental condition and the predicted protein interactions between the significantly altered genes by each strain. We found that although these highlighted genes were similarly affected by all four strains, the downstream genes or their interaction partners were not necessarily equally affected, thus reinforcing the idea of the role of parasite background on host cell transcriptome. These new analyses provide further evidence to the mechanistic understanding of how distinct T. cruzi strains lead to diverse remodeling of host cell transcriptome.

Asunto(s)

Trypanosoma cruzi , Animales , Brasil , Ciclo Celular , Humanos , Mioblastos , Ratas , Transducción de Señal , Transcriptoma , Trypanosoma cruzi/genética

RESUMEN

Despite advances in assisted reproduction techniques, the poor quality and failures in embryo in vitro development remain as drawbacks resulting in low pregnancy rate. Mouse embryonic fibroblasts (MEFs) have been widely used to support embryonic stem cells. Mesenchymal cells (MSCs) have also been shown to release bioactive factors. In the present study, we have evaluated the ability of MSCs and MEFs to support early development of mouse embryos. The embryos were cultivated alone or in coculture with inactivated MSC or MEF for 4 d. After 4 d in culture, the percentage of blastocyst formation in coculture with MSC (91.7 ± 4.3%) or MEF (95.1 ± 3.3%) was higher than in the control group (72.2 ± 9.0%). We did not observe any difference in proliferation or apoptosis. However, the blastocysts cocultured with MSC or MEF presented a significantly higher number of cells within the inner cell mass per embryo when compared to the controls. The MSC and MEF groups presented also a higher cell number and diameter when compared to the control (CTRL). In summary, our data indicate that coculture with MSC or MEF improves early embryonic development and quality in vitro.

Asunto(s)

Blastocisto/citología , Desarrollo Embrionario , Fibroblastos/citología , Células Madre Mesenquimatosas/citología , Animales , Apoptosis , Recuento de Células , Proliferación Celular , Técnicas de Cocultivo , Femenino , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Masculino , Células Madre Mesenquimatosas/metabolismo , Ratones Endogámicos C57BL , Ratas Wistar , Trofoblastos/citología

RESUMEN

A rich, collaborative program funded by the US NIH Fogarty program in 2004 has provided for a decade of remarkable opportunities for scientific advancement through the training of Brazilian undergraduate, graduate and postdoctoral students from the Federal University and Oswaldo Cruz Foundation systems at Albert Einstein College of Medicine. The focus of the program has been on the development of trainees in the broad field of Infectious Diseases, with a particular focus on diseases of importance to the Brazilian population. Talented trainees from various regions in Brazil came to Einstein to learn techniques and study fungal, parasitic and bacterial pathogens. In total, 43 trainees enthusiastically participated in the program. In addition to laboratory work, these students took a variety of courses at Einstein, presented their results at local, national and international meetings, and productively published their findings. This program has led to a remarkable synergy of scientific discovery for the participants during a time of rapid acceleration of the scientific growth in Brazil. This collaboration between Brazilian and US scientists has benefitted both countries and serves as a model for future training programs between these countries.

Asunto(s)

Educación/historia , Educación/organización & administración , Cooperación Internacional/historia , Personal de Laboratorio/educación , Brasil , Educación/economía , Historia del Siglo XXI , Humanos , Personal de Laboratorio/economía , National Institutes of Health (U.S.) , Estados Unidos , Recursos Humanos

RESUMEN

Abstract A rich, collaborative program funded by the US NIH Fogarty program in 2004 has provided for a decade of remarkable opportunities for scientific advancement through the training of Brazilian undergraduate, graduate and postdoctoral students from the Federal University and Oswaldo Cruz Foundation systems at Albert Einstein College of Medicine. The focus of the program has been on the development of trainees in the broad field of Infectious Diseases, with a particular focus on diseases of importance to the Brazilian population. Talented trainees from various regions in Brazil came to Einstein to learn techniques and study fungal, parasitic and bacterial pathogens. In total, 43 trainees enthusiastically participated in the program. In addition to laboratory work, these students took a variety of courses at Einstein, presented their results at local, national and international meetings, and productively published their findings. This program has led to a remarkable synergy of scientific discovery for the participants during a time of rapid acceleration of the scientific growth in Brazil. This collaboration between Brazilian and US scientists has benefitted both countries and serves as a model for future training programs between these countries.(AU)

Asunto(s)

Brasil/economía , Brasil/educación

RESUMEN

Abstract A rich, collaborative program funded by the US NIH Fogarty program in 2004 has provided for a decade of remarkable opportunities for scientific advancement through the training of Brazilian undergraduate, graduate and postdoctoral students from the Federal University and Oswaldo Cruz Foundation systems at Albert Einstein College of Medicine. The focus of the program has been on the development of trainees in the broad field of Infectious Diseases, with a particular focus on diseases of importance to the Brazilian population. Talented trainees from various regions in Brazil came to Einstein to learn techniques and study fungal, parasitic and bacterial pathogens. In total, 43 trainees enthusiastically participated in the program. In addition to laboratory work, these students took a variety of courses at Einstein, presented their results at local, national and international meetings, and productively published their findings. This program has led to a remarkable synergy of scientific discovery for the participants during a time of rapid acceleration of the scientific growth in Brazil. This collaboration between Brazilian and US scientists has benefitted both countries and serves as a model for future training programs between these countries.

Asunto(s)

Brasil/economía , Brasil/educación , Brasil/historia , Brasil , Brasil/organización & administración , Educación/economía , Educación/educación , Educación/historia , Educación , Educación/organización & administración , /economía , /educación , /historia , /organización & administración , Humanos/economía , Humanos/educación , Humanos/historia , Humanos , Humanos/organización & administración , Cooperación Internacional/economía , Cooperación Internacional/educación , Cooperación Internacional/historia , Cooperación Internacional , Cooperación Internacional/organización & administración , Personal de Laboratorio/economía , Personal de Laboratorio/educación , Personal de Laboratorio/historia , Personal de Laboratorio , Personal de Laboratorio/organización & administración , National Institutes of Health (U.S.)/economía , National Institutes of Health (U.S.)/educación , National Institutes of Health (U.S.)/historia , National Institutes of Health (U.S.) , National Institutes of Health (U.S.)/organización & administración , Estados Unidos/economía , Estados Unidos/educación , Estados Unidos/historia , Estados Unidos , Estados Unidos/organización & administración

RESUMEN

Chagasic cardiomyopathy, resulting from infection with the parasite Trypanosoma cruzi, was discovered more than a century ago and remains an incurable disease. Due to the unique properties of mesenchymal stem cells (MSC) we hypothesized that these cells could have therapeutic potential for chagasic cardiomyopathy. Recently, our group pioneered use of nanoparticle-labeled MSC to correlate migration with its effect in an acute Chagas disease model. We expanded our investigation into a chronic model and performed more comprehensive assays. Infected mice were treated with nanoparticle-labeled MSC and their migration was correlated with alterations in heart morphology, metalloproteinase activity, and expression of several proteins. The vast majority of labeled MSC migrated to liver, lungs and spleen whereas a small number of cells migrated to chagasic hearts. Magnetic resonance imaging demonstrated that MSC therapy reduced heart dilatation. Additionally metalloproteinase activity was higher in heart and other organs of infected mice. Protein expression analyses revealed that connexin 43, laminin γ1, IL-10 and INF-γ were affected by the disease and recovered after cell therapy. Interestingly, MSC therapy led to upregulation of SDF-1 and c-kit in the hearts. The beneficial effect of MSC therapy in Chagas disease is likely due to an indirect action of the cells of the heart, rather than the incorporation of large numbers of stem cells into working myocardium.

Asunto(s)

Trasplante de Médula Ósea/métodos , Enfermedad de Chagas/patología , Enfermedad de Chagas/terapia , Trasplante de Células Madre Mesenquimatosas/métodos , Animales , Quimiocina CXCL12/análisis , Citocinas/sangre , Modelos Animales de Enfermedad , Corazón/diagnóstico por imagen , Imagen por Resonancia Magnética , Masculino , Ratones , Imagen Molecular , Miocardio/patología , Proteínas Proto-Oncogénicas c-kit/análisis , Radiografía , Resultado del Tratamiento

RESUMEN

Sepsis, a major cause of morbidity/mortality in intensive care units worldwide, is commonly associated with cardiac dysfunction, which worsens the prognosis dramatically for patients. Although in recent years the concept of septic cardiomyopathy has evolved, the importance of myocardial structural alterations in sepsis has not been fully explored. This study offers novel and mechanistic data to clarify subcellular events that occur in the pathogenesis of septic cardiomyopathy and myocardial dysfunction in severe sepsis. Cultured neonatal mice cardiomyocytes subjected to serum obtained from mice with severe sepsis presented striking increment of [Ca(2+)]i and calpain-1 levels associated with decreased expression of dystrophin and disruption and derangement of F-actin filaments and cytoplasmic bleb formation. Severe sepsis induced in mice led to an increased expression of calpain-1 in cardiomyocytes. Moreover, decreased myocardial amounts of dystrophin, sarcomeric actin, and myosin heavy chain were observed in septic hearts associated with depressed cardiac contractile dysfunction and a very low survival rate. Actin and myosin from the sarcomere are first disassembled by calpain and then ubiquitinated and degraded by proteasome or sequestered inside specialized vacuoles called autophagosomes, delivered to the lysosome for degradation forming autophagolysosomes. Verapamil and dantrolene prevented the increase of calpain-1 levels and preserved dystrophin, actin, and myosin loss/reduction as well cardiac contractile dysfunction associated with strikingly improved survival rate. These abnormal parameters emerge as therapeutic targets, which modulation may provide beneficial effects on future vascular outcomes and mortality in sepsis. Further studies are needed to shed light on this mechanism, mainly regarding specific calpain inhibitors.

Asunto(s)

Calcio/metabolismo , Homeostasis , Miocardio/metabolismo , Miocardio/ultraestructura , Miocitos Cardíacos/metabolismo , Sepsis/patología , Sepsis/fisiopatología , Actinas/metabolismo , Animales , Animales Recién Nacidos , Western Blotting , Calpaína/metabolismo , Ciego/efectos de los fármacos , Ciego/patología , Células Cultivadas , Dantroleno/farmacología , Distrofina/metabolismo , Técnica del Anticuerpo Fluorescente , Hemodinámica/efectos de los fármacos , Homeostasis/efectos de los fármacos , Espacio Intracelular/metabolismo , Ligadura , Ratones , Ratones Endogámicos C57BL , Miocardio/patología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Cadenas Pesadas de Miosina/metabolismo , Punciones , Sarcómeros/efectos de los fármacos , Sarcómeros/metabolismo , Volumen Sistólico/efectos de los fármacos , Análisis de Supervivencia , Verapamilo/farmacología

RESUMEN

BACKGROUND: Chagas disease, resulting from infection with the parasite Trypanosoma cruzi (T. cruzi), is a major cause of cardiomyopathy in Latin America. Drug therapy for acute and chronic disease is limited. Stem cell therapy with bone marrow mesenchymal cells (MSCs) has emerged as a novel therapeutic option for cell death-related heart diseases, but efficacy of MSC has not been tested in Chagas disease. METHODS AND RESULTS: We now report the use of cell-tracking strategies with nanoparticle labeled MSC to investigate migration of transplanted MSC in a murine model of Chagas disease, and correlate MSC biodistribution with glucose metabolism and morphology of heart in chagasic mice by small animal positron emission tomography (microPET). Mice were infected intraperitoneally with trypomastigotes of the Brazil strain of T. cruzi and treated by tail vein injection with MSC one month after infection. MSCs were labeled with near infrared fluorescent nanoparticles and tracked by an in vivo imaging system (IVIS). Our IVIS results two days after transplant revealed that a small, but significant, number of cells migrated to chagasic hearts when compared with control animals, whereas the vast majority of labeled MSC migrated to liver, lungs and spleen. Additionally, the microPET technique demonstrated that therapy with MSC reduced right ventricular dilation, a phenotype of the chagasic mouse model. CONCLUSIONS: We conclude that the beneficial effects of MSC therapy in chagasic mice arise from an indirect action of the cells in the heart rather than a direct action due to incorporation of large numbers of transplanted MSC into working myocardium.

Asunto(s)

Trasplante de Médula Ósea/métodos , Cardiomiopatía Chagásica/terapia , Animales , Disponibilidad Biológica , Células de la Médula Ósea/citología , Cardiomiopatía Chagásica/patología , Modelos Animales de Enfermedad , Hígado/patología , Pulmón/patología , Masculino , Ratones , Miocardio/patología , Bazo/patología , Coloración y Etiquetado

RESUMEN

Stem cell therapy has emerged as a potential therapeutic option for cell death-related heart diseases. Application of non-invasive cell tracking approaches is necessary to determine tissue distribution and lifetime of stem cells following their injection and will likely provide knowledge about poorly understood stem cells mechanisms of tissue repair. Magnetic resonance imaging (MRI) is a potentially excellent tool for high-resolution visualization of the fate of cells after transplantation and for evaluation of therapeutic strategies. The application of MRI for in vivo cell tracking requires contrast agents to achieve efficient cell labeling without causing any toxic cellular effects or eliciting any other side effects. For these reasons clinically approved contrast agents (e.g., ferumoxides) and incorporation facilitators (e.g., protamine) are currently the preferred materials for cell labeling and tracking. Here we describe how to use superparamagnetic iron oxide nanoparticles to label cells and to monitor cell fate in several disease models.

Asunto(s)

Rastreo Celular/métodos , Dextranos/química , Nanopartículas de Magnetita/química , Células Madre/citología , Imagen por Resonancia Magnética/métodos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Microscopía Fluorescente/métodos , Coloración y Etiquetado , Células Madre/metabolismo

RESUMEN

Chagas disease, caused by the parasite Trypanosoma cruzi, is an important cause of cardiac disease in endemic areas of Latin America. It is now being diagnosed in nonendemic areas because of immigration. Typical cardiac manifestations of Chagas disease include dilated cardiomyopathy, congestive heart failure, arrhythmias, cardioembolism, and stroke. Clinical and laboratory-based research to define the pathology resulting from T. cruzi infection has shed light on many of the cellular and molecular mechanisms leading to these manifestations. Antiparasitic treatment may not be appropriate for patients with advanced cardiac disease. Clinical management of Chagas heart disease is similar to that used for cardiomyopathies caused by other processes. Cardiac transplantation has been successfully performed in a small number of patients with Chagas heart disease.

Asunto(s)

Cardiomiopatía Chagásica , Animales , Cardiomiopatía Chagásica/diagnóstico , Cardiomiopatía Chagásica/epidemiología , Cardiomiopatía Chagásica/terapia , Desfibriladores Implantables , Modelos Animales de Enfermedad , Diagnóstico Precoz , Ecocardiografía , Eicosanoides/fisiología , Endotelina-1/biosíntesis , Endotelina-1/fisiología , Trasplante de Corazón , Humanos , Estadios del Ciclo de Vida , Angiografía por Resonancia Magnética , Ratones , Marcapaso Artificial , Ratas , Trasplante de Células Madre/métodos , Tripanocidas/uso terapéutico , Trypanosoma cruzi/crecimiento & desarrollo , Vasoconstricción/fisiología

RESUMEN

Although bone marrow-derived mononuclear cells (BMNC) have been extensively used in cell therapy for cardiac diseases, little mechanistic information is available to support reports of their efficacy. To address this shortcoming, we compared structural and functional recovery and associated global gene expression profiles in post-ischaemic myocardium treated with BMNC transplantation. BMNC suspensions were injected into cardiac scar tissue 10 days after experimental myocardial infarction. Six weeks later, mice undergoing BMNC therapy were found to have normalized antibody repertoire and improved cardiac performance measured by ECG, treadmill exercise time and echocardiography. After functional testing, gene expression profiles in cardiac tissue were evaluated using high-density oligonucleotide arrays. Expression of more than 18% of the 11981 quantified unigenes was significantly altered in the infarcted hearts. BMNC therapy restored expression of 2099 (96.2%) of the genes that were altered by infarction but led to altered expression of 286 other genes, considered to be a side effect of the treatment. Transcriptional therapeutic efficacy, a metric calculated using a formula that incorporates both recovery and side effect of treatment, was 73%. In conclusion, our results confirm a beneficial role for bone marrow-derived cell therapy and provide new information on molecular mechanisms operating after BMNC transplantation on post ischemic heart failure in mice.

Asunto(s)

Trasplante de Médula Ósea , Corazón/fisiopatología , Infarto del Miocardio/terapia , Miocardio/metabolismo , Transcriptoma , Animales , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Mediadores de Inflamación/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Infarto del Miocardio/inmunología , Infarto del Miocardio/metabolismo , Miocardio/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Consumo de Oxígeno , Esfuerzo Físico , Remodelación Ventricular

RESUMEN

Gap junction channels provide intercellular communication between cells. In the heart, these channels coordinate impulse propagation along the conduction system and through the contractile musculature, thereby providing synchronous and optimal cardiac output. As in other arrhythmogenic cardiac diseases, chagasic cardiomyopathy is associated with decreased expression of the gap junction protein connexin43 (Cx43) and its gene. Our studies of cardiac myocytes infected with Trypanosoma cruzi have revealed that synchronous contraction is greatly impaired and gap junction immunoreactivity is lost in infected cells. Such changes are not seen for molecules forming tight junctions, another component of the intercalated disc in cardiac myocytes. Transcriptomic studies of hearts from mouse models of Chagas disease and from acutely infected cardiac myocytes in vitro indicate profound remodelling of gene expression patterns involving heart rhythm determinant genes, suggesting underlying mechanisms of the functional pathology. One curious feature of the altered expression of Cx43 and its gene expression is that it is limited in both extent and location, suggesting that the more global deterioration in cardiac function may result in part from spread of damage signals from more seriously compromised cells to healthier ones.

Asunto(s)

Cardiomiopatía Chagásica/parasitología , Uniones Comunicantes/fisiología , Corazón/fisiología , Corazón/parasitología , Trypanosoma cruzi/patogenicidad , Animales , Células Cultivadas , Perfilación de la Expresión Génica , Interacciones Huésped-Patógeno , Humanos , Ratones , Células Musculares/parasitología , Células Musculares/fisiología

RESUMEN

BACKGROUND: Stem cell therapy has emerged as a promising addition to traditional treatments for a number of diseases. However, harnessing the therapeutic potential of stem cells requires an understanding of their fate in vivo. Non-invasive cell tracking can provide knowledge about mechanisms responsible for functional improvement of host tissue. Superparamagnetic iron oxide nanoparticles (SPIONs) have been used to label and visualize various cell types with magnetic resonance imaging (MRI). In this study we performed experiments designed to investigate the biological properties, including proliferation, viability and differentiation capacity of mesenchymal cells (MSCs) labeled with clinically approved SPIONs. RESULTS: Rat and mouse MSCs were isolated, cultured, and incubated with dextran-covered SPIONs (ferumoxide) alone or with poly-L-lysine (PLL) or protamine chlorhydrate for 4 or 24 hrs. Labeling efficiency was evaluated by dextran immunocytochemistry and MRI. Cell proliferation and viability were evaluated in vitro with Ki67 immunocytochemistry and live/dead assays. Ferumoxide-labeled MSCs could be induced to differentiate to adipocytes, osteocytes and chondrocytes. We analyzed ferumoxide retention in MSCs with or without mitomycin C pretreatment. Approximately 95% MSCs were labeled when incubated with ferumoxide for 4 or 24 hrs in the presence of PLL or protamine, whereas labeling of MSCs incubated with ferumoxide alone was poor. Proliferative capacity was maintained in MSCs incubated with ferumoxide and PLL for 4 hrs, however, after 24 hrs it was reduced. MSCs incubated with ferumoxide and protamine were efficiently visualized by MRI; they maintained proliferation and viability for up to 7 days and remained competent to differentiate. After 21 days MSCs pretreated with mitomycin C still showed a large number of ferumoxide-labeled cells. CONCLUSIONS: The efficient and long lasting uptake and retention of SPIONs by MSCs using a protocol employing ferumoxide and protamine may be applicable to patients, since both ferumoxides and protamine are approved for human use.

Asunto(s)

Médula Ósea/efectos de los fármacos , Hierro/administración & dosificación , Imagen por Resonancia Magnética/métodos , Nanopartículas de Magnetita/administración & dosificación , Células Madre Mesenquimatosas/efectos de los fármacos , Óxidos/administración & dosificación , Coloración y Etiquetado/métodos , Adipocitos/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Condrocitos/efectos de los fármacos , Masculino , Células Madre Mesenquimatosas/citología , Ratones , Ratones Endogámicos C57BL , Mitomicina/administración & dosificación , Osteocitos/efectos de los fármacos , Polilisina/administración & dosificación , Protaminas/administración & dosificación , Ratas , Ratas Wistar

RESUMEN

Chronic chagasic cardiomyopathy is a leading cause of heart failure in Latin American countries, being associated with intense inflammatory response and fibrosis. We have previously shown that bone marrow mononuclear cell (BMC) transplantation improves inflammation, fibrosis, and ventricular diameter in hearts of mice with chronic Chagas disease. Here we investigated the transcriptomic recovery induced by BMC therapy by comparing the heart transcriptomes of control, chagasic, and BMC transplanted mice. Out of the 9390 unique genes quantified in all samples, 1702 had their expression altered in chronic chagasic hearts compared to those of normal mice. Major categories of significantly upregulated genes were related to inflammation, fibrosis and immune responses, while genes involved in mitochondrion function were downregulated. When BMC-treated chagasic hearts were compared to infected mice, 96% of the alterations detected in infected hearts were restored to normal levels, although an additional 109 genes were altered by treatment. Transcriptomic recovery, a new measure that considers both resotrative and side effects of treatment, was remarkably high (84%). Immunofluorescence and morphometric analyses confirmed the effects of BMC therapy in the pattern of inflammatory-immune response and expression of adhesion molecules. In conclusion, by using large-scale gene profiling for unbiased assessment of therapeutic efficacy we demonstrate immunomodulatory effects of BMC therapy in chronic chagasic cardiomyopathy and identify potentially relevant factors involved in the pathogenesis of the disease that may provide new therapeutic targets.

Asunto(s)

Trasplante de Médula Ósea/patología , Cardiomiopatía Chagásica/genética , Regulación de la Expresión Génica/inmunología , Miocardio/inmunología , Miocardio/patología , Trypanosoma cruzi/inmunología , Animales , Trasplante de Médula Ósea/inmunología , Cardiomiopatía Chagásica/inmunología , Cardiomiopatía Chagásica/terapia , Enfermedad Crónica , Modelos Animales de Enfermedad , Femenino , Fibrosis , Galectina 3/genética , Perfilación de la Expresión Génica/métodos , Masculino , Ratones , Ratones Endogámicos C57BL , Miocardio/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Sindecano-4/genética , Trypanosoma cruzi/clasificación , Trypanosoma cruzi/patogenicidad , Factor de von Willebrand/genética

RESUMEN

We examined the extent to which different Trypanosoma cruzi strains induce transcriptomic changes in cultured L(6)E(9) myoblasts 72 hours after infection with Brazil (TC I), Y (TC II), CL (TC II), and Tulahuen (TC II) strains. Expression of 6,289 distinct, fully annotated unigenes was quantified with 27,000 rat oligonucleotide arrays in each of the four replicas of all control and infected RNA samples. Considering changes greater than 1.5-fold and P values < 0.05, the Tulahuen strain was the most disruptive to host transcriptome (17% significantly altered genes), whereas the Y strain altered only 6% of the genes. The significantly altered genes in the infected cells were largely different among the strains, and only 21 genes were similarly changed by all four strains. However, myoblasts infected with different strains showed proportional overall gene-expression alterations. These results indicate that infection with different parasite strains modulates similar but not identical pathways in the host cells.

Asunto(s)

Perfilación de la Expresión Génica , Mioblastos/metabolismo , Mioblastos/parasitología , Transcripción Genética , Trypanosoma cruzi , Animales , Línea Celular , Humanos , Ratas , Trypanosoma cruzi/clasificación , Trypanosoma cruzi/genética

RESUMEN

Caveolae are motile, membrane-bound compartments that contain a number of molecules that participate in cell signaling. Caveolins are protein markers of caveolae and function in a variety of biological processes. Caveolin-3 (Cav-3) is expressed in muscle cells and Cav-3 null mice display a cardiomyopathic phenotype. Ultrastructural cytochemistry, confocal microscopy and immunoblotting revealed a reduction in Cav-3 expression and an activation of ERK (extracellular-signal-regulated kinase) 48 hours after Trypanosoma cruzi infection of cultured cardiac myocytes. CD-1 mice infected with the Brazil strain of T. cruzi displayed reduced expression of Cav-3 and activation of ERK 66 days post infection (dpi).   By 180 dpi there was a normalization of these values. These data suggest that the reduction in Cav-3 expression and the activation of ERK during the early phase of infection may contribute to the pathogenesis of chagasic cardiomyopathy.

Asunto(s)

Caveolina 3/metabolismo , Enfermedad de Chagas/metabolismo , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/parasitología , Trypanosoma cruzi , Animales , Caveolas/metabolismo , Ratones , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Miocitos Cardíacos/ultraestructura

RESUMEN

P19 cells, a pluripotent cell line derived from a teratocarcinoma induced in C3H/HeHa mice, have been widely used as a model system to study cardiac differentiation. We have used these cells to evaluate the extent to which exposure to DMSO and/or cardiogenol C for 4 days in suspension culture enhanced their differentiation into cardiomyocytes. Cardiac differentiation was assessed by observing beating clusters and further confirmed using immunocytochemical, biochemical, and pharmacological approaches. The presence of functional gap junctions in differentiated P19 cells was identified through calcium wave analyses. Proliferation rate and cell death were analyzed by BrdU incorporation and activated caspase-3 immunodetection, respectively. Beating clusters of differentiated P19 cells were only found in cultures treated with DMSO. In addition, groups treated with DMSO up-regulated cardiac troponin-T expression. However, when DMSO was used together with cardiogenol C the up-regulation was less than that with DMSO alone, approximately 1.5 times. Moreover, P19 cells cultured in DMSO or DMSO plus 0.25 microM cardiogenol C had lower proliferation rates and higher numbers of activated caspase-3-positive cells. In summary, using several methodological approaches we have demonstrated that DMSO can induce cardiac differentiation of P19 cells but that cardiogenol C does not.

Asunto(s)

Compuestos de Anilina/farmacología , Diferenciación Celular/efectos de los fármacos , Dimetilsulfóxido/farmacología , Miocitos Cardíacos/efectos de los fármacos , Pirimidinas/farmacología , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Señalización del Calcio/efectos de los fármacos , Caspasa 3/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Sinergismo Farmacológico , Células Madre de Carcinoma Embrionario/metabolismo , Células Madre de Carcinoma Embrionario/patología , Activación Enzimática/efectos de los fármacos , Ratones , Ratones Endogámicos C3H , Microscopía Confocal , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/biosíntesis , Troponina T/biosíntesis , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

BACKGROUND: We investigated the effects of the signaling molecules, cyclic AMP (cAMP) and protein-kinase C (PKC), on gap junctional intercellular communication (GJIC) between thymic epithelial cells (TEC). RESULTS: Treatment with 8-Br-cAMP, a cAMP analog; or forskolin, which stimulates cAMP production, resulted in an increase in dye transfer between adjacent TEC, inducing a three-fold enhancement in the mean fluorescence of coupled cells, ascertained by flow cytometry after calcein transfer. These treatments also increased Cx43 mRNA expression, and stimulated Cx43 protein accumulation in regions of intercellular contacts. VIP, adenosine, and epinephrine which may also signal through cyclic nucleotides were tested. The first two molecules did not mimic the effects of 8-Br-cAMP, however epinephrine was able to increase GJIC suggesting that this molecule functions as an endogenous inter-TEC GJIC modulators. Stimulation of PKC by phorbol-myristate-acetate inhibited inter-TEC GJIC. Importantly, both the enhancing and the decreasing effects, respectively induced by cAMP and PKC, were observed in both mouse and human TEC preparations. Lastly, experiments using mouse thymocyte/TEC heterocellular co-cultures suggested that the presence of thymocytes does not affect the degree of inter-TEC GJIC. CONCLUSIONS: Overall, our data indicate that cAMP and PKC intracellular pathways are involved in the homeostatic control of the gap junction-mediated communication in the thymic epithelium, exerting respectively a positive and negative role upon cell coupling. This control is phylogenetically conserved in the thymus, since it was seen in both mouse and human TEC preparations. Lastly, our work provides new clues for a better understanding of how the thymic epithelial network can work as a physiological syncytium.

Asunto(s)

Comunicación Celular/fisiología , AMP Cíclico/metabolismo , Células Epiteliales/metabolismo , Proteína Quinasa C/metabolismo , Timo/citología , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Colforsina/farmacología , Conexina 43/genética , Conexina 43/metabolismo , Regulación hacia Abajo , Uniones Comunicantes/enzimología , Uniones Comunicantes/metabolismo , Humanos , Ratones , Ésteres del Forbol/farmacología

RESUMEN

Chagas disease which is caused by the parasite Trypanosoma cruzi is an important cause of cardiomyopathy in Latin America. In later stages chagasic cardiomyopathy is associated with congestive heart failure which is often refractory to medical therapy. In these individuals heart transplantation has been attempted. However, this procedure is fraught with many problems attributable to the surgery and the postsurgical administration of immunosuppressive drugs. Studies in mice suggest that the transplantation of bone-marrow-derived cells ameliorates the inflammation and fibrosis in the heart associated with this infection. Cardiac magnetic resonance imaging reveals that bone marrow transplantation ameliorates the infection induced right ventricular enlargement. On the basis of these animal studies the safety of autologous bone marrow transplantation has been assessed in patients with chagasic end-stage heart disease. The initial results are encouraging and more studies need to be performed.