RESUMEN
BACKGROUND: Revascularization of the heart after myocardial infarction (MI) using growth factors delivered by hydrogel-based microspheres represents a promising therapeutic approach for cardiac regeneration. Microspheres have tuneable degradation properties and support the prolonged release of soluble factors. Cardiac patches provide mechanical restraint, preventing dilatation associated with ventricular remodelling. METHODS: We combined these approaches and produced a compacted calcium-alginate microsphere patch, restrained by a chitosan sheet, to deliver vascular endothelial growth factor (VEGF) to the heart after myocardial injury in rats. RESULTS: Microspheres had an average diameter of 3.2µm, were nonporous, and characterized by a smooth dimpled surface. Microsphere patches demonstrated prolonged in vitro release characteristics compared to non-compacted microspheres and VEGF supernatants obtained from patches maintained their bioactivity for the 5day duration of the study in vitro. In vivo, patches were assessed with magnetic resonance imaging following MI, and demonstrated 50% degradation 25.6days after implantation. Both VEGF(-) and VEGF(+) microsphere patch-treated hearts had better cardiac function than unpatched (chitosan sheet only) controls. However, VEGF(+) microsphere-patched hearts had thicker scars characterized by higher capillary density in the border zone than did those treated with VEGF(-) patches. VEGF was detected in the patches 4weeks post-implantation. CONCLUSION: The condensed microsphere patch represents a new therapeutic platform for cytokine delivery and could be used as an adjuvant to current biomaterial and cell-based therapies to promote localized angiogenesis in the infarcted heart. STATEMENT OF SIGNIFICANCE: Following a heart attack, a lack of blood flow to the heart results in loss of heart cells. Growth factors may facilitate growth of blood vessels and heart tissue repair and prevent the onset of heart failure. Determining a way to deliver these growth factors directly to the heart is vital. Here, we combined two biomaterial-based approaches to deliver vascular endothelial growth factor (VEGF) to rat hearts after heart attack: a microsphere for prolonged release of VEGF, and a cardiac patch for mechanical restraint to prevent heart dysfunction. The feasibility of this microsphere patch was demonstrated by surgically implanting it over the infarct region of the heart post-injury. VEGF-patched hearts had better blood vessel growth, tissue repair, and heart function.
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Sistemas de Liberación de Medicamentos , Microesferas , Isquemia Miocárdica/tratamiento farmacológico , Factor A de Crecimiento Endotelial Vascular/uso terapéutico , Alginatos/química , Animales , Materiales Biocompatibles/química , Calcio/química , Femenino , Ácido Glucurónico/química , Ácidos Hexurónicos/química , Humanos , Implantes Experimentales , Isquemia Miocárdica/patología , Neovascularización Fisiológica/efectos de los fármacos , Pericardio/patología , Ratas Sprague-Dawley , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
BACKGROUND: Efficient cardiac function requires synchronous ventricular contraction. After myocardial infarction, the nonconductive nature of scar tissue contributes to ventricular dysfunction by electrically uncoupling viable cardiomyocytes in the infarct region. Injection of a conductive biomaterial polymer that restores impulse propagation could synchronize contraction and restore ventricular function by electrically connecting isolated cardiomyocytes to intact tissue, allowing them to contribute to global heart function. METHODS AND RESULTS: We created a conductive polymer by grafting pyrrole to the clinically tested biomaterial chitosan to create a polypyrrole (PPy)-chitosan hydrogel. Cyclic voltammetry showed that PPy-chitosan had semiconductive properties lacking in chitosan alone. PPy-chitosan did not reduce cell attachment, metabolism, or proliferation in vitro. Neonatal rat cardiomyocytes plated on PPy-chitosan showed enhanced Ca(2+) signal conduction in comparison with chitosan alone. PPy-chitosan plating also improved electric coupling between skeletal muscles placed 25 mm apart in comparison with chitosan alone, demonstrating that PPy-chitosan can electrically connect contracting cells at a distance. In rats, injection of PPy-chitosan 1 week after myocardial infarction decreased the QRS interval and increased the transverse activation velocity in comparison with saline or chitosan, suggesting improved electric conduction. Optical mapping showed increased activation in the border zone of PPy-chitosan-treated rats. Echocardiography and pressure-volume analysis showed improvement in load-dependent (ejection fraction, fractional shortening) and load-independent (preload recruitable stroke work) indices of heart function 8 weeks after injection. CONCLUSIONS: We synthesized a biocompatible conductive biomaterial (PPy-chitosan) that enhances biological conduction in vitro and in vivo. Injection of PPy-chitosan better maintained heart function after myocardial infarction than a nonconductive polymer.
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Materiales Biocompatibles/administración & dosificación , Conductividad Eléctrica , Hidrogel de Polietilenoglicol-Dimetacrilato/administración & dosificación , Infarto del Miocardio/terapia , Polímeros/administración & dosificación , Animales , Animales Recién Nacidos , Materiales Biocompatibles/química , Células Cultivadas , Quitosano/administración & dosificación , Quitosano/química , Conductividad Eléctrica/uso terapéutico , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Infarto del Miocardio/fisiopatología , Polímeros/química , Pirroles/administración & dosificación , Pirroles/química , Ratas , Ratas Sprague-DawleyRESUMEN
AIMS: A mismatch between adequate angiogenesis and overgrowth of myocytes may be a critical mechanism controlling the transition from adaptive hypertrophy to heart failure. Canopy 2 (CNPY2) was recently identified as a secreted, HIF-1α-regulated angiogenic growth factor. As angiogenic factors play important roles in the development of myocardial hypertrophy, we investigated the role of CNPY2 in molecular and functional changes during development of chronic heart failure using cardiac-specific transgenic (TG) mice that overexpress human CNPY2. METHODS AND RESULTS: We generated TG mice that constitutively express CNPY2 in the myocardium. Cardiomyopathy was induced in TG and wild-type (WT) mice by transverse aortic constriction (TAC). WT mice developed significant ventricular hypertrophy at 4 weeks and severe dilatation and heart failure at 12 weeks after TAC. However, TG mice preserved much better cardiac structure and function, with less severe ventricular dilatation and markedly reduced cardiac apoptosis and fibrosis following TAC. Excess CNPY2 in TG mice prevented significant loss of vasculature up to 12 weeks after TAC injury, resulting in a better local myocardial environment that facilitated myocyte survival and prevented excessive matrix remodelling compared with WT mice. TG mice had less accumulation of endogenous tumor suppressor p53 after TAC, indicating intrinsic activation of the p53-mediated repression of HIF-1α, and Cnpy2 was diminished in TG mice compared with WT controls. CONCLUSION: Our study showed a correlation between downregulation of endogenous mouse Cnpy2 and p53-mediated HIF-1α inhibition during late-stage hypertrophic development. Additional CNPY2 attenuated the transition from compensatory hypertrophic response to maladaptive ventricular dilatation and heart failure.
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Cardiomiopatía Hipertrófica/complicaciones , Insuficiencia Cardíaca/etiología , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas de la Membrana/fisiología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Aorta , Constricción , Ensayo de Inmunoadsorción Enzimática , Humanos , Etiquetado Corte-Fin in Situ , Ratones Endogámicos C57BL , Ratones Transgénicos , Neovascularización Fisiológica/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Función Ventricular/fisiologíaRESUMEN
AIMS: Ischaemic heart disease is a leading cause of mortality. After ischaemic injury, tissue hypoxia induces the activity of angiogenic factors that promote revascularization. Increased understanding of hypoxia-responsive genes and their role in angiogenesis will lead to new therapies for ischaemic injury. We delineated the function of Canopy 2 (CNPY2), a newly discovered, hypoxia-regulated gene. METHODS AND RESULTS: We found CNPY2 in a screen for genes induced by low oxygen in human smooth muscle cells (SMCs). CNPY2 protein co-localized with the endoplasmic reticulum and the Golgi. Treatment with Brefeldin A, which destroys Golgi stacks, resulted in CNPY2 accumulation in the endoplasmic reticulum. Secreted CNPY2 was detected in the blood of healthy mice and humans, and the medium of cultured SMCs. SMCs under hypoxia or treated with a prolyl-4-hydroxylase inhibitor stabilized HIF-1α protein and up-regulated CNPY2, while CNPY2 induction was lost after HIF-1α silencing. Chromatin immunoprecipitation demonstrated that HIF-1α binds to a hypoxia response element (HRE-1157) upstream of the human CNPY2 promoter, which was verified by a luciferase reporter driven by HRE-1157-containing constructs. CNPY2 stimulation activated Cdc42, PAK1, and FAK in SMCs, resulting in enhanced proliferation and migration in vitro, and dramatic aortic ring sprouting ex vivo. CNPY2 significantly increased revascularization of the mouse retina after reperfusion injury. CONCLUSIONS: CNPY2 is a HIF-1α-regulated, secreted angiogenic growth factor that promotes SMC migration, proliferation, and tissue revascularization. This new target may have a broader profile than currently available proteins.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Movimiento Celular , Proliferación Celular , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Neovascularización Fisiológica , Proteínas Adaptadoras Transductoras de Señales/sangre , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Sitios de Unión , Hipoxia de la Célula , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Células HEK293 , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Péptidos y Proteínas de Señalización Intracelular/sangre , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/sangre , Proteínas de la Membrana/genética , Ratones , Regiones Promotoras Genéticas , Ratas , Daño por Reperfusión/genética , Daño por Reperfusión/metabolismo , Daño por Reperfusión/fisiopatología , Neovascularización Retiniana/genética , Neovascularización Retiniana/metabolismo , Neovascularización Retiniana/fisiopatología , Transducción de Señal , Factores de Tiempo , Transcripción GenéticaRESUMEN
Interleukin-10 (IL-10) gene transduction into allogeneic smooth muscle cells (SMCs) was evaluated to improve the long-term benefits of allogeneic cell transplantation into infarcted myocardium. Allogeneic cells, including SMCs, have been demonstrated to restore cardiac function and repair the infarcted myocardium, but late rejection of the transplanted cells by the host immune system may reverse the benefits of cell therapy. In a rat myocardial infarction model, three groups of rats were injected with either unmodified autologous, unmodified allogeneic, or allogeneic + IL-10 SMCs into the infarct region. Three weeks later, most of the allogeneic cells were rejected, whereas autologous cells were engrafted in the myocardium. IL-10 gene transduction of the allogeneic SMCs significantly improved the cell survival. To understand the mechanism of this improved survival, we evaluated the host immune responses against the SMCs. Allogeneic SMCs expressing IL-10 decreased leukocyte-mediated cytotoxicity in coculture, decreased the number of cytotoxic CD8(+) T-cells, and increased the number of CD4(+)CD25(+) regulatory T-cells in vitro and in vivo. Furthermore, IL-10 prevented the production of antidonor antibodies by the recipients against the allogeneic SMCs. Transplantation of unmodified autologous SMCs, but not unmodified allogeneic SMCs, significantly improved fractional shortening and left ventricular dimensions compared to the media-injected control group. However, IL-10 gene-enhanced allogeneic SMCs improved ventricular function, increased wall thickness, and decreased scar length in association with their enhanced survival. We conclude that IL-10 gene-enhanced cell therapy with allogeneic SMCs prevents detrimental alloimmune responses in the recipient, thereby increasing the survival of transplanted allogeneic SMCs and more effectively restoring cardiac function.
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Rechazo de Injerto/inmunología , Interleucina-10/uso terapéutico , Infarto del Miocardio/inmunología , Infarto del Miocardio/terapia , Miocitos del Músculo Liso/trasplante , Función Ventricular/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Antígeno CTLA-4/metabolismo , Muerte Celular , Técnicas de Cocultivo , Femenino , Regulación de la Expresión Génica , Pruebas de Función Cardíaca , Humanos , Tolerancia Inmunológica/genética , Tolerancia Inmunológica/inmunología , Leucocitos/metabolismo , Recuento de Linfocitos , Masculino , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Ratas Endogámicas Lew , Ratas Wistar , Linfocitos T Reguladores/inmunologíaRESUMEN
The epicardium supports cardiomyocyte proliferation early in development and provides fibroblasts and vascular smooth muscle cells to the developing heart. The epicardium has been shown to play an important role during tissue remodeling after cardiac injury, making access to this cell lineage necessary for the study of regenerative medicine. Here we describe the generation of epicardial lineage cells from human pluripotent stem cells by stage-specific activation of the BMP and WNT signaling pathways. These cells display morphological characteristics and express markers of the epicardial lineage, including the transcription factors WT1 and TBX18 and the retinoic acid-producing enzyme ALDH1A2. When induced to undergo epithelial-to-mesenchymal transition, the cells give rise to populations that display characteristics of the fibroblast and vascular smooth muscle lineages. These findings identify BMP and WNT as key regulators of the epicardial lineage in vitro and provide a model for investigating epicardial function in human development and disease.
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Linaje de la Célula/fisiología , Pericardio/citología , Células Madre Pluripotentes/citología , Aldehído Deshidrogenasa/metabolismo , Animales , Proteína Morfogenética Ósea 4/metabolismo , Transición Epitelial-Mesenquimal/fisiología , Humanos , Ratones , Miocitos Cardíacos/citología , Vía de Señalización Wnt/fisiologíaRESUMEN
The goal of cardiac tissue engineering is to restore function to the damaged myocardium with regenerative constructs. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) can produce viable, contractile, three-dimensional grafts that function in vivo. We sought to enhance the viability and functional maturation of cardiac tissue constructs by cyclical stretch. hESC-CMs seeded onto gelatin-based scaffolds underwent cyclical stretching. Histological analysis demonstrated a greater proportion of cardiac troponin T-expressing cells in stretched than non-stretched constructs, and flow sorting demonstrated a higher proportion of cardiomyocytes. Ultrastructural assessment showed that cells in stretched constructs had a more mature phenotype, characterized by greater cell elongation, increased gap junction expression, and better contractile elements. Real-time PCR revealed enhanced mRNA expression of genes associated with cardiac maturation as well as genes encoding cardiac ion channels. Calcium imaging confirmed that stretched constructs contracted more frequently, with shorter calcium cycle duration. Epicardial implantation of constructs onto ischemic rat hearts demonstrated the feasibility of this platform, with enhanced survival and engraftment of transplanted cells in the stretched constructs. This uniaxial stretching system may serve as a platform for the production of cardiac tissue-engineered constructs for translational applications.
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Diferenciación Celular , Células Madre Embrionarias/citología , Miocitos Cardíacos/citología , Estrés Mecánico , Ingeniería de Tejidos/métodos , Animales , Calcio/metabolismo , Diferenciación Celular/genética , Línea Celular , Conexina 43/metabolismo , Modelos Animales de Enfermedad , Células Madre Embrionarias/ultraestructura , Estudios de Factibilidad , Regulación de la Expresión Génica , Humanos , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/terapia , Miocardio/patología , Ratas , Ratas Desnudas , Andamios del Tejido/química , Cicatrización de HeridasRESUMEN
Following a myocardial infarction (MI), fibroblasts differentiate to myofibroblasts, which possess some of the characteristics of smooth muscle cells (SMCs) and contribute to wound healing. Previous studies suggested that the miR-143/-145 cluster plays a critical role in SMC differentiation. Therefore, we determined whether miR-145 promoted differentiation of cardiac fibroblasts to myofibroblasts. Following coronary occlusion in mice, myocardial miR-145 expression was downregulated at 3 days but was restored at 7 days. In vitro studies showed that hypoxia also downregulated miR-145 in cardiac fibroblasts. The number of α-smooth muscle actin (α-SMA) positive cells in fibroblast cultures was employed to determine their transdifferentiation to cardiac myofibroblasts and was increased by 73.5% after transient transfection with miR-145. Ultrastructural analysis of α-SMA stress fibers revealed that ~95% of the α-SMA(+) cells treated with miR-145 organized their actin-filament bundles with a specific orientation compared to only 15% in the scrambled control group. This orientation of the SMA bundles and their integration with the filamentous actin fibers of the cytoskeleton permit infarct wound contraction. Structural and functional studies showed that miR-145 induced a myofibroblast phenotype, and miR-145 also potentiated the production of mature collagen by myofibroblasts. Repression of KLF5, a target of miR-145, was validated by a chimeric luciferase construct tagged with the full-length 3'-UTR of KLF5. A dramatic decrease in KLF5 and a corresponding increase in myocardin expression were observed after transfecting cultured fibroblasts with miR-145. Similar results were found in vivo: the transient decrease in miR-145 expression 3 days post-MI was associated with an increase in KLF5 and a decrease in myocardin. In addition, in vivo delivery of a miR-145 antagomir 1 day prior to and 2 and 6 days after MI decreased myofibroblast formation and increased scar size. The antagomir also reversed the suppressed expression of KLF5 protein in the scar region at day 7 after MI. In summary, we describe a novel association between miR-145 and fibroblast differentiation toward myofibroblasts. These observations provide a new approach to promote endogenous scar healing and contracture by stimulating the transdifferentiation of cardiac fibroblasts to myofibroblasts.
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Fibroblastos/metabolismo , MicroARNs/genética , Miocardio/metabolismo , Repitelización/fisiología , Actinas/genética , Actinas/metabolismo , Animales , Diferenciación Celular , Transdiferenciación Celular , Colágeno/genética , Colágeno/metabolismo , Oclusión Coronaria/patología , Vasos Coronarios/cirugía , Femenino , Fibroblastos/patología , Expresión Génica , Genes Reporteros , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Ratones Endogámicos C57BL , MicroARNs/antagonistas & inhibidores , MicroARNs/metabolismo , Miocardio/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Miofibroblastos/metabolismo , Miofibroblastos/patología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oligonucleótidos Antisentido/genética , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
BACKGROUND: Allogeneic mesenchymal stem cells (MSCs) were immunoprivileged early after cardiac implantation and improved heart function in preclinical and clinical studies. However, long-term preclinical studies demonstrated that allogeneic MSCs lost their immunoprivilege and were rejected in the injured myocardium, resulting in recurrent ventricular dysfunction. This study identifies some of the mechanisms responsible for the immune switch in MSCs and suggests a new treatment to maintain immunoprivilege and preserve heart function. METHODS AND RESULTS: Rat MSC immunoprivilege was mediated by prostaglandin E2 (PGE2)-induced secretion of 2 critical chemokines, CCL12 and CCL5. These chemokines stimulated the chemoattraction of T cells toward MSCs, suppressed cytotoxic T-cell proliferation, and induced the production of T regulatory cells. MSCs treated with 5-azacytidine for 24 hours differentiated into myogenic cells after 2 weeks, which was associated with decreased PGE2 and chemokine production and the loss of immunoprivilege. Treatment of differentiated MSCs with PGE2 restored chemokine levels and preserved MSC immunoprivilege. In a rat myocardial infarction model, allogeneic MSCs (3 × 10(6) cells/rat) were injected into the infarct region with or without a biodegradable hydrogel that slowly released PGE2. Five weeks later, the transplanted MSCs expressed myogenic lineage markers and were rejected in the control group, but in the PGE2-treated group, the transplanted cells survived and heart function improved. CONCLUSIONS: Allogeneic MSCs maintained immunoprivilege by PGE2-induced secretion of chemokines CCL12 and CCL5. Differentiation of MSCs decreased PGE2 levels, and immunoprivilege was lost. Maintaining PGE2 levels preserved immunoprivilege after differentiation, prevented rejection of implanted MSCs, and restored cardiac function.
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Dinoprostona/metabolismo , Rechazo de Injerto/prevención & control , Trasplante de Células Madre Mesenquimatosas/métodos , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/metabolismo , Función Ventricular/fisiología , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Técnicas de Cocultivo , Dinoprostona/fisiología , Dinoprostona/uso terapéutico , Femenino , Rechazo de Injerto/metabolismo , Rechazo de Injerto/fisiopatología , Masculino , Infarto del Miocardio/patología , Distribución Aleatoria , Ratas , Ratas Endogámicas Lew , Ratas Wistar , Trasplante Homólogo , Función Ventricular/efectos de los fármacosRESUMEN
AIMS: Despite the diverse developmental origins of vascular smooth muscle cells (VSMCs), recent attempts to generate VSMCs from human embryonic stem cells (hESCs) differentiated along various lineages did not yield distinct cell phenotypes. The aim of this study was to derive and characterize functional coronary-like VSMCs from hESCs using serum-free cardiac-directed differentiation. METHODS AND RESULTS: Embryoid bodies (EBs) from three pluripotent stem cell lines subjected to cardiac-directed differentiation in defined media were characterized over 30 days for VSMC-specific gene expression by qRT-PCR, immunofluorescence microscopy and fluorescence-activated cell sorting (FACS). EBs composed of cardiomyocytes, endothelial cells (ECs), fibroblasts, and VSMCs underwent FACS on d28 to reveal that the VSMCs form a distinct subpopulation, which migrate with ECs in an in vitro angiogenesis assay. To enrich for VSMCs, d28 EBs were dissociated and cultured as monolayers. Over several passages, mRNA and protein levels of cardiomyocyte, endothelial, and fibroblast markers were abolished, whereas those of mature VSMCs were unchanged. Vascular endothelial growth factor and basic fibroblast growth factor were critical for the separation of the cardiac and VSMC lineages in EBs, and for the enrichment of functional VSMCs in monolayer cultures. Calcium cycling and cell shortening responses to vasoconstrictors in hESC-derived VSMCs in vitro were indistinguishable from primary human coronary artery SMCs, and distinct from bladder and aorta SMCs. VSMCs identically derived from green fluorescent protein -expressing hESCs integrated in and contributed to new vessel formation in vivo. CONCLUSION: The ability to generate hESC-derived functional human coronary-like VSMCs in serum-free conditions has implications for disease modelling, drug screening, and regenerative therapies.
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Diferenciación Celular , Vasos Coronarios/citología , Células Madre Embrionarias/citología , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Actinas/análisis , Animales , Calcio/metabolismo , Células Cultivadas , Medio de Cultivo Libre de Suero , Factor 2 de Crecimiento de Fibroblastos/fisiología , Humanos , Ratones , Ratones SCID , Neovascularización Fisiológica , ARN Mensajero/análisis , Factor A de Crecimiento Endotelial Vascular/fisiología , Vasoconstricción/efectos de los fármacosRESUMEN
Autologous stem cell therapy has not been as effective as forecasted from preclinical studies. Patient age was reported as an important contributing factor. The goal of this study was to uncover age-dependent mechanisms of stem cell dysfunction and to investigate possible means to restore the cellular function. Bone marrow mesenchymal stem cells (MSCs) were isolated from cardiovascular patients. Cell proliferation and number of colonies were inversely correlated with patient age. Myogenic differentiation of MSCs in culture was induced with 5-azacytidine. Differentiation correlated with age, with less differentiation in MSCs from aged patients. We performed real-time PCR to identify genes in the WNT/ß-catenin signaling network and found that transcript levels of CTNNB1, LEF1, FZD8, WNT3A, and SFRP4 were negatively correlated with age, whereas FOSL1, LRP6, and FZD6 were positively correlated with age. Protein evaluation showed that ß-catenin nuclear translocation correlated with age and was lower in aged MSCs. Aged MSCs treated with lithium chloride-to increase the bioavailability of ß-catenin-recovered their capacity for myogenic differentiation through myocyte enhancer factor 2C but not with the knockdown of ß-catenin using small-interfering RNA. This study may be the first to relate reduced nuclear ß-catenin bioavailability in MSCs from aged patients. Most important, this abnormality was potentially recoverable, providing a target for improving the function of bone marrow stem cells and their clinical utility in aged patients.
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Envejecimiento/patología , Enfermedades Cardiovasculares/patología , Diferenciación Celular , Senescencia Celular , Células Madre Mesenquimatosas/patología , Desarrollo de Músculos , Vía de Señalización Wnt , Adulto , Anciano , Anciano de 80 o más Años , Envejecimiento/genética , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/genética , Linaje de la Célula/efectos de los fármacos , Linaje de la Célula/genética , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Senescencia Celular/genética , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Litio/farmacología , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Persona de Mediana Edad , Desarrollo de Músculos/efectos de los fármacos , Desarrollo de Músculos/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Donantes de Tejidos , Vía de Señalización Wnt/efectos de los fármacos , Vía de Señalización Wnt/genética , beta Catenina/metabolismoRESUMEN
The Kv11.1 (hERG) K+ channel plays a fundamental role in cardiac repolarization. Missense mutations in KCNH2, the gene encoding Kv11.1, cause long QT syndrome (LQTS) and frequently cause channel trafficking-deficiencies. This study characterized the properties of a novel KCNH2 mutation discovered in a LQT2 patient resuscitated from a ventricular fibrillation arrest. Proband genotyping was performed by SSCP and DNA sequencing. The electrophysiological and biochemical properties of the mutant channel were investigated after expression in HEK293 cells. The proband manifested a QTc of 554 ms prior to electrolyte normalization. Mutation analysis revealed an autosomal dominant frameshift mutation at proline 1086 (P1086fs+32X; 3256InsG). Co-immunoprecipitation demonstrated that wild-type Kv11.1 and mutant channels coassemble. Western blot showed that the mutation did not produce mature complex-glycosylated Kv11.1 channels and coexpression resulted in reduced channel maturation. Electrophysiological recordings revealed mutant channel peak currents to be similar to untransfected cells. Co-expression of channels in a 1â¶1 ratio demonstrated dominant negative suppression of peak Kv11.1 currents. Immunocytochemistry confirmed that mutant channels were not present at the plasma membrane. Mutant channel trafficking rescue was attempted by incubation at reduced temperature or with the pharmacological agents E-4031. These treatments did not significantly increase peak mutant currents or induce the formation of mature complex-glycosylated channels. The proteasomal inhibitor lactacystin increased the protein levels of the mutant channels demonstrating proteasomal degradation, but failed to induce mutant Kv11.1 protein trafficking. Our study demonstrates a novel dominant-negative Kv11.1 mutation, which results in degraded non-functional channels leading to a LQT2 phenotype.
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Canales de Potasio Éter-A-Go-Go/genética , Canales de Potasio Éter-A-Go-Go/metabolismo , Síndrome de QT Prolongado/genética , Síndrome de QT Prolongado/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Adulto , Western Blotting , Membrana Celular/metabolismo , Canal de Potasio ERG1 , Electrofisiología , Canales de Potasio Éter-A-Go-Go/química , Femenino , Genotipo , Células HEK293 , Humanos , Inmunohistoquímica , Inmunoprecipitación , Microscopía Confocal , Modelos Biológicos , Mutación , Piperidinas/farmacología , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/genética , Piridinas/farmacologíaRESUMEN
Coordinated cardiac ion channel gating is fundamental for generation of action potential and excitability throughout the myocardium. The interaction of pore-forming ion channels with auxiliary subunits can regulate surface expression, localization and anchoring of these channels to plasma membrane. SNARE (soluble N-ethylmaleimide sensitive factors attachment protein or SNAP receptor) proteins mediate the targeting, docking, and fusion of intracellular vesicles for exocytotic release of neurotransmitters and hormones. In secretory neurons and neuroendocrine cells, some voltage-gated channels are physically coupled with SNARE proteins, resulting in alterations in channel gating and trafficking. Coupling of SNARE proteins to membrane ion channels is however not unique to secretory cells. We have demonstrated the expression of SNARE proteins in rodent myocardial tissue, and more importantly, functional interaction of SNARE proteins with cardiac K(ATP) and K(v) (K(v)1.2, K(v)2.1, K(v)4.2, K(v)4.3, and K(v)11.1) channels. SNARE proteins, therefore, have similar fundamental functions in ion channel trafficking and regulation per se, independent of secretion. We now review the body of work of SNARE protein regulation on membrane ion channels in the heart.
Asunto(s)
Factor Natriurético Atrial/metabolismo , Corazón/fisiología , Miocardio/metabolismo , Canales de Potasio/metabolismo , Proteínas SNARE/metabolismo , Animales , HumanosRESUMEN
Late after a myocardial infarction (MI), surgical ventricular restoration (SVR) can reduce left ventricular volumes, but an enhanced cardiac patch may be required to restore function. We developed a new, biodegradable patch (modified gelfoam, MGF) consisting of a spongy inner core (gelfoam) to encourage cell engraftment and an outer coating (poly epsilon-caprolactone) to provide sufficient strength to permit ventricular repair. Two weeks after coronary ligation in rats, SVR was performed using one of the following: gelfoam, MGF, MGF patches with hydrogel alone, or with hydrogel and cytokines (stem cell factor, stromal cell-derived factor-1alpha), bone marrow mesenchymal stem cells, or both. Cardiac function and morphology were evaluated by echocardiography, conduction catheterization, magnetic resonance imaging, and histology. Animals whose hearts were repaired with untreated gelfoam died of ventricular rupture. The MGF groups had significantly improved myocardial systolic function vs. MI controls. Enhancement with cytokines and/or cells promoted more alpha-smooth muscle actin-positive cells, more capillaries, greater wall thickness, a more ellipsoid shape, greater fractional shortening, and better-preserved systolic elastance than MGF alone. This combination of the new, reinforced, biodegradable biomaterial and cytokine/cell treatment created a viable tissue after SVR and produced better functional outcomes than un-reinforced gelfoam or MGF alone.
Asunto(s)
Citocinas/metabolismo , Ventrículos Cardíacos , Infarto del Miocardio , Ingeniería de Tejidos , Andamios del Tejido/química , Animales , Materiales Biocompatibles/química , Materiales Biocompatibles/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/fisiología , Citocinas/química , Femenino , Esponja de Gelatina Absorbible/química , Esponja de Gelatina Absorbible/metabolismo , Ventrículos Cardíacos/patología , Ventrículos Cardíacos/cirugía , Humanos , Hidrogeles/química , Hidrogeles/metabolismo , Ensayo de Materiales , Estructura Molecular , Infarto del Miocardio/patología , Infarto del Miocardio/cirugía , Poliésteres/química , Poliésteres/metabolismo , Ratas , Ratas Sprague-Dawley , Células del Estroma/citología , Células del Estroma/fisiología , Ingeniería de Tejidos/instrumentación , Ingeniería de Tejidos/métodos , Función Ventricular IzquierdaRESUMEN
Insulin secretion from pancreatic beta-cells is mediated by the opening of voltage-gated Ca2+ channels (CaV) and exocytosis of insulin dense core vesicles facilitated by the secretory soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein machinery. We previously observed that beta-cell exocytosis is sensitive to the acute removal of membrane cholesterol. However, less is known about the chronic changes in endogenous cholesterol and its biosynthesis in regulating beta-cell stimulus-secretion coupling. We examined the effects of inhibiting endogenous beta-cell cholesterol biosynthesis by using the squalene epoxidase inhibitor, NB598. The expression of squalene epoxidase in primary and clonal beta-cells was confirmed by RT-PCR. Cholesterol reduction of 36-52% was observed in MIN6 cells, mouse and human pancreatic islets after a 48-h incubation with 10 mum NB598. A similar reduction in cholesterol was observed in the subcellular compartments of MIN6 cells. We found NB598 significantly inhibited both basal and glucose-stimulated insulin secretion from mouse pancreatic islets. CaV channels were markedly inhibited by NB598. Rapid photolytic release of intracellular caged Ca2+ and simultaneous measurements of the changes in membrane capacitance revealed that NB598 also inhibited exocytosis independently from CaV channels. These effects were reversed by cholesterol repletion. Our results indicate that endogenous cholesterol in pancreatic beta-cells plays a critical role in regulating insulin secretion. Moreover, chronic inhibition of cholesterol biosynthesis regulates the functional activity of CaV channels and insulin secretory granule mobilization and membrane fusion. Dysregulation of cellular cholesterol may cause impairment of beta-cell function, a possible pathogenesis leading to the development of type 2 diabetes.