RESUMO
During heart development, epicardial cells residing within the outer layer undergo epithelial-mesenchymal transition (EMT) and migrate into the underlying myocardium to support organ growth and morphogenesis. Disruption of epicardial EMT results in embryonic lethality, yet its regulation is poorly understood. Here, we report epicardial EMT within the mesothelial layer of the mouse embryonic heart at ultra-high resolution using scanning electron microscopy combined with immunofluorescence analyses. We identified morphologically active EMT regions that associated with key components of the extracellular matrix, including the basement membrane-associated proteoglycan agrin. Deletion of agrin resulted in impaired EMT and compromised development of the epicardium, accompanied by downregulation of Wilms' tumor 1. Agrin enhanced EMT in human embryonic stem cell-derived epicardial-like cells by decreasing ß-catenin and promoting pFAK localization at focal adhesions, and promoted the aggregation of dystroglycan within the Golgi apparatus in murine epicardial cells. Loss of agrin resulted in dispersal of dystroglycan in vivo, disrupting basement membrane integrity and impairing EMT. Our results provide new insights into the role of the extracellular matrix in heart development and implicate agrin as a crucial regulator of epicardial EMT.
Assuntos
Agrina/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Proteínas da Matriz Extracelular/metabolismo , Coração/embriologia , Coração/crescimento & desenvolvimento , Organogênese/fisiologia , Animais , Feminino , Heterogeneidade Genética , Complexo de Golgi , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/metabolismo , Pericárdio/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
The adult mammalian heart is non-regenerative owing to the post-mitotic nature of cardiomyocytes. The neonatal mouse heart can regenerate, but only during the first week of life. Here we show that changes in the composition of the extracellular matrix during this week can affect cardiomyocyte growth and differentiation in mice. We identify agrin, a component of neonatal extracellular matrix, as required for the full regenerative capacity of neonatal mouse hearts. In vitro, recombinant agrin promotes the division of cardiomyocytes that are derived from mouse and human induced pluripotent stem cells through a mechanism that involves the disassembly of the dystrophin-glycoprotein complex, and Yap- and ERK-mediated signalling. In vivo, a single administration of agrin promotes cardiac regeneration in adult mice after myocardial infarction, although the degree of cardiomyocyte proliferation observed in this model suggests that there are additional therapeutic mechanisms. Together, our results uncover a new inducer of mammalian heart regeneration and highlight fundamental roles of the extracellular matrix in cardiac repair.
Assuntos
Agrina/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Coração/fisiologia , Regeneração , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Animais Recém-Nascidos , Proteínas de Ciclo Celular , Proliferação de Células , Distroglicanas/metabolismo , Feminino , Camundongos , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miocárdio/citologia , Miocárdio/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Sinalização YAPRESUMO
BACKGROUND: Ischemic heart diseases are leading causes of death and reduced life quality worldwide. Although revascularization strategies significantly reduce mortality after acute myocardial infarction (MI), a large number of patients with MI develop chronic heart failure over time. We previously reported that a fragment of the extracellular matrix protein agrin promotes cardiac regeneration after MI in adult mice. METHODS: To test the therapeutic potential of agrin in a preclinical porcine model, we performed ischemia-reperfusion injuries using balloon occlusion for 60 minutes followed by a 3-, 7-, or 28-day reperfusion period. RESULTS: We demonstrated that local (antegrade) delivery of recombinant human agrin to the infarcted pig heart can target the affected regions in an efficient and clinically relevant manner. A single dose of recombinant human agrin improved heart function, infarct size, fibrosis, and adverse remodeling parameters 28 days after MI. Short-term MI experiments along with complementary murine studies revealed myocardial protection, improved angiogenesis, inflammatory suppression, and cell cycle reentry as agrin's mechanisms of action. CONCLUSIONS: A single dose of agrin is capable of reducing ischemia-reperfusion injury and improving heart function, demonstrating that agrin could serve as a therapy for patients with acute MI and potentially heart failure.
Assuntos
Agrina/farmacologia , Infarto do Miocárdio/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Recuperação de Função Fisiológica/efeitos dos fármacos , Animais , Humanos , Camundongos , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/fisiopatologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Proteínas Recombinantes/farmacologia , SuínosAssuntos
Coração , Miocárdio , Humanos , Coração/diagnóstico por imagem , Matriz Extracelular , Regeneração , Miócitos CardíacosAssuntos
Senescência Celular , Fibroblastos/metabolismo , Traumatismos Cardíacos/metabolismo , Pericárdio/metabolismo , Regeneração , Proteína Supressora de Tumor p53/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Agrina/farmacologia , Animais , Senescência Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Traumatismos Cardíacos/genética , Traumatismos Cardíacos/patologia , Traumatismos Cardíacos/fisiopatologia , Cinética , Pericárdio/efeitos dos fármacos , Pericárdio/patologia , Regeneração/efeitos dos fármacos , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Limb amputation in salamanders yields a wound response that ultimately leads to replacement of the missing part. This unique-among-tetrapod trait involves the migration and recruitment of multiple cell types including epithelium, immune cells, axonal growth cones, and connective tissue cells to build the blastema which contains the proliferating stem and progenitor cells to rebuild the limb tissues. A number of the signaling and cell biological events have been defined. They point to the intimate coordination of physical events such as osmotic pressure, cell migration, and cell-cell communication with changes in cell identity such as dedifferentiation into embryonic-like epithelial and mesenchymal cells.
Assuntos
Extremidades , Urodelos , Animais , Transdução de Sinais , Células-TroncoRESUMO
The discovery of endogenous regenerative potential of the heart in zebrafish and neonatal mice has shifted the cardiac regenerative field towards the utilization of intrinsic regenerative mechanisms in the mammalian heart. The goal of these studies is to understand, and eventually apply, the neonatal regenerative mechanisms into adulthood. To facilitate these studies, the last two decades have seen advancements in the development of injury models in adult mice representative of the diversity of cardiac diseases. Here, we provide an overview for a selection of the most common cardiac ischemic injury models and describe a set of methods used to accurately analyze and quantify cardiac outcomes. Importantly, a comprehensive understanding of cardiac regeneration and repair requires a combination of multiple functional, histological, and molecular analyses.
Assuntos
Coração/fisiopatologia , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Regeneração , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICRRESUMO
The adult mammalian heart regenerates poorly after injury and, as a result, ischemic heart diseases are among the leading causes of death worldwide. The recovery of the injured heart is dependent on orchestrated repair processes including inflammation, fibrosis, cardiomyocyte survival, proliferation, and contraction properties that could be modulated in patients. In this work we designed an automated high-throughput screening system for small molecules that induce cardiomyocyte proliferation in vitro and identified the small molecule Chicago Sky Blue 6B (CSB). Following induced myocardial infarction, CSB treatment reduced scar size and improved heart function of adult mice. Mechanistically, we show that although initially identified using in vitro screening for cardiomyocyte proliferation, in the adult mouse CSB promotes heart repair through (i) inhibition of CaMKII signaling, which improves cardiomyocyte contractility; and (ii) inhibition of neutrophil and macrophage activation, which attenuates the acute inflammatory response, thereby contributing to reduced scarring. In summary, we identified CSB as a potential therapeutic agent that enhances cardiac repair and function by suppressing postinjury detrimental processes, with no evidence for cardiomyocyte renewal.
Assuntos
Coração/efeitos dos fármacos , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos , Azul Tripano/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Cicatriz/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos ICR , Miocárdio/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismoRESUMO
Abnormal architectures of collagen fibers in the extracellular matrix (ECM) are hallmarks of many invasive diseases, including cancer. Targeting specific stages of collagen assembly in vivo presents a great challenge due to the involvement of various crosslinking enzymes in the multistep, hierarchical process of ECM build-up. Using advanced microscopic tools, we monitored stages of fibrillary collagen assembly in a native fibroblast-derived 3D matrix system and identified anti-lysyl oxidase-like 2 (LOXL2) antibodies that alter the natural alignment and width of endogenic fibrillary collagens without affecting ECM composition. The disrupted collagen morphologies interfered with the adhesion and invasion properties of human breast cancer cells. Treatment of mice bearing breast cancer xenografts with the inhibitory antibodies resulted in disruption of the tumorigenic collagen superstructure and in reduction of primary tumor growth. Our approach could serve as a general methodology to identify novel therapeutics targeting fibrillary protein organization to treat ECM-associated pathologies. Cancer Res; 76(14); 4249-58. ©2016 AACR.
Assuntos
Neoplasias da Mama/patologia , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Aminoácido Oxirredutases/antagonistas & inibidores , Aminoácido Oxirredutases/fisiologia , Animais , Anticorpos Monoclonais/imunologia , Linhagem Celular Tumoral , Proliferação de Células , Proteínas da Matriz Extracelular/análise , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Invasividade Neoplásica , Microambiente TumoralRESUMO
Tumor-associated macrophages (TAMs) promote tumor development, invasion, and dissemination by various mechanisms. In this study, using an orthotopic colorectal cancer (CRC) model, we found that monocyte-derived TAMs advance tumor development by the remodeling of its extracellular matrix (ECM) composition and structure. Unbiased transcriptomic and proteomic analyses of (a) TAM-abundant and -deficient tumor tissues and (b) sorted tumor-associated and -resident colonic macrophage subpopulations defined a distinct TAM-induced ECM molecular signature composed of an ensemble of matricellular proteins and remodeling enzymes they provide to the tumor microenvironment. Remarkably, many of these ECM proteins are specifically increased in human CRC versus healthy colon. Specifically, we demonstrate that although differentiating into TAMs, monocytes up-regulate matrix-remodeling programs associated with the synthesis and assembly of collagenous ECM, specifically collagen types I, VI, and XIV. This finding was further established by advanced imaging showing that TAMs instruct the deposition, cross-linking, and linearization of collagen fibers during tumor development, especially at areas of tumor invasiveness. Finally, we show that cancer-associated fibroblasts are significantly outnumbered by TAMs in this model and that their expression of collagen XIV and I is reduced by TAM deficiency. Here, we outline a novel TAM protumoral function associated with building of the collagenous ECM niche.
Assuntos
Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Matriz Extracelular/metabolismo , Colágenos Fibrilares/metabolismo , Macrófagos/patologia , Animais , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Carcinogênese/metabolismo , Carcinogênese/patologia , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/ultraestrutura , Modelos Animais de Doenças , Matriz Extracelular/ultraestrutura , Proteínas da Matriz Extracelular/metabolismo , Colágenos Fibrilares/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Proteômica , Receptores CCR2/deficiência , Receptores CCR2/metabolismo , Transcriptoma/genética , Microambiente TumoralRESUMO
Mitochondrial carrier homolog 2 (MTCH2) is a repressor of mitochondrial oxidative phosphorylation (OXPHOS), and its locus is associated with increased BMI in humans. Here, we demonstrate that mice deficient in muscle MTCH2 are protected from diet-induced obesity and hyperinsulinemia and that they demonstrate increased energy expenditure. Deletion of muscle MTCH2 also increases mitochondrial OXPHOS and mass, triggers conversion from glycolytic to oxidative fibers, increases capacity for endurance exercise, and increases heart function. Moreover, metabolic profiling of mice deficient in muscle MTCH2 reveals a preference for carbohydrate utilization and an increase in mitochondria and glycolytic flux in muscles. Thus, MTCH2 is a critical player in muscle biology, modulating metabolism and mitochondria mass as well as impacting whole-body energy homeostasis.
Assuntos
Metaboloma/genética , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Músculo Esquelético/metabolismo , Obesidade/genética , Animais , Composição Corporal , Dieta Hiperlipídica , Modelos Animais de Doenças , Metabolismo Energético , Expressão Gênica , Glicólise/genética , Humanos , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/patologia , Proteínas de Transporte da Membrana Mitocondrial/deficiência , Músculo Esquelético/patologia , Obesidade/etiologia , Obesidade/metabolismo , Obesidade/patologia , Fosforilação Oxidativa , Condicionamento Físico AnimalRESUMO
The murine neonatal heart can regenerate after injury through cardiomyocyte (CM) proliferation, although this capacity markedly diminishes after the first week of life. Neuregulin-1 (NRG1) administration has been proposed as a strategy to promote cardiac regeneration. Here, using loss- and gain-of-function genetic tools, we explore the role of the NRG1 co-receptor ERBB2 in cardiac regeneration. NRG1-induced CM proliferation diminished one week after birth owing to a reduction in ERBB2 expression. CM-specific Erbb2 knockout revealed that ERBB2 is required for CM proliferation at embryonic/neonatal stages. Induction of a constitutively active ERBB2 (caERBB2) in neonatal, juvenile and adult CMs resulted in cardiomegaly, characterized by extensive CM hypertrophy, dedifferentiation and proliferation, differentially mediated by ERK, AKT and GSK3ß/ß-catenin signalling pathways. Transient induction of caERBB2 following myocardial infarction triggered CM dedifferentiation and proliferation followed by redifferentiation and regeneration. Thus, ERBB2 is both necessary for CM proliferation and sufficient to reactivate postnatal CM proliferative and regenerative potentials.
Assuntos
Desdiferenciação Celular , Proliferação de Células , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Receptor ErbB-2/metabolismo , Regeneração , Transdução de Sinais , Fatores Etários , Animais , Animais Recém-Nascidos , Desdiferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Imageamento por Ressonância Magnética , Camundongos Knockout , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Neuregulina-1/metabolismo , Neuregulina-1/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor ErbB-2/agonistas , Receptor ErbB-2/deficiência , Receptor ErbB-2/genética , Regeneração/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Imagem com Lapso de Tempo , beta Catenina/metabolismoRESUMO
In light of the increasing need for differentiated primary cells for cell therapy and the rapid dedifferentiation occurring during standard in vitro cultivation techniques, there is an urgent need for developing three-dimensional in vitro systems in which expanded cells display in vivo-like differentiated phenotypes. It is becoming clear that the natural microenvironment provides the optimal conditions for achieving this aim. To this end, we prepared natural decellularized scaffolds of microscopic dimensions that would allow appropriate diffusion of gases and nutrients to all seeded cells. Scaffolds from either the lung or the liver were analyzed for their ability to support growth and differentiation of progenitor alveolar cells and hepatocytes. We observed that progenitor alveolar cells that have been expanded on plastic culture and thus dedifferentiated grew within the lung-derived scaffolds into highly organized structures and regained differentiation markers classical for type I and type II alveolar cells. The cells generated proper alveolar structures, and only 15%-30% of them secreted surfactant proteins in a localized manner for extended periods. Vice versa, liver-derived scaffolds supported the differentiation state of primary hepatocytes. We further demonstrate that the natural scaffolds are organ specific, that is, only cells derived from the same organ become properly differentiated. A proteomic analysis shows significant different composition of lung and liver scaffolds, for example, decorin, thrombospondin 1, vimentin, and various laminin isoforms are especially enriched in the lung. Altogether, our data demonstrate that complex interactions between the seeded cells and a highly organized, organ-specific stroma are required for proper localized cell differentiation. Thus, our novel in vitro culture system can be used for ex vivo differentiation and organization of expanded primary cells.