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1.
Front Cell Dev Biol ; 12: 1388378, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38699159

RESUMO

In heart disease patients, myocyte loss or malfunction invariably leads to fibrosis, involving the activation and accumulation of cardiac fibroblasts that deposit large amounts of extracellular matrix. Apart from the vital replacement fibrosis that follows myocardial infarction, ensuring structural integrity of the heart, cardiac fibrosis is largely considered to be maladaptive. Much work has focused on signaling pathways driving the fibrotic response, including TGF-ß signaling and biomechanical strain. However, currently there are very limited options for reducing cardiac fibrosis, with most patients suffering from chronic fibrosis. The adult heart has very limited regenerative capacity. However, cardiac regeneration has been reported in humans perinatally, and reproduced experimentally in neonatal mice. Furthermore, model organisms such as the zebrafish are able to fully regenerate their hearts following massive cardiac damage into adulthood. Increasing evidence points to a transient immuno-fibrotic response as being key for cardiac regeneration to occur. The mechanisms at play in this context are changing our views on fibrosis, and could be leveraged to promote beneficial remodeling in heart failure patients. This review summarizes our current knowledge of fibroblast properties associated with the healthy, failing or regenerating heart. Furthermore, we explore how cardiac fibroblast activity could be targeted to assist future therapeutic approaches.

2.
Int J Mol Sci ; 24(21)2023 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-37958548

RESUMO

Cornelia de Lange Syndrome (CdLS) patients, who frequently carry a mutation in NIPBL, present an increased incidence of outflow tract (OFT)-related congenital heart defects (CHDs). Nipbl+/- mice recapitulate a number of phenotypic traits of CdLS patients, including a small body size and cardiac defects, but no study has specifically focused on the valves. Here, we show that adult Nipbl+/- mice present aortic valve thickening, a condition that has been associated with stenosis. During development, we observed that OFT septation and neural crest cell condensation was delayed in Nipbl+/- embryos. However, we did not observe defects in the deployment of the main lineages contributing to the semilunar valves. Indeed, endocardial endothelial-to-mesenchymal transition (EndMT), analysed via outflow tract explants, and neural crest migration, analysed via genetic lineage tracing, did not significantly differ in Nipbl+/- mice and their wild-type littermates. Our study provides the first direct evidence for valve formation defects in Nipbl+/- mice and points to specific developmental defects as an origin for valve disease in patients.


Assuntos
Proteínas de Ciclo Celular , Síndrome de Cornélia de Lange , Cardiopatias Congênitas , Animais , Humanos , Camundongos , Valva Aórtica , Proteínas de Ciclo Celular/genética , Síndrome de Cornélia de Lange/genética , Haploinsuficiência , Cardiopatias Congênitas/genética , Mutação
4.
J Mol Cell Biol ; 14(10)2023 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-36271843

RESUMO

Understanding how certain animals are capable of regenerating their hearts will provide much needed insights into how this process can be induced in humans in order to reverse the damage caused by myocardial infarction. Currently, it is becoming increasingly evident that cardiac interstitial cells play crucial roles during cardiac regeneration. To understand how interstitial cells behave during this process, we performed single-cell RNA sequencing of regenerating zebrafish hearts. Using a combination of immunohistochemistry, chemical inhibition, and novel transgenic animals, we were able to investigate the role of cell type-specific mechanisms during cardiac regeneration. This approach allowed us to identify a number of important regenerative processes within the interstitial cell populations. Here, we provide detailed insight into how interstitial cells behave during cardiac regeneration, which will serve to increase our understanding of how this process could eventually be induced in humans.


Assuntos
Infarto do Miocárdio , Miócitos Cardíacos , Animais , Humanos , Peixe-Zebra , Animais Geneticamente Modificados , Proliferação de Células
5.
Circ Res ; 126(10): 1330-1342, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32175811

RESUMO

RATIONALE: Fibro-fatty infiltration of subepicardial layers of the atrial wall has been shown to contribute to the substrate of atrial fibrillation. OBJECTIVE: Here, we examined if the epicardium that contains multipotent cells is involved in this remodeling process. METHODS AND RESULTS: One hundred nine human surgical right atrial specimens were evaluated. There was a relatively greater extent of epicardial thickening and dense fibro-fatty infiltrates in atrial tissue sections from patients aged over 70 years who had mitral valve disease or atrial fibrillation when compared with patients aged less than 70 years with ischemic cardiomyopathy as indicated using logistic regression adjusted for age and gender. Cells coexpressing markers of epicardial progenitors and fibroblasts were detected in fibro-fatty infiltrates. Such epicardial remodeling was reproduced in an experimental model of atrial cardiomyopathy in rat and in Wilms tumor 1 (WT1)CreERT2/+;ROSA-tdT+/- mice. In the latter, genetic lineage tracing demonstrated the epicardial origin of fibroblasts within fibro-fatty infiltrates. A subpopulation of human adult epicardial-derived cells expressing PDGFR (platelet-derived growth factor receptor)-α were isolated and differentiated into myofibroblasts in the presence of Ang II (angiotensin II). Furthermore, single-cell RNA-sequencing analysis identified several clusters of adult epicardial-derived cells and revealed their specification from adipogenic to fibrogenic cells in the rat model of atrial cardiomyopathy. CONCLUSIONS: Epicardium is reactivated during the formation of the atrial cardiomyopathy. Subsets of adult epicardial-derived cells, preprogrammed towards a specific cell fate, contribute to fibro-fatty infiltration of subepicardium of diseased atria. Our study reveals the biological basis for chronic atrial myocardial remodeling that paves the way of atrial fibrillation.


Assuntos
Tecido Adiposo/patologia , Fibrilação Atrial/etiologia , Remodelamento Atrial , Cardiomiopatias/complicações , Átrios do Coração/patologia , Miocárdio/patologia , Pericárdio/patologia , Potenciais de Ação , Adipócitos/metabolismo , Adipócitos/patologia , Tecido Adiposo/metabolismo , Idoso , Animais , Fibrilação Atrial/metabolismo , Fibrilação Atrial/patologia , Fibrilação Atrial/fisiopatologia , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Cardiomiopatias/fisiopatologia , Linhagem da Célula , Modelos Animais de Doenças , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Átrios do Coração/metabolismo , Átrios do Coração/fisiopatologia , Frequência Cardíaca , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miocárdio/metabolismo , Pericárdio/metabolismo , Pericárdio/fisiopatologia , Ratos Wistar , Células-Tronco/metabolismo , Células-Tronco/patologia , Proteínas WT1/genética , Proteínas WT1/metabolismo
6.
Nat Commun ; 10(1): 1929, 2019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-31028265

RESUMO

Genetically modified mice have advanced our understanding of valve development and disease. Yet, human pathophysiological valvulogenesis remains poorly understood. Here we report that, by combining single cell sequencing and in vivo approaches, a population of human pre-valvular endocardial cells (HPVCs) can be derived from pluripotent stem cells. HPVCs express gene patterns conforming to the E9.0 mouse atrio-ventricular canal (AVC) endocardium signature. HPVCs treated with BMP2, cultured on mouse AVC cushions, or transplanted into the AVC of embryonic mouse hearts, undergo endothelial-to-mesenchymal transition and express markers of valve interstitial cells of different valvular layers, demonstrating cell specificity. Extending this model to patient-specific induced pluripotent stem cells recapitulates features of mitral valve prolapse and identified dysregulation of the SHH pathway. Concurrently increased ECM secretion can be rescued by SHH inhibition, thus providing a putative therapeutic target. In summary, we report a human cell model of valvulogenesis that faithfully recapitulates valve disease in a dish.


Assuntos
Células Endoteliais/patologia , Proteínas Hedgehog/genética , Prolapso da Valva Mitral/patologia , Valva Mitral/patologia , Células-Tronco Pluripotentes/patologia , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Proteína Morfogenética Óssea 2/farmacologia , Proteínas Relacionadas a Caderinas , Caderinas/genética , Caderinas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Embrião de Mamíferos , Endocárdio/metabolismo , Endocárdio/patologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/transplante , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fator de Transcrição GATA5/genética , Fator de Transcrição GATA5/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Átrios do Coração/metabolismo , Átrios do Coração/patologia , Proteínas Hedgehog/metabolismo , Humanos , Camundongos , Valva Mitral/metabolismo , Prolapso da Valva Mitral/genética , Prolapso da Valva Mitral/metabolismo , Prolapso da Valva Mitral/terapia , Modelos Biológicos , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Cultura Primária de Células , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Proteína Wnt3A/farmacologia
7.
Physiol Rev ; 98(4): 2453-2475, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30156497

RESUMO

The heart is the first organ to be functional in the fetus. Heart formation is a complex morphogenetic process regulated by both genetic and epigenetic mechanisms. Congenital heart diseases (CHD) are the most prominent congenital diseases. Genetics is not sufficient to explain these diseases or the impact of them on patients. Epigenetics is more and more emerging as a basis for cardiac malformations. This review brings the essential knowledge on cardiac biology of development. It further provides a broad background on epigenetics with a focus on three-dimensional conformation of chromatin. Then, we summarize the current knowledge of the impact of epigenetics on cardiac cell fate decision. We further provide an update on the epigenetic anomalies in the genesis of CHD.


Assuntos
Epigênese Genética/genética , Epigênese Genética/fisiologia , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/fisiopatologia , Coração/crescimento & desenvolvimento , Animais , Epigenômica/métodos , Feto/fisiologia , Humanos
8.
Circ Res ; 122(4): 583-590, 2018 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-29269349

RESUMO

RATIONALE: Myocardial infarction is a major cause of adult mortality worldwide. The origin(s) of cardiac fibroblasts that constitute the postinfarct scar remain controversial, in particular the potential contribution of bone marrow lineages to activated fibroblasts within the scar. OBJECTIVE: The aim of this study was to establish the origin(s) of infarct fibroblasts using lineage tracing and bone marrow transplants and a robust marker for cardiac fibroblasts, the Collagen1a1-green fluorescent protein reporter. METHODS AND RESULTS: Using genetic lineage tracing or bone marrow transplant, we found no evidence for collagen-producing fibroblasts derived from hematopoietic or bone marrow lineages in hearts subjected to permanent left anterior descending coronary artery ligation. In fact, fibroblasts within the infarcted area were largely of epicardial origin. Intriguingly, collagen-producing fibrocytes from hematopoietic lineages were observed attached to the epicardial surface of infarcted and sham-operated hearts in which a suture was placed around the left anterior descending coronary artery. CONCLUSIONS: In this controversial field, our study demonstrated that the vast majority of infarct fibroblasts were of epicardial origin and not derived from bone marrow lineages, endothelial-to-mesenchymal transition, or blood. We also noted the presence of collagen-producing fibrocytes on the epicardial surface that resulted at least in part from the surgical procedure.


Assuntos
Células da Medula Óssea/citologia , Linhagem da Célula , Infarto do Miocárdio/terapia , Miofibroblastos/citologia , Animais , Células da Medula Óssea/metabolismo , Transplante de Medula Óssea/efeitos adversos , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/patologia , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Pericárdio/citologia
9.
Cell Stem Cell ; 20(3): 345-359.e5, 2017 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-28111199

RESUMO

Pericytes are widely believed to function as mesenchymal stem cells (MSCs), multipotent tissue-resident progenitors with great potential for regenerative medicine. Cultured pericytes isolated from distinct tissues can differentiate into multiple cell types in vitro or following transplantation in vivo. However, the cell fate plasticity of endogenous pericytes in vivo remains unclear. Here, we show that the transcription factor Tbx18 selectively marks pericytes and vascular smooth muscle cells in multiple organs of adult mouse. Fluorescence-activated cell sorting (FACS)-purified Tbx18-expressing cells behaved as MSCs in vitro. However, lineage-tracing experiments using an inducible Tbx18-CreERT2 line revealed that pericytes and vascular smooth muscle cells maintained their identity in aging and diverse pathological settings and did not significantly contribute to other cell lineages. These results challenge the current view of endogenous pericytes as multipotent tissue-resident progenitors and suggest that the plasticity observed in vitro or following transplantation in vivo arises from artificial cell manipulations ex vivo.


Assuntos
Células-Tronco Mesenquimais/citologia , Especificidade de Órgãos , Pericitos/citologia , Adipócitos/citologia , Envelhecimento/genética , Linhagem da Célula , Cicatriz/patologia , Fibroblastos/citologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/metabolismo , Humanos , Integrases/metabolismo , Células-Tronco Mesenquimais/metabolismo , Desenvolvimento Muscular , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , Neurônios/citologia , Pericitos/metabolismo , Fenótipo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo
10.
Proc Natl Acad Sci U S A ; 114(5): E771-E780, 2017 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-28096344

RESUMO

The abundance of epicardial adipose tissue (EAT) is associated with atrial fibrillation (AF), the most frequent cardiac arrhythmia. However, both the origin and the factors involved in EAT expansion are unknown. Here, we found that adult human atrial epicardial cells were highly adipogenic through an epithelial-mesenchymal transition both in vitro and in vivo. In a genetic lineage tracing the WT1CreERT2+/-RosatdT+/- mouse model subjected to a high-fat diet, adipocytes of atrial EAT derived from a subset of epicardial progenitors. Atrial myocardium secretome induces the adipogenic differentiation of adult mesenchymal epicardium-derived cells by modulating the balance between mesenchymal Wingless-type Mouse Mammary Tumor Virus integration site family, member 10B (Wnt10b)/ß-catenin and adipogenic ERK/MAPK signaling pathways. The adipogenic property of the atrial secretome was enhanced in AF patients. The atrial natriuretic peptide secreted by atrial myocytes is a major adipogenic factor operating at a low concentration by binding to its natriuretic peptide receptor A (NPRA) receptor and, in turn, by activating a cGMP-dependent pathway. Hence, our data indicate cross-talk between EAT expansion and mechanical function of the atrial myocardium.


Assuntos
Adipogenia/fisiologia , Tecido Adiposo/metabolismo , Fator Natriurético Atrial/metabolismo , Átrios do Coração/metabolismo , Pericárdio/metabolismo , Adipócitos/citologia , Idoso , Animais , Células Cultivadas , Dieta Hiperlipídica , Transição Epitelial-Mesenquimal , Feminino , Átrios do Coração/citologia , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Miócitos Cardíacos/metabolismo , Pericárdio/citologia , Proteínas Proto-Oncogênicas/metabolismo , Células-Tronco/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo
11.
J Mol Cell Cardiol ; 91: 1-5, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26748307

RESUMO

Cardiac fibroblasts produce the extracellular matrix (ECM) scaffold within which the various cellular components of the heart are organized. As well as providing structural support, it is becoming evident that the quality and quantity of ECM is a key factor for determining cardiac cell behavior during development and in pathological contexts such as heart failure involving fibrosis. Cardiac fibroblasts have long remained a poorly characterized cardiac lineage. Well characterized markers are now paving the way for a better understanding of the roles of these cells in various developmental and disease contexts. Notably, the relevance of processes including endothelial-tomesenchymal transition and the recruitment of circulating fibroblast progenitors in heart failure has been challenged. This review describes the latest findings on cardiac fibroblast markers and developmental origins, and discusses their importance in myocardial remodeling. Effective modulation of cardiac fibroblast activity would likely contribute to successful treatment of various cardiac disorders.


Assuntos
Linhagem da Célula/fisiologia , Fibroblastos/patologia , Insuficiência Cardíaca/patologia , Miocárdio/patologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biomarcadores/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Receptores com Domínio Discoidina , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibrose , Expressão Gênica , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Humanos , Miocárdio/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores Mitogênicos/genética , Receptores Mitogênicos/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo
12.
Stem Cells ; 34(1): 34-43, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26352327

RESUMO

Heart failure is still a major cause of hospitalization and mortality in developed countries. Many clinical trials have tested the use of multipotent stem cells as a cardiac regenerative medicine. The benefit for the patients of this therapeutic intervention has remained limited. Herein, we review the pluripotent stem cells as a cell source for cardiac regeneration. We more specifically address the various challenges of this cell therapy approach. We question the cell delivery systems, the immune tolerance of allogenic cells, the potential proarrhythmic effects, various drug mediated interventions to facilitate cell grafting and, finally, we describe the pathological conditions that may benefit from such an innovative approach. As members of a transatlantic consortium of excellence of basic science researchers and clinicians, we propose some guidelines to be applied to cell types and modes of delivery in order to translate pluripotent stem cell cardiac derivatives into safe and effective clinical trials.


Assuntos
Insuficiência Cardíaca/terapia , Miocárdio/citologia , Células-Tronco Pluripotentes/citologia , Transplante de Células-Tronco , Animais , Diferenciação Celular , Modelos Animais de Doenças , Insuficiência Cardíaca/fisiopatologia , Humanos
13.
J Mol Med (Berl) ; 93(8): 823-30, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26169532

RESUMO

Cardiac fibroblasts are a major cell population of the heart and are characterized by their capacity to produce extracellular matrix (ECM). In hearts subjected to pressure overload, excessive fibroblast accumulation is responsible for fibrosis of the myocardium, a major clinical issue. Hence, understanding mechanisms generating fibroblasts in this context has become a key question in the cardiovascular field. Recent studies now point to the activation of resident fibroblasts as the underlying cause of fibrosis. However, de novo generation of fibroblasts from endothelium and circulating hematopoietic cells has also been proposed to significantly contribute to fibrosis. Here, we discuss the latest findings on fibroblast origins, with a particular emphasis on the pressure overload model, and the implication of these findings for the development of anti-fibrotic therapies that are currently lacking.


Assuntos
Fibroblastos/citologia , Fibroblastos/patologia , Insuficiência Cardíaca/patologia , Miocárdio/citologia , Miocárdio/patologia , Animais , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Fibroblastos/metabolismo , Fibrose/metabolismo , Fibrose/patologia , Coração/embriologia , Insuficiência Cardíaca/metabolismo , Humanos , Miocárdio/metabolismo
14.
Genesis ; 53(5): 337-45, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25950518

RESUMO

Heart valve development begins with the endothelial-to-mesenchymal transition (EMT) of endocardial cells. Although lineage studies have demonstrated contributions from cardiac neural crest and epicardium to semilunar and atrioventricular (AV) valve formation, respectively, most valve mesenchyme derives from the endocardial EMT. Specific Cre mouse lines for fate-mapping analyses of valve endocardial cells are limited. Msx1 displayed expression in AV canal endocardium and cushion mesenchyme between E9.5 and E11.5, when EMT is underway. Additionally, previous studies have demonstrated that deletion of Msx1 and its paralog Msx2 results in hypoplastic AV cushions and impaired endocardial signaling. A knock-in tamoxifen-inducible Cre line was recently generated (Msx1CreERT2) and characterized during embryonic development and after birth, and was shown to recapitulate the endogenous Msx1 expression pattern. Here, we further analyze this knock-in allele and track the Msx1-expressing cells and their descendants during cardiac development with a particular focus on their contribution to the valves and their precursors. Thus, Msx1CreERT2 mice represent a useful model for lineage tracing and conditional gene manipulation of endocardial and mesenchymal cushion cells essential to understand mechanisms of valve development and remodeling.


Assuntos
Alelos , Técnicas de Introdução de Genes , Valvas Cardíacas/embriologia , Valvas Cardíacas/metabolismo , Integrases/genética , Fator de Transcrição MSX1/genética , Receptores de Estrogênio/genética , Animais , Endocárdio/enzimologia , Endocárdio/metabolismo , Transição Epitelial-Mesenquimal/genética , Regulação da Expressão Gênica no Desenvolvimento , Integrases/metabolismo , Fator de Transcrição MSX1/metabolismo , Camundongos , Organogênese/genética , Receptores de Estrogênio/metabolismo
17.
Proc Natl Acad Sci U S A ; 111(32): E3297-305, 2014 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-25074909

RESUMO

Hepatic myofibroblasts are activated in response to chronic liver injury of any etiology to produce a fibrous scar. Despite extensive studies, the origin of myofibroblasts in different types of fibrotic liver diseases is unresolved. To identify distinct populations of myofibroblasts and quantify their contribution to hepatic fibrosis of two different etiologies, collagen-α1(I)-GFP mice were subjected to hepatotoxic (carbon tetrachloride; CCl4) or cholestatic (bile duct ligation; BDL) liver injury. All myofibroblasts were purified by flow cytometry of GFP(+) cells and then different subsets identified by phenotyping. Liver resident activated hepatic stellate cells (aHSCs) and activated portal fibroblasts (aPFs) are the major source (>95%) of fibrogenic myofibroblasts in these models of liver fibrosis in mice. As previously reported using other methodologies, hepatic stellate cells (HSCs) are the major source of myofibroblasts (>87%) in CCl4 liver injury. However, aPFs are a major source of myofibroblasts in cholestatic liver injury, contributing >70% of myofibroblasts at the onset of injury (5 d BDL). The relative contribution of aPFs decreases with progressive injury, as HSCs become activated and contribute to the myofibroblast population (14 and 20 d BDL). Unlike aHSCs, aPFs respond to stimulation with taurocholic acid and IL-25 by induction of collagen-α1(I) and IL-13, respectively. Furthermore, BDL-activated PFs express high levels of collagen type I and provide stimulatory signals to HSCs. Gene expression analysis identified several novel markers of aPFs, including a mesothelial-specific marker mesothelin. PFs may play a critical role in the pathogenesis of cholestatic liver fibrosis and, therefore, serve as an attractive target for antifibrotic therapy.


Assuntos
Cirrose Hepática/patologia , Fígado/patologia , Miofibroblastos/patologia , Animais , Tetracloreto de Carbono/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Colestase/complicações , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Modelos Animais de Doenças , Proteínas Ligadas por GPI/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Fígado/metabolismo , Cirrose Hepática/etiologia , Cirrose Hepática/metabolismo , Mesotelina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miofibroblastos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vitamina A/metabolismo
18.
J Clin Invest ; 124(7): 2921-34, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24937432

RESUMO

Activation and accumulation of cardiac fibroblasts, which result in excessive extracellular matrix deposition and consequent mechanical stiffness, myocyte uncoupling, and ischemia, are key contributors to heart failure progression. Recently, endothelial-to-mesenchymal transition (EndoMT) and the recruitment of circulating hematopoietic progenitors to the heart have been reported to generate substantial numbers of cardiac fibroblasts in response to pressure overload-induced injury; therefore, these processes are widely considered to be promising therapeutic targets. Here, using multiple independent murine Cre lines and a collagen1a1-GFP fusion reporter, which specifically labels fibroblasts, we found that following pressure overload, fibroblasts were not derived from hematopoietic cells, EndoMT, or epicardial epithelial-to-mesenchymal transition. Instead, pressure overload promoted comparable proliferation and activation of two resident fibroblast lineages, including a previously described epicardial population and a population of endothelial origin. Together, these data present a paradigm for the origins of cardiac fibroblasts during development and in fibrosis. Furthermore, these data indicate that therapeutic strategies for reducing pathogenic cardiac fibroblasts should shift from targeting presumptive EndoMT or infiltrating hematopoietically derived fibroblasts, toward common pathways upregulated in two endogenous fibroblast populations.


Assuntos
Insuficiência Cardíaca/patologia , Miocárdio/patologia , Animais , Biomarcadores/metabolismo , Pressão Sanguínea , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Linhagem da Célula , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Endocárdio/metabolismo , Endocárdio/patologia , Transição Epitelial-Mesenquimal , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Perfilação da Expressão Gênica , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/fisiopatologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Miocárdio/metabolismo , Pericárdio/metabolismo , Pericárdio/patologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
19.
PLoS Genet ; 10(2): e1004114, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24586179

RESUMO

Recent interest has focused on the importance of the nucleus and associated nucleoskeleton in regulating changes in cardiac gene expression in response to biomechanical load. Mutations in genes encoding proteins of the inner nuclear membrane and nucleoskeleton, which cause cardiomyopathy, also disrupt expression of a biomechanically responsive gene program. Furthermore, mutations in the outer nuclear membrane protein Nesprin 1 and 2 have been implicated in cardiomyopathy. Here, we identify for the first time a role for the outer nuclear membrane proteins, Nesprin 1 and Nesprin 2, in regulating gene expression in response to biomechanical load. Ablation of both Nesprin 1 and 2 in cardiomyocytes, but neither alone, resulted in early onset cardiomyopathy. Mutant cardiomyocytes exhibited altered nuclear positioning, shape, and chromatin positioning. Loss of Nesprin 1 or 2, or both, led to impairment of gene expression changes in response to biomechanical stimuli. These data suggest a model whereby biomechanical signals are communicated from proteins of the outer nuclear membrane, to the inner nuclear membrane and nucleoskeleton, to result in changes in gene expression required for adaptation of the cardiomyocyte to changes in biomechanical load, and give insights into etiologies underlying cardiomyopathy consequent to mutations in Nesprin 1 and 2.


Assuntos
Cardiomiopatias/genética , Miocárdio/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Animais , Fenômenos Biomecânicos , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Núcleo Celular/metabolismo , Proteínas do Citoesqueleto , Regulação da Expressão Gênica , Humanos , Camundongos , Mutação , Miócitos Cardíacos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Membrana Nuclear/genética , Membrana Nuclear/metabolismo , Matriz Nuclear/metabolismo , Proteínas Nucleares/metabolismo
20.
J Vis Exp ; (79)2013 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-24056408

RESUMO

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes. The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.


Assuntos
Técnicas Citológicas/métodos , Miócitos Cardíacos/citologia , Animais , Animais Recém-Nascidos , Técnicas de Cultura de Células/métodos , Camundongos
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