RESUMO
Mechanotransduction plays a crucial role in vascular biology. One example of this is the local regulation of vascular resistance via flow-mediated dilation (FMD). Impairment of this process is a hallmark of endothelial dysfunction and a precursor to a wide array of vascular diseases, such as hypertension and atherosclerosis. Yet the molecules responsible for sensing flow (shear stress) within endothelial cells remain largely unknown. We designed a 384-well screening system that applies shear stress on cultured cells. We identified a mechanosensitive cell line that exhibits shear stress-activated calcium transients, screened a focused RNAi library, and identified GPR68 as necessary and sufficient for shear stress responses. GPR68 is expressed in endothelial cells of small-diameter (resistance) arteries. Importantly, Gpr68-deficient mice display markedly impaired acute FMD and chronic flow-mediated outward remodeling in mesenteric arterioles. Therefore, GPR68 is an essential flow sensor in arteriolar endothelium and is a critical signaling component in cardiovascular pathophysiology.
Assuntos
Mecanotransdução Celular , Interferência de RNA , Receptores Acoplados a Proteínas G/fisiologia , Animais , Materiais Biocompatíveis , Cálcio/metabolismo , Linhagem Celular Tumoral , Células Endoteliais/fisiologia , Endotélio Vascular/citologia , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Concentração de Íons de Hidrogênio , Artérias Mesentéricas/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/metabolismo , RNA Interferente Pequeno/metabolismo , Receptores Acoplados a Proteínas G/genética , Resistência ao Cisalhamento , Estresse Mecânico , Resistência VascularRESUMO
BACKGROUND: Adult mammalian cardiomyocytes (CMs) have the potential to proliferate, but this is not sufficient to generate adequate CMs after myocardial infarction (MI). The transcription factor Tbx20 is required for CM proliferation during development and adult CM homeostasis. The ability of Tbx20 overexpression (Tbx20(OE)) to promote adult CM proliferation and to improve cardiac function after MI was examined. METHODS AND RESULTS: Tbx20(OE) was induced specifically in adult mouse differentiated CMs. Increased CM proliferation and fetal-like characteristics were found in Tbx20(OE) hearts compared with controls without causing pathology 4 weeks after Tbx20(OE) at baseline. Moreover, Tbx20(OE) in adult CM after MI significantly improved survival, cardiac function, and infarct size 4 weeks after MI. Improved cardiac repair, as indicated by increased CM proliferation and capillary density, was observed in the MI border zone of Tbx20(OE) hearts compared with controls. Expression of proliferation activator (cyclin D1, E1, and IGF1) and fetal contractile protein (ssTNI, ßMHC) mRNA was increased whereas negative cell-cycle regulators (p21, Meis1) were decreased in Tbx20(OE) hearts compared with controls under both baseline and MI conditions. Tbx20(OE) in adult hearts activates multiple proproliferation pathways, including Akt, YAP and BMP. Interestingly, p21, Meis1, and a novel cell-cycle inhibitory gene, Btg2, are directly bound and repressed by Tbx20 with induction of proliferation in neonatal CM. CONCLUSIONS: Tbx20(OE), specifically in adult CM, activates multiple cardiac proliferative pathways, directly represses cell-cycle inhibitory genes p21, Meis1, and Btg2, promotes adult CM proliferation; and preserves cardiac performance after MI.
Assuntos
Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas com Domínio T/fisiologia , Animais , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Divisão Celular , Tamanho Celular/efeitos dos fármacos , Eletrocardiografia , Feminino , Proteínas Fetais/biossíntese , Proteínas Fetais/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Genes cdc/efeitos dos fármacos , Coração/fisiopatologia , Proteínas Imediatamente Precoces/biossíntese , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/fisiologia , Camundongos , Camundongos Transgênicos , Proteínas Musculares/biossíntese , Proteínas Musculares/genética , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , Miócitos Cardíacos/patologia , Tamanho do Órgão/efeitos dos fármacos , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Distribuição Aleatória , Transdução de Sinais/efeitos dos fármacos , Método Simples-Cego , Proteínas com Domínio T/biossíntese , Proteínas com Domínio T/genética , Tamoxifeno/farmacologia , Proteínas Supressoras de Tumor/biossíntese , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/fisiologiaRESUMO
The majority of cardiac fibroblasts in a mature mammalian heart are derived from the epicardium during prenatal development and reactivate developmental programs during the progression of fibrotic disease. In addition, epicardial activation, proliferation, and fibrosis occur with ischemic, but not hypertensive injury. Here we review cellular and molecular mechanisms that control epicardium-derived cell lineages during development and disease with a focus on cardiac fibroblasts. This article is part of a special issue entitled "Fibrosis and Myocardial Remodeling".
Assuntos
Fibroblastos/patologia , Infarto do Miocárdio/patologia , Miócitos Cardíacos/patologia , Pericárdio/patologia , Animais , Diferenciação Celular , Linhagem da Célula/fisiologia , Proliferação de Células , Fibroblastos/metabolismo , Fibrose , Humanos , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Organogênese/fisiologia , Pericárdio/metabolismoRESUMO
AIMS: Hypoplastic coronary artery disease is a rare congenital abnormality that is associated with sudden cardiac death. However, molecular mechanisms responsible for this disease are not clear. The aim of the present study was to assess the role of nitric oxide synthase-3 (NOS3) in the pathogenesis of hypoplastic coronary arteries. METHODS AND RESULTS: Wild-type (WT), NOS3(-/-), and a novel cardiac-specific NOS3 overexpression mouse model were employed. Deficiency in NOS3 resulted in coronary artery hypoplasia in foetal mice and spontaneous myocardial infarction in postnatal hearts. Coronary artery diameters, vessel density, and volume were significantly decreased in NOS3(-/-) mice at postnatal day 0. In addition, NOS3(-/-) mice showed a significant increase in the ventricular wall thickness, myocardial volume, and cardiomyocyte cell size compared with WT mice. Lack of NOS3 also down-regulated the expression of Gata4, Wilms tumour-1, vascular endothelial growth factor, basic fibroblast growth factor and erythropoietin, and inhibited migration of epicardial cells. These abnormalities and hypoplastic coronary arteries in the NOS3(-/-) mice were completely rescued by the cardiac-specific overexpression of NOS3. CONCLUSION: Nitric oxide synthase-3 is required for coronary artery development and deficiency in NOS3 leads to hypoplastic coronary arteries.
Assuntos
Anomalias dos Vasos Coronários/enzimologia , Infarto do Miocárdio/enzimologia , Óxido Nítrico Sintase Tipo III/deficiência , Animais , Animais Recém-Nascidos , Anomalias dos Vasos Coronários/embriologia , Camundongos Transgênicos , Infarto do Miocárdio/embriologia , Análise de SobrevidaRESUMO
Significance: Cardiovascular disease is a major contributor to human mortality and morbidity. The cardiac tissue undergoes fibrotic healing after injury because of the limited regenerative capacity of adult mammalian cardiomyocyte (CM). Extensive research has been performed to identify therapeutic targets for CM regeneration, as the success of promoting adult human CM regeneration to repair the injured heart is considered the Holy Grail in the field. Recent Advances: To date, more than 30 target genes have been shown to regulate adult mammalian CM proliferation. More than 20 targets have been validated in adult mouse myocardial infarction (MI) model in a therapeutic setting. In this review, the translational efficacy readouts from 17 selected pharmaceutical targets are summarized, among which the Hippo-yes-associated protein (Yap) pathway is the most extensively investigated and fits the criteria for a promising target for pro-CM-regeneration therapy development. Critical Issues and Future Directions: As the pro-CM-regeneration potential of current drug treatment for cardiovascular patients is limited, to help identify and fill the gap between basic research and drug discovery in this specific field, details regarding target identification, validation in mouse MI models, high-throughput screening assay development, and preclinical in vivo efficacy model optimization are discussed. Finally, suggestions and recommendations are also provided to help establish a common guideline for in vivo translational studies for drug discovery focusing on CM regeneration. Antioxid. Redox Signal. 39, 1070-1087.
Assuntos
Infarto do Miocárdio , Miócitos Cardíacos , Camundongos , Animais , Humanos , Miócitos Cardíacos/metabolismo , Transdução de Sinais/fisiologia , Coração/fisiologia , Infarto do Miocárdio/metabolismo , Modelos Animais de Doenças , Regeneração/fisiologia , Descoberta de Drogas , Proliferação de Células , MamíferosRESUMO
Introduction: Melanoma is a common and aggressive type of skin cancer with rising incidence rate globally. Gender is one of the determining factors, and overall males have a higher risk of developing melanoma as well as worse prognosis. Emerging evidence show that GPR68, a G protein-coupled receptor that is sensitive to acid and mechanical stimulations for cellular microenvironment, plays an important role in tumor biology. However, whether GPR68 is involved in gender-dependent regulation of tumor growth is unclear. Methods: We established a syngeneic melanoma model in Gpr68-deficient mice and investigated tumor growth in males and females. The GPR68 activation-induced cellular responses of melanocytes, including intracellular calcium dynamics, proliferation and migration were measured. The landscape of tumor-infiltrating immune cells were analyzed by flow cytometry and the expression various cytokines were checked by qRT-PCR. Results: GPR68 is required for melanoma growth in males but dispensable in females. GPR68 is expressed and functional in B16-F10 melanocytes, but the activity of the receptor does not directly contribute to proliferation and migration of the cells. GPR68 inhibits infiltration of CD45+ lymphocytes, CD8+ T cells and NK cells in melanoma in male mice, but has no apparent effect in females. Furthermore, GPR68 functionally inhibits the expression of IFNγ in the tumor infiltrating CD8+ T cells and NK cells as well as the inflammatory cytokine expression in the spleen in male mice but not in females. Our results show the gender-dependent modulatory effect of GPR68 on tumor-infiltrating immune cells and their tumor-killing capacity. Discussion: GPR68 is sensor for acid and mechanical stimulations, which are two important factors in the microenvironment associated with tumor growth and metastasis. Our results suggest a prominent role of the receptor molecules in tumor biology in a gender-dependent manner. Since GPCRs are more feasible to develop small molecule drugs compared to transcription factors, our study demonstrates the potential of GPR68 as a novel druggable therapeutic target for melanoma in male patients.
RESUMO
Emerging evidence suggests ginseng has therapeutic potential in cardiovascular disease. The aim of this study was to investigate the role of endothelial nitric oxide synthase (eNOS) in the cardioprotective effects of ginseng during myocardial ischemia and reperfusion (I/R). Treatment with ginseng extract significantly increased Akt phosphorylation and eNOS protein levels in cultured neonatal cardiomyocytes. Upregulation of eNOS was blocked by LY294002, a PI3-kinase inhibitor, suggesting a PI3-kinase/Akt-dependent mechanism. To simulate I/R, cultured neonatal cardiomyocytes from eNOS(-/-) and wild-type (WT) mice were subjected to anoxia and reoxygenation (A/R). Ginseng treatment inhibited A/R-induced apoptosis in WT, but not in either eNOS(-/-) cardiomyocytes or WT cardiomyocytes treated with LY294002. To further study the cardioprotective effects of ginseng in vivo, WT and eNOS(-/-) mice were pretreated with ginseng extract (50mg/kg/day, oral gavage) for 7 days before they were subjected to myocardial I/R. Treatment with ginseng significantly increased Akt phosphorylation and eNOS protein levels in the myocardium. Furthermore, ginseng-induced myocardial eNOS expression was inhibited by LY294002. Strikingly, ginseng treatment significantly decreased infarct size and myocardial apoptosis following I/R in WT mice, but not in either eNOS(-/-) mice or WT mice treated with LY294002. We conclude that ginseng treatment protects the heart from I/R injury via upregulation of eNOS expression. Our study suggests that ginseng may serve as a potential therapeutic agent to limit myocardial I/R injury.
Assuntos
Cardiotônicos/uso terapêutico , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Óxido Nítrico Sintase Tipo III/genética , Panax/química , Fitoterapia , Preparações de Plantas/uso terapêutico , Regulação para Cima/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Cardiotônicos/isolamento & purificação , Células Cultivadas , Deleção de Genes , Coração/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , América do Norte , Fosforilação/efeitos dos fármacos , Preparações de Plantas/isolamento & purificação , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
BACKGROUND: Soluble stem cell factor (SCF) has been shown to mobilize bone marrow stem cells and improve cardiac repair after myocardial infarction (MI). However, the effect of membrane-associated SCF on cardiac remodeling after MI is not known. The present study investigated the effects of cardiomyocyte-specific overexpression of the membrane-associated isoform of human SCF (hSCF) on cardiac function after MI. METHODS AND RESULTS: A novel mouse model with tetracycline-inducible and cardiac-specific overexpression of membrane-associated hSCF was generated. MI was induced by left coronary artery ligation. Thirty-day mortality after MI was decreased in hSCF/tetracycline transactivator (tTA) compared with wild-type mice. In vivo cardiac function was significantly improved in hSCF/tTA mice at 5 and 30 days after MI compared with wild-type mice. Endothelial progenitor cell recruitment and capillary density were increased and myocardial apoptosis was decreased in the peri-infarct area of hSCF/tTA mice. Myocyte size was decreased in hSCF/tTA mice 30 days after MI compared with WT mice. Furthermore, hSCF overexpression promoted de novo angiogenesis as assessed by matrigel implantation into the left ventricular myocardium. CONCLUSIONS: Cardiomyocyte-specific overexpression of hSCF improves myocardial function and survival after MI. These beneficial effects of hSCF may result from increases in endothelial progenitor cell recruitment and neovascularization and decreases in myocardial apoptosis and cardiac remodeling.
Assuntos
Infarto do Miocárdio/terapia , Miócitos Cardíacos/metabolismo , Fator de Células-Tronco/fisiologia , Animais , Apoptose , Doxiciclina/farmacologia , Humanos , Hipertrofia Ventricular Esquerda/etiologia , Camundongos , Camundongos Transgênicos , Infarto do Miocárdio/mortalidade , Infarto do Miocárdio/fisiopatologia , Neovascularização Fisiológica , Proteínas Proto-Oncogênicas c-kit/fisiologia , Fator de Células-Tronco/genética , Células-Tronco/fisiologia , Tetraciclina/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Função Ventricular EsquerdaRESUMO
BACKGROUND: Neuronal nitric oxide synthase (nNOS) is expressed in cardiomyocytes and plays a role in regulating cardiac function and Ca2+ homeostasis. However, the role of nNOS in cardiac electrophysiology after myocardial infarction (MI) is unclear. We hypothesized that nNOS deficiency increases ventricular arrhythmia and mortality after MI. METHODS AND RESULTS: MI was induced in wild-type (WT) or nNOS(-/-) mice by ligation of the left coronary artery. Thirty-day mortality was significantly higher in nNOS(-/-) compared with WT mice. Additionally, nNOS(-/-) mice had impaired cardiac function 2 days after MI. Telemetric ECG monitoring showed that compared with WT, nNOS(-/-) mice had significantly more ventricular arrhythmias and were more likely to develop ventricular fibrillation after MI. Treatment with the L-type Ca2+ channel blocker verapamil reduced the incidence of arrhythmia and ventricular fibrillation in nNOS(-/-) mice after MI. To assess the role of nNOS in Ca2+ handling, patch-clamp and Ca2+ fluorescence techniques were used. Ca2+ transients and L-type Ca2+ currents were higher in nNOS(-/-) compared with WT cardiomyocytes. Additionally, nNOS(-/-) cardiomyocytes exhibited significantly higher systolic and diastolic Ca2+ over a range of pacing frequencies. Treatment with the NO donor S-nitroso N-acetyl-penicillamine decreased Ca2+ transients and L-type Ca2+ current in both nNOS(-/-) and WT cardiomyocytes. Furthermore, S-nitrosylation of Ca2+ handling proteins was significantly decreased in nNOS(-/-) myocardium after MI. CONCLUSIONS: Deficiency in nNOS increases ventricular arrhythmia and mortality after MI in mice. The antiarrhythmic effect of nNOS involves inhibition of L-type Ca2+ channel activity and regulation of Ca2+ handling proteins via S-nitrosylation.
Assuntos
Arritmias Cardíacas/etiologia , Arritmias Cardíacas/prevenção & controle , Infarto do Miocárdio/complicações , Infarto do Miocárdio/prevenção & controle , Animais , Apoptose , Arritmias Cardíacas/mortalidade , Cálcio/metabolismo , Canais de Cálcio Tipo L/fisiologia , Vasos Coronários/patologia , Eletrocardiografia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monitorização Fisiológica , Isquemia Miocárdica/fisiopatologia , Óxido Nítrico Sintase Tipo I/deficiência , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo I/metabolismo , Superóxidos/metabolismo , Telemetria , Disfunção Ventricular Esquerda/etiologiaRESUMO
The aim of this study was to investigate the role of endothelial nitric oxide synthase (eNOS) in the host myocardium on bone marrow mesenchymal stromal cells (MSC) migration to the ischemic myocardium and whether stromal cell-derived factor-1alpha (SDF-1alpha) contributes to eNOS-mediated MSC migration. MSCs and coronary microvascular endothelial cells were isolated from adult wild-type (WT) mouse bone marrow and hearts, respectively. Cultured neonatal cardiomyocytes from WT, eNOS(-/-), and eNOS overexpressing transgenic (Tg) mice were subjected to anoxia and reoxygenation (A/R), and the conditioned medium was used as a chemoattractant for in vitro transendothelial migration assay. MSC migration was decreased in the presence of conditioned medium derived from eNOS(-/-) cardiomyocytes but increased in the presence of eNOS-Tg conditioned medium. SDF-1alpha expression was decreased in eNOS(-/-) but increased in eNOS-Tg cardiomyocytes following A/R and in the myocardium following ischemia/reperfusion (I/R). SDF-1alpha expression was cGMP-dependent as inhibition of soluble guanylyl cyclase decreased SDF-1alpha expression in WT cardiomyocytes. MSCs expressed very low levels of eNOS proteins compared with the adult myocardium. To examine MSC migration in vivo, MSCs derived from mice expressing enhanced green fluorescence protein (EGFP(+)) were intravenously administered to WT mice subjected to myocardial I/R. EGFP(+) cells in the ischemic region were decreased in eNOS(-/-) but increased in eNOS-Tg compared with WT hearts. MSC treatment improved cardiac function following I/R in WT but not in eNOS(-/-) mice. In conclusion, eNOS in the host myocardium promotes MSC migration to the ischemic myocardium and improves cardiac function through cGMP-dependent increases in SDF-1alpha expression.
Assuntos
Movimento Celular/fisiologia , Quimiocina CXCL12/biossíntese , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Multipotentes/metabolismo , Isquemia Miocárdica/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Animais , Células da Medula Óssea/metabolismo , GMP Cíclico/metabolismo , Citometria de Fluxo , Imunofluorescência , Camundongos , Camundongos Transgênicos , Miocárdio/enzimologia , Óxido Nítrico/biossíntese , Receptores CXCR4/metabolismo , Células Estromais/metabolismo , Regulação para CimaRESUMO
Erythropoietin (EPO) is a potent cardioprotective agent in models of myocardial ischemia and reperfusion (I/R). It has been suggested recently that EPO may also reduce ventricular arrhythmia after I/R. The present study investigated the role of neuronal nitric oxide synthase (nNOS) on the antiarrhythmic effects of EPO. EPO treatment increased nNOS expression in isolated neonatal mouse ventricular myocytes. Cotreatment with the phosphatidylinositol 3 (PI3)-kinase inhibitor, LY294002 [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride], or treatment of cardiomyocytes infected with a dominant negative adenovirus targeted to Akt1 (ADV-dnAkt1) blocked the effects of EPO on nNOS expression, suggesting that EPO regulates nNOS expression via PI3-kinase and Akt. To examine the in vivo antiarrhythmic effects of EPO, wild-type (WT) and nNOS(-/-) mice were anesthetized and, after a baseline measurement, subjected to myocardial I/R to provoke ventricular arrhythmias. Pretreatment with EPO 24 h before ischemia increased nNOS expression and significantly reduced the number of premature ventricular contractions (PVCs) and the incidence of ventricular tachycardia (VT) in WT mice. In contrast, treatment with EPO had no effect on PVCs or the incidence of VT in nNOS(-/-) mice. Furthermore, EPO treatment after ischemia significantly reduced the threshold dose of cesium chloride (CsCl) to induce VT. We conclude that EPO via nNOS protects the heart from spontaneous and CsCl-induced ventricular arrhythmia during myocardial I/R.
Assuntos
Arritmias Cardíacas/prevenção & controle , Eritropoetina/farmacologia , Traumatismo por Reperfusão Miocárdica/complicações , Óxido Nítrico Sintase Tipo I/fisiologia , Animais , Arritmias Cardíacas/induzido quimicamente , Arritmias Cardíacas/etiologia , Células Cultivadas , Césio/farmacologia , Cloretos/farmacologia , Eletrocardiografia , Inibidores Enzimáticos/farmacologia , Eritropoetina/uso terapêutico , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Coração/efeitos dos fármacos , Coração/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/patologia , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Taquicardia Ventricular/induzido quimicamente , Taquicardia Ventricular/fisiopatologia , Taquicardia Ventricular/prevenção & controle , Complexos Ventriculares Prematuros/fisiopatologia , Complexos Ventriculares Prematuros/prevenção & controleRESUMO
Cardiac fibrosis is characterized by excessive extracellular matrix deposition that contributes to compromised cardiac function and potentially heart failure. Cardiac pressure overload resulting from trans-aortic constriction in mice leads to cardiac fibrosis and increased Wnt/ß-catenin signaling in cardiac fibroblasts. Here, we conditionally induce ß-catenin loss of function in resident cardiac fibroblasts using Tcf21 MerCreMer or in activated cardiac fibroblasts using periostin (Postn) MerCreMer . We show that ß-catenin loss of function in cardiac fibroblasts after trans-aortic constriction significantly preserves cardiac function, and reduces interstitial fibrosis but does not alter the numbers of activated or differentiated cardiac fibroblasts in vivo. However, ß-catenin is specifically required in resident cardiac fibroblasts for fibrotic excessive extracellular matrix gene expression and binds Col3a1 and Postn gene sequences in cultured cardiac fibroblasts after induction of Wnt signaling. Moreover, cardiomyocyte hypertrophy is blunted with cardiac fibroblast-specific loss of ß-catenin after trans-aortic constriction in vivo. Thus, Wnt/ß-catenin signaling in resident cardiac fibroblasts is required for excessive extracellular matrix gene expression and collagen deposition after trans-aortic constriction.Understanding the mechanisms causing cardiac fibrosis is key to prevention and therapy development of many heart diseases. Here, the authors show that Wnt/ß-catenin signaling in resident cardiac fibroblasts is required for deposition of fibrotic extracellular matrix and the regulation of cardiomyocyte hypertrophy in a mouse model of heart fibrosis.
Assuntos
Fibroblastos/metabolismo , Cardiopatias/metabolismo , beta Catenina/metabolismo , Animais , Aorta/metabolismo , Aorta/fisiopatologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Colágeno/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Feminino , Fibroblastos/patologia , Fibrose/genética , Fibrose/metabolismo , Fibrose/patologia , Fibrose/fisiopatologia , Coração/fisiopatologia , Cardiopatias/genética , Cardiopatias/patologia , Cardiopatias/fisiopatologia , Humanos , Masculino , Camundongos , Camundongos Knockout , Pressão , Via de Sinalização Wnt , beta Catenina/genéticaRESUMO
BACKGROUND: The adult epicardium is a potential source of cardiac progenitors after myocardial infarction (MI). We tested the hypothesis that cardiomyocyte-specific overexpression of membrane-associated human stem cell factor (hSCF) enhances epicardial activation, epicardium-derived cells (EPDCs) production, and myocardial arteriogenesis post MI. METHODS AND RESULTS: Wild-type and the inducible cardiac-specific hSCF transgenic (hSCF/tetracycline transactivator) mice were subjected to MI. Wilms tumor-1 (Wt1)-positive epicardial cells were higher in hSCF/tetracycline transactivator compared with wild-type mice 3 days post MI. Arteriole density was significantly higher in the peri-infarct area of hSCF/tetracycline transactivator mice compared with wild-type mice 5 days post MI. In cultured EPDCs, adenoviral hSCF treatment significantly increased cell proliferation and growth factor expression. Furthermore, adenoviral hSCF treatment in wild-type cardiomyocytes significantly increased EPDC migration. These effects of hSCF overexpression on EPDC proliferation and growth factor expression were all abrogated by ACK2, a neutralizing antibody against c-kit. Finally, lineage tracing using ROSA(mTmG);Wt1(CreER) mice showed that adenoviral hSCF treatment increased Wt1(+) lineage-derived EPDC migration into the infarcted myocardium 5 days post MI, which was inhibited by ACK2. CONCLUSIONS: Cardiomyocyte-specific overexpression of hSCF promotes epicardial activation and myocardial arteriogenesis post MI.
Assuntos
Vasos Coronários/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Infarto do Miocárdio/genética , Miócitos Cardíacos/metabolismo , Pericárdio/metabolismo , RNA Mensageiro/genética , Fator de Células-Tronco/genética , Animais , Animais Recém-Nascidos , Western Blotting , Proliferação de Células , Células Cultivadas , Vasos Coronários/patologia , Modelos Animais de Doenças , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miócitos Cardíacos/patologia , Neovascularização Patológica , Pericárdio/patologia , Reação em Cadeia da Polimerase em Tempo Real , Fator de Células-Tronco/biossínteseRESUMO
Recent studies have investigated histological staining compounds as micro-computed tomography (micro-CT) contrast agents, delivered by soaking tissue specimens in stain and relying on passive diffusion for agent uptake. This study describes a perfusion approach using iodine or phosphotungstic acid (PTA) stains, delivered to an intact mouse, to capitalize on the microvasculature as a delivery conduit for parenchymal staining and direct contact for staining artery walls. Twelve C57BL/6 mice, arterially perfused with either 25% Lugol's solution or 5% PTA solution were scanned intact and reconstructed with 26 µm isotropic voxels. The animals were fixed and the heart and surrounding vessels were excised, embedded and scanned; isolated heart images were reconstructed with 13 µm isotropic voxels. Myocardial enhancement and artery diameters were measured. Both stains successfully enhanced the myocardium and vessel walls. Interestingly, Lugol's solution provided a significantly higher enhancement of the myocardium than PTA [2502 ± 437 vs 656 ± 178 Hounsfield units (HU); p < 0.0001], delineating myofiber architecture and orientation. There was no significant difference in vessel wall enhancement (Lugol's, 1036 ± 635 HU; PTA, 738 ± 124 HU; p = 0.29), but coronary arteries were more effectively segmented from the PTA-stained hearts, enabling segmented imaging of fifth- order coronary artery branches. The combination of whole mouse perfusion delivery and use of heavy metal-containing stains affords high-resolution imaging of the mouse heart and vasculature by micro-CT. The differential imaging patterns of Lugol's- and PTA-stained tissues reveals new opportunities for micro-analyses of cardiac and vascular tissues.
Assuntos
Imageamento Tridimensional/métodos , Iodo , Ácido Fosfotúngstico/química , Animais , Meios de Contraste/química , Vasos Coronários/diagnóstico por imagem , Coração/diagnóstico por imagem , Iodo/química , Camundongos , Tomografia Computadorizada por Raios XRESUMO
BACKGROUND: Cardiomyocyte-specific overexpression of human membrane-associated stem cell factor (hSCF) improves cardiac function post-myocardial infarction. However, whether hSCF overexpression protects the heart from ischemia and reperfusion (I/R) injury is unknown. We aimed to investigate the effects of cardiomyocyte-specific overexpression of hSCF on cardiac injury after acute myocardial I/R and related cellular and molecular signaling mechanisms. METHODS AND RESULTS: Wild-type (WT) and hSCF/tetracycline transactivator (tTA) transgenic mice (hSCF/tTA) were subjected to myocardial ischemia for 45 min followed by 3 h of reperfusion. Infarct size and myocardial apoptosis were decreased in hSCF/tTA compared to WT mice (P<0.05). Furthermore, these cardioprotective effects in the hSCF/tTA mice were abrogated by doxycycline, which turned off hSCF overexpression, and by a PI3 kinase inhibitor LY294002. Myocardial expression of insulin-like growth factor (IGF)-1 and hepatocyte growth factor (HGF), which are upstream activators of Akt signaling, was significantly increased in hSCF/tTA compared to WT mice after I/R (P<0.05), and was associated with higher number of c-kit(+) cardiac stem cells (CSCs) (P<0.05). Inhibition of c-kit signaling by ACK2 treatment abolished these protective effects in hSCF/tTA mice. CONCLUSIONS: Cardiomyocyte-specific overexpression of hSCF protects the heart from I/R injury. The cardioprotective effects of hSCF overexpression are mediated by increased c-kit(+) CSCs, enhanced growth factor expression and activation of Akt signaling pathway.
Assuntos
Regulação da Expressão Gênica , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/metabolismo , Fator de Células-Tronco/biossíntese , Fator de Células-Tronco/genética , Animais , Cardiotônicos/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Isquemia Miocárdica/genética , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/prevenção & controle , Traumatismo por Reperfusão Miocárdica/genética , Miócitos Cardíacos/patologiaRESUMO
Nitric oxide synthase-3 (NOS3) has recently been shown to promote endothelial-to-mesenchymal transition (EndMT) in the developing atrioventricular (AV) canal. The present study was aimed to investigate the role of NOS3 in embryonic development of AV valves. We hypothesized that NOS3 promotes embryonic development of AV valves via EndMT. To test this hypothesis, morphological and functional analysis of AV valves were performed in wild-type (WT) and NOS3(-/-) mice at postnatal day 0. Our data show that the overall size and length of mitral and tricuspid valves were decreased in NOS3(-/-) compared with WT mice. Echocardiographic assessment showed significant regurgitation of mitral and tricuspid valves during systole in NOS3(-/-) mice. These phenotypes were all rescued by cardiac specific NOS3 overexpression. To assess EndMT, immunostaining of Snail1 was performed in the embryonic heart. Both total mesenchymal and Snail1(+) cells in the AV cushion were decreased in NOS3(-/-) compared with WT mice at E10.5 and E12.5, which was completely restored by cardiac specific NOS3 overexpression. In cultured embryonic hearts, NOS3 promoted transforming growth factor (TGFß), bone morphogenetic protein (BMP2) and Snail1expression through cGMP. Furthermore, mesenchymal cell formation and migration from cultured AV cushion explants were decreased in the NOS3(-/-) compared with WT mice. We conclude that NOS3 promotes AV valve formation during embryonic heart development and deficiency in NOS3 results in AV valve insufficiency.
Assuntos
Desenvolvimento Embrionário/fisiologia , Valvas Cardíacas/embriologia , Óxido Nítrico Sintase Tipo III/metabolismo , Animais , Proteína Morfogenética Óssea 2/metabolismo , GMP Cíclico/metabolismo , Valvas Cardíacas/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos/embriologia , Camundongos Transgênicos/metabolismo , Fatores de Transcrição da Família Snail , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Conductance catheters (CC) represent an established method of determining cardiac function in mice; however, the potentially detrimental effects a catheter may have on the mouse heart have never been evaluated. The present study takes advantage of rapid three-dimensional (3D) microcomputed tomography (CT) to compare simultaneously acquired micro-CT and CC measurements of left ventricular (LV) volumes in healthy and infarcted mice and to determine changes in LV volume and function associated with CC insertion. LV volumes were measured in C57BL/6 mice (10 healthy, 10 infarcted, 2% isoflurane anesthesia) using a 1.4-Fr Millar CC. 3D micro-CT images of each mouse were acquired before CC insertion as well as during catheterization. Each CT scan produced high-resolution images throughout the entire cardiac cycle in <1 min, enabling accurate volume measurements as well as direct visualization of the CC within the LV. Bland-Altman analysis demonstrated that CC measurements underestimate volume compared with CT measurements in both healthy [bias of -18.4 and -28.9 µl for end-systolic (ESV) and end-diastolic volume (EDV), respectively] and infarcted mice (ESV = -51.6 µl and EDV = -71.7 µl); underestimation was attributed to the off-center placement of the catheter. Individual evaluation of each heart revealed LV dilation following CC insertion in 40% of mice in each group. No change in ejection fraction was observed, suggesting the enlargement was caused by volume overload associated with disruption of the papillary muscles or chords. The enlargement witnessed was not significant; however, the results suggest the potential for CC insertion to detrimentally affect mouse myocardium, necessitating further investigation.
Assuntos
Cateterismo Cardíaco/efeitos adversos , Cateterismo Cardíaco/métodos , Função Ventricular Esquerda/fisiologia , Microtomografia por Raio-X/métodos , Animais , Ventrículos do Coração/anatomia & histologia , Ventrículos do Coração/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/fisiopatologia , Volume Sistólico/fisiologiaRESUMO
Stanniocalcin-1 (STC-1) is made by kidney collecting duct cells for autocrine and paracrine targeting of nephron cell mitochondria. Here, the ligand stimulates respiratory uncoupling and calcium uniport activity. However, the underlying purpose of these actions and how the renal gene is regulated are poorly understood. In a previous study, we described the time-dependent, stimulatory effects of water deprivation on renal STC-1 mRNA levels in both rats and mice. In cortical kidney, STC-1 mRNA levels were increased 8-fold by 72h of water deprivation, whereas the gene response in outer and inner medulla was less pronounced (2-4 fold). Gene induction occurred equally in males and females and was accompanied by increased mitochondrial STC-1 protein levels. As water deprivation increases extracellular fluid (ECF) tonicity and at the same time reduces ECF volume, the present study examined the individual effects of hypertonicity and hypovolemia on renal gene activity in rats. Hypertonicity, whether induced by mannitol, glucose or NaCl, uniquely stimulated the cortical gene, to the extent that transcript levels were positively correlated with serum osmolality. This was in contrast to high dietary sodium, which had no bearing on cortical or medullary transcript levels. The situation was reversed in the case of hypovolemia. Inner medullary gene expression was uniquely induced by hypovolemia (low sodium diet or polyethylene glycol) such that transcript levels were positively correlated with hematocrit, while cortical gene activity was unaffected or reduced. Hence, the cortical and medullary genes proved to be differentially regulated by changing ECF tonicity and volume, respectively. The findings are therefore indicative of cortical and medullary STC-1 having separate roles in the renal control of ECF balance.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Glicoproteínas/genética , Soluções Hipertônicas/farmacologia , Hipovolemia/genética , Rim/efeitos dos fármacos , Rim/metabolismo , Animais , Glicemia/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Feminino , Glicoproteínas/metabolismo , Hematócrito , Hipovolemia/fisiopatologia , Rim/fisiopatologia , Córtex Renal/efeitos dos fármacos , Córtex Renal/metabolismo , Córtex Renal/fisiopatologia , Medula Renal/efeitos dos fármacos , Medula Renal/metabolismo , Medula Renal/fisiopatologia , Masculino , Camundongos , Concentração Osmolar , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Cloreto de Sódio/farmacologia , Fatores de TempoRESUMO
BACKGROUND: The common γ chain (γc) plays a critical role in regulating proliferation, differentiation, and apoptosis of peripheral T-cells. It was previously confirmed that blocking the γc signal can successfully induce transplant tolerance in a murine model. Here we investigated the potential mechanism. METHODS: Splenocytes from C57BL/6 mice were transfused into T-cell deficient Balb/c nude mice that were reconstituted with syngeneic wild-type T-cells labeled with 5-carboxyfluorescein diacetate succinimidyl ester (CFSE). After 24 hours, recipients received i.p. injection of mixture of anti-γc mAbs, or with isotype control IgG2a. The labeled T-cells were harvested from recipient spleens after 12 and 48 hours. T-cell proliferation and apoptosis were detected by flow cytometry. RESULTS: T-cell proliferation was markedly inhibited and apoptotic T cells could be detected at 12 hours after the mAbs injection. Proliferation was inhibited at 48 hours, but the proportion of apoptotic T-cells was not more than at 12 hours. In the control group, however, T-cells actively proliferated and no significant apoptosis was detected at either time point. CONCLUSIONS: The results suggested that blockade of γc signals can synergize with donor splenocyte transfusion and lead to inhibition of antigen-specific T-cell proliferation and induction of apoptotic T-cell death. This protocol may develop a novel approach to induce donor-specific tolerance.
Assuntos
Apoptose/efeitos dos fármacos , Tolerância Imunológica/efeitos dos fármacos , Subunidade gama Comum de Receptores de Interleucina/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Baço/citologia , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Animais , Anticorpos Monoclonais/farmacologia , Células Cultivadas , Citometria de Fluxo , Fluoresceínas , Subunidade gama Comum de Receptores de Interleucina/antagonistas & inibidores , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Transdução de Sinais/efeitos dos fármacos , SuccinimidasRESUMO
OBJECTIVE: The role of NOX2-containing NADPH oxidase in the development of diabetes is not fully understood. We hypothesized that NOX2 deficiency decreases reactive oxygen species (ROS) production and immune response and protects against streptozotocin (STZ)-induced ß-cell destruction and development of diabetes in mice. RESEARCH DESIGN AND METHODS: Five groups of mice--wild-type (WT), NOX2(-/-), WT treated with apocynin, and WT adoptively transferred with NOX2(-/-) or WT splenocytes--were treated with multiple-low-dose STZ. Blood glucose and insulin levels were monitored, and an intraperitoneal glucose tolerance test was performed. Isolated WT and NOX2(-/-) pancreatic islets were treated with cytokines for 48 h. RESULTS: Significantly lower blood glucose levels, higher insulin levels, and better glucose tolerance was observed in NOX2(-/-) mice and in WT mice adoptively transferred with NOX2(-/-) splenocytes compared with the respective control groups after STZ treatment. Compared with WT, ß-cell apoptosis, as determined by TUNEL staining, and insulitis were significantly decreased, whereas ß-cell mass was significantly increased in NOX2(-/-) mice. In response to cytokine stimulation, ROS production was significantly decreased, and insulin secretion was preserved in NOX2(-/-) compared with WT islets. Furthermore, proinflammatory cytokine release induced by concanavalin A was significantly decreased in NOX2(-/-) compared with WT splenocytes. CONCLUSIONS: NOX2 deficiency decreases ß-cell destruction and preserves islet function in STZ-induced diabetes by reducing ROS production, immune response, and ß-cell apoptosis.