RESUMEN
INTRODUCTION: Recent evidence suggests that transcriptional reprogramming is involved in the pathogenesis of cardiac remodeling (cardiomyocyte hypertrophy and fibrosis) and the development of heart failure. 5-Azacytidine (5aza), an inhibitor of DNA methylation approved for hematological malignancies, has previously demonstrated beneficial effects on cardiac remodeling in hypertension. The aim of our work was to investigate whether pressure overload is associated with alterations in DNA methylation and if intervention with low-dose 5aza can attenuate the associated pathological changes. METHODS AND RESULTS: C57Bl6/J mice underwent surgical constriction of the aortic arch for 8 weeks. Mice began treatment 4 weeks post-surgery with either vehicle or 5aza (5 mg/kg). Cardiac structure and function was examined in vivo using echocardiography followed by post mortem histological assessment of hypertrophy and fibrosis. Global DNA methylation was examined by immunostaining for 5-methylcytosine (5MeC) and assessment of DNA methyltransferase expression. The results highlighted that pressure overload-induced pathological cardiac remodeling is associated with increased DNA methylation (elevated cardiac 5MeC positivity and Dnmt1 expression). Administration of 5aza attenuated pathological remodeling and diastolic dysfunction. These beneficial changes were mirrored by a treatment-related reduction in global 5MeC levels and expression of Dnmt1 and Dnmt3B in the heart. CONCLUSION: DNA methylation plays an important role in the pathogenesis of pressure overload-induced cardiac remodeling. Therapeutic intervention with 5aza, at a dose 5 times lower than clinically given for oncology treatment, attenuated myocardial hypertrophy and fibrosis. Our work supports the rationale for its potential use in cardiac pathologies associated with aberrant cardiac wound healing.
Asunto(s)
Azacitidina/farmacología , Cardiomegalia/prevención & control , Cardiomegalia/fisiopatología , Metilación de ADN/efectos de los fármacos , Animales , Azacitidina/uso terapéutico , Reposicionamiento de Medicamentos , Electrocardiografía , Neoplasias Hematológicas/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: The development of heart failure is associated with changes in the size, shape, and structure of the heart that has a negative impact on cardiac function. These pathological changes involve excessive extracellular matrix deposition within the myocardial interstitium and myocyte hypertrophy. Alterations in fibroblast phenotype and myocyte activity are associated with reprogramming of gene transcriptional profiles that likely requires epigenetic alterations in chromatin structure. The aim of our work was to investigate the potential of a currently licensed anticancer epigenetic modifier as a treatment option for cardiac diseases associated with hypertension-induced cardiac hypertrophy and fibrosis. METHODS AND RESULTS: The effects of DNA methylation inhibition with 5-azacytidine (5-aza) were examined in a human primary fibroblast cell line and in a spontaneously hypertensive rat (SHR) model. The results from this work allude to novel in vivo antifibrotic and antihypertrophic actions of 5-aza. Administration of the DNA methylation inhibitor significantly improved several echocardiographic parameters associated with hypertrophy and diastolic dysfunction. Myocardial collagen levels and myocyte size were reduced in 5-aza-treated SHRs. These findings are supported by beneficial in vitro effects in cardiac fibroblasts. Collagen I, collagen III, and α-smooth muscle actin were reduced in a human ventricular cardiac fibroblast cell line treated with 5-aza. CONCLUSION: These findings suggest a role for epigenetic modifications in contributing to the profibrotic and hypertrophic changes evident during disease progression. Therapeutic intervention with 5-aza demonstrated favorable effects highlighting the potential use of this epigenetic modifier as a treatment option for cardiac pathologies associated with hypertrophy and fibrosis.
Asunto(s)
Azacitidina/farmacología , Cardiomegalia/prevención & control , Metilación de ADN/efectos de los fármacos , Metilasas de Modificación del ADN/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Epigénesis Genética/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Hipertensión/tratamiento farmacológico , Miocitos Cardíacos/efectos de los fármacos , Actinas/metabolismo , Animales , Cardiomegalia/enzimología , Cardiomegalia/genética , Cardiomegalia/patología , Cardiomegalia/fisiopatología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Células Cultivadas , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Metilasas de Modificación del ADN/metabolismo , Modelos Animales de Enfermedad , Fibroblastos/enzimología , Fibroblastos/patología , Fibrosis , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Hipertensión/enzimología , Hipertensión/genética , Hipertensión/patología , Hipertensión/fisiopatología , Masculino , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/patología , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Factor de Crecimiento Transformador beta1/farmacología , Remodelación Ventricular/efectos de los fármacosRESUMEN
The potential for serum amyloid P-component (SAP) to prevent cardiac remodeling and identify worsening diastolic dysfunction (DD) was investigated. The anti-fibrotic potential of SAP was tested in an animal model of hypertensive heart disease (spontaneously hypertensive rats treated with SAP [SHR - SAP] × 12 weeks). Biomarker analysis included a prospective study of 60 patients with asymptomatic progressive DD. Compared with vehicle-treated Wistar-Kyoto rats (WKY-V), the vehicle-treated SHRs (SHR-V) exhibited significant increases in left ventricular mass, perivascular collagen, cardiomyocyte size, and macrophage infiltration. SAP administration was associated with significantly lower left ventricular mass (p < 0.01), perivascular collagen (p < 0.01), and cardiomyocyte size (p < 0.01). Macrophage infiltration was significantly attenuated in the SHR-SAP group. Biomarker analysis showed significant decreases in SAP concentration over time in patients with progressive DD (p < 0.05). Our results indicate that SAP prevents cardiac remodeling by inhibiting recruitment of pro-fibrotic macrophages and that depleted SAP levels identify patients with advancing DD suggesting a role for SAP therapy.
Asunto(s)
Hipertensión/tratamiento farmacológico , Hipertrofia Ventricular Izquierda/tratamiento farmacológico , Componente Amiloide P Sérico/administración & dosificación , Remodelación Ventricular/efectos de los fármacos , Animales , Biopsia con Aguja , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Hipertensión/complicaciones , Hipertensión/fisiopatología , Hipertrofia Ventricular Izquierda/etiología , Hipertrofia Ventricular Izquierda/patología , Inmunohistoquímica , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Distribución Aleatoria , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Valores de ReferenciaRESUMEN
Fibrosis is a progressive and potentially fatal process that can occur in numerous organ systems. Characterised by the excessive deposition of extracellular matrix proteins such as collagens and fibronectin, fibrosis affects normal tissue architecture and impedes organ function. Although a considerable amount of research has focused on the mechanisms underlying disease pathogenesis, current therapeutic options do not directly target the pro-fibrotic process. As a result, there is a clear unmet clinical need to develop new agents. Novel findings implicate a role for epigenetic modifications contributing to the progression of fibrosis by alteration of gene expression profiles. This review will focus on DNA methylation; its association with fibroblast differentiation and activation and the consequent buildup of fibrotic scar tissue. The potential use of therapies that modulate this epigenetic pathway for the treatment of fibrosis in several organ systems is also discussed.
RESUMEN
Ischemia caused by coronary artery disease and myocardial infarction leads to aberrant ventricular remodeling and cardiac fibrosis. This occurs partly through accumulation of gene expression changes in resident fibroblasts, resulting in an overactive fibrotic phenotype. Long-term adaptation to a hypoxic insult is likely to require significant modification of chromatin structure in order to maintain the fibrotic phenotype. Epigenetic changes may play an important role in modulating hypoxia-induced fibrosis within the heart. Therefore, the aim of the study was to investigate the potential pro-fibrotic impact of hypoxia on cardiac fibroblasts and determine whether alterations in DNA methylation could play a role in this process. This study found that within human cardiac tissue, the degree of hypoxia was associated with increased expression of collagen 1 and alpha-smooth muscle actin (ASMA). In addition, human cardiac fibroblast cells exposed to prolonged 1% hypoxia resulted in a pro-fibrotic state. These hypoxia-induced pro-fibrotic changes were associated with global DNA hypermethylation and increased expression of the DNA methyltransferase (DNMT) enzymes DNMT1 and DNMT3B. Expression of these methylating enzymes was shown to be regulated by hypoxia-inducible factor (HIF)-1α. Using siRNA to block DNMT3B expression significantly reduced collagen 1 and ASMA expression. In addition, application of the DNMT inhibitor 5-aza-2'-deoxycytidine suppressed the pro-fibrotic effects of TGFß. Epigenetic modifications and changes in the epigenetic machinery identified in cardiac fibroblasts during prolonged hypoxia may contribute to the pro-fibrotic nature of the ischemic milieu. Targeting up-regulated expression of DNMTs in ischemic heart disease may prove to be a valuable therapeutic approach.
Asunto(s)
Metilación de ADN , Epigenómica , Fibrosis/etiología , Corazón/fisiopatología , Hipoxia/complicaciones , Miofibroblastos/patología , Anciano , Western Blotting , Células Cultivadas , Colágeno/genética , Colágeno/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Femenino , Fibrosis/metabolismo , Fibrosis/patología , Citometría de Flujo , Humanos , Hipoxia/fisiopatología , Técnicas para Inmunoenzimas , Masculino , Miofibroblastos/metabolismo , Fenotipo , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , ADN Metiltransferasa 3BRESUMEN
Chronic hypoxia causes pulmonary hypertension associated with structural alterations in pulmonary vessels and sustained vasoconstriction. The transcriptional mechanisms responsible for these distinctive changes are unclear. We have previously reported that CREB1 is activated in the lung in response to alveolar hypoxia but not in other organs. To directly investigate the role of α and Δ isoforms of CREB1 in the regulation of pulmonary vascular resistance we examined the responses of mice in which these isoforms of CREB1 had been inactivated by gene mutation, leaving only the ß isoform intact (CREB(αΔ) mice). Here we report that expression of CREB regulated genes was altered in the lungs of CREB(αΔ) mice. CREB(αΔ) mice had greater pulmonary vascular resistance than wild types, both basally in normoxia and following exposure to hypoxic conditions for three weeks. There was no difference in rho kinase mediated vasoconstriction between CREB(αΔ) and wild type mice. Stereological analysis of pulmonary vascular structure showed characteristic wall thickening and lumen reduction in hypoxic wild-type mice, with similar changes observed in CREB(αΔ). CREB(αΔ) mice had larger lungs with reduced epithelial surface density suggesting increased pulmonary compliance. These findings show that α and Δ isoforms of CREB1 regulate homeostatic gene expression in the lung and that normal activity of these isoforms is essential to maintain low pulmonary vascular resistance in both normoxic and hypoxic conditions and to maintain the normal alveolar structure. Interventions that enhance the actions of α and Δ isoforms of CREB1 warrant further investigation in hypoxic lung diseases.
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Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Isoformas de Proteínas/metabolismo , Resistencia Vascular/fisiología , Animales , Western Blotting , Peso Corporal/fisiología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Femenino , Hematócrito , Humanos , Hipertensión Pulmonar/metabolismo , Masculino , Ratones , Isoformas de Proteínas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Resistencia Vascular/genéticaRESUMEN
BACKGROUND: Pulmonary fibrosis is a debilitating and lethal disease with no effective treatment options. Understanding the pathological processes at play will direct the application of novel therapeutic avenues. Hypoxia has been implicated in the pathogenesis of pulmonary fibrosis yet the precise mechanism by which it contributes to disease progression remains to be fully elucidated. It has been shown that chronic hypoxia can alter DNA methylation patterns in tumour-derived cell lines. This epigenetic alteration can induce changes in cellular phenotype with promoter methylation being associated with gene silencing. Of particular relevance to idiopathic pulmonary fibrosis (IPF) is the observation that Thy-1 promoter methylation is associated with a myofibroblast phenotype where loss of Thy-1 occurs alongside increased alpha smooth muscle actin (α-SMA) expression. The initial aim of this study was to determine whether hypoxia regulates DNA methylation in normal human lung fibroblasts (CCD19Lu). As it has been reported that hypoxia suppresses Thy-1 expression during lung development we also studied the effect of hypoxia on Thy-1 promoter methylation and gene expression. METHODS: CCD19Lu were grown for up to 8 days in hypoxia and assessed for global changes in DNA methylation using flow cytometry. Real-time PCR was used to quantify expression of Thy-1, α-SMA, collagen I and III. Genomic DNA was bisulphite treated and methylation specific PCR (MSPCR) was used to examine the methylation status of the Thy-1 promoter. RESULTS: Significant global hypermethylation was detected in hypoxic fibroblasts relative to normoxic controls and was accompanied by increased expression of myofibroblast markers. Thy-1 mRNA expression was suppressed in hypoxic cells, which was restored with the demethylating agent 5-aza-2'-deoxycytidine. MSPCR revealed that Thy-1 became methylated following fibroblast exposure to 1% O2. CONCLUSION: These data suggest that global and gene-specific changes in DNA methylation may play an important role in fibroblast function in hypoxia.
Asunto(s)
Metilación de ADN , Fibroblastos/metabolismo , Pulmón/metabolismo , Regiones Promotoras Genéticas , Fibrosis Pulmonar/genética , Antígenos Thy-1/genética , Actinas/metabolismo , Azacitidina/análogos & derivados , Azacitidina/farmacología , Hipoxia de la Célula , Línea Celular , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Metilación de ADN/efectos de los fármacos , Metilasas de Modificación del ADN/antagonistas & inhibidores , Metilasas de Modificación del ADN/metabolismo , Decitabina , Inhibidores Enzimáticos/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Citometría de Flujo , Regulación de la Expresión Génica , Humanos , Pulmón/efectos de los fármacos , Pulmón/patología , Miofibroblastos/metabolismo , Miofibroblastos/patología , Fenotipo , Regiones Promotoras Genéticas/efectos de los fármacos , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Antígenos Thy-1/metabolismo , Factores de TiempoRESUMEN
BACKGROUND: Mechanical overload of the heart is associated with excessive deposition of extracellular matrix proteins and the development of cardiac fibrosis. This can result in reduced ventricular compliance, diastolic dysfunction, and heart failure. Extracellular matrix synthesis is regulated primarily by cardiac fibroblasts, more specifically, the active myofibroblast. The influence of mechanical stretch on human cardiac fibroblasts' response to pro-fibrotic stimuli, such as transforming growth factor beta (TGFß), is unknown as is the impact of stretch on B-type natriuretic peptide (BNP) and natriuretic peptide receptor A (NPRA) expression. BNP, acting via NPRA, has been shown to play a role in modulation of cardiac fibrosis. METHODS AND RESULTS: The effect of cyclical mechanical stretch on TGFß induction of myofibroblast differentiation in primary human cardiac fibroblasts and whether differences in response to stretch were associated with changes in the natriuretic peptide system were investigated. Cyclical mechanical stretch attenuated the effectiveness of TGFß in inducing myofibroblast differentiation. This finding was associated with a novel observation that mechanical stretch can increase BNP and NPRA expression in human cardiac fibroblasts, which could have important implications in modulating myocardial fibrosis. Exogenous BNP treatment further reduced the potency of TGFß on mechanically stretched fibroblasts. CONCLUSION: We postulate that stretch induced up-regulation of the natriuretic peptide system may contribute to the observed reduction in myofibroblast differentiation.