RESUMEN
UNLABELLED: Cardiac c-Kit+ cells have a modest cardiogenic potential that could limit their efficacy in heart disease treatment. The present study was designed to augment the cardiogenic potential of cardiac c-Kit+ cells through class I histone deacetylase (HDAC) inhibition and evaluate their therapeutic potency in the chronic heart failure (CHF) animal model. Myocardial infarction (MI) was created by coronary artery occlusion in rats. c-Kit+ cells were treated with mocetinostat (MOCE), a specific class I HDAC inhibitor. At 3 weeks after MI, CHF animals were retrogradely infused with untreated (control) or MOCE-treated c-Kit+ cells (MOCE/c-Kit+ cells) and evaluated at 3 weeks after cell infusion. We found that class I HDAC inhibition in c-Kit+ cells elevated the level of acetylated histone H3 (AcH3) and increased AcH3 levels in the promoter regions of pluripotent and cardiac-specific genes. Epigenetic changes were accompanied by increased expression of cardiac-specific markers. Transplantation of CHF rats with either control or MOCE/c-Kit+ cells resulted in an improvement in cardiac function, retardation of CHF remodeling made evident by increased vascularization and scar size, and cardiomyocyte hypertrophy reduction. Compared with CHF infused with control cells, infusion of MOCE/c-Kit+ cells resulted in a further reduction in left ventricle end-diastolic pressure and total collagen and an increase in interleukin-6 expression. The low engraftment of infused cells suggests that paracrine effects might account for the beneficial effects of c-Kit+ cells in CHF. In conclusion, selective inhibition of class I HDACs induced expression of cardiac markers in c-Kit+ cells and partially augmented the efficacy of these cells for CHF repair. SIGNIFICANCE: The study has shown that selective class 1 histone deacetylase inhibition is sufficient to redirect c-Kit+ cells toward a cardiac fate. Epigenetically modified c-Kit+ cells improved contractile function and retarded remodeling of the congestive heart failure heart. This study provides new insights into the efficacy of cardiac c-Kit+ cells in the ischemic heart failure model.
Asunto(s)
Epigénesis Genética , Insuficiencia Cardíaca/terapia , Infarto del Miocardio/terapia , Miocitos Cardíacos/trasplante , Proteínas Proto-Oncogénicas c-kit/genética , Acetilación , Animales , Benzamidas/farmacología , Biomarcadores/metabolismo , Diferenciación Celular , Células Cultivadas , Colágeno/genética , Colágeno/metabolismo , Modelos Animales de Enfermedad , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Inhibidores de Histona Desacetilasas/farmacología , Histonas/genética , Histonas/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , Infarto del Miocardio/genética , Infarto del Miocardio/metabolismo , Infarto del Miocardio/fisiopatología , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Pirimidinas/farmacología , Ratas , Ratas Sprague-Dawley , Recuperación de la Función , Técnicas de Cultivo de Tejidos , Función Ventricular Izquierda , Remodelación VentricularRESUMEN
BACKGROUND: Recent studies have linked histone deacetylases (HDAC) to remodeling of the heart and cardiac fibrosis in heart failure. However, the molecular mechanisms linking chromatin remodeling events with observed anti-fibrotic effects are unknown. Here, we investigated the molecular players involved in anti-fibrotic effects of HDAC inhibition in congestive heart failure (CHF) myocardium and cardiac fibroblasts in vivo. METHODS AND RESULTS: MI was created by coronary artery occlusion. Class I HDACs were inhibited in three-week post MI rats by intraperitoneal injection of Mocetinostat (20 mg/kg/day) for duration of three weeks. Cardiac function and heart tissue were analyzed at six week post-MI. CD90+ cardiac fibroblasts were isolated from ventricles through enzymatic digestion of heart. In vivo treatment of CHF animals with Mocetinostat reduced CHF-dependent up-regulation of HDAC1 and HDAC2 in CHF myocardium, improved cardiac function and decreased scar size and total collagen amount. Moreover, expression of pro-fibrotic markers, collagen-1, fibronectin and Connective Tissue Growth Factor (CTGF) were reduced in the left ventricle (LV) of Mocetinostat-treated CHF hearts. Cardiac fibroblasts isolated from Mocetinostat-treated CHF ventricles showed a decrease in expression of collagen I and III, fibronectin and Timp1. In addition, Mocetinostat attenuated CHF-induced elevation of IL-6 levels in CHF myocardium and cardiac fibroblasts. In parallel, levels of pSTAT3 were reduced via Mocetinostat in CHF myocardium. CONCLUSIONS: Anti-fibrotic effects of Mocetinostat in CHF are associated with the IL-6/STAT3 signaling pathway. In addition, our study demonstrates in vivo regulation of cardiac fibroblasts via HDAC inhibition.
Asunto(s)
Benzamidas/farmacología , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Interleucina-6/metabolismo , Isquemia Miocárdica/complicaciones , Pirimidinas/farmacología , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Cicatriz , Colágeno/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/genética , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Modelos Animales de Enfermedad , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Fibrosis , Expresión Génica , Insuficiencia Cardíaca/patología , Histona Desacetilasas/metabolismo , Ratas , Función Ventricular/efectos de los fármacosRESUMEN
BACKGROUND: Interstitial fibrosis and fibrotic scar formation contribute to cardiac remodeling and loss of cardiac function in myocardial infarction (MI) and heart failure. Recent studies showed that histone deacetylase (HDAC) inhibitors retard fibrosis formation in acute MI settings. However, it is unknown whether HDAC inhibition can reverse cardiac fibrosis in ischemic heart failure. In addition, specific HDAC isoforms involved in cardiac fibrosis and myofibroblast activation are not well defined. Thus, the purpose of this study is to determine the effects of selective class I HDAC inhibition on cardiac fibroblasts activation and cardiac fibrosis in a congestive heart failure (CHF) model secondary to MI. METHODS: MI was created by left anterior descending (LAD) coronary artery occlusion. Class I HDACs were selectively inhibited via Mocetinostat in CD90+ fibroblasts isolated from atrial and ventricular heart tissue in vitro. In vivo, Class I HDACs were inhibited in 3 weeks post MI rats by injecting Mocetinostat for the duration of 3 weeks. Cardiac function and heart tissue were analyzed at 6 weeks post MI. RESULTS: In sham hearts, HDAC1 and HDAC2 displayed differential expression patterns where HDAC1 mainly expressed in cardiac fibroblast and HDAC2 in cardiomyocytes. On the other hand, we showed that HDAC1 and 2 were upregulated in CHF hearts, and were found to co-localize with CD90+ cardiac fibroblasts. In vivo treatment of CHF animals with Mocetinostat improved left ventricle end diastolic pressure and dp/dt max and decreased the total collagen amount. In vitro treatment of CD90+ cells with Mocetinostat reversed myofibroblast phenotype as indicated by a decrease in α-Smooth muscle actin (α-SMA), Collagen III, and Matrix metalloproteinase-2 (MMP2). Furthermore, Mocetinostat increased E-cadherin, induced ß-catenin localization to the membrane, and reduced Akt/GSK3ß signaling in atrial cardiac fibroblasts. In addition, Mocetinostat treatment of atrial CD90+ cells upregulated cleaved-Caspase3 and activated the p53/p21 axis. CONCLUSIONS: Taken together, our results demonstrate upregulation of HDAC1 and 2 in CHF. In addition, HDAC inhibition reverses interstitial fibrosis in CHF. Possible anti-fibrotic actions of HDAC inhibition include reversal of myofibroblast activation and induction of cell cycle arrest/apoptosis.
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BACKGROUND: Progenitor cells isolated from cardiac explant-derived cells improve cardiac function after myocardial infarction (MI). To fully realize the therapeutic potential of these cells, it is essential to develop a safe and efficient delivery method. Therefore, the objective of this study was to determine the efficacy of our newly developed approach to retrograde coronary vein (RCV) infusion of cardiac c-Kit(+) cells in a small-animal model of congestive heart failure (CHF). METHODS: Sprague-Dawley rats underwent experimental MI. After 21 days, cardiac explant-derived c-Kit(+) cells were delivered to both sham and CHF animals using RCV delivery. Vehicle-treated (serum-free medium) sham and CHF animals were used as controls. Cardiac function and heart tissues were evaluated 21 days post-transplantation. RESULTS: RCV-delivered cells were retained in infarcted hearts for at least 21 days after transplantation. At 21 days post-RCV infusion, the majority of transplanted c-Kit(+)/GFP(+) cells were localized in the left ventricle. Compared with vehicle-treated CHF animals, RCV-treated rats showed a significant improvement in cardiac function. Furthermore, RCV-treated rats exhibited an increase in capillary density, a decrease in total heart collagen, and a reduction in both infarct size and cardiomyocyte hypertrophy when compared with vehicle-treated CHF rats. CONCLUSIONS: Our study showed that the RCV infusion approach is an efficient technique for targeted cell delivery to the infarcted myocardium. Cardiac c-Kit(+) cells, delivered using RCV infusion ameliorated progression of heart failure, improved cardiac function and retarded myocardial remodeling in heart failure rats.
Asunto(s)
Insuficiencia Cardíaca/terapia , Infarto del Miocardio/complicaciones , Miocitos Cardíacos/trasplante , Proteínas Proto-Oncogénicas c-kit , Trasplante de Células Madre/métodos , Animales , Vasos Coronarios , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/patología , Infusiones Intravenosas , Masculino , Infarto del Miocardio/patología , Infarto del Miocardio/terapia , Ratas Sprague-Dawley , Remodelación VentricularRESUMEN
BACKGROUND: Cardiac c-Kit+ cells isolated from cardiac explant-derived cells modestly improve cardiac functions after myocardial infarction; however, their full potential has not yet been realized. For instance, the majority of potential candidates for cell therapy suffer from chronic heart failure (CHF), and it is unclear how this disease affects the explant-derived progenitor cells. Therefore, the objective of this study was to determine the effect of CHF on the number and phenotype of cardiac explant c-Kit+ progenitors and elucidate mechanisms of their regulation. METHODS AND RESULTS: Myocardial infarction was created by left anterior descending coronary artery occlusion. Sham-operated animals were used as a control group. CHF-developed infarcted animals were selected on the basis of left ventricle end-diastolic pressure ≥ 20 mm Hg and scar size ≥ 30%. Here, we found that CHF atrial explants produced less c-Kit+ cells than sham explants. CHF-derived c-Kit+ cells exhibited upregulated transforming growth factor-ß (TGF-ß) signaling, increased level of epithelial to mesenchymal transition markers, and diminished expression of pluripotency markers compared with shams. We show that intervention with TGF-ß signaling by inhibiting TGF-ß receptor type I or Smad 2/3 using small-molecule inhibitors improved c-Kit+ cell yield, attenuated epithelial to mesenchymal transition markers, stimulated the pluripotency marker Nanog, and improved efficiency of c-Kit+ cell differentiation toward cardiomyocyte-like cells in vitro. CONCLUSIONS: Taken together, our findings suggest that TGF-ß inhibition positively modulates c-Kit+ cell phenotype and function in vitro, and this strategy may be considered in optimizing cardiac progenitor function and cell expansion protocols for clinical application.
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Atrios Cardíacos/citología , Insuficiencia Cardíaca/patología , Factor de Crecimiento Transformador beta/fisiología , Animales , Células Cultivadas , Enfermedad Crónica , Ratas , Ratas Sprague-DawleyRESUMEN
Cardiovascular (CV) diseases are known to have a negative impact on the brain and neurocognition, and contribute to the development of vascular dementia and neurodegenerative diseases such as Alzheimer's disease (AD). Among CV diseases, congestive heart failure (CHF) after myocardial infarction (MI) is a condition where the ability of the left ventricle to eject blood to the circulation is impaired. As a consequence, CHF triggers inflammation and results in reduced cerebral blood flow which are considered among the risk factors for development of AD. However, biochemical alterations in the brain following MI and CHF remain unknown. To address this issue, we investigated microglia activation; levels of BACE1, the key rate-limiting enzyme involved in the pathogenesis of AD; and VEGF levels in the hippocampus and cortex following MI. We created MI by the ligation of the left anterior descending coronary artery in Sprague-Dawley male rats and collected brains either 3 days after MI (AMI) or 21 days after MI (CHF). We investigated microglia activation in AMI and CHF brains by immunohistochemistry and immunoblotting using macrophage/microglia marker Ionized calcium binding adaptor molecule 1 (Iba-1), and observed activated morphology of microglia in the cortex of rats in both AMI and CHF. We also showed the levels of BACE1 were increased in the cortex and hippocampus of CHF rats. To determine whether hypoxia occurs in the CHF brain, we assessed levels of VEGF in the hippocampus and cortex. Western blotting analysis showed up-regulation of VEGF in the hippocampus of CHF brains. These results suggest that neuroinflammation takes place secondary to myocardial infarction. In addition, CHF-induced hypoxia might play a role in the elevation of BACE1 and VEGF levels.
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Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Insuficiencia Cardíaca/metabolismo , Animales , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/fisiopatología , Hipocampo/metabolismo , Hipocampo/patología , Masculino , Microglía/metabolismo , Infarto del Miocardio/metabolismo , Infarto del Miocardio/fisiopatología , Ratas , Ratas Sprague-Dawley , Factor A de Crecimiento Endotelial Vascular/metabolismo , Función Ventricular IzquierdaRESUMEN
BACKGROUND: Progenitor cell therapy is emerging as a novel treatment for heart failure. However the molecular mechanisms regulating the generation of cardiac progenitor cells is not fully understood. We hypothesized that cardiac progenitor cells are generated from cardiac explant via a process similar to epithelial to mesenchymal transition (EMT). METHODS/FINDINGS: Explant-derived cells were generated from partially digested atrial tissue. After 21 days in culture, c-Kit+ cells were isolated from cell outgrowth. The majority of explant-originated c-Kit+ cells expressed the epicardial marker Wt1. Cardiac cell outgrowth exhibits a temporal up-regulation of EMT-markers. Notch stimulation augmented, while Notch inhibition suppressed, mesenchymal transition in both c-Kit+ and c-Kit- cells. In c-Kit+ cells, Notch stimulation reduced, while Notch inhibition up-regulated pluripotency marker expressions such as Nanog and Sox2. Notch induction was associated with degradation of ß-catenin in c-Kit- cells. In contrast, Notch inhibition resulted in ß-catenin accumulation, acquisition of epitheloid morphology, and up-regulation of Wnt target genes in c-Kit- cells. CONCLUSION: Our study suggests that Notch-mediated reversible EMT process is a mechanism that regulates explant-derived c-Kit+ and c-Kit- cells.