RESUMEN
AIMS: The myocardium accumulates intracellular lipids under ischaemic conditions, and myocardial fat deposition is closely associated with cardiac dysfunction. Our aims were to analyse the effect of hypoxia on low-density lipoprotein receptor-related protein 1 (LRP1) expression in neonatal rat ventricular myocytes (NRVM) and cardiac-derived HL-1 cells and the molecular mechanisms involved in this effect, to determine the role of LRP1 in the very low density lipoprotein (VLDL) uptake by hypoxic cardiomyocytes, and to study the effect of hypoxia on lipoprotein receptor expression and myocardial lipid profile in an in vivo porcine experimental model of acute myocardial infarction. METHODS AND RESULTS: Thin-layer chromatography after lipid extraction showed that VLDL exposure leads to cholesteryl ester (CE) and triglyceride (TG) accumulation in a dose-dependent manner and that hypoxic conditions further increased VLDL-derived intracellular lipid accumulation in HL-1 cells. Knockdown of LRP1 through lentiviral-mediated interfering RNA specifically prevented hypoxia-induced VLDL-CE internalization in HL-1 cells and NRVM. Lipopolysaccharide (LPS)-induced LRP1 overexpression specifically increased VLDL-CE accumulation in NRVM. In addition, using double-radiolabelled [(3)H]CE-[(14)C]TG-VLDL, we found that LRP1 deficiency specifically prevented hypoxia-induced VLDL-[(3)H]CE uptake. Finally, in an in vivo porcine model of infarcted myocardium, ischaemic areas exhibited LRP1 protein up-regulation and intramyocardial CE overaccumulation. CONCLUSION: Our results demonstrate that hypoxia increases LRP1 expression through HIF-1α and that LRP1 overexpression mediates hypoxia-induced VLDL-CE uptake and accumulation in cardiomyocytes.
Asunto(s)
Ésteres del Colesterol/metabolismo , VLDL-Colesterol/metabolismo , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Isquemia Miocárdica/metabolismo , Miocitos Cardíacos/metabolismo , Receptores de LDL/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Animales Recién Nacidos/metabolismo , Hipoxia de la Célula/fisiología , Línea Celular , Ésteres del Colesterol/genética , Técnicas de Silenciamiento del Gen , Metabolismo de los Lípidos/fisiología , Lipoproteínas VLDL/genética , Lipoproteínas VLDL/metabolismo , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Ratones , Isquemia Miocárdica/prevención & control , Ratas , Ratas Wistar , Receptores de LDL/genética , Porcinos/metabolismo , Proteínas Supresoras de Tumor/genéticaRESUMEN
AIMS: Coronary artery occlusion is associated with the risk of ventricular remodelling, heart failure, and cardiogenic shock. Novel strategies are sought to treat these ominous complications. We examined the effect of a pericardial-derived fat flap secured over an acute infarct caused by coronary occlusion. METHODS AND RESULTS: A novel intervention consisting of the pericardial isolation of a vascularized adipose flap and its transposition fully covering acute infarcted myocardium was developed in the swine model of coronary artery ligation (n= 52). Left ventricular (LV) ejection fraction and LV end-diastolic and end-systolic volumes were assessed using magnetic resonance imaging (MRI). Infarct size and gene expression analysis were performed on Day 6 and 1 month. Histological changes, collagen volume fraction (CVF), and vascular density were also evaluated on postmortem sections. One month after the intervention, a 18.8% increase in LV ejection fraction (P= 0.007), and significant reductions in LV end-systolic (P= 0.009) and LV end-diastolic volumes (P= 0.03) were found in treated animals compared with the control-MI group. At Day 6, histopathology confirmed a significant infarct size reduction (P= 0.018), the presence of vascular connections at the flap-myocardium interface, and less apoptosis in the infarct border zone compared with control animals (P< 0.001). Up-regulation of genes involved in cell cycle progression, cellular growth and proliferation, and angiogenesis were identified within the flap. CONCLUSIONS: Our results indicate that a vascular fat flap exerts beneficial effects on LV function and limits myocardial remodelling. Future studies must confirm whether these findings provide an alternative therapeutic approach for myocardial salvage after infarction.
Asunto(s)
Infarto del Miocardio/cirugía , Colgajos Quirúrgicos , Animales , Colágeno/análisis , Oclusión Coronaria/complicaciones , Femenino , Regulación de la Expresión Génica , Infarto del Miocardio/fisiopatología , Pericardio , Volumen Sistólico , Porcinos , Función Ventricular IzquierdaRESUMEN
It is known that myocardium suffers serious alterations under ischemic conditions such as lipid overloading and electrophysiological alterations. However, it is unknown whether intracellular lipid accumulation and calcium dysfunction share common pathophysiological mechanisms under ischemia. The aims of this study were 1) to analyze the effect of normal and high doses of very low density lipoproteins (VLDL) on lipid content and calcium handling; 2) to investigate whether hypoxia modulates the effect of high VLDL doses; and 3) to identify potentially underlying mechanisms in cardiomyocytes. For this purpose, neonatal rat ventricular myocytes cultures were prepared from hearts of 3-4-day-old rats. High doses of VLDL that induced cholesteryl ester (CE) and triglyceride (TG) accumulation strongly reduced sarco(endo)plasmic reticulum Ca ATPase-2 (SERCA-2) expression, calcium transient amplitude and sarcoplasmic reticulum (SR) calcium loading. Interestingly, hypoxia, by upregulating VLDL-receptor expression (4.5-fold at 16h) increased CE (1.5-fold) and TG (3-fold) cardiomyocyte content and exacerbated the negative effect of VLDL on SERCA-2 expression. Functionally, the hypoxic exacerbation of VLDL-mediated SERCA-2 downregulation was translated into a stronger decrease in calcium transient amplitude and SR calcium loading in myocytes exposed simultaneously to hypoxia and high VLDL. In conclusion, high VLDL doses alter calcium handling in cardiomyocytes and SERCA-2 play a pivotal role in the hypoxic exacerbation of VLDL-mediated effects on cardiac calcium handling. Potentiation of VLDL's effects under hypoxia is explained, at least in part, by hypoxic upregulation of the expression of VLDL-receptor.
Asunto(s)
Calcio/metabolismo , Lipoproteínas VLDL/metabolismo , Miocitos Cardíacos/metabolismo , Animales , Animales Recién Nacidos , Western Blotting , Hipoxia de la Célula/fisiología , Células Cultivadas , Humanos , Reacción en Cadena de la Polimerasa , Ratas , Ratas WistarRESUMEN
Myocardial infarction caused by vascular occlusion results in the formation of nonfunctional fibrous tissue. Cumulative evidence indicates that cell therapy modestly improves cardiac function; thus, novel cell sources with the potential to repair injured tissue are actively sought. Here, we identify and characterize a cell population of cardiac adipose tissue-derived progenitor cells (ATDPCs) from biopsies of human adult cardiac adipose tissue. Cardiac ATDPCs express a mesenchymal stem cell-like marker profile (strongly positive for CD105, CD44, CD166, CD29 and CD90) and have immunosuppressive capacity. Moreover, cardiac ATDPCs have an inherent cardiac-like phenotype and were able to express de novo myocardial and endothelial markers in vitro but not to differentiate into adipocytes. In addition, when cardiac ATDPCs were transplanted into injured myocardium in mouse and rat models of myocardial infarction, the engrafted cells expressed cardiac (troponin I, sarcomeric α-actinin) and endothelial (CD31) markers, vascularization increased, and infarct size was reduced in mice and rats. Moreover, significant differences between control and cell-treated groups were found in fractional shortening and ejection fraction, and the anterior wall remained significantly thicker 30days after cardiac delivery of ATDPCs. Finally, cardiac ATDPCs secreted proangiogenic factors under in vitro hypoxic conditions, suggesting a paracrine effect to promote local vascularization. Our results indicate that the population of progenitor cells isolated from human cardiac adipose tissue (cardiac ATDPCs) may be valid candidates for future use in cell therapy to regenerate injured myocardium.
Asunto(s)
Tejido Adiposo/citología , Infarto del Miocardio/terapia , Miocardio/citología , Trasplante de Células Madre , Células Madre/citología , Anciano , Inductores de la Angiogénesis/metabolismo , Animales , Capilares/patología , Diferenciación Celular , Linaje de la Célula , Separación Celular , Células Cultivadas , Técnicas de Cocultivo , Células Endoteliales/citología , Células Endoteliales/metabolismo , Pruebas de Función Cardíaca , Humanos , Ratones , Infarto del Miocardio/diagnóstico por imagen , Infarto del Miocardio/patología , Neovascularización Fisiológica , Ratas , UltrasonografíaRESUMEN
BACKGROUND: Cell-based therapies offer a promising approach to reducing the short-term mortality rate associated with heart failure after a myocardial infarction. The aim of the study was to analyze histological and functional effects of adipose tissue-derived stem cells (ADSCs) after myocardial infarction and compare 2 types of administration pathways. METHODS AND RESULTS: ADSCs from 28 pigs were labeled by transfection. Animals that survived myocardial infarction (n = 19) received: intracoronary culture media (n = 4); intracoronary ADSCs (n = 5); transendocardial culture media (n = 4); or transendocardial ADSCs (n = 6). At 3 weeks' follow-up, intracoronary and transendocardial administration of ADSCs resulted in similar rates of engrafted cells (0.85 [0.19-1.97] versus 2 [1-2] labeled cells/cm(2), respectively; P = NS) and some of those cells expressed smooth muscle cell markers. The intracoronary administration of ADSCs was more effective in increasing the number of small vessels than transendocardial administration (223 +/- 40 versus 168 +/- 35 vessels/mm(2); P < .05). Ejection fraction was not modified by stem cell therapy. CONCLUSIONS: This is the first study to compare intracoronary and transendocardial administration of autologous ADSCs in a porcine model of myocardial infarction. Both pathways of ADSCs delivery are feasible, producing a similar number of engrafted and differentiated cells, although intracoronary administration was more effective in increasing neovascularization.
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Tejido Adiposo/trasplante , Endocardio/cirugía , Infarto del Miocardio/cirugía , Trasplante de Células Madre/métodos , Tejido Adiposo/citología , Animales , Células Cultivadas , Endocardio/patología , Femenino , Estudios de Seguimiento , Infarto del Miocardio/patología , Porcinos , Factores de TiempoRESUMEN
The ability of human umbilical cord blood-derived mesenchymal stem cells (UCBMSCs) to transdifferentiate towards cardiomyocytes remains unclear. The aim of this study was to direct UCBMSCs to the cardiac lineage by exposure to: (1) 5-azacytidine (AZ) or dimethyl sulfoxide (DMSO); (2) a combination of growth factors involved in early cardiomyogenesis (BMP-2 + bFGF + IGF-1); (3) the Wnt signaling activators lithium chloride (LiCl) and phorbol-12-myristate-13-acetate (PMA); and (4) direct contact with neonatal rat cardiomyocytes. Expression of cardiomyocyte-specific proteins and beta-catenin were assessed by quantitative RT-PCR, immunofluorescence and Western blot. Cocultures of human UCBMSCs with neonatal rat cardiomyocytes were also analyzed for the presence of calcium oscillations and changes in electrical potential using Fura Red and di-4-ANEPPS confocal imaging, respectively. Induction of cardiac-specific proteins was not detected in 5-AZ- or DMSO-treated cells. Following DMSO addition, beta-catenin cytoplasmic expression increased, but did not translocate into cell nuclei to promote cardiac gene activation. Likewise, neither co-stimulation with BMP-2 + bFGF + IGF-1, nor exposure to LiCl and PMA resulted in the acquisition of a cardiac phenotype by UCBMSCs. Direct contact with neonatal rat cardiomyocytes promoted neither the expression of cardiomyocyte-specific proteins, nor the presence of calcium rhythmic oscillations and potential-dependent fluorescence emission in UCBMSCs. The cardiomyogenic stimuli investigated in this study failed to transdifferentiate human UCBMSCs. Alternative strategies or regulatory factors and signaling pathways may be better suited to recruit UCBMSCs into cardiac cell lineage.
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Azacitidina/farmacología , Diferenciación Celular/efectos de los fármacos , Dimetilsulfóxido/farmacología , Sangre Fetal/citología , Péptidos y Proteínas de Señalización Intercelular/farmacología , Células Madre Mesenquimatosas/citología , Miocitos Cardíacos/citología , Tejido Adiposo/citología , Tejido Adiposo/fisiología , Animales , Proteína Morfogenética Ósea 2/farmacología , Células Cultivadas , Técnicas de Cocultivo , Sangre Fetal/efectos de los fármacos , Sangre Fetal/fisiología , Factores de Crecimiento de Fibroblastos/farmacología , Humanos , Factor I del Crecimiento Similar a la Insulina/farmacología , Cloruro de Litio/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/fisiología , Miocitos Cardíacos/fisiología , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Células Madre/citología , Células Madre/fisiología , Acetato de Tetradecanoilforbol/farmacología , Proteínas Wnt/efectos de los fármacos , Proteínas Wnt/fisiologíaRESUMEN
Human bone marrow-derived mesenchymal stem cells (MSCs) exhibit limited in vitro growth. Fibroblast growth factors (FGFs) elicit a variety of biological responses, such as cell proliferation, differentiation and migration. FGF-4 represents one of the FGFs with the highest cell mitogenic activity. We studied the effect of FGF-4 on MSCs growth and pluripotency. MSCs duplication time (Td) was significantly reduced with FGF-4 compared to controls (2.2 +/- 0.2 vs. 4.1 +/- 0.2 days, respectively; p = 0.03) while BMP-2 and SCF-1 did not exert a significant growth effect. MSC expression of surface markers, differentiation into adipogenic and osteogenic lineages, and baseline expression of cardiomyogenic genes were unaffected by FGF-4. In summary, exogenous FGF-4 increases the rate at which MSC proliferate and has no significant effect on MSC pluripotency.
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Células de la Médula Ósea/metabolismo , Factor 4 de Crecimiento de Fibroblastos/fisiología , Células Madre Mesenquimatosas/metabolismo , Adipocitos/metabolismo , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Proliferación Celular , Citometría de Flujo , Humanos , Modelos Biológicos , Miocitos Cardíacos/metabolismo , Osteogénesis , Fenotipo , Factores de TiempoRESUMEN
BACKGROUND: Ultrastructural findings of idiopathic dilated cardiomyopathy (IDCM) include myocyte atrophy and myofilament loss, yet little is known about the vascular abnormalities present in IDCM. METHODS AND RESULTS: Patients with IDCM and controls underwent multi-slice CT to examine length and diameter of epicardial vasculature. The levels of mobilizing cytokines and circulating EPCs were assessed by endothelial colony formation assay and flow cytometry. Immunohistochemistry and Western blot were used to examine microvessel density and expression of HIF-1alpha and beta-catenin. Main epicardial coronary arteries were shorter and smaller, and microvascular density was reduced in the epicardium in IDCM. Epicardial vessel paucity was associated with increased numbers of HIF-1alpha(+) cells (46.8+/-13.1% vs. 19.4+/-9.4%, p=0.006) indicating local epicardial hypoxia and elevation of circulating VEGF-A (394 pg/mL vs. 22 pg/mL, p=0.001). The number of mobilized progenitors CD133(+)/VEGF-R2(+) was 21-fold higher in IDCM compared with controls (6.5+/-3.3% vs. 0.3+/-0.2%; p<0.001). Moreover, this defective vascularization was associated with reduced myocardial expression of vascular beta-catenin, an important angiogenic regulator. CONCLUSIONS: This study shows defective vascularization and impaired vasculogenesis (the de novo vascular organization of mobilized endothelial progenitors) and angiogenesis (by which new blood vessels are formed from pre-existing mature endothelial cells) in human IDCM.
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Cardiomiopatía Dilatada/fisiopatología , Endotelio Vascular/fisiopatología , Neovascularización Patológica , Células Madre/patología , beta Catenina/fisiología , Adulto , Biomarcadores , Cardiomiopatía Dilatada/genética , Estudios de Casos y Controles , Citocinas , Femenino , Expresión Génica , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Factor A de Crecimiento Endotelial VascularRESUMEN
For decades, it has been widely accepted that the heart is a terminally differentiated organ that is unable to regenerate. Studies of recipients of hearts donated by other humans have shed light on the regenerative potential of the human heart. Investigators have been able to trace the Y chromosome by fluorescence in situ hybridization or polymerase chain reaction, or both, in sex-mismatched heart recipients. Cardiac chimerism has been reported, with concentrations of chimeric cells ranging from 0.04% to 10.0%. Cardiac chimerism after bone marrow or progenitor cell transplantation has also been reported to a low extent (approximately 0.20%), suggesting that a fraction of the extracardiac cells that colonize the myocardium are of bone marrow origin. Cardiac chimerism after pregnancy with male offspring (fetal cell microchimerism) has also been demonstrated. Cells of fetal origin have been shown to be capable of differentiating into myocardial cells. Collectively, we show that chimerism studies provide a proof of concept of a process that it is likely to be part of normal cardiac homeostasis in humans but apparently insufficient for cardiac repair in diseased hearts.
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Quimerismo , Corazón/fisiología , Regeneración , Trasplante de Células Madre , Quimera por Trasplante , Femenino , Humanos , Masculino , Miocitos Cardíacos , EmbarazoRESUMEN
Spontaneous calcium release from the sarcoplasmic reticulum in cardiac myocytes plays a central role in cardiac arrhythmogenesis. Compounds intended for therapeutical use that interfere with intracellular calcium handling may therefore have an undesired proarrhythmic potential. Here we have used isolated human atrial myocytes to compare the effect of the proarrhythmic antihistaminic drug terfenadine with the non-proarrhythmic antihistaminic drugs fexofenadine and rupatadine on intracellular calcium homeostasis. Perforated patch-clamp technique was used to measure ionic currents and to detect spontaneous calcium release from the sarcoplasmic reticulum. Our results show that the compound terfenadine, with known arrhythmogenic effects, inhibits L-type calcium current (I(Ca)) with an IC(50) of 185 nM when cells are stimulated at 1.0 Hz. The inhibitory effect of 0.3 muM terfenadine increased from 19+/-4% at stimulation frequency of 0.2 Hz to 63+/-6% at 2.0 Hz. Moreover, terfenadine also increased spontaneous calcium release from the sarcoplasmic reticulum. At a concentration of 1 muM, terfenadine significantly increased the spontaneous Na-Ca exchange current (I(NCX)) frequency from 0.48+/-0.25 to 1.93+/-0.67 s(-1). In contrast, fexofenadine and rupatadine did not change I(Ca) or the frequency of spontaneous I(NCX). We conclude that the proarrhythmic antihistaminic drug terfenadine alters intracellular calcium handling in isolated human atrial myocytes. This experimental model may be suitable to screen for potential arrhythmogenic side-effects of compounds intended for therapeutical use.
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Arritmias Cardíacas/inducido químicamente , Calcio/metabolismo , Antagonistas de los Receptores Histamínicos H1/farmacología , Miocitos Cardíacos/metabolismo , Terfenadina/farmacología , Canales de Calcio Tipo L/efectos de los fármacos , Ciproheptadina/análogos & derivados , Ciproheptadina/farmacología , Electrofisiología , Humanos , Procesamiento de Imagen Asistido por Computador , Técnicas In Vitro , Potenciales de la Membrana/efectos de los fármacos , Microscopía Confocal , Miocitos Cardíacos/efectos de los fármacos , Técnicas de Placa-Clamp , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/metabolismo , Terfenadina/análogos & derivadosRESUMEN
BACKGROUND: Whether aging modifies mesenchymal stem cell (MSC) properties is unknown. AIM: To compare the differentiation capacity of human CD105(+) MSCs obtained from young and elderly donors. METHODS AND RESULTS: Cells were obtained from young (n=10, 24+/-6.4 years) and elderly (n=9, 77+/-8.4 years) donors. Cell senescence was assessed by telomere length assays and lipofuscin accumulation. Cell pluripotentiality was analysed by adipogenic and osteogenic induction media, and myocyte phenotype was attempted with 5-azacytidine (5-AZ). Immunofluorescence, Western blot, transmission electron microscopy and fluo-4 confocal imaging were used to analyse the sarcomere, gap junctions and Ca(2+) dynamics. Cells obtained from young and elderly donors showed no significant differences in relative telomere length (40.1+/-6.4% and 40.3+/-3.6%, p=0.9) and lipofuscin accumulation. Adipogenic and osteogenic potential of CD105(+) MSCs was demonstrated. 5-AZ induced increased expression of sarcomeric proteins without complete sarcomere organization. Treated cells also showed increased presence of connexin-43 both in young and old donor-derived cells. Intercellular communications were verified by the observation of gap junctions and passage of Ca(2+) between neighbouring cells. Spontaneous Ca(2+) raises did not significantly increase after 5-AZ treatment in both age groups. CONCLUSION: Age does not influence the adipogenic and myogenic differentiation potential of human CD105(+) MSCs.