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The tissues or organs derived decellularized extracellular matrix carry immunogenicity and the risk of pathogen transmission, resulting in limited therapeutic effects. The cell derived dECM cultured in vitro can address these potential risks, but its impact on wound remodeling is still unclear. This study aimed to explore the role of decellularized extracellular matrix (dECM) extracted from adipose derived stem cells (ADSCs) in skin regeneration. Methods: ADSCs were extracted from human adipose tissue. Then we cultivated adipose-derived stem cell cells and decellularized ADSC-dECM for freeze-drying. Western blot (WB), enzyme-linked immunosorbent assay (ELISA) and mass spectrometry (MS) were conducted to analyzed the main protein components in ADSC-dECM. The cell counting assay (CCK-8) and scratch assay were used to explore the effects of different concentrations of ADSC-dECM on the proliferation and migration of human keratinocytes cells (HaCaT), human umbilical vein endothelia cells (HUVEC) and human fibroblasts (HFB), respectively. Moreover, we designed a novel ADSC-dECM-CMC patch which used carboxymethylcellulose (CMC) to load with ADSC-dECM; and we further investigated its effect on a mouse full thickness skin wound model. Results: ADSC-dECM was obtained after decellularization of in vitro cultured human ADSCs. Western blot, ELISA and mass spectrometry results showed that ADSC-dECM contained various bioactive molecules, including collagen, elastin, laminin, and various growth factors. CCK-8 and scratch assay showed that ADSC-dECM treatment could significantly promote the proliferation and migration of HaCaT, human umbilical vein endothelia cells, and human fibroblasts, respectively. To evaluate the therapeutic effect on wound healing in vivo, we developed a novel ADSC-dECM-CMC patch and transplanted it into a mouse full-thickness skin wound model. And we found that ADSC-dECM-CMC patch treatment significantly accelerated the wound closure with time. Further histology and immunohistochemistry indicated that ADSC-dECM-CMC patch could promote tissue regeneration, as confirmed via enhanced angiogenesis and high cell proliferative activity. Conclusion: In this study, we developed a novel ADSC-dECM-CMC patch containing multiple bioactive molecules and exhibiting good biocompatibility for skin reconstruction and regeneration. This patch provides a new approach for the use of adipose stem cells in skin tissue engineering.
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Background: Merkel cell carcinoma (MCC) is a rare type of invasive neuroendocrine skin malignancy with high mortality. However, with years of follow-up, what is the actual survival rate and how can we continually assess an individual's prognosis? The purpose of this study was to estimate conditional survival (CS) for MCC patients and establish a novel CS-based nomogram model. Methods: This study collected MCC patients from the Surveillance, Epidemiology, and End Results (SEER) database and divided these patients into training and validation groups at the ratio of 7:3. CS refers to the probability of survival for a specific timeframe (y years), based on the patient's survival after the initial diagnosis (x years). Then, we attempted to describe the CS pattern of MCCs. The Least absolute shrinkage and selection operator (LASSO) regression was employed to screen predictive factors. The Multivariate Cox regression analysis was applied to demonstrate these predictors' effect on overall survival and establish a novel CS-based nomogram. Results: A total of 3,843 MCC patients were extracted from the SEER database. Analysis of the CS revealed that the 7-year survival rate of MCC patients progressively increased with each subsequent year of survival. The rates progressed from an initial 41-50%, 61, 70, 78, 85%, and finally to 93%. And the improvement of survival rate was nonlinear. The LASSO regression identified five predictors including patient age, sex, AJCC stage, surgery and radiotherapy as predictors for CS-nomogram development. And this novel survival prediction model was successfully validated with good predictive performance. Conclusion: CS of MCC patients was dynamic and increased with time since the initial diagnosis. Our newly established CS-based nomogram can provide a dynamic estimate of survival, which has implications for follow-up guidelines and survivorship planning, enabling clinicians to guide treatment for these patients better.
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OBJECTIVES: To determine the therapeutic effect of tetrahedral framework nucleic acids (tFNAs) on diabetic wound healing and the underlying mechanism. MATERIALS AND METHODS: The tFNAs were characterized by polyacrylamide gel electrophoresis (PAGE), atomic force microscopy (AFM), transmission electron microscopy (TEM), dynamic light scattering (DLS) and zeta potential assays. Cell Counting Kit-8 (CCK-8) and migration assays were performed to evaluate the effects of tFNAs on cellular proliferation and migration. Quantitative polymerase chain reaction (Q-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect the effect of tFNAs on growth factors. The function and role of tFNAs in diabetic wound healing were investigated using diabetic wound models, histological analyses and western blotting. RESULTS: Cellular proliferation and migration were enhanced after treatment with tFNAs in a high-glucose environment. The expression of growth factors was also facilitated by tFNAs in vitro. During in vivo experiments, tFNAs accelerated the healing process in diabetic wounds and promoted the regeneration of the epidermis, capillaries and collagen. Moreover, tFNAs increased the secretion of growth factors and activated the Wnt pathway in diabetic wounds. CONCLUSIONS: This study indicates that tFNAs can accelerate diabetic wound healing and have potential for the treatment of diabetic wounds.
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Diabetes Mellitus , Ácidos Nucleicos , Humanos , Ácidos Nucleicos/farmacología , Vía de Señalización Wnt , Cicatrización de Heridas , Proliferación CelularRESUMEN
The aim of the present study was to develop and study a new model of left atrial thrombus (LAT) in rat with congestive heart failure (CHF). CHF was induced by aortic banding for 2 mo, followed by ischemia-reperfusion (I/R) and subsequent aortic debanding for 1 mo. Cardiac function and the presence of LAT were assessed by echocardiography. Masson's staining was performed for histological analysis. All CHF rats presented with significantly decreased cardiac function, fibrosis in remote myocardium, and pulmonary edema. The incidence rate of LAT was 18.8% in the rats. LAT was associated with severity of aortic constriction, aortic pressure gradient, aortic blood flow velocity, and pulmonary edema but not myocardial infarction or a degree of left ventricular depression. The progressive process of thrombogenesis was characterized by myocyte hypertrophy, fibrosis, and inflammation in the left atrial wall. Fibrin adhesion and clot formation were observed, whereas most LAT presented as a relatively hard "mass," likely attributable to significant fibrosis in the middle and outer layers. Some LAT mass showed focal necrosis as well as fibrin bulging. Most LAT occurred at the upper anterior wall of the left atrial appendage. Aortic debanding had no significant impact on large LATs (>5 mm2) that had formed, whereas small LATs (<5 mm2) regressed 1 mo after aortic release. LAT is found in a rat model of aortic banding plus I/R followed by aortic debanding. The model provides a platform to study molecular mechanisms and potential new pathways for LAT treatment. NEW & NOTEWORTHY It is critically important to have a rodent model to study the molecular mechanism of thrombogenesis in the left atrium. Left atrial thrombus (LAT) is not a simple fibrin clot like those seen in peripheral veins or arteries. Rather, LAT is a cellular mass that likely develops in conjunction with blood clotting. Studying this phenomenon will help us understand congestive heart failure and promote new therapies for LAT.
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Aorta/cirugía , Vasos Coronarios/cirugía , Atrios Cardíacos/patología , Insuficiencia Cardíaca/etiología , Daño por Reperfusión Miocárdica/etiología , Técnicas de Sutura , Trombosis/complicaciones , Animales , Aorta/fisiopatología , Función del Atrio Izquierdo , Remodelación Atrial , Coagulación Sanguínea , Vasos Coronarios/fisiopatología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Fibrosis , Atrios Cardíacos/fisiopatología , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/fisiopatología , Hemodinámica , Ligadura , Masculino , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/fisiopatología , Ratas Sprague-Dawley , Trombosis/sangre , Trombosis/patología , Trombosis/fisiopatología , Factores de TiempoRESUMEN
BACKGROUND: Despite its functional importance in various fundamental bioprocesses, studies of N6-methyladenosine (m6A) in the heart are lacking. Here, we show that the FTO (fat mass and obesity-associated protein), an m6A demethylase, plays a critical role in cardiac contractile function during homeostasis, remodeling, and regeneration. METHODS: We used clinical human samples, preclinical pig and mouse models, and primary cardiomyocyte cell cultures to study the functional role of m6A and FTO in the heart and in cardiomyocytes. We modulated expression of FTO by using adeno-associated virus serotype 9 (in vivo), adenovirus (both in vivo and in vitro), and small interfering RNAs (in vitro) to study its function in regulating cardiomyocyte m6A, calcium dynamics and contractility, and cardiac function postischemia. We performed methylated (m6A) RNA immunoprecipitation sequencing to map transcriptome-wide m6A, and methylated (m6A) RNA immunoprecipitation quantitative polymerase chain reaction assays to map and validate m6A in individual transcripts, in healthy and failing hearts, and in myocytes. RESULTS: We discovered that FTO has decreased expression in failing mammalian hearts and hypoxic cardiomyocytes, thereby increasing m6A in RNA and decreasing cardiomyocyte contractile function. Improving expression of FTO in failing mouse hearts attenuated the ischemia-induced increase in m6A and decrease in cardiac contractile function. This is performed by the demethylation activity of FTO, which selectively demethylates cardiac contractile transcripts, thus preventing their degradation and improving their protein expression under ischemia. In addition, we demonstrate that FTO overexpression in mouse models of myocardial infarction decreased fibrosis and enhanced angiogenesis. CONCLUSIONS: Collectively, our study demonstrates the functional importance of the FTO-dependent cardiac m6A methylome in cardiac contraction during heart failure and provides a novel mechanistic insight into the therapeutic mechanisms of FTO.
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Adenosina/análogos & derivados , Insuficiencia Cardíaca/enzimología , Infarto del Miocardio/enzimología , Miocitos Cardíacos/enzimología , Regeneración , Función Ventricular Izquierda , Remodelación Ventricular , Adenosina/metabolismo , Adulto , Anciano , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/genética , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Animales , Señalización del Calcio , Estudios de Casos y Controles , Línea Celular , Proliferación Celular , Desmetilación , Modelos Animales de Enfermedad , Femenino , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/fisiopatología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Infarto del Miocardio/genética , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Miocitos Cardíacos/patología , Procesamiento Postranscripcional del ARN , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Sus scrofaRESUMEN
Temporary or permanent left coronary artery (LCA) ligation is the most widely used model of heart failure. In the present protocol, we describe the materials necessary for the procedure, key steps of the LCA ligation, triphenyl tetrazolium chloride (TTC) staining, and calculation of myocardial infarction (MI) size after ischemia-reperfusion (I/R) injury (30 min/24 h) in rats and mice. We discuss precautions and tips regarding the operation before and after surgery, both in vivo and ex vivo. The aim of this chapter is to describe the details of LCA surgery and provide recommendations for current and future surgical operators.
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Modelos Animales de Enfermedad , Insuficiencia Cardíaca/patología , Isquemia Miocárdica/patología , Miocardio/patología , Animales , Vasos Coronarios/patología , Vasos Coronarios/cirugía , Insuficiencia Cardíaca/etiología , Ligadura , Masculino , Ratones , Ratones Endogámicos C57BL , Infarto del Miocardio/etiología , Infarto del Miocardio/patología , Isquemia Miocárdica/etiología , Ratas , Ratas Sprague-DawleyRESUMEN
Adeno-associated virus serotype 9 (AAV9) is an efficient vector for gene transfer to the myocardium. However, the use of ubiquitous promoters, such as the cytomegalovirus (CMV) promoter, can result in expression of the transgene in organs other than the heart. This study tested if the efficiency and specificity of cardiac transcription from a chicken cardiac troponin T (TnT) promoter could be further increased by incorporating a cardiomyocyte-specific transcriptional cis-regulatory motif from human calsequestrin 2 (CS-CRM4) into the expression cassette (Enh.TnT). The efficiency of luciferase expression from the TnT and Enh.TnT constructs was compared to expression of luciferase under the control of the CMV promoter in both adult and neonatal mice. Overall, expression levels of luciferase in the heart were similar in mice injected with AAV9.TnT.Luc, AAV9.Enh.TnT.Luc and AAV9.CMV.Luc. In contrast, expression levels of luciferase activity in nontarget organs, including the liver and muscle, was lower in mice injected with the AAV9.TnT.Luc compared to AAV9.CMV.Luc and was negligible with AAV9.Enh.TnT. In neonates, in organs other than the heart, luciferase expression levels were too low to be quantified for all constructs. Taken together, the data show that the AAV9 Enh.TnT constructs drives high levels of expression of the transgene in the myocardium, with insignificant expression in other organs. These properties reduce the risks associated with the AAV9-mediated expression of the therapeutic protein of interest in nontarget organs. The excellent cardiac specificity should allow for the use of higher vector doses than are currently used, which might be essential to achieve the levels of transgene expression necessary for therapeutic benefits. Taken together, the findings suggest that the Enh.TnT transcription unit is a potentially attractive tool for clinical cardiac gene therapy in adults.
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Dependovirus/genética , Terapia Genética , Cardiopatías/terapia , Miocardio/metabolismo , Transducción Genética , Animales , Animales Recién Nacidos , Calsecuestrina/genética , Pollos/genética , Regulación de la Expresión Génica/genética , Vectores Genéticos/genética , Vectores Genéticos/uso terapéutico , Cardiopatías/genética , Humanos , Ratones , Miocardio/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/virología , Regiones Promotoras Genéticas/genética , Troponina T/genéticaRESUMEN
OBJECTIVES: The purpose of this study was to evaluate the feasibility of imaging apoptosis in experimental ischemia-reperfusion model by technetium-99m (99mTc)-labeled Duramycin, and compare it to an established tracer, 99mTc-labeled Annexin-V, which has a relative disadvantage of high radiation burden to nontarget organs. BACKGROUND: During apoptosis, the cell membrane phospholipids-phosphatidylserine (PS) and phosphatidylethanolamine (PE) are exposed and can be targeted by Annexin-V and Duramycin, respectively, for in vivo imaging. Identification of a reversible cell death process should permit therapeutic intervention to help reduce myocyte loss and left ventricle dysfunction. METHODS: In a 40-min left coronary artery ischemia-reperfusion model in 17 rabbits, 7 mCi of 99mTc-labeled Duramycin (n = 10), 99mTc-linear Duramycin (a negative tracer control; n = 3), or 99mTc-Annexin-V (a positive tracer-control; n = 4) were intravenously administered 30 min after reperfusion. Of the 10 Duramycin group animals, 4 animals were treated with an antiapoptotic agent, minocycline at the time of reperfusion. In vivo and ex vivo micro-single-photon emission computed tomography (µSPECT) and micro-computed tomography (µCT) imaging was performed 3 h after reperfusion, followed by quantitative assessment of tracer uptake and pathological characterization. Fluorescent Duramycin and Annexin-V were injected in 4 rats to visualize colocalization in infarct areas in a 40-min left coronary artery occlusion and 30-min reperfusion model. RESULTS: Intense uptake of Duramycin and Annexin-V was observed in the apical (infarcted) areas. The percent injected dose per gram uptake of Duramycin in apical region (0.751 ± 0.262%) was significantly higher than remote area in same animals (0.045 ± 0.029%; p < 0.01). Duramycin uptake was insignificantly lower than Annexin-V uptake (1.23 ± 0.304%; p > 0.01) but demonstrated substantially lower radiation burden to kidneys (0.358 ± 0.210% vs. 1.58 ± 0.316%, respectively; p < 0.001). Fluorescence studies with Duramycin and Annexin V showed colocalization in infarct areas. Minocycline treatment substantially resolved Duramycin uptake (0.354% ± 0.0624%; p < 0.01). CONCLUSIONS: Duramycin is similarly effective in imaging apoptotic cell death as Annexin-V with lower nontarget organ radiation. Clinical feasibility of apoptosis imaging with a PE-seeking tracer should be tested.
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Anexina A5/administración & dosificación , Apoptosis , Bacteriocinas/administración & dosificación , Imagen Molecular/métodos , Infarto del Miocardio/diagnóstico por imagen , Daño por Reperfusión Miocárdica/diagnóstico por imagen , Miocardio/patología , Compuestos de Organotecnecio/administración & dosificación , Fosfatidiletanolaminas/metabolismo , Radiofármacos/administración & dosificación , Tomografía Computarizada de Emisión de Fotón Único , Animales , Anexina A5/toxicidad , Bacteriocinas/toxicidad , Modelos Animales de Enfermedad , Estudios de Factibilidad , Masculino , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/patología , Miocardio/metabolismo , Compuestos de Organotecnecio/toxicidad , Órganos en Riesgo , Valor Predictivo de las Pruebas , Conejos , Radiofármacos/toxicidad , Medición de Riesgo , Factores de Tiempo , Microtomografía por Rayos XRESUMEN
The aim of this study was to explore the role of abnormal coronary microvasculature morphology and hemodynamics in the development of congestive heart failure (CHF). CHF was induced in rats by aortic banding, followed by ischemia-reperfusion and later aortic debanding. Polymerized casts of coronary vasculature were imaged under a scanning electron microscope (SEM). Matrix Laboratory (MATLAB) software was used to calculate capillary structure index (CSI), a measure of structural alignment also called mean vector length (MVL), for 93 SEM images of coronary capillaries (CSIâ1 perfect linearity; CSIâ0 circular disarray). CSI was incorporated as a constant to represent tortuosity and nonlaminar flow in Poiseuille's equation to estimate the differences in capillary blood flow rate, velocity, and resistance for CHF vs. CONTROL: The morphology of CHF capillaries is significantly disordered and tortuous compared with control (CSI: 0.35 ± 0.02 for 61 images from 7 CHF rats; 0.58 ± 0.02 for 32 images from 7 control rats; P < 0.01). Estimated capillary resistance in CHF is elevated by 173% relative to control, while blood flow rate and blood velocity are 56 and 43% slower than control. Capillary resistance increased 67% due to the significantly narrower capillary diameter in CHF, while it increased an additional 105% due to tortuosity. The significant structural abnormalities of CHF coronary capillaries may drastically stagnate hemodynamics in myocardium and increase resistance to blood flow. This could play a role in the development of CHF. NEW & NOTEWORTHY In the present study, coronary capillary tortuosity was measured by applying Matrix Laboratory software to scanning electron microscope images of capillaries in a rat model of congestive heart failure. Stagnant blood flow in coronary capillaries may play a role in the development of congestive heart failure. The application of computer modeling to histological and physiological data to characterize the hemodynamics of coronary microcirculation is a new area of study.
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Capilares/fisiopatología , Circulación Coronaria , Insuficiencia Cardíaca/fisiopatología , Modelos Biológicos , Animales , Velocidad del Flujo Sanguíneo , Modelos Animales de Enfermedad , Masculino , Ratas Sprague-DawleyRESUMEN
Low reliability and reproducibility in heart failure models are well established. The purpose of the present study is to explore factors that affect model consistency of myocardial infarction (MI) in mice. MI was induced by left coronary artery (LCA) ligation. The coronary artery was casted with resin and visualized with fluorescent imaging ex vivo. LCA characteristics and MI size were analyzed individually in each animal, and MI size was correlated with left ventricular (LV) function by echocardiography. Coronary anatomy varies widely in mice, posing challenges for surgical ligation and resulting in inconsistent MI size postligation. The length of coronary arterial trunk, level of bifurcation, number of branches, and territory supplied by these branches are unique in each animal. When the main LCA trunk is ligated, this results in a large MI, but when a single branch is ligated, MI size is variable due to differing levels of LCA ligation and area supplied by the branches. During the ligation procedure, nearly 40% of LCAs are not grossly visible to the surgeon. In these situations, the surgeon blindly sutures a wider and deeper area of tissue in an attempt to catch the LCA. Paradoxically, these situations have greater odds of resulting in smaller MIs. In conclusion, variation in MI size and LV function after LCA ligation in mice is difficult to avoid. Anatomic diversity of the LCA in mice leads to inconsistency in MI size and functional parameters, and this is independent of potential technical modifications made by the operator.NEW & NOTEWORTHY In the present study, we demonstrate that left coronary artery diversity in mice is one of the primary causes of variable myocardial infarction size and cardiac functional parameters in the left coronary artery ligation model. Recognition of anatomic diversity is essential to improve reliability and reproducibility in heart failure research.
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Anomalías de los Vasos Coronarios/complicaciones , Vasos Coronarios/patología , Infarto del Miocardio/patología , Miocardio/patología , Animales , Anomalías de los Vasos Coronarios/patología , Anomalías de los Vasos Coronarios/fisiopatología , Vasos Coronarios/fisiopatología , Vasos Coronarios/cirugía , Modelos Animales de Enfermedad , Ligadura , Masculino , Ratones Endogámicos C57BL , Infarto del Miocardio/etiología , Infarto del Miocardio/fisiopatología , Imagen Óptica , Fenotipo , Técnicas de Réplica , Volumen Sistólico , Función Ventricular IzquierdaRESUMEN
In the present study, we explore the inherent variability that leads to overlaps in cardiac functional parameters between control and post-myocardial infarction (MI) mice. Heart failure was induced by Left Coronary Artery (LCA) ligation in mice. Average Ejection Fraction (EF) measured by echocardiography was lower in MI mice compared to control, but exhibited higher Standard Deviation (SD) and Standard Error (SEM), notably in 2D mode. Fractional Shortening (FS) showed a higher degree of overlap between MI and control mice even though the mean values were significantly different. Hemodynamic measurements of EF resulted in greater SD, SEM, ± 95% confidence intervals, and effect size. In comparing echocardiography at different time points, EF and FS were consistent by mean, but had apparent fluctuation in individual tracks, which were more obvious in MI than control mice. Hemodynamic measurements showed more complexity in data collection in mice in vivo. MI size showed variability that correlated with severity of cardiac function. These studies show that there is inherent variability in functional cardiac parameters after induction of heart failure by MI in mice. Analysis of these parameters by traditional statistical methods is insufficient, and we propose a more robust statistical analysis for proper data interpretation.
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BACKGROUND: Cardiac fibrosis (CF) is associated with increased ventricular stiffness and diastolic dysfunction and is an independent predictor of long-term clinical outcomes of patients with heart failure (HF). We previously showed that the matricellular CCN5 protein is cardioprotective via its ability to inhibit CF and preserve cardiac contractility. OBJECTIVES: This study examined the role of CCN5 in human heart failure and tested whether CCN5 can reverse established CF in an experimental model of HF induced by pressure overload. METHODS: Human hearts were obtained from patients with end-stage heart failure. Extensive CF was induced by applying transverse aortic constriction for 8 weeks, which was followed by adeno-associated virus-mediated transfer of CCN5 to the heart. Eight weeks following gene transfer, cellular and molecular effects were examined. RESULTS: Expression of CCN5 was significantly decreased in failing hearts from patients with end-stage heart failure compared to nonfailing hearts. Trichrome staining and myofibroblast content measurements revealed that the established CF had been reversed by CCN5 gene transfer. Anti-CF effects of CCN5 were associated with inhibition of the transforming growth factor beta signaling pathway. CCN5 significantly inhibited endothelial-mesenchymal transition and fibroblast-to-myofibroblast transdifferentiation, which are 2 critical processes for CF progression, both in vivo and in vitro. In addition, CCN5 induced apoptosis in myofibroblasts, but not in cardiomyocytes or fibroblasts, both in vivo and in vitro. CCN5 provoked the intrinsic apoptotic pathway specifically in myofibroblasts, which may have been due the ability of CCN5 to inhibit the activity of NFκB, an antiapoptotic molecule. CONCLUSIONS: CCN5 can reverse established CF by inhibiting the generation of and enhancing apoptosis of myofibroblasts in the myocardium. CCN5 may provide a novel platform for the development of targeted anti-CF therapies.
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Proteínas CCN de Señalización Intercelular/metabolismo , Miocardio/patología , Proteínas Represoras/metabolismo , Animales , Apoptosis , Proteínas CCN de Señalización Intercelular/genética , Transdiferenciación Celular , Dependovirus , Regulación hacia Abajo , Transición Epitelial-Mesenquimal , Fibrosis , Terapia Genética , Vectores Genéticos , Insuficiencia Cardíaca/metabolismo , Humanos , Ratones Transgénicos , Miocardio/metabolismo , Miofibroblastos/patología , Proteínas Represoras/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Nanoparticle-based delivery of nucleotides offers an alternative to viral vectors for gene therapy. We report highly efficient in vivo delivery of modified mRNA (modRNA) to rat and pig myocardium using formulated lipidoid nanoparticles (FLNP). Direct myocardial injection of FLNP containing 1-10 µg eGFPmodRNA in the rat (n = 3 per group) showed dose-dependent enhanced green fluorescent protein (eGFP) mRNA levels in heart tissue 20 hours after injection, over 60-fold higher than for naked modRNA. Off-target expression, including lung, liver, and spleen, was <10% of that in heart. Expression kinetics after injecting 5 µg FLNP/eGFPmodRNA showed robust expression at 6 hours that reduced by half at 48 hours and was barely detectable at 2 weeks. Intracoronary administration of 10 µg FLNP/eGFPmodRNA also proved successful, although cardiac expression of eGFP mRNA at 20 hours was lower than direct injection, and off-target expression was correspondingly higher. Findings were confirmed in a pilot study in pigs using direct myocardial injection as well as percutaneous intracoronary delivery, in healthy and myocardial infarction models, achieving expression throughout the ventricular wall. Fluorescence microscopy revealed GFP-positive cardiomyocytes in treated hearts. This nanoparticle-enabled approach for highly efficient, rapid and short-term mRNA expression in the heart offers new opportunities to optimize gene therapies for enhancing cardiac function and regeneration.
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Proteínas Fluorescentes Verdes/metabolismo , Infarto del Miocardio/metabolismo , Miocardio/metabolismo , Nanopartículas/química , ARN Mensajero/administración & dosificación , Animales , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Proteínas Fluorescentes Verdes/genética , Humanos , Inyecciones , Masculino , Nanopartículas/administración & dosificación , Especificidad de Órganos , Proyectos Piloto , Ratas , PorcinosRESUMEN
Identifying a bona fide population of cardiac stem cells (CSCs) is a critical step for developing cell-based therapies for heart failure patients. Previously, cardiac c-kit(+) cells were reported to be CSCs with a potential to become myocardial, endothelial and smooth muscle cells in vitro and after cardiac injury. Here we provide further insights into the nature of cardiac c-kit(+) cells. By targeting the c-kit locus with multiple reporter genes in mice, we find that c-kit expression rarely co-localizes with the expression of the cardiac progenitor and myogenic marker Nkx2.5, or that of the myocardial marker, cardiac troponin T (cTnT). Instead, c-kit predominantly labels a cardiac endothelial cell population in developing and adult hearts. After acute cardiac injury, c-kit(+) cells retain their endothelial identity and do not become myogenic progenitors or cardiomyocytes. Thus, our work strongly suggests that c-kit(+) cells in the murine heart are endothelial cells and not CSCs.
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Infarto del Miocardio/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Células Madre/metabolismo , Animales , Proliferación Celular , Células Cultivadas , Células Endoteliales/citología , Células Endoteliales/metabolismo , Femenino , Proteína Homeótica Nkx-2.5 , Proteínas de Homeodominio/metabolismo , Humanos , Masculino , Ratones , Infarto del Miocardio/genética , Infarto del Miocardio/fisiopatología , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Células Madre/citología , Factores de Transcripción/metabolismoRESUMEN
The aim of the present study is to explore the role of capillary disorder in coronary ischemic congestive heart failure (CHF). CHF was induced in rats by aortic banding plus ischemia-reperfusion followed by aortic debanding. Coronary arteries were perfused with plastic polymer containing fluorescent dye. Multiple fluorescent images of casted heart sections and scanning electric microscope of coronary vessels were obtained to characterize changes in the heart. Cardiac function was assessed by echocardiography and in vivo hemodynamics. Stenosis was found in all levels of the coronary arteries in CHF. Coronary vasculature volume and capillary density in remote myocardium were significantly increased in CHF compared with control. This occurred largely in microvessels with a diameter of ≤3 µm. Capillaries in CHF had a tortuous structure, while normal capillaries were linear. Capillaries in CHF had inconsistent diameters, with assortments of narrowed and bulged segments. Their surfaces appeared rough, potentially indicating endothelial dysfunction in CHF. Segments of main capillaries between bifurcations were significantly shorter in length in CHF than in control. Transiently increasing preload by injecting 50 µl of 30% NaCl demonstrated that the CHF heart had lower functional reserve; this may be associated with congestion in coronary microcirculation. Ischemic coronary vascular disorder is not limited to the main coronary arteries, as it occurs in arterioles and capillaries. Capillary disorder in CHF included stenosis, deformed structure, proliferation, and roughened surfaces. This disorder in the coronary artery architecture may contribute to the reduction in myocyte contractility in the setting of heart failure.
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Capilares/patología , Vasos Coronarios/patología , Insuficiencia Cardíaca/patología , Daño por Reperfusión Miocárdica/patología , Animales , Capilares/fisiopatología , Estenosis Coronaria/patología , Estenosis Coronaria/fisiopatología , Vasos Coronarios/fisiopatología , Reserva del Flujo Fraccional Miocárdico , Insuficiencia Cardíaca/fisiopatología , Masculino , Daño por Reperfusión Miocárdica/fisiopatología , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Phenotypic modulation or switching of vascular smooth muscle cells from a contractile/quiescent to a proliferative/synthetic phenotype plays a key role in vascular proliferative disorders such as atherosclerosis and restenosis. Although several calcium handling proteins that control differentiation of smooth muscle cells have been identified, the role of protein phosphatase inhibitor 1 (I-1) in the acquisition or maintenance of the contractile phenotype modulation remains unknown. METHODS AND RESULTS: In human coronary arteries, I-1 and sarco/endoplasmic reticulum Ca2+ -ATPase expression is specific to contractile vascular smooth muscle cells. In synthetic cultured human coronary artery smooth muscle cells, protein phosphatase inhibitor 1 (I-1 target) is highly expressed, leading to a decrease in phospholamban phosphorylation, sarco/endoplasmic reticulum Ca2+ -ATPase, and cAMP-responsive element binding activity. I-1 knockout mice lack phospholamban phosphorylation and exhibit vascular smooth muscle cell arrest in the synthetic state with excessive neointimal proliferation after carotid injury, as well as significant modifications of contractile properties and relaxant response to acetylcholine of femoral artery in vivo. Constitutively active I-1 gene transfer decreased neointimal formation in an angioplasty rat model by preventing vascular smooth muscle cell contractile to synthetic phenotype change. CONCLUSIONS: I-1 and sarco/endoplasmic reticulum Ca2+ -ATPase synergistically induce the vascular smooth muscle cell contractile phenotype. Gene transfer of constitutively active I-1 is a promising therapeutic strategy for preventing vascular proliferative disorders.
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Péptidos y Proteínas de Señalización Intracelular/metabolismo , Músculo Liso Vascular/metabolismo , Proteína Fosfatasa 1/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Vasoconstricción/fisiología , Animales , Aorta Torácica/citología , Aorta Torácica/fisiología , Señalización del Calcio/fisiología , Vasos Coronarios/citología , Vasos Coronarios/fisiología , Arteria Femoral/citología , Arteria Femoral/fisiología , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Arterias Mamarias/citología , Arterias Mamarias/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/citología , Fenotipo , Proteína Fosfatasa 1/genética , Proteínas/genética , Proteínas/metabolismo , Ratas , Ratas Sprague-Dawley , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Enfermedades Vasculares/genética , Enfermedades Vasculares/metabolismoRESUMEN
BACKGROUND: Galectin-3 is a ß-galactoside-binding lectin expressed in most of tissues in normal conditions and overexpressed in myocardium from early stages of heart failure (HF). It is an established biomarker associated with extracellular matrix (ECM) turnover during myocardial remodeling. The aim of this study is to test the ability of (123)I-galectin-3 (IG3) to assess cardiac remodeling in a model of myocardial infarction (MI) using imaging techniques. METHODS: Recombinant galectin-3 was labeled with iodine-123 and in vitro binding assays were conducted to test (123)I-galectin-3 ability to bind to ECM targets. For in vivo studies, a rat model of induced-MI was used. Animals were subjected to magnetic resonance and micro-SPETC/micro-CT imaging two (2 W-MI) or four (4 W-MI) weeks after MI. Sham rats were used as controls. Pharmacokinetic, biodistribution, and histological studies were also performed after intravenous administration of IG3. RESULTS: In vitro studies revealed that IG3 shows higher binding affinity (measured as counts per minute, cpm) (p < 0.05) to laminin (2.45 ± 1.67 cpm), fibronectin (4.72 ± 1.95 cpm), and collagen type I (1.88 ± 0.53 cpm) compared to bovine serum albumin (BSA) (0.88 ± 0.31 cpm). Myocardial quantitative IG3 uptake (%ID/g) was higher (p < 0.01) in the infarct of 2 W-MI rats (0.15 ± 0.04%) compared to control (0.05 ± 0.03%). IG3 infarct uptake correlates with the extent of scar (r s = 1, p = 0.017). Total collagen deposition in the infarct (percentage area) was higher (p < 0.0001) at 2 W-MI (24.2 ± 5.1%) and 4 W-MI (30.4 ± 7.5%) compared to control (1.9 ± 1.1%). However, thick collagen content in the infarct (square micrometer stained) was higher at 4 W-MI (20.5 ± 11.2 µm(2)) compared to control (4.7 ± 2.0 µm(2), p < 0.001) and 2 W-MI (10.6 ± 5.1 µm(2), p < 0.05). CONCLUSIONS: This study shows, although preliminary, enough data to consider IG3 as a potential contrast agent for imaging of myocardial interstitial changes in rats after MI. Labeling strategies need to be sought to improve in vivo IG3 imaging, and if proven, galectin-3 might be used as an imaging tool for the assessment and treatment of MI patients.
RESUMEN
Pathological left ventricle (LV) hypertrophy (LVH) results in reactive and replacement fibrosis. Volume overload LVH (VOH) is less profibrotic than pressure overload LVH (POH). Studies attribute subendocardial fibrosis in POH to ischaemia, and reduced fibrosis in VOH to collagen degradation favouring dilatation. However, the mechanical origin of the relative lack of fibrosis in VOH is incompletely understood. We hypothesized that reduced ischaemia propensity in VOH compared to POH accounted for the reduced replacement fibrosis, along with reduced reactive fibrosis. Rats with POH (ascending aortic banding) evolved into either compensated-concentric POH (POH-CLVH) or dilated cardiomyopathy (POH-DCM); they were compared to VOH (aorta-caval fistula). We quantified LV fibrosis, structural and haemodynamic factors of ischaemia propensity, and the activation of profibrotic pathways. Fibrosis in POH-DCM was severe, subendocardial and subepicardial, in contrast with subendocardial fibrosis in POH-CLVH and nearly no fibrosis in VOH. The propensity for ischaemia was more important in POH versus VOH, explaining different patterns of replacement fibrosis. LV collagen synthesis and maturation, and matrix metalloproteinase-2 expression, were more important in POH. The angiotensin II-transforming growth-factor ß axis was enhanced in POH, and connective tissue growth factor (CTGF) was overexpressed in all types of LVH. LV resistin expression was markedly elevated in POH, mildly elevated in VOH and independently reflected chronic ischaemic injury after myocardial infarction. In vitro, resistin is induced by angiotensin II and induces CTGF in cardiomyocytes. Based on these findings, we conclude that a reduced ischaemia propensity and attenuated upstream reactive fibrotic pathways account for the attenuated fibrosis in VOH versus POH.
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Hemodinámica , Isquemia Miocárdica/metabolismo , Resistina/metabolismo , Disfunción Ventricular Izquierda/metabolismo , Animales , Cardiomiopatía Dilatada/metabolismo , Cardiomiopatía Dilatada/patología , Cardiomiopatía Dilatada/fisiopatología , Colágeno/genética , Colágeno/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/genética , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Fibrosis/metabolismo , Fibrosis/patología , Fibrosis/fisiopatología , Masculino , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Isquemia Miocárdica/patología , Isquemia Miocárdica/fisiopatología , Miocitos Cardíacos/metabolismo , Ratas , Ratas Sprague-Dawley , Resistina/genética , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Disfunción Ventricular Izquierda/patología , Disfunción Ventricular Izquierda/fisiopatologíaRESUMEN
BACKGROUND: Clinical two-dimensional (2D) and clinical three-dimensional echocardiography are validated against cardiac magnetic resonance imaging (CMR), the gold standard for left ventricular (LV) volume measurement. In rodents, there is no widely accepted echocardiographic measure of whole LV volumes, and CMR measurements vary among studies. The aim of this study was to compare LV volumes by 2D echocardiography (using a hemisphere-cylinder [HC] model) with HC and full-volume (FV) CMR in normal and diseased rats to measure the impact of geometric models and imaging modalities. METHODS: Rats (n = 27) underwent ascending aortic banding, myocardial infarction induction by either permanent left anterior descending coronary artery ligation or ischemia-reperfusion, and sham thoracotomy. Subsequently, end-diastolic volume, end-systolic volume, and ejection fraction were measured using an HC 2D echocardiographic model combining parasternal short-axis and long-axis measurements, and these were compared with HC and FV CMR. RESULTS: Diseased groups showed LV dilatation and dysfunction. HC echocardiographic and FV CMR measures of end-diastolic volume, end-systolic volume, and ejection fraction were correlated. On Bland-Altman plots, end-diastolic volumes were concordant between both methods, while HC echocardiography underestimated end-systolic volumes, resulting in a modest overestimation of ejection fractions compared with FV CMR. Other 2D echocardiographic geometric models offered less concordance with FV CMR than HC. HC CMR overestimated LV volumes compared with FV CMR, while HC echocardiography underestimated HC CMR volumes. Echocardiography underestimated corresponding LV dimensions by CMR, particularly short axis. CONCLUSIONS: Concordant measures of LV volume and function were obtained using (1) a relatively simple HC model of the left ventricle inclusive of two orthogonal 2D echocardiographic planes and (2) FV CMR in normal and diseased rats. The HC model appeared to compensate for the underestimation of LV dimensions by echocardiography.
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Ecocardiografía Tridimensional/métodos , Ecocardiografía/métodos , Imagen por Resonancia Cinemagnética/métodos , Infarto del Miocardio/diagnóstico , Volumen Sistólico , Disfunción Ventricular Izquierda/diagnóstico , Animales , Masculino , Infarto del Miocardio/complicaciones , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Disfunción Ventricular Izquierda/etiologíaRESUMEN
This study investigates the impact of pressure overload on vascular changes after myocardial infarction (MI) in rats. To evaluate the effect of pressure overload, MI was induced in three groups: 1) left coronary artery ligation for 1 mo (MI-1m), 2) ischemia 30 min/reperfusion for 1 mo (I/R-1m), and 3) ischemia-reperfusion (I/R) was performed after pressure overload induced by aortic banding for 2 mo; 1 mo post-I/R, aortic constriction was released (Ab+I/R+DeAb). Heart function was assessed by echocardiography and in vivo hemodynamics. Resin casting and three-dimensional imaging with microcomputed tomography were used to characterize changes in coronary vasculature. TTC (triphenyltetrazohum chloride) staining and Masson's Trichrome were conducted in parallel experiments. In normal rats, MI induced by I/R and permanent occlusion was transmural or subendocardial. Occluded arterial branches vanished in MI-1m rats. A short residual tail was retained, distal to the occluded site in the ischemic area in I/R-1m hearts. Vascular pathological changes in transmural MI mostly occurred in ischemic areas and remote vasculature remained normal. In pressure overloaded rats, I/R injury induced a sub-MI in which ischemia was transmural, but myocardium in the involved area had survived. The ischemic arterial branches were preserved even though the capillaries were significantly diminished and the pathological changes were extended to remote areas, characterized by fibrosis, atrial thrombus, and pulmonary edema in the Ab+I/R+DeAb group. Pressure overload could increase vascular tolerance to I/R injury, but also trigger severe global ventricular fibrosis and results in atrial thrombus and pulmonary edema.