RESUMO
Wt1 encodes a zinc finger protein that is crucial for epicardium development. Although WT1 is also expressed in coronary endothelial cells (ECs), the abnormal heart development observed in Wt1 knockout mice is mainly attributed to its functions in the epicardium. Here, we have generated an inducible endothelial-specific Wt1 knockout mouse model (Wt1KOΔEC). Deletion of Wt1 in ECs during coronary plexus formation impaired coronary blood vessels and myocardium development. RNA-Seq analysis of coronary ECs from Wt1KOΔEC mice demonstrated that deletion of Wt1 exerted a major impact on the molecular signature of coronary ECs and modified the expression of several genes that are dynamically modulated over the course of coronary EC development. Many of these differentially expressed genes are involved in cell proliferation, migration and differentiation of coronary ECs; consequently, the aforementioned processes were affected in Wt1KOΔEC mice. The requirement of WT1 in coronary ECs goes beyond the initial formation of the coronary plexus, as its later deletion results in defects in coronary artery formation. Through the characterization of these Wt1KOΔEC mouse models, we show that the deletion of Wt1 in ECs disrupts physiological blood vessel formation.
Assuntos
Vasos Coronários , Células Endoteliais , Camundongos , Animais , Células Endoteliais/metabolismo , Vasos Coronários/metabolismo , Pericárdio/metabolismo , Proliferação de Células/genética , Neovascularização Fisiológica/genética , Modelos Animais de Doenças , Camundongos Knockout , Miocárdio/metabolismo , Proteínas WT1/genéticaRESUMO
Heart failure of ischemic origin is caused by the presence of a large scar resulting from an acute myocardial infarction. Acute myocardial infarction generally occurs when blood supply to the heart is blocked. Regenerative strategies that limit infarct injury would be able to prevent adverse post-ischemic remodelling and maintain the structural support necessary for effective cardiomyocyte contraction. Our understanding of endogenous cardiac regeneration and its biology has exposed a variety of targets for therapeutic approaches, such as non-coding RNAs, DNA methylation, histone modifications, direct cardiac reprogramming, cell transplantation, stimulation of resident cardiomyocytes, proliferation, and inhibition of cardiomyocyte death. In this review, we address the epigenetic mechanisms underlying these strategies and the use of therapeutic epigenetic molecules or epidrugs.
Assuntos
Reprogramação Celular/genética , Epigênese Genética/genética , Miócitos Cardíacos/efeitos dos fármacos , Regeneração/efeitos dos fármacos , HumanosRESUMO
Idiopathic dilated cardiomyopathy (IDCM) is a frequent cause of heart transplantation. Potentially valuable blood markers are being sought, and low-density lipoprotein receptor-related protein 1 (LRP1) has been linked to the underlying molecular basis of the disease. This study compared circulating levels of soluble LRP1 (sLRP1) in IDCM patients and healthy controls and elucidated whether sLRP1 is exported out of the myocardium through extracellular vesicles (EVs) to gain a better understanding of the pathogenesis of the disease. LRP1 α chain expression was analysed in samples collected from the left ventricles of explanted hearts using immunohistochemistry. sLRP1 concentrations were determined in platelet-free plasma by enzyme-linked immunosorbent assay. Plasma-derived EVs were extracted by size-exclusion chromatography (SEC) and characterized by nanoparticle tracking analysis and cryo-transmission electron microscopy. The distributions of vesicular (CD9, CD81) and myocardial (caveolin-3) proteins and LRP1 α chain were assessed in SEC fractions by flow cytometry. LRP1 α chain was preferably localized to blood vessels in IDCM compared to control myocardium. Circulating sLRP1 was increased in IDCM patients. CD9- and CD81-positive fractions enriched with membrane vesicles with the expected size and morphology were isolated from both groups. The LRP1 α chain was not present in these SEC fractions, which were also positive for caveolin-3. The increase in circulating sLRP1 in IDCM patients may be clinically valuable. Although EVs do not contribute to higher sLRP1 levels in IDCM, a comprehensive analysis of EV content would provide further insights into the search for novel blood markers.
Assuntos
Cardiomiopatia Dilatada/sangue , Vesículas Extracelulares/química , Ventrículos do Coração/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/sangue , Miocárdio/metabolismo , Idoso , Biomarcadores/sangue , Cardiomiopatia Dilatada/diagnóstico , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/cirurgia , Estudos de Casos e Controles , Caveolina 3/sangue , Caveolina 3/genética , Feminino , Regulação da Expressão Gênica , Transplante de Coração , Ventrículos do Coração/patologia , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Masculino , Pessoa de Meia-Idade , Miocárdio/patologia , Tetraspanina 28/sangue , Tetraspanina 28/genética , Tetraspanina 29/sangue , Tetraspanina 29/genéticaRESUMO
BACKGROUND: Despite recent advances, myocardial infarction (MI) remains the leading cause of death worldwide. Pre-clinical animal models that closely mimic human MI are pivotal for a quick translation of research and swine have similarities in anatomy and physiology. Here, we compared coronary surgical ligation versus coil embolization MI models in swine. METHODS: Fifteen animals were randomly distributed to undergo surgical ligation (n=7) or coil embolization (n=8). We evaluated infarct size, scar fibrosis, inflammation, myocardial vascularization, and cardiac function by magnetic resonance imaging (MRI). RESULTS: Thirty-five days after MI, there were no differences between the models in infarct size (P=0.53), left ventricular (LV) ejection fraction (P=0.19), LV end systolic volume (P=0.22), LV end diastolic volume (P=0.84), and cardiac output (P=0.89). Histologically, cardiac scars did not differ and the collagen content, collagen type I (I), collagen type III (III), and the I/III ratio were similar in both groups. Inflammation was assessed using specific anti-CD3 and anti-CD25 antibodies. There was similar activation of inflammation throughout the heart after coil embolization (P=0.78); while, there were more activated lymphocytes in the infarcted myocardium in the surgical occlusion model (P=0.02). Less myocardial vascularization in the infarction areas compared with the border and remote zones only in coil embolization animals was observed (P=0.004 and P=0.014, respectively). CONCLUSIONS: Our results support that surgical occlusion and coil embolization MI models generate similar infarct size, cardiac function impairment, and myocardial fibrosis; although, inflammation and myocardial vascularization levels were closer to those found in humans when coil embolization was performed.
Assuntos
Modelos Animais de Doenças , Infarto do Miocárdio/terapia , Animais , Feminino , Coração/fisiopatologia , Imageamento por Ressonância Magnética , Infarto do Miocárdio/fisiopatologia , Infarto do Miocárdio/cirurgia , SuínosRESUMO
Cardiac ATP production is tightly regulated in order to satisfy the evolving energetic requirements imposed by different cues during health and pathological conditions. In order to sustain high ATP production rates, cardiac cells are endowed with a vast mitochondrial network that is essentially acquired during the perinatal period. Nevertheless, adult cardiac cells also adapt their mitochondrial mass and oxidative function to changes in energy demand and substrate availability by fine-tuning the pathways and mitochondrial machinery involved in energy production. The reliance of cardiac cells on mitochondrial metabolism makes them particularly sensitive to alterations in proper mitochondrial function, so that deficiency in energy production underlies or precipitates the development of heart diseases. Mitochondrial biogenesis is a complex process fundamentally controlled at the transcriptional level by a network of transcription factors and co-regulators, sometimes with partially redundant functions, that ensure adequate energy supply to the working heart. Novel uncovered regulators, such as RIP140, PERM1, MED1 or BRD4 have been recently shown to modulate or facilitate the transcriptional activity of the PGC-1s/ERRs/PPARs regulatory axis, allowing cardiomyocytes to adapt to a variety of physiological or pathological situations requiring different energy provision. In this review, we summarize the current knowledge on the mechanisms that regulate cardiac mitochondrial biogenesis, highlighting the recent discoveries of new transcriptional regulators and describing the experimental models that have provided solid evidence of the relevant contribution of these factors to cardiac function in health and disease.
Assuntos
Metabolismo Energético , Animais , Metabolismo Energético/fisiologia , Metabolismo Energético/genética , Humanos , Transcrição Gênica/fisiologia , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/genética , Cardiopatias/metabolismo , Cardiopatias/genética , Miocárdio/metabolismo , Regulação da Expressão Gênica , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Modelos Animais de Doenças , Miócitos Cardíacos/metabolismoRESUMO
Fibrosis is present in an important proportion of myocardial disorders. Injury activates cardiac fibroblasts, which deposit excess extracellular matrix, increasing tissue stiffness, impairing cardiac function, and leading to heart failure. Clinical therapies that directly target excessive fibrosis are limited, and more effective treatments are needed. Immunotherapy based on chimeric antigen receptor (CAR) T cells is a novel technique that redirects T lymphocytes toward specific antigens to eliminate the target cells. It is currently used in haematological cancers but has demonstrated efficacy in mouse models of hypertensive cardiac fibrosis, with activated fibroblasts as the target cells. CAR T cell therapy is associated with significant toxicities, but CAR natural killer cells can overcome efficacy and safety limitations. The use of CAR immunotherapy offers a potential alternative to current therapies for fibrosis reduction and restoration of cardiac function in patients with myocardial fibrosis.
Assuntos
Cardiomiopatias , Neoplasias , Receptores de Antígenos Quiméricos , Animais , Camundongos , Imunoterapia/métodos , Linfócitos T , Imunoterapia Adotiva/efeitos adversos , Imunoterapia Adotiva/métodos , Cardiomiopatias/tratamento farmacológico , Fibrose , Neoplasias/tratamento farmacológicoRESUMO
Compelling evidence supports the therapeutic benefit of extracellular vesicles (EVs). EVs are nanostructures with a lipid bilayer membrane that are secreted by multiple cells, including mesenchymal stromal cells (MSCs), as means of cellular communication. MSC-EVs, resembling their MSC origin, carry protected immunomodulatory and pro-regenerative cargoes to targeted neighboring or distant cells and tissues. Though treatments focused on MSC-EVs have emerged as greatly versatile approaches to modulate multiple inflammatory-related conditions, crucial concerns, including the possibility of increasing therapeutic outcomes by pre-conditioning parental MSCs or engineering derived EVs and clarification of the most relevant mechanisms of action, remain. Here, we summarize the large amount of preclinical research surrounding the modulation of beneficial effects by MSC-EVs.
Assuntos
Vesículas Extracelulares/metabolismo , Células-Tronco Mesenquimais/metabolismo , Animais , Apoptose/fisiologia , Bioengenharia , Citocinas/metabolismo , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Imunomodulação/fisiologia , Técnicas In Vitro , Camundongos , MicroRNAs/metabolismo , NF-kappa B/metabolismo , Ratos , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Rationale: Extracellular vesicles (EVs) from mesenchymal stromal cell (MSC) are a potential therapy for cardiac healing after myocardial infarction (MI). Nevertheless, neither their efficient administration nor therapeutic mechanisms are fully elucidated. Here, we evaluate the preclinical efficacy of a tissue engineering approach to locally deliver porcine cardiac adipose tissue MSC-EV (cATMSC-EV) in an acute MI pig model. Methods: After MI by permanent ligation of the coronary artery, pigs (n = 24) were randomized to Untreated or treated groups with a decellularised pericardial scaffold filled with peptide hydrogel and cATMSC-EV purified by size exclusion chromatography (EV-Treated group) or buffer (Control group), placed over the post-infarcted myocardium. Results: After 30 days, cardiac MRI showed an improved cardiac function in EV-Treated animals, with significantly higher right ventricle ejection fraction (+20.8% in EV-Treated; p = 0.026), and less ventricle dilatation, indicating less myocardial remodelling. Scar size was reduced, with less fibrosis in the distal myocardium (-42.6% Col I in EV-Treated vs Untreated; p = 0.03), a 2-fold increase in vascular density (EV-Treated; p = 0.019) and less CCL2 transcription in the infarct core. EV-treated animals had less macrophage infiltration in the infarct core (-31.7% of CD163+ cells/field in EV-Treated; p = 0.026), but 5.8 times more expressing anti-inflammatory CD73 (p = 0.015). Systemically, locally delivered cATMSC-EV also triggered a systemic effect, doubling the circulating IL-1ra (p = 0.01), and reducing the PBMC rush 2d post-MI, the TNFα and GM-CSF levels at 30d post-MI, and modulating the CD73+ and CCR2+ monocyte populations, related to immunomodulation and fibrosis modulation. Conclusions: These results highlight the potential of cATMSC-EV in modulating hallmarks of ischemic injury for cardiac repair after MI.
Assuntos
Vesículas Extracelulares , Células-Tronco Mesenquimais , Infarto do Miocárdio , Animais , Modelos Animais de Doenças , Fibrose , Imunomodulação , Leucócitos Mononucleares , Infarto do Miocárdio/patologia , Miocárdio/patologia , Suínos , Remodelação VentricularRESUMO
Specific proteins and processes have been identified in post-myocardial infarction (MI) pathological remodeling, but a comprehensive understanding of the complete molecular evolution is lacking. We generated microarray data from swine heart biopsies at baseline and 6, 30, and 45 days after infarction to feed machine-learning algorithms. We cross-validated the results using available clinical and experimental information. MI progression was accompanied by the regulation of adipogenesis, fatty acid metabolism, and epithelial-mesenchymal transition. The infarct core region was enriched in processes related to muscle contraction and membrane depolarization. Angiogenesis was among the first morphogenic responses detected as being sustained over time, but other processes suggesting post-ischemic recapitulation of embryogenic processes were also observed. Finally, protein-triggering analysis established the key genes mediating each process at each time point, as well as the complete adverse remodeling response. We modeled the behaviors of these genes, generating a description of the integrative mechanism of action for MI progression. This mechanistic analysis overlapped at different time points; the common pathways between the source proteins and cardiac remodeling involved IGF1R, RAF1, KPCA, JUN, and PTN11 as modulators. Thus, our data delineate a structured and comprehensive picture of the molecular remodeling process, identify new potential biomarkers or therapeutic targets, and establish therapeutic windows during disease progression.
Assuntos
Adipogenia/genética , Transição Epitelial-Mesenquimal/genética , Infarto do Miocárdio/genética , Miocárdio/metabolismo , Algoritmos , Animais , Biópsia , Aprendizado Profundo , Modelos Animais de Doenças , Ácidos Graxos/genética , Ácidos Graxos/metabolismo , Humanos , Análise em Microsséries , Modelos Moleculares , Contração Muscular/genética , Infarto do Miocárdio/patologia , Miocárdio/patologia , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-raf/genética , Receptor IGF Tipo 1/genética , Suínos/genéticaRESUMO
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.
Assuntos
Tecido Adiposo/citologia , Infarto do Miocárdio/terapia , Miocárdio/citologia , Transplante de Células-Tronco , Células-Tronco/citologia , Idoso , Indutores da Angiogênese/metabolismo , Animais , Capilares/patologia , Diferenciação Celular , Linhagem da Célula , Separação Celular , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Testes de Função Cardíaca , Humanos , Camundongos , Infarto do Miocárdio/diagnóstico por imagem , Infarto do Miocárdio/patologia , Neovascularização Fisiológica , Ratos , UltrassonografiaRESUMO
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.
Assuntos
Azacitidina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Dimetil Sulfóxido/farmacologia , Sangue Fetal/citologia , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Células-Tronco Mesenquimais/citologia , Miócitos Cardíacos/citologia , Tecido Adiposo/citologia , Tecido Adiposo/fisiologia , Animais , Proteína Morfogenética Óssea 2/farmacologia , Células Cultivadas , Técnicas de Cocultura , Sangue Fetal/efeitos dos fármacos , Sangue Fetal/fisiologia , Fatores de Crescimento de Fibroblastos/farmacologia , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Cloreto de Lítio/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/fisiologia , Miócitos Cardíacos/fisiologia , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Células-Tronco/citologia , Células-Tronco/fisiologia , Acetato de Tetradecanoilforbol/farmacologia , Proteínas Wnt/efeitos dos fármacos , Proteínas Wnt/fisiologiaRESUMO
OBJECTIVES: Prevention of postischaemic ventricular dilatation progressing towards pathological remodelling is necessary to decrease ventricular wall deterioration. Myocardial tissue engineering may play a therapeutic role due to its capacity to replace the extracellular matrix, thereby creating niches for cell homing. In this experimental animal study, a biomimetic cardiopatch was created with elastomeric scaffolds and nanotechnologies. METHODS: In an experimental animal study in 18 sheep, a cardiopatch was created with adipose tissue-derived progenitor cells seeded into an engineered bioimplant consisting of 3-dimensional bioabsorbable polycaprolactone scaffolds filled with a peptide hydrogel (PuraMatrix™). This patch was then transplanted to cover infarcted myocardium. Non-absorbable poly(ethyl) acrylate polymer scaffolds were used as controls. RESULTS: Fifteen sheep were followed with ultrasound scans at 6 months, including echocardiography scans, tissue Doppler and spectral flow analysis and speckle-tracking imaging, which showed a reduction in longitudinal left ventricular deformation in the cardiopatch-treated group. Magnetic resonance imaging (late gadolinium enhancement) showed reduction of infarct size relative to left ventricular mass in the cardiopatch group versus the controls. Histopathological analysis at 6 months showed that the cardiopatch was fully anchored and integrated to the infarct area with minimal fibrosis interface, thereby promoting angiogenesis and migration of adipose tissue-derived progenitor cells to surrounding tissues. CONCLUSIONS: This study shows the feasibility and effectiveness of a cardiopatch grafted onto myocardial infarction scars in an experimental animal model. This treatment decreased fibrosis, limited infarct scar expansion and reduced postischaemic ventricular deformity. A capillary network developed between our scaffold and the heart. The elastomeric cardiopatch seems to have a positive impact on ventricular remodelling and performance in patients with heart failure.
Assuntos
Meios de Contraste , Alicerces Teciduais , Animais , Gadolínio , Humanos , Miocárdio , Ovinos , Engenharia Tecidual , Remodelação VentricularRESUMO
BACKGROUND: Low-density lipoprotein receptor-related protein 1 (LRP1) plays a key role in fatty acid metabolism and glucose homeostasis. In the context of dyslipemia, LRP1 is upregulated in the heart. Our aim was to evaluate the impact of cardiomyocyte LRP1 deficiency on high fat diet (HFD)-induced cardiac and metabolic alterations, and to explore the potential mechanisms involved. METHODS: We used TnT-iCre transgenic mice with thoroughly tested suitability to delete genes exclusively in cardiomyocytes to generate an experimental mouse model with conditional Lrp1 deficiency in cardiomyocytes (TNT-iCre+-LRP1flox/flox). FINDINGS: Mice with Lrp1-deficient cardiomyocytes (cm-Lrp1-/-) have a normal cardiac function combined with a favorable metabolic phenotype against HFD-induced glucose intolerance and obesity. Glucose intolerance protection was linked to higher hepatic fatty acid oxidation (FAO), lower liver steatosis and increased whole-body energy expenditure. Proteomic studies of the heart revealed decreased levels of cardiac pro-atrial natriuretic peptide (pro-ANP), which was parallel to higher ANP circulating levels. cm-Lrp1-/- mice showed ANP signaling activation that was linked to increased fatty acid (FA) uptake and increased AMPK/ ACC phosphorylation in the liver. Natriuretic peptide receptor A (NPR-A) antagonist completely abolished ANP signaling and metabolic protection in cm-Lrp1-/- mice. CONCLUSIONS: These results indicate that an ANP-dependent axis controlled by cardiac LRP1 levels modulates AMPK activity in the liver, energy homeostasis and whole-body metabolism.
Assuntos
Resistência à Insulina/genética , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Miócitos Cardíacos/metabolismo , Obesidade/genética , Adenilato Quinase/metabolismo , Animais , Fator Natriurético Atrial/metabolismo , Células Cultivadas , Dieta Hiperlipídica , Intolerância à Glucose/genética , Intolerância à Glucose/metabolismo , Intolerância à Glucose/patologia , Metabolismo dos Lipídeos/genética , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/deficiência , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Camundongos Transgênicos , Miócitos Cardíacos/patologia , Obesidade/metabolismo , Obesidade/patologiaRESUMO
The loss and degeneration of spinal cord motor neurons result in muscle denervation in spinal muscular atrophy (SMA), but whether there are primary pathogenetic abnormalities of muscle in SMA is not known. We previously detected increased DNA fragmentation and downregulation of Bcl-2 and Bcl-X(L) expression but no morphological changes in spinal motor neurons of SMA fetuses. Here, we performed histological and morphometric analysis of myotubes and assessed DNA fragmentation and Bcl-2/Bcl-X(L) expression in skeletal muscle from fetuses with type I SMA (at approximately 12 and 15 weeks' gestational ages, n = 4) and controls (at approximately 10-15 weeks' gestational ages, n = 7). Myotubes were smaller in the SMA than in control samples at all ages analyzed (p < 0.001) and were often arranged in clusters close to isolated and larger myotubes. Numbers of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells in control and SMA fetuses were similar, and no differences in Bcl-2 or Bcl-X(L) immunostaining between control and SMA muscle were identified. Areas with smaller myotubes and the morphometric analysis suggested a delay in growth and maturation in SMA muscle. These results suggest that spinal motor neurons and skeletal muscle undergo different pathogenetic processes in SMA during development; they imply that muscle as well as motor neurons may be targets for early therapeutic intervention in SMA.
Assuntos
Fibras Musculares Esqueléticas/patologia , Músculo Esquelético , Atrofia Muscular Espinal/patologia , Fatores Etários , Fragmentação do DNA , Feto/patologia , Feto/fisiopatologia , Humanos , Marcação In Situ das Extremidades Cortadas , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Músculo Esquelético/embriologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Medula Espinal/embriologia , Medula Espinal/metabolismo , Medula Espinal/patologiaRESUMO
Cardiovascular diseases are the number one cause of death worldwide and greatly impact quality of life and medical costs. Enormous effort has been made in research to obtain new tools for efficient and quick diagnosis and predicting the prognosis of these diseases. Discoveries of epigenetic mechanisms have related several pathologies, including cardiovascular diseases, to epigenetic dysregulation. This has implications on disease progression and is the basis for new preventive strategies. Advances in methodology and big data analysis have identified novel mechanisms and targets involved in numerous diseases, allowing more individualized epigenetic maps for personalized diagnosis and treatment. This paves the way for what is called pharmacoepigenetics, which predicts the drug response and develops a tailored therapy based on differences in the epigenetic basis of each patient. Similarly, epigenetic biomarkers have emerged as a promising instrument for the consistent diagnosis and prognosis of cardiovascular diseases. Their good accessibility and feasible methods of detection make them suitable for use in clinical practice. However, multicenter studies with a large sample population are required to determine with certainty which epigenetic biomarkers are reliable for clinical routine. Therefore, this review focuses on current discoveries regarding epigenetic biomarkers and its controversy aiming to improve the diagnosis, prognosis, and therapy in cardiovascular patients.
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The existence of radio- and chemotherapy-surviving cancer stem cells is currently believed to explain the inefficacy of anti-glioblastoma (GBM) therapies. The aim of this study was to determine if a therapeutic strategy specifically targeting GBM stem cells (GSC) would completely eradicate a GBM tumor. In both the in vitro and the in vivo models, ganciclovir therapy targeting proliferating GSC promotes the survival of a quiescent, stem-like cell pool capable of reproducing the tumor upon release of the therapeutic pressure. Images of small niches of therapy-surviving tumor cells show organized networks of vascular-like structures formed by tumor cells expressing CD133 or OCT4/SOX2. These results prompted the investigation of tumor cells differentiated to endothelial and pericytic lineages as a potential reservoir of tumor-initiating capacity. Isolated tumor cells with pericyte and endothelial cell lineage characteristics, grown under tumorsphere forming conditions and were able to reproduce tumors after implantation in mice.
Assuntos
Antígeno AC133/genética , Glioma/genética , Glioma/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fatores de Transcrição SOXB1/genética , Antígeno AC133/metabolismo , Animais , Biomarcadores , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Imunofluorescência , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Glioma/tratamento farmacológico , Glioma/patologia , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Camundongos , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/metabolismoRESUMO
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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Mechanical conditioning is incompletely characterized for stimulating therapeutic cells within the physiological range. We sought to unravel the mechanism of action underlying mechanical conditioning of adipose tissue-derived progenitor cells (ATDPCs), both in vitro and in silico. Cardiac ATDPCs, grown on 3 different patterned surfaces, were mechanically stretched for 7 days at 1 Hz. A custom-designed, magnet-based, mechanical stimulator device was developed to apply ~10% mechanical stretching to monolayer cell cultures. Gene and protein analyses were performed for each cell type and condition. Cell supernatants were also collected to analyze secreted proteins and construct an artificial neural network. Gene and protein modulations were different for each surface pattern. After mechanostimulation, cardiac ATDPCs increased the expression of structural genes and there was a rising trend on cardiac transcription factors. Finally, secretome analyses revealed upregulation of proteins associated with both myocardial infarction and cardiac regeneration, such as regulators of the immune response, angiogenesis or cell adhesion. To conclude, mechanical conditioning of cardiac ATDPCs enhanced the expression of early and late cardiac genes in vitro. Additionally, in silico analyses of secreted proteins showed that mechanical stimulation of cardiac ATDPCs was highly associated with myocardial infarction and repair.
Assuntos
Tecido Adiposo/citologia , Células-Tronco/metabolismo , Estresse Mecânico , Células Cultivadas , Conexina 43/metabolismo , Humanos , Fatores de Transcrição MEF2/metabolismo , Miocárdio/citologia , Miocárdio/metabolismo , Redes Neurais de Computação , Proteoma/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Células-Tronco/citologia , Fatores de Transcrição/metabolismoRESUMO
Some conditions are well known to be directly associated with stent failure, including in-stent re-occlusion and stent fracture. Currently, identification of these high-risk conditions requires invasive and complex procedures. This study aims to assess microwave spectrometry (MWS) for monitoring stents non-invasively. Preliminary ex vivo data are presented to move to in vivo validation. Fifteen mice were assigned to receive subcutaneous stent implantations (n = 10) or sham operations (n = 5). MWS measurements were carried out at 0, 2, 4, 7, 14, 22, and 29 days of follow-up. Additionally, 5 stented animals were summited to micro-CT analyses at the same time points. At 29 days, 3 animals were included into a stent fracture subgroup and underwent a last MWS and micro-CT analysis. MWS was able to identify stent position and in-stent stenosis over time, also discerning significant differences from baseline measures (P < 0.001). Moreover, MWS identified fractured vs. non-fractured stents in vivo. Taken together, MWS emerges as a non-invasive, non-ionizing alternative for stent monitoring. MWS analysis clearly distinguished between in-stent stenosis and stent fracture phenomena.
Assuntos
Análise de Falha de Equipamento/métodos , Micro-Ondas , Análise Espectral/métodos , Stents , Animais , CamundongosRESUMO
A preclinical model of glioblastoma (GB) bystander cell therapy using human adipose mesenchymal stromal cells (hAMSCs) is used to address the issues of cell availability, quality, and feasibility of tumor cure. We show that a fast proliferating variety of hAMSCs expressing thymidine kinase (TK) has therapeutic capacity equivalent to that of TK-expressing hAMSCs and can be used in a multiple-inoculation procedure to reduce GB tumors to a chronically inhibited state. We also show that up to 25% of unmodified hAMSCs can be tolerated in the therapeutic procedure without reducing efficacy. Moreover, mimicking a clinical situation, tumor debulking previous to cell therapy inhibits GB tumor growth. To understand these striking results at a cellular level, we used a bioluminescence imaging strategy and showed that tumor-implanted therapeutic cells do not proliferate, are unaffected by GCV, and spontaneously decrease to a stable level. Moreover, using the CLARITY procedure for tridimensional visualization of fluorescent cells in transparent brains, we find therapeutic cells forming vascular-like structures that often associate with tumor cells. In vitro experiments show that therapeutic cells exposed to GCV produce cytotoxic extracellular vesicles and suggest that a similar mechanism may be responsible for the in vivo therapeutic effectiveness of TK-expressing hAMSCs.