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Advanced therapy products, considered special medications, require Anvisa approval for use and commercialization in Brazil. They include advanced cellular therapy products, tissue engineering products, and gene therapy products, which due to their complexity involve innovation and risks, optimized regulatory channels for their development and life cycle monitoring. The scientific elements and the compliance with applicable regulatory aspects are fundamental pillars for the advancement of clinical trials, the positive evidence of the benefit-risk profile, and the definition of the critical quality attributes, from the perspective of making safe, efficacy, and high-quality products available to the population. The approval models of these products in Brazil adapt to the specificities and characteristics of the technology and the patient target population, with accelerated regulatory analyses, use in emergency situations by risk controls and specific monitoring mechanisms, principally those related to rare diseases without other therapeutic alternatives. The opportune access to the advance therapy product with safety, efficacy, and quality involves innovative normative elements that include the long-term follow-up of the safety and efficacy and of the adaptive pharmacovigilance requisites, as well as the traceability mechanisms for starting materials, products, and patients.
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Terapia Baseada em Transplante de Células e Tecidos , Terapia Genética , Humanos , Brasil , Medição de Risco , Engenharia TecidualRESUMO
In this review of cell therapies in Chagas disease, we cover aspects related to the disease, its treatment and world demographics, before proceeding to describe the preclinical and clinical trials performed using cell therapies in the search for an alternative therapy for the most severe and lethal form of this disease, chronic chagasic cardiomyopathy.
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Terapia Baseada em Transplante de Células e Tecidos/métodos , Doença de Chagas/terapia , Animais , Transplante de Medula Óssea/métodos , Cardiomiopatia Chagásica/terapia , Doença de Chagas/epidemiologia , Doença de Chagas/etiologia , Ensaios Clínicos como Assunto , Modelos Animais de Doenças , Transplante de Coração , Humanos , CamundongosRESUMO
BACKGROUND: Chagas disease is a public health problem caused by infection with the protozoan Trypanosoma cruzi. There is currently no effective therapy for Chagas disease. Although there is some evidence for the beneficial effect of bone marrow-derived cells in chagasic disease, the mechanisms underlying their effects in the heart are unknown. Reports have suggested that bone marrow cells are recruited to the chagasic heart; however, studies using chimeric mouse models of chagasic cardiomyopathy are rare. OBJECTIVES: The aim of this study was to investigate the migration of bone marrow cells to the heart after T. cruzi infection in a model of chagasic disease in chimeric mice. METHODS: To obtain chimerical mice, wild-type (WT) C57BL6 mice were exposed to full body irradiation (7 Gy), causing bone marrow ablation. Then, bone marrow cells from green fluorescent protein (GFP)-transgenic mice were infused into the mice. Graft effectiveness was confirmed by flow cytometry. Experimental mice were divided into four groups: (i) infected chimeric (iChim) mice; (ii) infected WT (iWT) mice, both of which received 3 × 104 trypomastigotes of the Brazil strain; (iii) non-infected chimeric (Chim) mice; and (iv) non-infected WT mice. FINDINGS: At one-month post-infection, iChim and iWT mice showed first degree atrioventricular block with decreased heart rate and treadmill exercise parameters compared to those in the non-infected groups. MAIN CONCLUSIONS: iChim mice showed an increase in parasitaemia, myocarditis, and the presence of amastigote nests in the heart tissue compared to iWT mice. Flow cytometry analysis did not detect haematopoietic progenitor cells in the hearts of infected mice. Furthermore, GFP+ cardiomyocytes were not detected in the tissues of chimeric mice.
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Células da Medula Óssea/fisiologia , Movimento Celular , Doença de Chagas/parasitologia , Miocárdio/citologia , Doença Aguda , Animais , Transplante de Medula Óssea/métodos , Cardiomiopatia Chagásica/parasitologia , Quimera , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Trypanosoma cruzi/fisiologiaRESUMO
We review the evidence for the presence of stem/progenitor cells in the heart and the preclinical and clinical data using diverse cell types for the therapy of cardiac diseases. We highlight the failure of adult stem/progenitor cells to ameliorate heart function in most cardiac diseases, with the possible exception of refractory angina. The use of pluripotent stem cell-derived cardiomyocytes is analysed as a viable alternative therapeutic option but still needs further research at preclinical and clinical stages. We also discuss the use of direct reprogramming of cardiac fibroblasts into cardiomyocytes and the use of extracellular vesicles as therapeutic agents in ischemic and non-ischemic cardiac diseases. Finally, gene therapies and genome editing for the treatment of hereditary cardiac diseases, ablation of genes responsible for atherosclerotic disease, or modulation of gene expression in the heart are discussed.
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Terapia Genética , Humanos , Terapia Genética/métodos , Animais , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/citologia , Cardiopatias/terapia , Cardiopatias/genética , Terapia Baseada em Transplante de Células e Tecidos/métodos , Edição de Genes , Cardiologia/métodos , Transplante de Células-Tronco/métodosRESUMO
Sarcopenia, a clinical syndrome primarily associated with reduced muscle mass in the elderly, has a negative impact on quality of life and survival. It can occur secondarily to other diseases such as heart failure (HF), a complex clinical syndrome with high morbidity and mortality. The simultaneous occurrence of these two conditions can worsen the prognosis of their carriers, especially in the most severe cases of HF, as in patients with reduced left ventricular ejection fraction (LVEF). However, due to the heterogeneous diagnostic criteria for sarcopenia, estimates of its prevalence present a wide variation, leading to new criteria having been recently proposed for its diagnosis, emphasizing muscle strength and function rather than skeletal muscle mass. The primary objective of this study is to evaluate the prevalence of sarcopenia and/or dynapenia in individuals with HF with reduced LVEF according to the most recent criteria, and compare the gene and protein expression of those patients with and without sarcopenia. The secondary objectives are to evaluate the association of sarcopenia and/or dynapenia with the risk of clinical events and death, quality of life, cardiorespiratory capacity, ventilatory efficiency, and respiratory muscle strength. The participants will answer questionnaires to evaluate sarcopenia and quality of life, and will undergo the following tests: handgrip strength, gait speed, dual-energy X-ray absorptiometry, respiratory muscle strength, cardiopulmonary exercise, as well as genomic and proteomic analysis, and dosage of N-terminal pro-B-type natriuretic peptide and growth differentiation factor-15. An association between sarcopenia and/or dynapenia with unfavorable clinical evolution is expected to be found, in addition to reduced quality of life, cardiorespiratory capacity, ventilatory efficiency, and respiratory muscle strength.
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Insuficiência Cardíaca , Sarcopenia , Humanos , Idoso , Sarcopenia/complicações , Sarcopenia/epidemiologia , Sarcopenia/diagnóstico , Volume Sistólico , Força da Mão/fisiologia , Prevalência , Qualidade de Vida , Proteômica , Função Ventricular Esquerda , Força Muscular/fisiologia , Insuficiência Cardíaca/complicações , Insuficiência Cardíaca/epidemiologia , Músculo Esquelético , Estudos Observacionais como AssuntoRESUMO
Up to 25% of pediatric cataract cases are inherited. There is sparse information in the literature regarding the cost of whole-exome sequencing (WES) for suspected hereditary pediatric cataracts. Molecular diagnosis of suspected hereditary pediatric cataracts is important for comprehensive genetic counseling. We performed a partial economic evaluation with a mixed costing analysis, using reimbursement data and microcosting approach with a bottom-up technique to estimate the cost of using WES for genetic diagnosis of suspected hereditary pediatric cataracts from the perspective of the Brazilian governmental health care system. One hundred and ten participants from twenty-nine families in Rio de Janeiro (RJ) were included. Costs of consumables, staff and equipment were calculated. Two scenarios were created: (1) The reference scenario included patients from RJ with suspected hereditary pediatric cataracts plus two family members. (2) The alternative scenario considered other genetic diseases, resulting in 5,280 exams per month. Sensitivity analysis was also performed. In the reference scenario, the total cost per exam was 700.09 United States dollars (USD), and in the alternative scenario, the total cost was 559.23 USD. The cost of WES alone was 527.85 USD in the reference scenario and 386.98 USD in the alternative scenario. Sensitivity analysis revealed that the largest costs were associated with consumables in both scenarios. Economic evaluations can help inform policy decisions, especially in middle-income countries such as Brazil.
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RATIONALE: Chronic rejection, accelerated coronary atherosclerosis, myocardial infarction, and ischemic heart failure determine the unfavorable evolution of the transplanted heart in humans. OBJECTIVE: Here we tested whether the pathological manifestations of the transplanted heart can be corrected partly by a strategy that implements the use of cardiac progenitor cells from the recipient to repopulate the donor heart with immunocompatible cardiomyocytes and coronary vessels. METHODS AND RESULTS: A large number of cardiomyocytes and coronary vessels were created in a rather short period of time from the delivery, engraftment, and differentiation of cardiac progenitor cells from the recipient. A proportion of newly formed cardiomyocytes acquired adult characteristics and was integrated structurally and functionally within the transplant. Similarly, the regenerated arteries, arterioles, and capillaries were operative and contributed to the oxygenation of the chimeric myocardium. Attenuation in the extent of acute damage by repopulating cardiomyocytes and vessels decreased significantly the magnitude of myocardial scarring preserving partly the integrity of the donor heart. CONCLUSIONS: Our data suggest that tissue regeneration by differentiation of recipient cardiac progenitor cells restored a significant portion of the rejected donor myocardium. Ultimately, immunosuppressive therapy may be only partially required improving quality of life and lifespan of patients with cardiac transplantation.
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Rejeição de Enxerto/patologia , Transplante de Coração , Histocompatibilidade , Miócitos Cardíacos/citologia , Regeneração/imunologia , Células-Tronco/citologia , Animais , Sequência de Bases , Diferenciação Celular/fisiologia , Fusão Celular , Vasos Coronários/citologia , Cães , Feminino , Genótipo , Rejeição de Enxerto/tratamento farmacológico , Rejeição de Enxerto/imunologia , Proteínas de Fluorescência Verde/genética , Insuficiência Cardíaca/imunologia , Insuficiência Cardíaca/patologia , Imunossupressores/uso terapêutico , Masculino , Dados de Sequência Molecular , Miocárdio/patologia , Células-Tronco/fisiologiaRESUMO
BACKGROUND: The objective of this study was to evaluate the therapeutic potential of bone marrow cells (BMCs) obtained from cirrhotic donors in a model of chronic liver disease. METHODS: Chronic liver injury was induced in female Wistar rats by the association of an alcoholic diet with intraperitoneal injections of carbon tetrachloride. BMCs obtained from cirrhotic donors or placebo were injected through the portal vein. Blood analysis of alanine aminotransferase (ALT) and albumin levels, ultrasound assessment including the measurement of the portal vein diameter (PVD) and liver echogenicity, histologic evaluation with hematoxylin and eosin and Sirius red staining, and quantification of collagen deposition were performed. RESULTS: ALT and albumin blood levels showed no significant differences between the experimental groups two months after injection. Additionally, no significant variation in PVD and liver echogenicity was found. Histological analysis also showed no significant variation in collagen deposition two months after placebo or BMC injection. CONCLUSION: This study suggests that, even though BMC therapy using cells from healthy donors has previously shown to be effective, this is not the case when BMCs are obtained from cirrhotic animals. This result has major clinical implications when considering the use of autologous BMCs from patients with chronic liver diseases.
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Células da Medula Óssea/citologia , Transplante de Medula Óssea , Modelos Animais de Doenças , Doença Hepática Terminal/complicações , Doença Hepática Terminal/terapia , Cirrose Hepática/etiologia , Cirrose Hepática/patologia , Animais , Células da Medula Óssea/metabolismo , Tetracloreto de Carbono/toxicidade , Feminino , Testes de Função Hepática , Ratos , Ratos WistarRESUMO
Cardiovascular diseases represent the world's leading cause of death. In this heterogeneous group of diseases, ischemic cardiomyopathies are the most devastating and prevalent, estimated to cause 17.9 million deaths per year. Despite all biomedical efforts, there are no effective treatments that can replace the myocytes lost during an ischemic event or progression of the disease to heart failure. In this context, cell therapy is an emerging therapeutic alternative to treat cardiovascular diseases by cell administration, aimed at cardiac regeneration and repair. In this review, we will cover more than 30 years of cell therapy in cardiology, presenting the main milestones and drawbacks in the field and signaling future challenges and perspectives. The outcomes of cardiac cell therapies are discussed in three distinct aspects: The search for remuscularization by replacement of lost cells by exogenous adult cells, the endogenous stem cell era, which pursued the isolation of a progenitor with the ability to induce heart repair, and the utilization of pluripotent stem cells as a rich and reliable source of cardiomyocytes. Acellular therapies using cell derivatives, such as microvesicles and exosomes, are presented as a promising cell-free therapeutic alternative.
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Decellularized cardiac extracellular matrix scaffolds with preserved composition and architecture can be used in tissue engineering to reproduce the complex cardiac extracellular matrix. However, evaluating the extent of cardiomyocyte repopulation of decellularized cardiac extracellular matrix scaffolds after recellularization attempts is challenging. Here, we describe a unique combination of biochemical, biomechanical, histological, and physiological parameters for quantifying recellularization efficiency of tissue-engineered cardiac patches compared with native cardiac tissue. Human embryonic stem cell-derived cardiomyocytes were seeded into rat heart atrial and ventricular decellularized cardiac extracellular matrix patches. Confocal and atomic force microscopy showed cell integration within the extracellular matrix basement membrane that was accompanied by restoration of native cardiac tissue passive mechanical properties. Multi-electrode array and immunostaining (connexin 43) were used to determine synchronous field potentials with electrical coupling. Myoglobin content (~60%) and sarcomere length measurement (>45% vs 2D culture) were used to evaluate cardiomyocyte maturation of integrated cells. The combination of these techniques allowed us to demonstrate that as cellularization efficiency improves, cardiomyocytes mature and synchronize electrical activity, and tissue mechanical/biochemical properties improve toward those of native tissue.
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Hemodynamic collapse and myocardial dysfunction are among the major causes of death in severe sepsis. The purpose of this study was to assess the role played by toll-like receptor 4 and by the NLRP3 inflammasome in the cardiac dysfunction that occurs after high-grade polymicrobial sepsis. We performed the colon ascendens stent peritonitis (CASP) surgery in Tlr4, Nlrp3, and caspase-1 mice. We also assessed for the first time the electrical heart function in the colon ascendens stent peritonitis (CASP) model. The QJ interval was increased in wild-type C57BL/6J mice after CASP when compared with sham controls, a result paralleled by an increase in the cardiac action potential (AP) duration (APD). The decreases in ejection fraction (EF), left ventricle end diastolic volume, stroke volume, and cardiac output found after CASP were similar among all groups of mice. Similar heart response was found when Nlrp3 mice were submitted to high-grade cecal ligation and puncture. Despite developing cardiac dysfunction similar to wild types after CASP, Nlrp3 mice had reduced circulating levels of interleukin (IL)-1ß, IL-6 and tumor necrosis factor-α. Our results demonstrate that the genetic ablation of Tlr4, Nlrp3, and caspase-1 does not prevent the cardiac dysfunction, despite preventing the increase in pro-inflammatory cytokines, indicating that these are not feasible targets to therapy in high-grade sepsis.
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Caspase 1/metabolismo , Colo/metabolismo , Cardiopatias/metabolismo , Interleucina-1beta/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Peritonite/complicações , Peritonite/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Ecocardiografia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Função Ventricular Esquerda/fisiologiaRESUMO
BACKGROUND: Doxorubicin (Dox) is a chemotherapy drug with limited application due to cardiotoxicity that may progress to heart failure. This study aims to evaluate the role of cardiomyocytes derived from mouse embryonic stem cells (CM-mESCs) in the treatment of Dox-induced cardiomyopathy (DIC) in mice. METHODS: The mouse embryonic stem cell (mESC) line E14TG2A was characterized by karyotype analysis, gene expression using RT-PCR and immunofluorescence. Cells were transduced with luciferase 2 and submitted to cardiac differentiation. Total conditioned medium (TCM) from the CM-mESCs was collected for proteomic analysis. To establish DIC in CD1 mice, Dox (7.5 mg/kg) was administered once a week for 3 weeks, resulting in a cumulative Dox dose of 22.5 mg/kg. At the fourth week, a group of animals was injected intramyocardially with CM-mESCs (8 × 105 cells). Cells were tracked by a bioluminescence assay, and the body weight, echocardiogram, electrocardiogram and number of apoptotic cardiomyocytes were evaluated. RESULTS: mESCs exhibited a normal karyotype and expressed pluripotent markers. Proteomic analysis of TCM showed proteins related to the negative regulation of cell death. CM-mESCs presented ventricular action potential characteristics. Mice that received Dox developed heart failure and showed significant differences in body weight, ejection fraction (EF), end-systolic volume (ESV), stroke volume (SV), heart rate and QT and corrected QT (QTc) intervals when compared to the control group. After cell or placebo injection, the Dox + CM-mESC group showed significant increases in EF and SV when compared to the Dox + placebo group. Reduction in ESV and QT and QTc intervals in Dox + CM-mESC-treated mice was observed at 5 or 30 days after cell treatment. Cells were detected up to 11 days after injection. The Dox + CM-mESC group showed a significant reduction in the percentage of apoptotic cardiomyocytes in the hearts of mice when compared to the Dox + placebo group. CONCLUSIONS: CM-mESC transplantation improves cardiac function in mice with DIC.
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Cardiomiopatias/terapia , Doxorrubicina/efeitos adversos , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/transplante , Cardiomiopatias/induzido quimicamente , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Linhagem Celular , Doxorrubicina/uso terapêutico , Células-Tronco Embrionárias Humanas/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Miócitos Cardíacos/patologiaRESUMO
The aim of the present study was to investigate whether feeder layers composed of human hair follicle-derived mesenchymal stem cells (hHFDCs) are able to support human embryonic stem cells (hESCs). hHFDCs and mouse embryonic fibroblasts (MEFs) were isolated and cultured in Dulbecco's modified Eagle's medium (DMEM)/F-12 and low-glucose DMEM, respectively. hHFDCs were passaged three times and subsequently characterized. hHFDCs and MEFs were mitotically inactivated with mitomycin C for 3 h prior to co-culture with H9-hESCs. hESCs were initially established on a mouse feeder layer, subsequently transferred onto a human feeder layer and split every 5 days. Cell morphology, expression of specific 'undifferentiation' markers and growth factors, and the differentiation capacity of hESCs grown on the hHFDC feeder layer were analyzed. hHFDCs are adherent to plastic, possess the classic mesenchymal stem cell phenotype [they express cluster of differentiation (CD)90, CD73 and CD105] and are able to differentiate into adipocytes, chondroblasts and osteocytes, indicating that these cells are multipotent. Population-doubling time analysis revealed that hHFDCs rapidly proliferate over 34.5 h. As a feeder layer, hHFDC behaved similarly to MEF in maintaining the morphology of hESCs. The results of alkaline phosphatase activity, reverse transcription-quantitative polymerase chain reaction analysis of the expression of pluripotency transcription factors [octamer-binding transcription factor 4 (Oct4), Nanog and sex determining region Y-box 2], and immunofluorescence assays of markers (stage-specific embryonic antigen-4 and Oct4) in hESCs co-cultured over hHFDC, indicated that the undifferentiated state of hESCs was preserved. No change in the level of growth factor transcripts (bone morphogenetic protein 4, fibroblast growth factor-2, vascular endothelial growth factor, Pigment epithelium-derived factor and transforming growth factor-ß1) was detected for either feeder layer prior to or following inactivation. Similar phenotypes of embryoid body formation, size and morphology were observed in the hHFDC and MEF feeders. In conclusion, hHFDC maintained hESCs in an undifferentiated state comparable to MEF in standard conditions, which may be an important finding regarding the establishment of stem cell-based translational applications.
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Chronic administration of anabolic androgenic steroids (AAS) in adult rats results in cardiac hypertrophy and increased susceptibility to myocardial ischemia/reperfusion (IR) injury. Molecular analyses demonstrated that hyperactivation of type 1 angiotensin II (AT1) receptor mediates cardiac hypertrophy induced by AAS and also induces down-regulation of myocardial ATP-sensitive potassium channel (KATP), resulting in loss of exercise-induced cardioprotection. Exposure to AAS during adolescence promoted long-term cardiovascular dysfunctions, such as dysautonomia. We tested the hypothesis that chronic AAS exposure in the pre/pubertal phase increases the susceptibility to myocardial ischemia/reperfusion (IR) injury in adult rats. Male Wistar rats (26day old) were treated with vehicle (Control, n=12) or testosterone propionate (TP) (AAS, 5mgkg-1 n=12) 5 times/week during 5 weeks. At the end of AAS exposure, rats underwent 23days of washout period and were submitted to euthanasia. Langendorff-perfused hearts were submitted to IR injury and evaluated for mechanical dysfunctions and infarct size. Molecular analysis was performed by mRNA levels of α-myosin heavy chain (MHC), ßMHC and brain-derived natriuretic peptide (BNP), ryanodine receptor (RyR2) and sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) by quantitative RT-PCR (qRT-PCR). The expression of AT1 receptor and KATP channel subunits (Kir6.1 and SURa) was analyzed by qRT-PCR and Western Blot. NADPH oxidase (Nox)-related reactive oxygen species generation was assessed by spectrofluorimetry. The expression of antioxidant enzymes was measured by qRT-PCR in order to address a potential role of redox unbalance. AAS exposure promoted long-term cardiac hypertrophy characterized by increased expression of ßMHC and ßMHC/αMHC ratio. Baseline derivative of pressure (dP/dt) was impaired by AAS exposure. Postischemic recovery of mechanical properties was impaired (decreased left ventricle [LV] developed pressure and maximal dP/dt; increased LV end-diastolic pressure and minimal dP/dt) and infarct size was larger in the AAS group. Catalase mRNA expression was significantly decreased in the AAS group. In conclusion, chronic administration of AAS during adolescence promoted long-term pathological cardiac hypertrophy and persistent increase in the susceptibility to myocardial IR injury possible due to disturbances on catalase expression.
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Envelhecimento , Anabolizantes/toxicidade , Androgênios/toxicidade , Cardiomegalia/induzido quimicamente , Vasos Coronários/efeitos dos fármacos , Coração/efeitos dos fármacos , Isquemia Miocárdica/induzido quimicamente , Traumatismo por Reperfusão Miocárdica/induzido quimicamente , Anabolizantes/administração & dosagem , Androgênios/administração & dosagem , Animais , Cardiomegalia/fisiopatologia , Catalase/antagonistas & inibidores , Catalase/genética , Catalase/metabolismo , Vasos Coronários/fisiopatologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Coração/fisiopatologia , Injeções Intramusculares , Masculino , Isquemia Miocárdica/patologia , Isquemia Miocárdica/fisiopatologia , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/enzimologia , Miocárdio/metabolismo , Miocárdio/patologia , Cadeias Pesadas de Miosina/química , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Distribuição Aleatória , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Propionato de Testosterona/administração & dosagem , Propionato de Testosterona/toxicidade , Fatores de TempoRESUMO
BACKGROUND: Heart failure represents an important public health issue due to its high costs and growing incidence worldwide. Evidence showing the regenerative potential of postmitotic heart tissue has suggested the existence of endogenous cardiac stem cells in adult hearts. Cardiosphere-derived cells (CDC) constitute a candidate pool of such cardiac stem cells. Previous studies using acute myocardial infarction (MI) models in rodents demonstrated an improvement in cardiac function after cell therapy with CDC. We evaluated the therapeutic potential of CDC 60 days after MI in a rat model. METHODS: CDC were obtained from human discarded myocardial tissue and rat hearts by enzymatic digestion with collagenase II. At 10-15 days after isolation, small, round, phase-bright cells (PBCs) appeared on top of the adherent fibroblast-like cells. The PBCs were collected and placed on a nonadherent plate for 2 days, where they formed cardiospheres which were then transferred to adherent plates, giving rise to CDC. These CDC were characterized by flow cytometry. Wistar rats were submitted to MI through permanent occlusion of the anterior descending coronary artery. After 60 days, they were immunosuppressed with cyclosporine A during 10 days. On the third day, infarcted animals were treated with 5 × 105 human CDC (hCDC) or placebo through intramyocardial injection guided by echocardiogram. Another group of animals was treated with rat CDC (rCDC) without immunosuppression. hCDC and rCDC were stably transduced with a viral construct expressing luciferase under control of a constitutive promoter. CDC were then used in a bioluminescence assay. Functional parameters were evaluated by echocardiogram 90 and 120 days after MI and by Langendorff at 120 days. RESULTS: CDC had a predominantly mesenchymal phenotype. Cell tracking by bioluminescence demonstrated over 85% decrease in signal at 5-7 days after cell therapy. Cardiac function evaluation by echocardiography showed no differences in ejection fraction, end-diastolic volume, or end-systolic volume between groups receiving human cells, rat cells, or placebo. Hemodynamic analyses and infarct area quantification confirmed that there was no improvement in cardiac remodeling after cell therapy with CDC. CONCLUSION: Our study challenges the effectiveness of CDC in post-ischemic heart failure.
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Oclusão Coronária/terapia , Hospedeiro Imunocomprometido , Infarto do Miocárdio/terapia , Esferoides Celulares/transplante , Animais , Oclusão Coronária/diagnóstico por imagem , Oclusão Coronária/imunologia , Oclusão Coronária/fisiopatologia , Vasos Coronários/diagnóstico por imagem , Vasos Coronários/patologia , Vasos Coronários/fisiopatologia , Ciclosporina/administração & dosagem , Modelos Animais de Doenças , Ecocardiografia , Testes de Função Cardíaca , Humanos , Injeções Intralesionais , Infarto do Miocárdio/diagnóstico por imagem , Infarto do Miocárdio/imunologia , Infarto do Miocárdio/fisiopatologia , Ratos , Ratos Wistar , Esferoides Celulares/citologia , Esferoides Celulares/fisiologia , Células-Tronco/citologia , Células-Tronco/fisiologia , Falha de TratamentoRESUMO
Diabetes mellitus (DM) encompasses a multitude of secondary disorders, including heart disease. One of the most frequent and potentially life threatening disorders of DM-induced heart disease is ventricular tachycardia (VT). Here we show that toll-like receptor 2 (TLR2) and NLRP3 inflammasome activation in cardiac macrophages mediate the production of IL-1ß in DM mice. IL-1ß causes prolongation of the action potential duration, induces a decrease in potassium current and an increase in calcium sparks in cardiomyocytes, which are changes that underlie arrhythmia propensity. IL-1ß-induced spontaneous contractile events are associated with CaMKII oxidation and phosphorylation. We further show that DM-induced arrhythmias can be successfully treated by inhibiting the IL-1ß axis with either IL-1 receptor antagonist or by inhibiting the NLRP3 inflammasome. Our results establish IL-1ß as an inflammatory connection between metabolic dysfunction and arrhythmias in DM.
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Diabetes Mellitus Experimental/imunologia , Interleucina-1beta/imunologia , Macrófagos/imunologia , Miócitos Cardíacos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Taquicardia Ventricular/imunologia , Receptor 2 Toll-Like/imunologia , Potenciais de Ação , Animais , Antirreumáticos/farmacologia , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/imunologia , Arritmias Cardíacas/metabolismo , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Caspase 1/metabolismo , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Inflamassomos/antagonistas & inibidores , Proteína Antagonista do Receptor de Interleucina 1/farmacologia , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Camundongos , Camundongos Transgênicos , Contração Miocárdica , Miócitos Cardíacos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Potássio/metabolismo , Receptores de Interleucina-1/antagonistas & inibidores , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/imunologia , Taquicardia Ventricular/etiologia , Taquicardia Ventricular/metabolismo , Receptor 2 Toll-Like/genéticaRESUMO
Chagas disease is caused by Trypanosoma cruzi and can lead to a dilated cardiomyopathy decades after the prime infection by the parasite. As with other dilated cardiomyopathies, conventional pharmacologic therapies are not always effective and as heart failure progresses patients need heart transplantation. Therefore alternative therapies are highly desirable and cell-based therapies have been investigated in preclinical and clinical studies. In this paper we review the main findings of such studies and discuss future directions for stem cell-based therapies in chronic chagasic cardiomyopathy.
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Cardiomiopatia Chagásica/terapia , Transplante de Células-Tronco , Animais , Ensaios Clínicos como Assunto , Modelos Animais de Doenças , Humanos , Pesquisa Translacional BiomédicaRESUMO
The human ihFib3.2 iPS cell line was generated from dermal fibroblasts obtained from a healthy donor. Lentiviral particles were produced with the polycistronic hSTEMCCA vector with Oct4, Sox2, cMyc and Klf4 as reprogramming factors.
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Fibroblastos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Linhagem Celular , Células Cultivadas , Humanos , Fator 4 Semelhante a KruppelRESUMO
INTRODUCTION: The increasing interest in 99m-technetium ((99m)Tc)-labeled stem cells encouraged us to study the (99m)Tc binding sites in stem cell compartments. METHODS: Bone marrow mononuclear cells were collected from femurs and tibia of rats. Cells were labeled with (99m)Tc by a direct method, in which reduced molecules react with (99m)Tc with the use of chelating agents, and lysed carefully in an ultrasonic apparatus. The organelles were separated by means of differential centrifugation. At the end of this procedure, supernatants and pellets were counted, and the percentages of radioactivity (in megabecquerels) bound to the different cellular fractions were determined. Percentages were calculated by dividing the radioactivity in each fraction by total radioactivity in the sample. The pellets were separated and characterized by their morphology on electron microscopy. RESULTS: The labeling procedure did not affect viability of bone marrow mononuclear cells. Radioactivity distributions in bone marrow mononuclear cell organelles, obtained in five independent experiments, were approximately 38.5 % in the nuclei-rich fraction, 5.3 % in the mitochondria-rich fraction, 2.2 % in microsomes, and 54 % in the cytosol. Our results showed that most of the radioactivity remained in the cytosol; therefore, this is an intracellular labeling procedure that has ribosomes unbound to membrane and soluble molecules as targets. However, approximately 39 % of the radioactivity remained bound to the nuclei-rich fraction. To confirm that cell disruption and organelle separation were efficient, transmission electron microscopy assays of all pellets were performed. CONCLUSIONS: Our results showed that most of the radioactivity was present in the cytosol fraction. More studies to elucidate the mechanisms involved in the cellular uptake of (99m)Tc in bone marrow cells are ongoing.