Advanced cell and gene therapies in cardiology.

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.

Toll-Like Receptor 4 and NLRP3 Caspase 1- Interleukin-1ß-Axis are Not Involved in Colon Ascendens Stent Peritonitis-Associated Heart Disease.

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.

Embryonic stem cell-derived cardiomyocytes for the treatment of doxorubicin-induced cardiomyopathy.

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.

Cell therapies for Chagas disease.

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.

Bone marrow cell migration to the heart in a chimeric mouse model of acute chagasic disease.

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.

Bone marrow cell migration to the heart in a chimeric mouse model of acute chagasic disease

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.

Macrophage-dependent IL-1ß production induces cardiac arrhythmias in diabetic mice.

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.

Stem Cell-Based Therapies in Chagasic Cardiomyopathy.

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.

99m-Technetium binding site in bone marrow mononuclear cells.

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.

Isolamento, cultivo e caracterização de células derivadas de cardioesferas de camundongos CD1 / Isolation, culture and characterization of cardiospheres-derived cells from CD1 mice

No Brasil, como em todo o mundo, as doenças cardiovasculares têm sido uma das principais causas de morte. A alta mortalidade e as poucas alternativas terapêuticas para esta doença têm estimulado a investigação no campo das células estaminais. Recentemente, alguns grupos têm mostrado a presença de células-tronco/progenitoras residentes no coração. Estas poderiam ser cultivadas diretamente a partir de tecidos cardíacos produzindo aglomerados esféricos denominados Cardioesferas estas, contém células proliferativas que dão origem, após o plaqueamento, a uma população heterogênea denominada: células derivadas de cardioesferas (CDCs). O objetivo deste estudo foi isolar, cultivar e caracterizar as CDCs de camundongos da linhagem CD1. Para isto, as células primárias foram isoladas a partir de corações de camundongos adultos da linhagem CD1 após a digestão de pequenos fragmentos do órgão em 420U/ml utilizando colagenase tipo II por 20 minutos 37°C. Nas análises por Citometria de Fluxo (FACS) foram observadas baixa expressão das moléculas de CD19 (0,4%), CD45 (0,5%) e CD90 (4,77%), e alta expressão das moléculas CD73 (71,47%), CD105 (25,1%), CD14 (25,17%). Nos ensaios de imunofluorescência foi possível observar a expressão das proteínas no citoplasma dos cardiomiócitos: vimentina, desmina e alfa actina de músculo liso, além da expressão do filamento intermediário nestina. Ao analisar a expansão celular por population doubling time foi observado que as CDCs duplicaram sua população original em cerca de 1,8 dias. Estes resultados sugerem que as CDCs isoladas a partir de camundongos da linhagem CD1, são células que apresentam características de células mesenquimais, constituindo uma população celular a ser testada nos estudos em terapias celulares. Estes resultados, motiva a estabelecer protocolos mais efetivos a fim de investigar possíveis efeitos parácrinos benéficos, bem como o potencial angiogênico e cardiogênico destas células.

In Brazil, as elsewhere in the world, cardiovascular diseases have been a major cause of death. The high mortality and few therapeutic alternatives for this disease have stimulated research in the field of stem cells. Recently, some groups have shown the presence of stem cells residents at heart. These could be grown directly from tissue cardiac producing spherical agglomerates called cardiospheres these contains proliferating cells that give rise after plating, a heterogeneous population named: cells derived from cardiospheres (CDC). Our goal in this study was to isolate and characterize the cultivar CDC CD1 strain of mice. For this purpose, primary cells were isolated from hearts of adult mice of the CD1 strain after digestion of the organ into small fragments using 420U/ml collagenase type II for 20 minutes 37 ° C. In analysis by Flow Cytometry (FACS) were observed low expression of CD19 molecules (0.4%), CD45 (0.5%) and CD90 (4.77%), and high expression of the molecules CD73 (71.47%), CD105 (25.1%), CD14 (25.17%). In the immunofluorescence assays was possible to observe the expression of the proteins in the cytoplasm of cardiomyocytes: vimentin, desmin and smooth muscle alpha actin, and expression of the intermediate filament nestin. By analyzing the cellular expansion team for Population doubling was observed that the original CDC doubled its population in about 1.8 days. These results suggest that CDCs isolated from CD1 mouse strain to be have characteristics of mesenchymal cells, constituting a potential population studied in cellular therapies, motivating us to establish more effective protocols to investigate possible beneficial paracrine effects and their angiogenic and cardiogenic potential.

Bone marrow cells obtained from cirrhotic rats do not improve function or reduce fibrosis in a chronic liver disease model.

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.

Progenitor cells from the explanted heart generate immunocompatible myocardium within the transplanted donor heart.

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.