Aspirin Affects MDA-MB-231 Vesicle Production and Their Capacity to Induce Fibroblasts towards a Pro-Invasive State.

Long-term administration of aspirin (ASA, acetylsalicylic acid) in oncogenic patients has been related to a reduction in cancer risk incidence, but its precise mechanism of action is unclear. The activation of cancer-associated fibroblasts (CAFs) is a key element in tumor progression and can be triggered by cancer-derived extracellular vesicles (EVs). Targeting the communication between cancer cells and the surrounding tumor microenvironment (TME) may control cancer progression. Our aim was to investigate the effect of ASA on breast cancer cells, focusing on EV secretion and their effect on the biological properties of CAFs. As a result, ASA was shown to reduce the amount and alter the size distribution of EVs produced by MDA-MB-231 tumor cells. Fibroblasts stimulated with EVs derived from MDA-MB-231 treated with ASA (EV-ASA) showed a lower expression of alpha-smooth muscle actin (α-SMA), matrix metalloproteinase-2 (MMP2) but not fibroblast activation protein (FAP) in respect to the ones stimulated with EVs from untreated breast cancer cells (EV-CTR). Furthermore, invasion assays using a three-dimensional (3D) fibroblast spheroid model showed reduced MDA-MB-231 invasion towards fibroblast spheroids pretreated with EV-ASA as compared to spheroids prepared with EV-CTR-stimulated fibroblasts. This suggests that ASA partially inhibits the ability of tumor EVs to stimulate CAFs to promote cancer invasion. In conclusion, ASA can interfere with tumor communication by reducing EV secretion by breast tumor cells as well as by interfering with their capacity to stimulate fibroblasts to become CAFs.

Adipose Tissue-Derived Mesenchymal Stromal Cells from Ex-Morbidly Obese Individuals Instruct Macrophages towards a M2-Like Profile In Vitro.

Obesity, which continues to increase worldwide, was shown to irreversibly impair the differentiation potential and angiogenic properties of adipose tissue mesenchymal stromal cells (ADSCs). Because these cells are intended for regenerative medicine, especially for the treatment of inflammatory conditions, and the effects of obesity on the immunomodulatory properties of ADSCs are not yet clear, here we investigated how ADSCs isolated from former obese subjects (Ex-Ob) would influence macrophage differentiation and polarization, since these cells are the main instructors of inflammatory responses. Analysis of the subcutaneous adipose tissue (SAT) of overweight (OW) and Ex-Ob subjects showed the maintenance of approximately twice as many macrophages in Ex-Ob SAT, contained within the CD68＋/FXIII-A- inflammatory pool. Despite it, in vitro, coculture experiments revealed that Ex-Ob ADSCs instructed monocyte differentiation into a M2-like profile, and under inflammatory conditions induced by LPS treatment, inhibited HLA-DR upregulation by resting M0 macrophages, originated a similar percentage of TNF-α＋ cells, and inhibited IL-10 secretion, similar to OW-ADSCs and BMSCs, which were used for comparison, as these are the main alternative cell types available for therapeutic purposes. Our results showed that Ex-Ob ADSCs mirrored OW-ADSCs in macrophage education, favoring the M2 immunophenotype and a mixed (M1/M2) secretory response. These results have translational potential, since they provide evidence that ADSCs from both Ex-Ob and OW subjects can be used in regenerative medicine in eligible therapies. Further in vivo studies will be fundamental to validate these observations.

Proteomic Analysis of HCC-1954 and MCF-7 Cell Lines Highlights Crosstalk between αv and ß1 Integrins, E-Cadherin and HER-2.

Overexpression of human epidermal growth factor receptor-2 (HER-2) occurs in 20% of all breast cancer subtypes, especially those that present the worst prognostic outcome through a very invasive and aggressive tumour. HCC-1954 (HER-2+) is a highly invasive, metastatic cell line, whereas MCF-7 is mildly aggressive and non-invasive. We investigated membrane proteins from both cell lines that could have a pivotal biological significance in metastasis. Membrane protein enrichment for HCC-1954 and MCF-7 proteomic analysis was performed. The samples were analysed and quantified by mass spectrometry. High abundance membrane proteins were confirmed by Western blot, immunofluorescence, and flow cytometry. Protein interaction prediction and correlations with the Cancer Genome Atlas (TCGA) patient data were conducted by bioinformatic analysis. In addition, ß1 integrin expression was analysed by Western blot in cells upon trastuzumab treatment. The comparison between HCC-1954 and MCF-7 membrane-enriched proteins revealed that proteins involved in cytoskeleton organisation, such as HER-2, αv and ß1 integrins, E-cadherin, and CD166 were more abundant in HCC-1954. ß1 integrin membrane expression was higher in the HCC-1954 cell line resistant after trastuzumab treatment. TCGA data analysis showed a trend toward a positive correlation between HER-2 and ß1 integrin in HER-2+ breast cancer patients. Differences in protein profile and abundance reflected distinctive capabilities for aggressiveness and invasiveness between HCC-1954 and MCF-7 cell line phenotypes. The higher membrane ß1 integrin expression after trastuzumab treatment in the HCC-1954 cell line emphasised the need for investigating the contribution of ß1 integrin modulation and its effect on the mechanism of trastuzumab resistance.

Human mesenchymal stromal/stem cells recruit resident pericytes and induce blood vessels maturation to repair experimental spinal cord injury in rats.

Angiogenesis is considered to mediate the beneficial effects of mesenchymal cell therapy in spinal cord injury. After a moderate balloon-compression injury in rats, injections of either human adipose tissue-derived stromal/stem cells (hADSCs) or their conditioned culture media (CM-hADSC) elicited angiogenesis around the lesion site. Both therapies increased vascular density, but the presence of hADSCs in the tissue was required for the full maturation of new blood vessels. Only animals that received hADSC significantly improved their open field locomotion, assessed by the BBB score. Animals that received CM-hADSC only, presented haemorrhagic areas and lack pericytes. Proteomic analyses of human angiogenesis-related factors produced by hADSCs showed that both pro- and anti-angiogenic factors were produced by hADSCs in vitro, but only those related to vessel maturation were detectable in vivo. hADSCs produced PDGF-AA only after insertion into the injured spinal cord. hADSCs attracted resident pericytes expressing NG2, α-SMA, PDGF-Rß and nestin to the lesion, potentially contributing to blood vessel maturation. We conclude that the presence of hADSCs in the injured spinal cord is essential for tissue repair.

Neutrophil Extracellular Traps (NETs) Promote Pro-Metastatic Phenotype in Human Breast Cancer Cells through Epithelial-Mesenchymal Transition.

Neutrophil extracellular traps (NETs) have been associated with several steps of tumor progression, including primary growth and metastasis. One of the key features for the acquisition of the metastatic ability is the epithelial-mesenchymal transition (EMT), a complex cellular program. In this study, we evaluated the ability of isolated NETs in modulating the pro-metastatic phenotype of human breast cancer cells. Tumor cells were treated with isolated NETs and then samples were generated for cell migration, quantitative RT-PCR, western blotting, immunofluorescence, and flow cytometry assays. RNA-seq data from The Cancer Genome Atlas (TCGA) database were assessed. NETs changed the typical epithelial morphology of MCF7 cells into a mesenchymal phenotype, a process that was accompanied by enhanced migratory properties. Additional EMT traits were observed: increased expression of N-cadherin and fibronectin, while the E-cadherin expression was repressed. Notably, NETs positively regulated the gene expression of several factors linked to the pro-inflammatory and pro-metastatic properties. Analyses of TCGA data showed that samples from breast cancer patients exhibit a significant correlation between pro-tumoral and neutrophil signature gene expression, including several EMT and pro-metastatic factors. Therefore, NETs drive pro-metastatic phenotype in human breast cancer cells through the activation of the EMT program.

Lymphatic vessels in human adipose tissue.

Despite being considered present in most vascularised tissues, lymphatic vessels have not been properly shown in human adipose tissue (AT). Our goal in this study is to investigate an unanswered question in AT biology, regarding lymphatic network presence in tissue parenchyma. Using human subcutaneous (S-) and visceral (V-) AT samples with whole mount staining for lymphatic specific markers and three-dimensional imaging, we showed lymphatic capillaries and larger lymphatic vessels in the human VAT. Conversely, in the human SAT, microcirculatory lymphatic vascular structures were rarely detected and no initial lymphatics were found.

The two faces of titanium dioxide nanoparticles bio-camouflage in 3D bone spheroids.

Titanium (Ti) and its alloys are widely used in dental implants and hip-prostheses due to their excellent biocompatibility. Growing evidence support that surface degradation due to corrosion and wear processes, contribute to implant failure, since the release of metallic ions and wear particles generate local tissue reactions (peri-implant inflammatory reactions). The generated ions and wear debris (particles at the micron and nanoscale) stay, in a first moment, at the interface implant-bone. However, depending on their size, they can enter blood circulation possibly contributing to systemic reactions and toxicities. Most of the nanotoxicological studies with titanium dioxide nanoparticles (TiO2 NPs) use conventional two-dimensional cell culture monolayers to explore macrophage and monocyte activation, where limited information regarding bone cells is available. Recently three-dimensional models have been gaining prominence since they present a greater anatomical and physiological relevance. Taking this into consideration, in this work we developed a human osteoblast-like spheroid model, which closely mimics bone cell-cell interactions, providing a more realistic scenario for nanotoxicological studies. The treatment of spheroids with different concentrations of TiO2 NPs during 72 h did not change their viability significantly. Though, higher concentrations of TiO2 NPs influenced osteoblast cell cycle without interfering in their ability to differentiate and mineralize. For higher concentration of TiO2 NPs, collagen deposition and pro-inflammatory cytokine, chemokine and growth factor secretion (involved in osteolysis and bone homeostasis) increased. These results raise the possible use of this model in nanotoxicological studies of osseointegrated devices and demonstrate a possible therapeutic potential of this TiO2 NPs to prevent or reverse bone resorption.

Increased extracellular matrix deposition during chondrogenic differentiation of dental pulp stem cells from individuals with neurofibromatosis type 1: an in vitro 2D and 3D study.

BACKGROUND: Neurofibromatosis 1 (NF1) presents a wide range of clinical manifestations, including bone alterations. Studies that seek to understand cellular and molecular mechanisms underlying NF1 orthopedic problems are of great importance to better understand the pathogenesis and the development of new therapies. Dental pulp stem cells (DPSCs) are being used as an in vitro model for several diseases and appear as a suitable model for NF1. The aim of this study was to evaluate in vitro chondrogenic differentiation of DPSCs from individuals with NF1 using two-dimensional (2D) and three-dimensional (3D) cultures. RESULTS: To fulfill the criteria of the International Society for Cellular Therapy, DPSCs were characterized by surface antigen expression and by their multipotentiality, being induced to differentiate towards adipogenic, osteogenic, and chondrogenic lineages in 2D cultures. Both DPSCs from individuals with NF1 (NF1 DPSCs) and control cultures were positive for CD90, CD105, CD146 and negative for CD13, CD14, CD45 and CD271, and successfully differentiated after the protocols. Chondrogenic differentiation was evaluated in 2D and in 3D (pellet) cultures, which were further evaluated by optical microscopy and transmission electron microscopy (TEM). 2D cultures showed greater extracellular matrix deposition in NF1 DPSCs comparing with controls during chondrogenic differentiation. In semithin sections, control pellets hadhomogenous-sized intra and extracelullar matrix vesicles, whereas NF1 cultures had matrix vesicles of different sizes. TEM analysis showed higher amount of collagen fibers in NF1 cultures compared with control cultures. CONCLUSION: NF1 DPSCs presented increased extracellular matrix deposition during chondrogenic differentiation, which could be related to skeletal changes in individuals with NF1.

Leishmania amazonensis isolated from human visceral leishmaniasis: histopathological analysis and parasitological burden in different inbred mice.

Leishmania amazonensis is a major etiological agent of human cutaneous leishmaniasis in the Americas; nevertheless there are some reports of this species causing visceral disease in dogs and men. In the present work we have studied a Leishmania strain isolated from a human case of visceral leishmaniasis. We have infected different mouse strains and analyzed the development of the disease, studying the parasite's ability to visceralize and whether this ability is influenced by host genetics. Female BALB/c, C57BL/6, C57BL/10, CBA, DBA/2, and C3H/He mice were subcutaneously infected with 104 L. amazonensis amastigotes. BALB/c, C57BL/6 and C57BL/10 mice were found to be very susceptible to infection, showing lesions that developed to necrosis and ulceration. CBA mice developed a late but severe lesion. DBA/2 mice developed only discrete lesions, while C3H/He mice did not develop any lesions. All mouse strains except C3H/He showed some degree of visceralization, presenting parasites in the spleen, while BALB/c, C57BL/6 and CBA presented parasites also in the liver. Moreover, most of the strains presented high parasite load at the infection site, whereas DBA and C3H/He mice showed low or no parasite load 90 days after infection, respectively. Histopathology corroborates the results, showing that susceptible mice presented an inflammatory reaction with parasites in the skin, lymph nodes and spleen, while strains that are more resistant presented low parasitism and discrete inflammatory reaction. Results indicate that this isolate is extremely virulent, can easily visceralize and that the pathogenesis of leishmaniasis is, at least in part, related to the genetic background of the host.

PS1/γ-Secretase-Mediated Cadherin Cleavage Induces ß-Catenin Nuclear Translocation and Osteogenic Differentiation of Human Bone Marrow Stromal Cells.

Bone marrow stromal cells (BMSCs) are considered a promising tool for bone bioengineering. However, the mechanisms controlling osteoblastic commitment are still unclear. Osteogenic differentiation of BMSCs requires the activation of ß-catenin signaling, classically known to be regulated by the canonical Wnt pathway. However, BMSCs treatment with canonical Wnts in vitro does not always result in osteogenic differentiation and evidence indicates that a more complex signaling pathway, involving cadherins, would be required to induce ß-catenin signaling in these cells. Here we showed that Wnt3a alone did not induce TCF activation in BMSCs, maintaining the cells at a proliferative state. On the other hand, we verified that, upon BMSCs osteoinduction with dexamethasone, cadherins were cleaved by the PS1/γ-secretase complex at the plasma membrane, and this event was associated with an enhanced ß-catenin translocation to the nucleus and signaling. When PS1/γ-secretase activity was inhibited, the osteogenic process was impaired. Altogether, we provide evidence that PS1/γ-secretase-mediated cadherin cleavage has as an important role in controlling ß-catenin signaling during the onset of BMSCs osteogenic differentiation, as part of a complex signaling pathway responsible for cell fate decision. A comprehensive map of these pathways might contribute to the development of strategies to improve bone repair.

Human mesenchymal cells from adipose tissue deposit laminin and promote regeneration of injured spinal cord in rats.

Cell therapy is a promising strategy to pursue the unmet need for treatment of spinal cord injury (SCI). Although several studies have shown that adult mesenchymal cells contribute to improve the outcomes of SCI, a description of the pro-regenerative events triggered by these cells is still lacking. Here we investigated the regenerative properties of human adipose tissue derived stromal cells (hADSCs) in a rat model of spinal cord compression. Cells were delivered directly into the spinal parenchyma immediately after injury. Human ADSCs promoted functional recovery, tissue preservation, and axonal regeneration. Analysis of the cord tissue showed an abundant deposition of laminin of human origin at the lesion site and spinal midline; the appearance of cell clusters composed of neural precursors in the areas of laminin deposition, and the appearance of blood vessels with separated basement membranes along the spinal axis. These effects were also observed after injection of hADSCs into non-injured spinal cord. Considering that laminin is a well-known inducer of axonal growth, as well a component of the extracellular matrix associated to neural progenitors, we propose that it can be the paracrine factor mediating the pro-regenerative effects of hADSCs in spinal cord injury.

Intraperitoneal but not intravenous cryopreserved mesenchymal stromal cells home to the inflamed colon and ameliorate experimental colitis.

BACKGROUND AND AIMS: Mesenchymal stromal cells (MSCs) were shown to have immunomodulatory activity and have been applied for treating immune-mediated disorders. We compared the homing and therapeutic action of cryopreserved subcutaneous adipose tissue (AT-MSCs) and bone marrow-derived mesenchymal stromal cells (BM-MSCs) in rats with trinitrobenzene sulfonic acid (TNBS)-induced colitis. METHODS: After colonoscopic detection of inflammation AT-MSCs or BM-MSCs were injected intraperitoneally. Colonoscopic and histologic scores were obtained. Density of collagen fibres and apoptotic rates were evaluated. Cytokine levels were measured in supernatants of colon explants. For cell migration studies MSCs and skin fibroblasts were labelled with Tc-99m or CM-DiI and injected intraperitonealy or intravenously. RESULTS: Intraperitoneal injection of AT-MSCs or BM-MSCs reduced the endoscopic and histopathologic severity of colitis, the collagen deposition, and the epithelial apoptosis. Levels of TNF-α and interleukin-1ß decreased, while VEGF and TGF-ß did not change following cell-therapy. Scintigraphy showed that MSCs migrated towards the inflamed colon and the uptake increased from 0.5 to 24 h. Tc-99m-MSCs injected intravenously distributed into various organs, but not the colon. Cm-DiI-positive MSCs were detected throughout the colon wall 72 h after inoculation, predominantly in the submucosa and muscular layer of inflamed areas. CONCLUSIONS: Intraperitoneally injected cryopreserved MSCs home to and engraft into the inflamed colon and ameliorate TNBS-colitis.

Osteoblasts and bone marrow mesenchymal stromal cells control hematopoietic stem cell migration and proliferation in 3D in vitro model.

BACKGROUND: Migration, proliferation, and differentiation of hematopoietic stem cells (HSCs) are dependent upon a complex three-dimensional (3D) bone marrow microenvironment. Although osteoblasts control the HSC pool, the subendosteal niche is complex and its cellular composition and the role of each cell population in HSC fate have not been established. In vivo models are complex and involve subtle species-specific differences, while bidimensional cultures do not reflect the 3D tissue organization. The aim of this study was to investigate in vitro the role of human bone marrow-derived mesenchymal stromal cells (BMSC) and active osteoblasts in control of migration, lodgment, and proliferation of HSCs. METHODOLOGY/PRINCIPAL FINDINGS: A complex mixed multicellular spheroid in vitro model was developed with human BMSC, undifferentiated or induced for one week into osteoblasts. A clear limit between the two stromal cells was established, and deposition of extracellular matrix proteins fibronectin, collagens I and IV, laminin, and osteopontin was similar to the observed in vivo. Noninduced BMSC cultured as spheroid expressed higher levels of mRNA for the chemokine CXCL12, and the growth factors Wnt5a and Kit ligand. Cord blood and bone marrow CD34(+) cells moved in and out the spheroids, and some lodged at the interface of the two stromal cells. Myeloid colony-forming cells were maintained after seven days of coculture with mixed spheroids, and the frequency of cycling CD34(+) cells was decreased. CONCLUSIONS/SIGNIFICANCE: Undifferentiated and one-week osteo-induced BMSC self-assembled in a 3D spheroid and formed a microenvironment that is informative for hematopoietic progenitor cells, allowing their lodgment and controlling their proliferation.

Dissecting coronary angiogenesis: 3D co-culture of cardiomyocytes with endothelial or mesenchymal cells.

In embryogenesis, coronary blood vessels are formed by vasculogenesis from epicardium-derived progenitors. Subsequently, growing or regenerating myocardium increases its vasculature by angiogenesis, forming new vessels from the pre-existing ones. Recently, cell therapies for myocardium ischemia that used different protocols have given promising results, using either extra-cardiac blood vessel cell progenitors or stimulating the cardiac angiogenesis. We have questioned whether cardiomyocytes could sustain both vasculogenesis and angiogenesis. We used a 3D culture model of tissue-like spheroids in co-cultures of cardiomyocytes supplemented either with endothelial cells or with bone marrow-derived mesenchymal stroma cells. Murine foetal cardiomyocytes introduced into non-adherent U-wells formed 3D contractile structures. They were coupled by gap junctions. Cardiomyocytes segregated inside the 3D structure into clumps separated by connective tissue septa, rich in fibronectin. Three vascular endothelial growth factor isoforms were produced (VEGF 120, 164 and 188). When co-cultured with human umbilical cord endothelial cells, vascular structures were produced in fibronectin-rich external layer and in radial septa, followed by angiogenic sprouting into the cardiomyocyte microtissue. Presence of vascular structures led to the maintenance of long-term survival and contractile capacity of cardiac microtissues. Conversely, bone marrow mesenchymal cells formed isolated cell aggregates, which progressively expressed the endothelial markers von Willebrand's antigen and CD31. They proceeded to typical vasculogenesis forming new blood vessels organised in radial pattern. Our results indicate that the in vitro 3D model of cardiomyocyte spheroids provides the two basic elements for formation of new blood vessels: fibronectin and VEGF. Within the myocardial environment, endothelial and mesenchymal cells can proceed to formation of new blood vessels either through angiogenesis or vasculogenesis, respectively.

Adipose tissue of control and ex-obese patients exhibit differences in blood vessel content and resident mesenchymal stem cell population.

BACKGROUND: The normal function of white adipose tissue is disturbed in obesity. After weight loss that follows bariatric surgery, ex-obese patients undergo plastic surgery to remove residual tissues and it is not known whether their adipose tissue returns to its original state. The aim of this study was to compare the white adipose tissue composition of ex-obese with control patients with regard to blood vessels and resident mesenchymal stem cells (MSC). METHODS: Quantification of blood vessels was performed on histological sections of adipose tissue stained with hematoxylin and eosin and for von Willebrand antigen. MSC were induced to the adipogenic and osteogenic lineages by specific inductive culture media. Expression of PPARgamma2 was analyzed by reverse transcription polymerase chain reaction. RESULTS: Ex-obese adipose tissue showed a higher number (p = 0.0286) of small (107.3 +/- 22.0) and large (22.5 +/- 6.4) blood vessels, when compared to control patients (42.0 +/- 24.4 and 7.2 +/- 2.2, respectively) and they also occupied a larger area (control versus ex-obese, p = 0.0286). Adipose tissue MSC from both groups of patients expressed PPARgamma2 and were equally able to differentiate to the osteogenic lineage, but ex-obese MSC showed a higher adipogenic potential when induced in vitro (p < 0.05). CONCLUSIONS: The higher number of adipose tissue blood vessels in ex-obese patients explains the excessive bleeding observed during their plastic surgery. The presence of more committed cells to the adipogenic lineage may favor the easy weight regain that occurs in ex-obese patients. These results show that, after extensive weight loss, adipose tissue cell composition was not totally restored.

Terapias celulares do miocárdio com células da medula óssea: critérios de qualidade e perspectivas / Myocardial cell therapy with bone marrow cells: criteria for quality and future perspectives

A terapia celular pode ser uma nova opção terapêutica para pacientes cardíacos, modificando o processo de remodelamento cardíaco e prevenindo a falência cardíaca pós-infarto. Estudos clínicos até o presente usaram células mononucleadas de medula óssea, isoladas por centrifugação em gradiente de densidade a partir de aspirados de medula óssea da crista ilíaca. Embora esta nova estratégia revolucionária pareça ser segura e melhorar a função cardíaca, resultados negativos surgiram desafiando o futuro de terapias baseadas em células para o reparo cardíaco. Aqui discutimos alguns resultados laboratoriais que podem explicar, pelo menos parcialmente, as diferenças obtidas em protocolos similares. Uma análise da correlação entre a composição celular da fração mononuclear do aspirado da medula óssea e o êxito clínico da terapia indicou que os linfócitos não favorecem o reparo do miocárdio. Uma seleção negativa eliminando as linhagens do sistema imunitário pode ser proposta para melhorar a terapia celular do miocárdio.

Cell therapy may provide a novel therapeutic option for cardiac patients, modifying myocardium remodeling processes and preventing post-infarction heart failure. Currently clinical studies predominantly use bone marrow mononuclear cells isolated by density gradient centrifugation of iliac crest bone marrow aspirates. Although this revolutionary new strategy seems to be safe and to improve myocardial function, negative data have emerged challenging the future of cell-based therapy for heart repair. Here we discuss some laboratory data that might explain, at least in part, variations in outcomes using similar protocols. Analysis of the correlation between the cell composition of the mononuclear fraction of bone marrow aspirates and the clinical outcome of the therapy has indicated that cells of the lymphocyte lineage are not beneficial in myocardial regeneration. A proposal of selection to eliminate these cells may improve cell therapy of infarcted myocardium.

Padrão de distribuição de células mononucleares de medula óssea em tecido cardíaco sadio por diferentes vias de infusão / Interaction between infusion route and bone marrow mononuclear cell distribution patterns in normal cardiac tissue

Introdução: A medicina regenerativa tem ganho grande importância nos últimos anos em decorrência da possibilidade de certas células se diferenciarem em linhagens celulares distintas e, assim, reconstruírem o tecido lesado. As células-tronco têm despontado como forma alternativa de tratamento para doenças pela sua capacidade de diferenciação nos mais de 100 tipos de tecido. A medula óssea contém células-tronco adultas, hematopoéticas e mesenquimais, que auxiliam na limitação do remodelamento cardíaco. Método: Foram utilizados 9 cães com peso entre 25 kg e 30 kg, divididos em três grupos: intracoronária, intramiocárdica-transendocárdica e retrógrada venosa. Células mononucleares da medula óssea foram coletadas por densidade Ficoll, marcadas com fluorocromo Hoechst e infundidas nas diferentes vias citadas anteriormente...

Background: Regenerative medicine has become increasingly important in recent years due to the possibility of certain cells to differentiate into different cell lines and thus recover the damaged tissue. The stem cell has emerged as an alternative treatment for diseases as a result of their ability to differentiate in more than 100 types of tissue. Bone marrow contains adult stem cells, hematopoietic and mesenchymal cells, which limit heart remodeling. Methods: Nine dogs weighing between 25 and 30 kg were divided into three groups: intracoronary group, intramyocardial-transendocardial group and retrograde venous group. Mononuclear cells were collected from bone marrow by Ficoll density, stained with Hoechst fluorocrom and infused through the different routes mentioned above...

Fibrosis and hypertrophy induced by Trypanosoma cruzi in a three-dimensional cardiomyocyte-culture system.

Cardiac damages caused by in vivo infection with Trypanosoma cruzi are still not fully clarified. Here we describe for the first time an in vitro model of fibrosis, hypertrophy, and remodeling induced by T. cruzi in cardiomyocyte spheroids (cardiac microtissues). In this new 3-dimensional system, cardiac spheroids showed spontaneous contractility, with typical cardiac morphology and production of extracellular matrix components. There were 4- and 6-fold increases, respectively, in the area and the volume of T. cruzi-infected cardiomyocytes and whole microtissues, together with a 50% reduction of the cell population. Immunofluorescence showed increased expression of fibronectin, collagen IV, and laminin in the microtissues 144 h after infection. T. cruzi infection induced an increase in both the cellular area and the extracellular matrix components in cardiac spheroids, which contributed to an increase in total microtissue volume, making this a powerful 3-dimensional in vitro model for the study of cardiac-tissue hypertrophy, fibrosis, and remodeling.

Bone-marrow mononuclear cell therapy of severe ischemic heart failure.

We describe cell therapy for severe ischemic heart failure using transendocardial injection of autologous bone-marrow-derived mononuclear cells. The treated patients had significantly less heart failure and angina, sustained significant improvement of pumping power, exercise capacity, cardiac muscle irrigation, and blood supply to the body. Electrical and mechanical mappings of the myocardium before and after the therapy, and anatomopathological examination of the myocardium of one of the patients that had deceased of a stroke eleven months after the treatment indicated sustained neoangiogenesis and improvement of activity and quantity of cardiomyocytes in the injected regions. Post-hoc analyses of injected cell phenotype and improvement of myocardial function indicate that presence of CD8+ and CD56+ cells does not correlate with good prognostics, suggesting a possibility of cell selection. For 'no-option' severe cardiac patients, significant benefits of cell therapy and absence of adverse effects may justify the application of bone-marrow-derived cell therapy.

A unique CD72 epitope suggests a potential interaction with Fc gamma RII/CD32 on B lineage lymphocytes.

It has long been known that ligation of the transmembrane CD72 glycoprotein delivers signals to B lymphocytes, with the outcome depending on context. Of particular interest is its ability to function as a counter-receptor/ ligand for the CD100 semaphorin protein. We have now obtained evidence that CD72 physically interacts on the lymphocyte membrane with Fcgamma receptor II (CD32). The association was first revealed with a new monoclonal antibody that recognizes polymorphic determinants on murine CD72. Although the specificity for CD72 was clear from immunoblotting, transfection and other experiments, staining with this reagent was inhibited when cells were pretreated with an Fc receptor-blocking antibody (CD16/CD32 specific). Furthermore, confocal microscopy revealed that the two molecules co-distributed on viable B cells. We also used the antibody to determine when CD72 becomes available to maturing lymphocytes. The marker is first acquired as large pre-B cells and enter the IL-7 independent phase of maturation within bone marrow. Subsequent interactions between CD72 and CD32 may cooperatively deliver negative signals that modulate humoral immune responses.