RESUMEN
Aldehyde dehydrogenase 1A1 (ALDH) activity is one hallmark of human bone marrow (BM), umbilical cord blood (UCB), and peripheral blood (PB) primitive progenitors presenting high reconstitution capacities in vivo. In this study, we have identified ALDH(+) cells within human skeletal muscles, and have analyzed their phenotypical and functional characteristics. Immunohistofluorescence analysis of human muscle tissue sections revealed rare endomysial cells. Flow cytometry analysis using the fluorescent substrate of ALDH, Aldefluor, identified brightly stained (ALDH(br)) cells with low side scatter (SSC(lo)), in enzymatically dissociated muscle biopsies, thereafter abbreviated as SMALD(+) (for skeletal muscle ALDH(+)) cells. Phenotypical analysis discriminated two sub-populations according to CD34 expression: SMALD(+)/CD34(-) and SMALD(+)/CD34(+) cells. These sub-populations did not initially express endothelial (CD31), hematopoietic (CD45), and myogenic (CD56) markers. Upon sorting, however, whereas SMALD(+)/CD34(+) cells developed in vitro as a heterogeneous population of CD56(-) cells able to differentiate in adipoblasts, the SMALD(+)/CD34(-) fraction developed in vitro as a highly enriched population of CD56(+) myoblasts able to form myotubes. Moreover, only the SMALD(+)/CD34(-) population maintained a strong myogenic potential in vivo upon intramuscular transplantation. Our results suggest that ALDH activity is a novel marker for a population of new human skeletal muscle progenitors presenting a potential for cell biology and cell therapy.
Asunto(s)
Aldehído Deshidrogenasa/metabolismo , Células Musculares/citología , Desarrollo de Músculos/fisiología , Músculo Esquelético/citología , Adipogénesis/fisiología , Diferenciación Celular/fisiología , Células Cultivadas , Citometría de Flujo , Humanos , Inmunohistoquímica , Técnicas In Vitro , FenotipoRESUMEN
Endostatin has been considered a highly specific inhibitor of endothelial cell proliferation and/or migration. To explore the use of endostatin in antiangiogenic gene therapy, we generated a recombinant adenovirus, AdEndo, carrying the gene for mouse endostatin. Injection of 10(9) PFU of AdEndo resulted in a low but significant suppression (25%) of preestablished tumor growth in murine models involving murine Lewis lung carcinoma (LLC) and human breast cancer MDA-MB-231 tumors. Greater anticancer activity was observed when the same dose of AdEndo was injected into two other preestablished murine models involving C51 murine colon cancer and HT29 human colon cancer (55 and 47% tumor growth reduction, respectively). In vitro, endostatin derived from AdEndo-infected MRC-5 fibroblasts inhibited the growth of C51 and HT29 cell lines (72 and 61%, respectively). The extent of this inhibition was comparable to that observed in endothelial cells: 75% for microcapillary endothelial cell line HMEC-1, 52% for human dermal microvascular endothelial cells, 46% for human umbilical vein endothelial cells, and 67% for calf pulmonary arterial endothelial cells. Both endothelial and colon cancer cells showed a clear increase in cell apoptosis (4- to 5-fold for endothelial cells and 5- to 10-fold for colon cancer cells) and an accumulation in the G(1) phase of the cell cycle. This antiproliferative activity was not observed in other tumor cell lines: LLC, MDA-MB-231, murine colon adenocarcinoma MC38, human prostate cancer cell line DU145, and human breast cancer cell line CAL51. Taken together, these results provide evidence that, in addition to its antiangiogenic activity, endostatin exerts a direct anticancer action that appears to be restricted to some tumor cell lines. Thus, endostatin could be used in some colon cancer treatments and its clinical efficacy would depend on the response of tumor cells themselves.
Asunto(s)
Adenoviridae/genética , Inhibidores de la Angiogénesis , Antineoplásicos , Colágeno/genética , Neoplasias del Colon/terapia , Fragmentos de Péptidos/genética , Animales , Apoptosis , Ciclo Celular , División Celular , Neoplasias del Colon/irrigación sanguínea , Neoplasias del Colon/patología , Endostatinas , Endotelio Vascular/citología , Femenino , Citometría de Flujo , Vectores Genéticos , Humanos , Integrina alfa5beta1/genética , Integrina alfa5beta1/metabolismo , Integrina alfaV/genética , Integrina alfaV/metabolismo , Ratones , Ratones Endogámicos BALB C , Microscopía Confocal , Neovascularización Patológica , Células Tumorales CultivadasRESUMEN
AIMS: The transcription factor Islet-1 (ISL1) is a marker of cardiovascular progenitors and is essential for mammalian cardiogenesis. An ISL1 haplotype has recently been associated with congenital heart disease. In this study we evaluated whether ISL1 variants are associated with hypertrophic (HCM), dilated (DCM), arrhythmogenic right ventricular cardiomyopathy (ARVC), or with Emery-Dreifuss muscular dystrophy (EDMD). METHODS AND RESULTS: The six exon and intron boundaries of ISL1 were screened for genetic variants in a cohort of 454 index cases. Eleven exonic variants were identified in HCM, DCM, ARVC, and/or EDMD. Out of the five novel variants, two are located in the 5'-untranslated region, two are silent (p.Arg171Arg and p.Asn189Asn), and one is a missense (p.Asn252Ser). The latter was identified in the homozygous state in one DCM patient, and in the heterozygous state in 11 relatives, who did not present with DCM but often with cardiovascular features. This variant was found in one HCM patient also carrying a MYH7 mutation and in 3/96 North-African Caucasian control individuals, but was absent in 138 European Caucasian control individuals. We investigated the effect of the ISL1 wild type and p.Asn252Ser mutant on myocyte enhancer factor 2C (Mef2c) promoter activity, an established ISL1 target. Mef2c promoter activity was â¼4-fold higher in the presence of wild-type and â¼6-fold higher in the presence of mutant ISL1 in both HEK and CHO cells. CONCLUSION: This study describes a new gain-of-function p.Asn252Ser variant in the human ISL1 gene, which could potentially lead to greater activation of downstream targets involved in cardiac development, dilation, and hypertrophy.
Asunto(s)
Cardiomiopatías/genética , Proteínas con Homeodominio LIM/genética , Proteínas de Dominio MADS/metabolismo , Distrofia Muscular de Emery-Dreifuss/genética , Factores Reguladores Miogénicos/metabolismo , Factores de Transcripción/genética , Regiones no Traducidas 5'/genética , Adulto , Animales , Displasia Ventricular Derecha Arritmogénica/genética , Células CHO , Cardiomiopatía Dilatada/genética , Cardiomiopatía Hipertrófica/genética , Estudios de Casos y Controles , Estudios de Cohortes , Cricetinae , Cricetulus , Exones , Femenino , Técnicas de Transferencia de Gen , Predisposición Genética a la Enfermedad , Células HEK293 , Heterocigoto , Homocigoto , Humanos , Intrones , Factores de Transcripción MEF2 , Masculino , Mutación Missense , Linaje , Polimorfismo de Nucleótido Simple , Regiones Promotoras GenéticasRESUMEN
Islet-1 expression identifies populations of progenitor cells in embryonic, fetal, and newborn murine hearts that are able to give rise to all cardiac cell lineages ex vivo and in vivo. Using systematic immunohistochemistry, we investigated whether islet-1-positive cells are present in adult mouse heart from the perspective of their potential therapeutic utility. The presence, localization, and nature of islet-1-positive cells were assessed in mice of different strains, ages, and conditions. Islet-1-positive cells were present in mouse heart from postnatal day 1 to young adulthood. Depending on the strain, these cells were organized in either 1 or 2 types of clusters localized to restricted areas, at a distance of 6%-35% of the heart length from the base. The first type of cluster was present in all strains and consisted of neural crest-derived cells that formed cardiac ganglia. The number of cells remained stable (a few hundred) from neonatal up to adult ages, and variations were noted between strains regarding their long-term persistency. The second type of cluster was essentially present in 129SvJ or Balb/C strains and absent from the other strains tested (C57BL/6J, C3H, SJL). It consisted of cells expressing highly ordered sarcomeric actin, consistent with their having cardiomyocyte identity. These cells disappeared in animals older than 4 months. Neither the number nor the type of islet-1-positive cells varied with time in a mouse model of dilated cardiomyopathy. Our studies demonstrate that islet-1-positive cells are relatively few in number in adult murine heart, being localized in restricted and rather inaccessible areas, and can represent both neural crest and cardiomyocyte lineages.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Miocardio/citología , Animales , Biomarcadores/metabolismo , Agregación Celular , Recuento de Células , Proliferación Celular , Técnicas de Sustitución del Gen , Proteínas con Homeodominio LIM , Ratones , Ratones Endogámicos , Modelos Animales , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Proteínas de Neurofilamentos/metabolismo , Especificidad de Órganos , Factores de Transcripción , beta-Galactosidasa/metabolismoRESUMEN
As relações familiares constituem objeto das ações do Estado que, visando a promoção do cuidado e garantia dos direitos infantojuvenis, atende por meio de abrigos crianças e adolescentes que habitaram a rua ou passaram, por diversas razões, pelo processo de deserção familiar. O presente estudo teve como objetivo analisar o processo de reinserção familiar de crianças que habitaram a rua e instituições de acolhimento por meio de narrativas de profissionais de abrigos, famílias e das próprias crianças. Adicionalmente, objetivou-se investigar o potencial benéfico e os grandes desafios deste processo que, não raras vezes, incorre em repetidas reinstitucionalizações de crianças previamente reinseridas em suas famílias. A metodologia aplicada foi definida a partir de contribuições teóricas da etnografia e da entrevista etnográfica. Foi realizado acompanhamento de 2 famílias atendidas por uma instituição de acolhimento infantojuvenil por meio de entrevistas etnográficas, visitas presenciais e registros em diários de campo. Também foram analisados documentos da instituição de acolhimento para contribuir com o levantamento da trajetória e das experiências das crianças e familiares entrevistados. A análise de dados ocorreu em articulação teórica com a perspectiva dos Estudos Sociais da Infância, visando gerar contribuições enriquecedoras para o campo de pesquisa, diante de um cenário social permeado por conquistas contemporâneas, bem como pelos desafios das políticas públicas voltadas à defesa do direito à convivência familiar e comunitária da criança e do adolescente. Com os resultados obtidos, foi possível concluir que o manejo e a qualidade de escuta das crianças e familiares por parte da equipe técnica do serviço de acolhimento e de toda a rede de garantia de direitos pode oferecer subsídios ou obstáculos ao processo de reinserção familiar de acordo com as concepções de família que os profissionais adotam e norteiam a sua atuação prática.
Family relations are the object of the State's actions, which, aiming at promoting care and guaranteeing children's rights, provide childcare services to children and adolescents who lived in the streets or, for various reasons, went through the process of family desertion. This study analyzed the process of family reintegration of children who lived in the streets and childcare institutions through narratives of shelter professionals, families and the children themselves. Additionally, this research aimed to investigate the beneficial potential and the main challenges of this process that often incurs repeated reinstitutionalizations of children previously reinserted in their families. The applied methodology was defined from theoretical contributions of ethnography and ethnographic interview. Two families assisted by a childcare institution were accompanied through ethnographic interviews, presential visits and field diary records. Documents from the host institution were also analyzed to contribute to the researched children and families' trajectory and experiences mapping. Data analysis was conducted in theoretical articulation with the perspective of the Social Studies of Childhood, aiming to generate enriching contributions to this research field, facing a social scenario permeated by contemporary achievements, as well as the challenges of public policies aimed to defending the right to family and community life of children and adolescents. With the results obtained, it was concluded that the management and listening quality of children and family members by the technical staff of the care services can offer subsidies or obstacles to the process of family reintegration according to the family conceptions that professionals adopt and with which guide their practice.