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1.
EMBO J ; 36(13): 1946-1962, 2017 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-28515121

RESUMO

Control of stem cell fate to either enter terminal differentiation versus returning to quiescence (self-renewal) is crucial for tissue repair. Here, we showed that AMP-activated protein kinase (AMPK), the master metabolic regulator of the cell, controls muscle stem cell (MuSC) self-renewal. AMPKα1-/- MuSCs displayed a high self-renewal rate, which impairs muscle regeneration. AMPKα1-/- MuSCs showed a Warburg-like switch of their metabolism to higher glycolysis. We identified lactate dehydrogenase (LDH) as a new functional target of AMPKα1. LDH, which is a non-limiting enzyme of glycolysis in differentiated cells, was tightly regulated in stem cells. In functional experiments, LDH overexpression phenocopied AMPKα1-/- phenotype, that is shifted MuSC metabolism toward glycolysis triggering their return to quiescence, while inhibition of LDH activity rescued AMPKα1-/- MuSC self-renewal. Finally, providing specific nutrients (galactose/glucose) to MuSCs directly controlled their fate through the AMPKα1/LDH pathway, emphasizing the importance of metabolism in stem cell fate.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Diferenciação Celular , Autorrenovação Celular , Homeostase , L-Lactato Desidrogenase/metabolismo , Músculos/citologia , Células-Tronco/metabolismo , Animais , Glicólise , Camundongos , Camundongos Knockout
2.
Genome Res ; 27(6): 1016-1028, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28424354

RESUMO

The evolutionary origin of the striking genome size variations found in eukaryotes remains enigmatic. The effective size of populations, by controlling selection efficacy, is expected to be a key parameter underlying genome size evolution. However, this hypothesis has proved difficult to investigate using empirical data sets. Here, we tested this hypothesis using 22 de novo transcriptomes and low-coverage genomes of asellid isopods, which represent 11 independent habitat shifts from surface water to resource-poor groundwater. We show that these habitat shifts are associated with higher transcriptome-wide [Formula: see text] After ruling out the role of positive selection and pseudogenization, we show that these transcriptome-wide [Formula: see text] increases are the consequence of a reduction in selection efficacy imposed by the smaller effective population size of subterranean species. This reduction is paralleled by an important increase in genome size (25% increase on average), an increase also confirmed in subterranean decapods and mollusks. We also control for an adaptive impact of genome size on life history traits but find no correlation between body size, or growth rate, and genome size. We show instead that the independent increases in genome size measured in subterranean isopods are the direct consequence of increasing invasion rates by repeat elements, which are less efficiently purged out by purifying selection. Contrary to selection efficacy, polymorphism is not correlated to genome size. We propose that recent demographic fluctuations and the difficulty of observing polymorphism variation in polymorphism-poor species can obfuscate the link between effective population size and genome size when polymorphism data are used alone.


Assuntos
Especiação Genética , Tamanho do Genoma , Isópodes/genética , Filogenia , Seleção Genética , Animais , Decápodes/classificação , Decápodes/genética , Sequenciamento de Nucleotídeos em Larga Escala , Isópodes/classificação , Repetições de Microssatélites , Moluscos/classificação , Moluscos/genética , Polimorfismo Genético , Transcriptoma
3.
Cell Tissue Res ; 380(3): 565-579, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32043208

RESUMO

The RNASET2 ribonuclease, belonging to the highly conserved RH/T2/s RNase gene family, has been recently shown to modulate inflammatory processes in both vertebrates and invertebrates. Indeed, the RNASET2 protein acts as a chemoattractor for macrophages in both in vitro and in vivo experimental settings and its expression significantly increases following bacterial infections. Moreover, we recently observed that injection of human recombinant RNASET2 protein in the body wall of the medicinal leech (a consolidated invertebrate model for both immune response and tissue regeneration) not only induced immune cell recruitment but also apparently triggered massive connective tissue remodelling as well. Based on these data, we evaluate here a possible role of leech recombinant RNASET2 protein (rHvRNASET2) in connective tissue remodelling by characterizing the cell types involved in this process through histochemical, morphological and immunofluorescent assays. Moreover, a time-course expression analysis of newly synthesized pro-collagen1α1 (COL1α1) and basic FGF receptor (bFGFR, a known fibroblast marker) following rHvRNASET2 injection in the leech body wall further supported the occurrence of rHvRNASET2-mediated matrix remodelling. Human MRC-5 fibroblast cells were also investigated in order to evaluate their pattern of collagen neosynthesis driven by rHvRNASET2 injection.Taken together, the data reported in this work provide compelling evidence in support of a pleiotropic role for RNASET2 in orchestrating an evolutionarily conserved crosstalk between inflammatory response and regenerative process, based on macrophage recruitment and fibroblast activation, coupled to a massive extracellular reorganization.


Assuntos
Colágeno Tipo I/metabolismo , Tecido Conjuntivo/efeitos dos fármacos , Hirudo medicinalis/efeitos dos fármacos , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Proteínas Recombinantes/farmacologia , Ribonucleases/farmacologia , Animais , Linhagem Celular , Cadeia alfa 1 do Colágeno Tipo I , Tecido Conjuntivo/fisiologia , Fibroblastos/efeitos dos fármacos , Humanos
4.
Methods ; 134-135: 50-55, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29269151

RESUMO

Muscle stem cells or satellite cells are required for skeletal muscle regeneration. It has been shown that the satellite cell microenvironment, including neighboring cells such as endothelial cells, macrophages or fibroblasts are essential for complete and efficient regeneration. A deficient behavior of these cells compromises regeneration. Therefore, there is a strong interest in understanding the cellular and molecular interactions at work between these cell types during muscle regeneration. Fluorescence-activated cell sorting allows to isolate these four cell types at different time points of regeneration, for further high throughput or behavioral experiments. We present here a method for the concomitant isolation of 4 cell types present in the regenerating skeletal muscle: muscle stem cells, endothelial cells, fibro-adipogenic precursor cells and macrophages.


Assuntos
Separação Celular/métodos , Citometria de Fluxo/métodos , Músculo Esquelético/diagnóstico por imagem , Células Satélites de Músculo Esquelético/citologia , Adipogenia/genética , Animais , Diferenciação Celular/genética , Humanos , Camundongos , Desenvolvimento Muscular/genética , Músculo Esquelético/metabolismo , Regeneração/genética , Células Satélites de Músculo Esquelético/metabolismo
5.
Cell Death Discov ; 9(1): 224, 2023 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-37402716

RESUMO

Lack of dystrophin expression is the underlying genetic basis for Duchenne muscular dystrophy (DMD). However, disease severity varies between patients, based on specific genetic modifiers. D2-mdx is a model for severe DMD that exhibits exacerbated muscle degeneration and failure to regenerate even in the juvenile stage of the disease. We show that poor regeneration of juvenile D2-mdx muscles is associated with an enhanced inflammatory response to muscle damage that fails to resolve efficiently and supports the excessive accumulation of fibroadipogenic progenitors (FAPs), leading to increased fibrosis. Unexpectedly, the extent of damage and degeneration in juvenile D2-mdx muscle is significantly reduced in adults, and is associated with the restoration of the inflammatory and FAP responses to muscle injury. These improvements enhance regenerative myogenesis in the adult D2-mdx muscle, reaching levels comparable to the milder B10-mdx model of DMD. Ex vivo co-culture of healthy satellite cells (SCs) with juvenile D2-mdx FAPs reduces their fusion efficacy. Wild-type juvenile D2 mice also manifest regenerative myogenic deficit and glucocorticoid treatment improves their muscle regeneration. Our findings indicate that aberrant stromal cell responses contribute to poor regenerative myogenesis and greater muscle degeneration in juvenile D2-mdx muscles and reversal of this reduces pathology in adult D2-mdx muscle, identifying these responses as a potential therapeutic target for the treatment of DMD.

6.
bioRxiv ; 2023 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-37034785

RESUMO

Lack of dystrophin is the genetic basis for the Duchenne muscular dystrophy (DMD). However, disease severity varies between patients, based on specific genetic modifiers. D2- mdx is a model for severe DMD that exhibits exacerbated muscle degeneration and failure to regenerate even in the juvenile stage of the disease. We show that poor regeneration of juvenile D2- mdx muscles is associated with enhanced inflammatory response to muscle damage that fails to resolve efficiently and supports excessive accumulation of fibroadipogenic progenitors (FAPs). Unexpectedly, the extent of damage and degeneration of juvenile D2- mdx muscle is reduced in adults and is associated with the restoration of the inflammatory and FAP responses to muscle injury. These improvements enhance myogenesis in the adult D2- mdx muscle, reaching levels comparable to the milder (B10- mdx ) mouse model of DMD. Ex vivo co-culture of healthy satellite cells (SCs) with the juvenile D2- mdx FAPs reduced their fusion efficacy and in vivo glucocorticoid treatment of juvenile D2 mouse improved muscle regeneration. Our findings indicate that aberrant stromal cell response contributes to poor myogenesis and greater muscle degeneration in dystrophic juvenile D2- mdx muscles and reversal of this reduces pathology in adult D2- mdx mouse muscle, identifying these as therapeutic targets to treat dystrophic DMD muscles.

7.
Blood ; 116(23): 4795-805, 2010 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-20733157

RESUMO

This study investigated the role of the ETS transcription factor Fli-1 in adult myelopoiesis using new transgenic mice allowing inducible Fli-1 gene deletion. Fli-1 deletion in adult induced mild thrombocytopenia associated with a drastic decrease in large mature megakaryocytes number. Bone marrow bipotent megakaryocytic-erythrocytic progenitors (MEPs) increased by 50% without increase in erythrocytic and megakaryocytic common myeloid progenitor progeny, suggesting increased production from upstream stem cells. These MEPs were almost unable to generate pure colonies containing large mature megakaryocytes, but generated the same total number of colonies mainly identifiable as erythroid colonies containing a reduced number of more differentiated cells. Cytological and fluorescence-activated cell sorting analyses of MEP progeny in semisolid and liquid cultures confirmed the drastic decrease in large mature megakaryocytes but revealed a surprisingly modest (50%) reduction of CD41-positive cells indicating the persistence of a megakaryocytic commitment potential. Symmetrical increase and decrease of monocytic and granulocytic progenitors were also observed in the progeny of purified granulocytic-monocytic progenitors and common myeloid progenitors. In summary, this study indicates that Fli-1 controls several lineages commitment decisions at the stem cell, MEP, and granulocytic-monocytic progenitor levels, stimulates the proliferation of committed erythrocytic progenitors at the expense of their differentiation, and is a major regulator of late stages of megakaryocytic differentiation.


Assuntos
Diferenciação Celular/genética , Linhagem da Célula/genética , Eritrócitos/citologia , Eritropoese/genética , Megacariócitos/citologia , Proteína Proto-Oncogênica c-fli-1/genética , Animais , Western Blotting , Proliferação de Células , Separação Celular , Citometria de Fluxo , Deleção de Genes , Células-Tronco Hematopoéticas/citologia , Camundongos , Camundongos Transgênicos , Células Mieloides/citologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
8.
Neurology ; 98(21): e2108-e2119, 2022 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-35351794

RESUMO

BACKGROUND AND OBJECTIVES: The idiopathic inflammatory myopathy dermatomyositis is an acquired disease that involves muscle, lung, and skin impairments. Patients with dermatomyositis show a wide range of severity of proximal skeletal muscle weakness, associated with inflammatory infiltrates, vasculitis, capillary dropout, and perifascicular myofiber atrophy. Muscles of patients with dermatomyositis show signs of muscle regeneration. Because muscle stem cells (MuSCs) are responsible for myofiber repair, we wondered whether the proliferative properties of MuSCs are altered in dermatomyositis muscle. We investigated the role of type I interferon (IFN-I) in this process because dermatomyositis is associated with sustained inflammation with high IFN-I levels. METHODS: MuSCs isolated from normal muscles and those from adult and juvenile patients with dermatomyositis were grown in culture and analyzed in vitro for their proliferating properties, myogenic capacities, and senescence. Gain- and loss-of-function experiments were performed to assess the role of IFN-I signaling in the proliferative capacities of MuSCs. RESULTS: MuSCs derived from 8 adult patients with dermatomyositis (DM-MuSCs) (5 severe form and 3 mild form, established from histologic evaluation), from 3 patients with juvenile dermatomyositis, and from normal muscle were used to analyze their myogenesis in vitro. DM-MuSCs exhibited strongly reduced proliferating capacities as compared with healthy MuSCs (-31% to -43% for mild and severe dermatomyositis, respectively), leading to poor myotube formation (-36% to -71%). DM-MuSCs were enriched in senescent, ß-galactosidase-positive cells, partly explaining the proliferation defect. Gain- and loss-of-function experiments were performed to assess the role of IFN-I on the proliferative capacity of MuSCs. High concentrations of IFN-I decreased the proliferation of healthy MuSCs. Similarly, conditioned medium from DM-MuSCs decreased the proliferation of healthy MuSCs (-15% to -22%), suggesting the delivery of an autocrine effector. Pharmacologic blockade of IFN signaling (using ruxolitinib or anti-IFN receptor antibodies) in DM-MuSCs rescued their proliferation up to the control values. DISCUSSION: These results show that autocrine IFN-I signaling prevents MuSC expansion, leading to muscle repair deficit. This process may explain the persistent muscle weakness observed in patients with severe dermatomyositis.


Assuntos
Dermatomiosite , Interferon Tipo I , Adulto , Proliferação de Células , Dermatomiosite/patologia , Humanos , Debilidade Muscular/patologia , Músculo Esquelético/patologia , Transdução de Sinais
9.
Antioxidants (Basel) ; 11(8)2022 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-36009247

RESUMO

Atherosclerosis is associated with low-grade inflammation involving circulating monocytes. It has been shown that the levels of intermediate pro-inflammatory monocytes are associated with cardiovascular mortality and risk of ischemic stroke. It also has been shown that physical activity (PA) decreases inflammation markers, incidence of strokes, and mortality. In this cross-sectional study, we tested the effect of PA on circulating monocytes phenotype rate. A total of 29 patients with a carotid stenosis > 50% were recruited. Levels of physical activity (MET.min/week) were measured by the GPAQ questionnaire, arterial samples of blood were collected to analyze monocyte phenotype (classical, intermediate and non-classical) assessed by flow cytometry, and venous blood samples were used to dose antioxidant activity and oxidative damage. Antioxidant capacity was reduced and oxidative damage increased in patients. There was a significant decrease in the percentage of classical and intermediate monocytes in moderately active patients as compared with non-active and highly active patients. Inversely, the rate of non-classical monocytes increased in moderately active patients. Intense PA appears to blunt the beneficial effects of moderate PA. Our study also suggests that PA could be beneficial in such patients by reducing the rate of intermediate monocytes known to predict the risk of ischemic stroke and by increasing the non-classical monocytes involved in lesions' healing. Nevertheless, a longitudinal study would be necessary to confirm this hypothesis.

10.
Trials ; 23(1): 145, 2022 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-35164816

RESUMO

BACKGROUND: Carotid atherosclerotic plaques remain silent until their rupture, which may lead to detrimental ischemic events such as strokes. This is due, in part, to intraplaque hemorrhages (IPH) and the resulting inflammatory processes, which may promote carotid plaque vulnerability. Currently, the benefits of carotid endarterectomy remain unclear for asymptomatic patients. Interestingly, the completion of physical activity (PA) may have beneficial effects; however, the paucity of current data warrants robust longitudinal interventions. We therefore aim to study the effects of a 6-month longitudinal personalized home-based PA program on IPH, biological, and inflammatory markers in asymptomatic stroke patients. METHODS: Eighty patients (≥ 18 years old) will be recruited for the Physical Activity and Carotid Atherosclerotic Plaque Hemorrhage (PACAPh) clinical trial from the Hospices Civils de Lyon. Patients will be eligible if they present with carotid stenosis ≥ 50% and are asymptomatic from any ischemic events for at least 6 months. Recruited patients will be randomized into either a PA or a control group, and assessed at baseline and after 6 months. At both time points, all patients will be assessed using magnetic resonance imaging to assess IPH, blood sampling to measure inflammatory markers and monocytic phenotyping, PA and sedentary behavior questionnaires, 6-min walking test, and maximal isometric quadricep contraction test. The randomized PA intervention will consist of reaching a daily walking step goal individually tailored to each patient. Steps will be collected using a wirelessly connected wristband. The number of steps completed by individuals in the PA group will be re-evaluated bimonthly to encourage walking habits. DISCUSSION: The PACAPh study is the first of its kind representing a feasible, easily accessible therapeutic strategy for asymptomatic stroke patients. We hypothesize that the personalized home-based PA program will reduce IPH and modulate inflammatory and biological parameters in patients presenting with carotid plaques. If the results of the PACAPh study prove to be beneficial on such health parameters, the implementation of such kind of intervention in the daily treatment of these patients would be an advantageous and cost-effective practice to adopt globally. TRIAL REGISTRATION: This study has been approved by the National Ethics Committee (IDRCB:2019-A01543-54/SI:19.06.21.40640). ClinicalTrials.gov NCT04053166.


Assuntos
Estenose das Carótidas , Endarterectomia das Carótidas , Placa Aterosclerótica , Acidente Vascular Cerebral , Adolescente , Adulto , Artérias Carótidas , Estenose das Carótidas/diagnóstico por imagem , Estenose das Carótidas/cirurgia , Exercício Físico , Humanos , Imageamento por Ressonância Magnética , Ensaios Clínicos Controlados Aleatórios como Assunto , Acidente Vascular Cerebral/diagnóstico , Acidente Vascular Cerebral/etiologia
11.
iScience ; 24(3): 102210, 2021 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-33733070

RESUMO

Transcription factors are key players in a broad range of cellular processes such as cell-fate decision. Understanding how they act to control these processes is of critical importance for therapy purposes. FLI-1 controls several hematopoietic lineage differentiation including megakaryopoiesis and erythropoiesis. Its aberrant expression is often observed in cancer and is associated with poor prognosis. We showed that FLI-1 interacts with the LDB1 complex, which also plays critical roles in erythropoiesis and megakaryopoiesis. In this study, we aimed to unravel how FLI-1 and the LDB1 complex act together in murine erythroleukemia cells and in megakaryocyte. Combining omics techniques, we show that FLI-1 enables the recruitment of the LDB1 complex to regulatory sequences of megakaryocytic genes and to enhancers. We show as well for the first time that FLI-1 is able to modulate the 3D chromatin organization by promoting chromatin looping between enhancers and promoters most likely through the LDB1 complex.

12.
FEMS Yeast Res ; 10(6): 757-68, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20662936

RESUMO

Saccharomyces cerevisiae has proved to be an interesting model for studies of evolution, with whole-genome duplication shown to have played an important role in the evolution of this species. This phenomenon depends on the formation of a transient stable polyploid state. Previous studies have reported polyploidy to be an unstable state in yeast, but here, we describe a polyploid population of S. cerevisiae. The evolution of higher eukaryotes has also involved the development of different systems of sexual reproduction, the choice between self-fertilization and out-crossing becoming a key issue. Saccharomyces cerevisiae is a hermaphrodite eukaryote, despite the theoretical genetic disadvantages of this strategy, in which self-fertilization occurs. We describe, for the first time, a near-dioecious (with separate sexes) population in this species. Mating type and the MAT locus display complex segregations. Essentially, each strain produces, by meiosis, spores of only one mating type: mata or matalpha. Moreover, strains are heterothallic, and diploid nonmating clones generated from a single spore do not sporulate. These three properties limit self-fertilization and strongly favour out-crossing. We suggest that the shift in sexual strategy, from hermaphroditism to dioecy, is specific to the brewing process, which overcomes the sexual isolation probably found in natural biotopes.


Assuntos
Meiose , Poliploidia , Recombinação Genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/genética , Segregação de Cromossomos , Genes Fúngicos Tipo Acasalamento , Esporos Fúngicos/genética
13.
Methods Mol Biol ; 2002: 121-128, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30242569

RESUMO

Angiogenesis, the growth of new blood vessels, is crucial for efficient skeletal muscle regeneration. Myogenesis and angiogenesis take place concomitantly during muscle regeneration. Myogenic precursor cells (MPCs) are in close proximity to vessels and interact with neighboring endothelial cells (ECs) to expand and differentiate. To demonstrate functional interplay between the two cell types, we established a robust and predictive ex vivo assay to evaluate activity of MPCs on angiogenesis and vice-et-versa, of ECs on myogenesis. Here, we describe an optimized three-dimensional co-culture protocol for the assessment of biological interactions between MPCs and ECs during skeletal muscle regeneration.


Assuntos
Técnicas de Cultura de Células/métodos , Técnicas de Cocultura/métodos , Células Endoteliais/citologia , Músculo Esquelético/citologia , Neovascularização Fisiológica , Regeneração , Células-Tronco/citologia , Humanos , Desenvolvimento Muscular
14.
Methods Mol Biol ; 1732: 539-549, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29480498

RESUMO

In this chapter, we describe the methods to isolate and culture muscle stem cells (MuSCs) from murine skeletal muscle in order to decipher the intrinsic effect of AMP-activated kinase activity on MuSC fate. Culture of MuSCs is a powerful model to recapitulate every step of stem cell behavior observed in vivo: activation, proliferation, differentiation, fusion and also self-renewal. We provide the detailed procedures to isolate pure MuSCs by a flow cytometry-based method using the selection of a combination of specific markers and to characterize MuSC fate (quiescence, activation, and differentiation) in response to AMPK activity modulation by assessing of the expression of stem cell (e.g., Pax7) and myogenic marker (e.g., MyoD).


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Ativação Enzimática/fisiologia , Desenvolvimento Muscular/fisiologia , Mioblastos/fisiologia , Animais , Biomarcadores/análise , Biomarcadores/metabolismo , Diferenciação Celular/fisiologia , Separação Celular/instrumentação , Separação Celular/métodos , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Ativadores de Enzimas/farmacologia , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo/instrumentação , Citometria de Fluxo/métodos , Antígeno Ki-67/análise , Antígeno Ki-67/metabolismo , Camundongos , Desenvolvimento Muscular/efeitos dos fármacos , Músculo Esquelético/citologia , Proteína MyoD/análise , Proteína MyoD/metabolismo , Fator de Transcrição PAX7/análise , Fator de Transcrição PAX7/metabolismo , Cultura Primária de Células/instrumentação , Cultura Primária de Células/métodos
15.
Arthritis Rheumatol ; 70(1): 134-145, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28941175

RESUMO

OBJECTIVE: Juvenile dermatomyositis (JDM) is an inflammatory pediatric myopathy characterized by focal capillary loss in muscle, followed by progressive recovery upon adequate treatment with immunomodulating drugs, although some patients remain refractory to treatment. While the underlying mechanism of capillary depletion remains uncertain, recent studies have identified an up-regulation of type I interferon (IFN) expression specific to JDM. Given that myogenic precursor cells (MPCs) exert proangiogenic activity during normal skeletal muscle regeneration, we hypothesized that they may also modulate vascular remodeling/angiogenesis during JDM. The aim of this study was to investigate that hypothesis. METHODS: Human cell cocultures were used to analyze angiogenic properties in patients with JDM, patients with Duchenne's muscular dystrophy (DMD) (control patients for vascular remodeling), and healthy control subjects. Transcriptome analysis was used to examine muscle-derived MPCs. Histologic analysis of type I IFN in muscle biopsy samples was also performed. RESULTS: Using human cell cocultures, we showed highly angiogenic properties of MPCs from JDM patients in association with the expression of an angiogenic molecular signature. Transcriptome analysis of MPCs freshly isolated from muscle samples revealed type I IFN as the master regulator of the most up-regulated genes in JDM-derived MPCs. Functionally, treatment of normal MPCs with type I IFN recapitulated the molecular pattern and the proangiogenic functions of JDM-derived MPCs. In vivo histologic investigation showed that MPCs synthesized type I IFN and major proangiogenic molecules in JDM muscle. Moreover, MPCs derived from JDM muscles that were characterized by strong vasculopathy produced higher levels of type I IFN, confirming MPCs as a cellular source of type I IFN during JDM, and this correlated with the severity of the disease. CONCLUSION: These results demonstrate a new type I IFN pathway in JDM that activates the production of angiogenic effectors by MPCs, triggering their proangiogenic function to promote vessel recovery and muscle reconstruction.


Assuntos
Dermatomiosite/patologia , Interferon Tipo I/metabolismo , Músculo Esquelético/patologia , Neovascularização Patológica/metabolismo , Células-Tronco/metabolismo , Técnicas de Cultura de Células , Ensaios de Migração Celular , Proliferação de Células , Criança , Pré-Escolar , Dermatomiosite/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Imuno-Histoquímica , Masculino , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/patologia , Células-Tronco/patologia
16.
Stem Cell Reports ; 9(6): 2018-2033, 2017 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-29198825

RESUMO

In skeletal muscle, new functions for vessels have recently emerged beyond oxygen and nutrient supply, through the interactions that vascular cells establish with muscle stem cells. Here, we demonstrate in human and mouse that endothelial cells (ECs) and myogenic progenitor cells (MPCs) interacted together to couple myogenesis and angiogenesis in vitro and in vivo during skeletal muscle regeneration. Kinetics of gene expression of ECs and MPCs sorted at different time points of regeneration identified three effectors secreted by both ECs and MPCs. Apelin, Oncostatin M, and Periostin were shown to control myogenesis/angiogenesis coupling in vitro and to be required for myogenesis and vessel formation during muscle regeneration in vivo. Furthermore, restorative macrophages, which have been previously shown to support myogenesis in vivo, were shown in a 3D triculture model to stimulate myogenesis/angiogenesis coupling, notably through Oncostatin M production. Our data demonstrate that restorative macrophages orchestrate muscle regeneration by controlling myogenesis/angiogenesis coupling.


Assuntos
Diferenciação Celular/genética , Desenvolvimento Muscular/genética , Músculo Esquelético/crescimento & desenvolvimento , Neovascularização Fisiológica/genética , Regeneração/genética , Animais , Apelina/genética , Vasos Sanguíneos/crescimento & desenvolvimento , Vasos Sanguíneos/metabolismo , Moléculas de Adesão Celular/genética , Movimento Celular/genética , Células Progenitoras Endoteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Macrófagos/metabolismo , Camundongos , Mioblastos/citologia , Mioblastos/metabolismo , Oncostatina M/genética , Células-Tronco/citologia , Células-Tronco/metabolismo , Cicatrização
18.
PLoS One ; 11(4): e0153860, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27089435

RESUMO

This study aimed at reinvestigating the controversial contribution of Notch signaling to megakaryocytic lineage development. For that purpose, we combined colony assays and single cells progeny analyses of purified megakaryocyte-erythroid progenitors (MEP) after short-term cultures on recombinant Notch ligand rDLL1. We showed that Notch activation stimulated the SCF-dependent and preferential amplification of Kit+ erythroid and bipotent progenitors while favoring commitment towards the erythroid at the expense of megakaryocytic lineage. Interestingly, we also identified a CD9High MEP subset that spontaneously generated almost exclusively megakaryocytic progeny mainly composed of single megakaryocytes. We showed that Notch activation decreased the extent of polyploidization and maturation of megakaryocytes, increased the size of megakaryocytic colonies and surprisingly restored the generation of erythroid and mixed colonies by this CD9High MEP subset. Importantly, the size increase of megakaryocytic colonies occurred at the expense of the production of single megakaryocytes and the restoration of colonies of alternative lineages occurred at the expense of the whole megakaryocytic progeny. Altogether, these results indicate that Notch activation is able to extend the number of divisions of MK-committed CD9High MEPs before terminal maturation while allowing a fraction of them to generate alternative lineages. This unexpected plasticity of MK-committed progenitors revealed upon Notch activation helps to better understand the functional promiscuity between megakaryocytic lineage and hematopoietic stem cells.


Assuntos
Diferenciação Celular , Linhagem da Célula , Hematopoese/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Células Progenitoras de Megacariócitos/citologia , Receptores Notch/metabolismo , Tetraspanina 29/metabolismo , Animais , Antígenos CD34/genética , Antígenos CD34/metabolismo , Proteínas de Ligação ao Cálcio , Ciclo Celular , Proliferação de Células , Células Cultivadas , Células Precursoras Eritroides/citologia , Células Precursoras Eritroides/metabolismo , Feminino , Citometria de Fluxo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Células Progenitoras de Megacariócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores Notch/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tetraspanina 29/genética
19.
J Leukoc Biol ; 99(2): 311-9, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26336156

RESUMO

M-CSF and G-CSF are instructive cytokines that specifically induce differentiation of bipotent myeloid progenitors into macrophages and granulocytes, respectively. Through morphology and colony assay studies, flow cytometry analysis of specific markers, and expression of myeloid transcription factors, we show here that the Eger/Fms cell line is composed of cells whose differentiation fate is instructed by M-CSF and G-CSF, thus representing a good in vitro model of myeloid bipotent progenitors. Consistent with the essential role of ERK1/2 during macrophage differentiation and defects of macrophagic differentiation in native ERK1(-/-) progenitors, ERK signaling is strongly activated in Eger/Fms cells upon M-CSF-induced macrophagic differentiation but only to a very small extent during G-CSF-induced granulocytic differentiation. Previous in vivo studies indicated a key role of Fli-1 in myeloid differentiation and demonstrated its weak expression during macrophagic differentiation with a strong expression during granulocytic differentiation. Here, we demonstrated that this effect could be mediated by a differential regulation of protein kinase Cδ (PKCd) on Fli-1 expression in response to M-CSF and G-CSF. With the use of knockdown of PKCd by small interfering RNA, we demonstrated that M-CSF activates PKCd, which in turn, inhibits Fli-1 expression and granulocytic differentiation. Finally, we studied the connection between ERK and PKCd and showed that in the presence of the MEK inhibitor U0126, PKCd expression is decreased, and Fli-1 expression is increased in response to M-CSF. Altogether, we demonstrated that in bipotent myeloid cells, M-CSF promotes macrophagic over granulocytic differentiation by inducing ERK activation but also PKCd expression, which in turn, down-regulates Fli-1 expression and prevents granulocytic differentiation.


Assuntos
Granulócitos/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/citologia , Células-Tronco Multipotentes/efeitos dos fármacos , Mielopoese/efeitos dos fármacos , Animais , Butadienos/farmacologia , Linhagem Celular , Ensaio de Unidades Formadoras de Colônias , Ativação Enzimática/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/farmacologia , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Knockout , Proteína Quinase 3 Ativada por Mitógeno/deficiência , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Mielopoese/fisiologia , Nitrilas/farmacologia , Proteína Quinase C-delta/genética , Proteína Quinase C-delta/fisiologia , Proteína Proto-Oncogênica c-fli-1/biossíntese , Proteína Proto-Oncogênica c-fli-1/genética , Interferência de RNA , RNA Interferente Pequeno/genética
20.
Sci Rep ; 6: 19967, 2016 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-26822159

RESUMO

Endosymbiotic associations constitute a driving force in the ecological and evolutionary diversification of metazoan organisms. Little is known about whether and how symbiotic cells are coordinated according to host physiology. Here, we use the nutritional symbiosis between the insect pest, Acyrthosiphon pisum, and its obligate symbiont, Buchnera aphidicola, as a model system. We have developed a novel approach for unculturable bacteria, based on flow cytometry, and used this method to estimate the absolute numbers of symbionts at key stages of aphid life. The endosymbiont population increases exponentially throughout nymphal development, showing a growing rate which has never been characterized by indirect molecular techniques. Using histology and imaging techniques, we have shown that the endosymbiont-bearing cells (bacteriocytes) increase significantly in number and size during the nymphal development, and clustering in the insect abdomen. Once adulthood is reached and the laying period has begun, the dynamics of symbiont and host cells is reversed: the number of endosymbionts decreases progressively and the bacteriocyte structure degenerates during insect aging. In summary, these results show a coordination of the cellular dynamics between bacteriocytes and primary symbionts and reveal a fine-tuning of aphid symbiotic cells to the nutritional demand imposed by the host physiology throughout development.


Assuntos
Afídeos/microbiologia , Simbiose , Animais , Carga Bacteriana , Buchnera/fisiologia , Citometria de Fluxo
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