RESUMO
Genome-wide analysis of transcription factors and epigenomic features is instrumental to shed light on DNA-templated regulatory processes such as transcription, cellular differentiation or to monitor cellular responses to environmental cues. Two decades of technological developments have led to a rich set of approaches progressively pushing the limits of epigenetic profiling towards single cells. More recently, disruptive technologies using innovative biochemistry came into play. Assays such as CUT&RUN, CUT&Tag and variations thereof show considerable potential to survey multiple TFs or histone modifications in parallel from a single experiment and in native conditions. These are in the path to become the dominant assays for genome-wide analysis of TFs and chromatin modifications in bulk, single-cell, and spatial genomic applications. The principles together with pros and cons are discussed.
Assuntos
Cromatina , Histonas , Humanos , Cromatina/genética , Fatores de Transcrição/genética , Genômica , Epigenômica/métodosRESUMO
Acute erythroleukemia (AEL or acute myeloid leukemia [AML]-M6) is a rare but aggressive hematologic malignancy. Previous studies showed that AEL leukemic cells often carry complex karyotypes and mutations in known AML-associated oncogenes. To better define the underlying molecular mechanisms driving the erythroid phenotype, we studied a series of 33 AEL samples representing 3 genetic AEL subgroups including TP53-mutated, epigenetic regulator-mutated (eg, DNMT3A, TET2, or IDH2), and undefined cases with low mutational burden. We established an erythroid vs myeloid transcriptome-based space in which, independently of the molecular subgroup, the majority of the AEL samples exhibited a unique mapping different from both non-M6 AML and myelodysplastic syndrome samples. Notably, >25% of AEL patients, including in the genetically undefined subgroup, showed aberrant expression of key transcriptional regulators, including SKI, ERG, and ETO2. Ectopic expression of these factors in murine erythroid progenitors blocked in vitro erythroid differentiation and led to immortalization associated with decreased chromatin accessibility at GATA1-binding sites and functional interference with GATA1 activity. In vivo models showed development of lethal erythroid, mixed erythroid/myeloid, or other malignancies depending on the cell population in which AEL-associated alterations were expressed. Collectively, our data indicate that AEL is a molecularly heterogeneous disease with an erythroid identity that results in part from the aberrant activity of key erythroid transcription factors in hematopoietic stem or progenitor cells.
Assuntos
Leucemia Eritroblástica Aguda/genética , Proteínas de Neoplasias/fisiologia , Fatores de Transcrição/fisiologia , Transcriptoma , Adulto , Animais , Transformação Celular Neoplásica/genética , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Dioxigenases , Eritroblastos/metabolismo , Eritropoese/genética , Feminino , Fator de Transcrição GATA1/deficiência , Fator de Transcrição GATA1/genética , Técnicas de Introdução de Genes , Heterogeneidade Genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Pessoa de Meia-Idade , Mutação , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/metabolismo , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , RNA-Seq , Quimera por Radiação , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Fatores de Transcrição/genética , Regulador Transcricional ERG/genética , Regulador Transcricional ERG/fisiologia , Sequenciamento do Exoma , Adulto JovemRESUMO
Lyl1 encodes a hematopoietic- and endothelial-specific bHLH transcription factor. Lyl1-deficient mice are viable, but they display mild hematopoietic and vascular defects. Specifically, LYL1 is required for the maturation and stabilization of blood vessel endothelial adherens junctions. Here, we report that young adult Lyl1-/- mice exhibit transient overweight associated with general expansion of adipose tissue, without signs of metabolic disorder and unrelated to food intake. The increased fat tissue development in Lyl1-/- mice resulted from earlier differentiation of adipose stem cells (ASCs) into adipocytes through noncell autonomous mechanisms. Specifically, we found that in Lyl1-/- mice, the adipose tissue vascular structures are immature, as indicated by their high permeability, reduced coverage by pericytes, lower recruitment of VE-cadherin and ZO1 at cell junctions, and more prone to angiogenesis. Together, our data show that in Lyl1-/- mice, the impaired vascular compartment of the adipose niche promotes ASC differentiation, leading to early adipocyte expansion and premature ASC depletion. Our study highlights the major structural role of the adipose tissue vascular niche in coordinating stem cell self-renewal and differentiation into adipocytes.
Assuntos
Tecido Adiposo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Proteínas de Neoplasias/deficiência , Neovascularização Patológica , Nicho de Células-Tronco , Células-Tronco/metabolismo , Tecido Adiposo/irrigação sanguínea , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/metabolismo , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Células-Tronco/patologiaRESUMO
Maturation of newly formed vessels is a multistep phenomenon during which functional endothelial barriers are established. Disruption of vessel integrity is an important feature in many physiological and pathological processes. We previously reported that lymphoblastic leukemia-derived sequence 1 (LYL1) is required for the late stages of postnatal angiogenesis to limit the formation of new blood vessels, notably by regulating the activity of the small GTPase Rap1. In this study, we show that LYL1 is also required during the formation of the mature endothelial barrier in the lungs of adult mice. Specifically, LYL1 knockdown in human endothelial cells downregulated the expression of ARHGAP21 and ARHGAP24, which encode two Rho GTPase-activating proteins, and this was correlated with increased RhoA activity and reorganization of the actin cytoskeleton into stress fibers. Importantly, in lungs of Lyl1-deficient mice, both vascular endothelial (VE)-cadherin and p120-catenin were poorly recruited to endothelial adherens junctions, indicative of defective cell-cell junctions. Consistent with this, higher Evans blue dye extravasation, edema, and leukocyte infiltration in the lung parenchyma of Lyl1-/- mice than in wild-type littermates confirmed that lung vascular permeability is constitutively elevated in Lyl1-/- adult mice. Our data show that LYL1 acts as a stabilizing signal for adherens junction formation by operating upstream of VE-cadherin and of the two GTPases Rap1 and RhoA. As increased vascular permeability is a key feature and a major mechanism of acute respiratory distress syndrome, molecules that regulate LYL1 activity could represent additional tools to modify the endothelial barrier permeability.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Permeabilidade da Membrana Celular , Endotélio Vascular/patologia , Células Endoteliais da Veia Umbilical Humana/patologia , Pulmão/patologia , Proteínas de Neoplasias/fisiologia , Actinas/genética , Actinas/metabolismo , Animais , Western Blotting , Células Cultivadas , Endotélio Vascular/metabolismo , Imunofluorescência , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Técnicas Imunoenzimáticas , Pulmão/irrigação sanguínea , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fibras de Estresse , Proteínas rap1 de Ligação ao GTP/genética , Proteínas rap1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Precise insertion of fluorescent tags by CRISPR-Cas9-mediated homologous recombination (HR) in mammalian genes is a powerful tool allowing to study gene function and protein gene products. Here, we present a protocol for efficient HR-mediated targeted insertion of fluorescent markers in the genome of hard-to-transfect erythroid cell lines MEL (mouse erythroleukemic) and MEDEP (mouse ES cell-derived erythroid progenitor line). We describe steps for plasmid construction, electroporation, amplification, and verification of genome editing. We then detail procedures for isolating positive clones and validating knockin clones. For complete details on the use and execution of this protocol, please refer to Deleuze et al.1.
Assuntos
Sistemas CRISPR-Cas , Células Eritroides , Edição de Genes , Técnicas de Introdução de Genes , Sistemas CRISPR-Cas/genética , Animais , Camundongos , Células Eritroides/metabolismo , Células Eritroides/citologia , Técnicas de Introdução de Genes/métodos , Edição de Genes/métodos , Transfecção/métodos , Linhagem Celular , Recombinação Homóloga/genética , Eletroporação/métodosRESUMO
CCR6 is a chemokine receptor that is expressed at the cell surface of Th17 cells, an IL-17- and IL-22-secreting population of CD4(+) T cells with antipathogenic, as well as inflammatory, properties. In the current study, we have determined the involvement of CCR6 in human Th17 lymphocyte migration toward inflamed tissue by analyzing the capacity of its ligands to induce arrest of these cells onto inflamed endothelium in vitro under flow conditions. We show that polarized, in situ-differentiated, skin-derived Th17 clones activated via the TCR-CD3 complex produce CCL20 in addition to IL-17 and IL-22. The latter cytokines induce, in a synergic fashion, the production of human ß-defensin (hBD)-2, but neither hBD-1 nor hBD-3, by epidermal keratinocytes. Both CCL20 and hBD-2 are capable of inducing the arrest of Th17 cells, but not Th1 or Th2 cells, on HUVEC in an CD54-dependent manner that is CCR6 specific and independent from the expression of CXCR4, reported to be an alternative receptor for hBD-2. In addition, Ag-specific activation induces a transient loss of CCR6 expression, both at the transcriptional and protein level, which occurs with slow kinetics and is not due to endogenous CCL20-mediated internalization of CCR6. Together, these results indicate that Ag-specific activation will initially contribute to CCR6-mediated Th17 cell trafficking toward and sequestration in inflamed tissue, but that it eventually results in a transitory state of nonresponsiveness to further stimulation of these cells with CCR6 ligands, thus permitting their subsequent migration out of the inflamed site.
Assuntos
Apoptose/imunologia , Velocidade do Fluxo Sanguíneo/imunologia , Quimiocina CCL20/fisiologia , Endotélio Vascular/imunologia , Mediadores da Inflamação/fisiologia , Interleucina-17/biossíntese , Linfócitos T Auxiliares-Indutores/imunologia , beta-Defensinas/fisiologia , Animais , Movimento Celular/imunologia , Células Cultivadas , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Células Epidérmicas , Epiderme/imunologia , Epiderme/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Interleucinas/biossíntese , Queratinócitos/citologia , Queratinócitos/imunologia , Queratinócitos/metabolismo , Células L , Ligantes , Ativação Linfocitária/imunologia , Camundongos , Receptores CCR6/biossíntese , Receptores CCR6/genética , Receptores CCR6/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Auxiliares-Indutores/patologia , Interleucina 22RESUMO
Targeted genome editing holds great promise in biology. However, efficient genome modification, including gene knock-in (KI), remains an unattained goal in multiple cell types and loci due to poor transfection efficiencies and low target genes expression, impeding the positive selection of recombined cells. Here, we describe a genome editing approach to achieve efficient gene targeting using hard to transfect erythroid cell lines. We demonstrate robust fluorescent protein KI efficiency in low expressed transcription factor (TF) genes (e.g., Myb or Zeb1). We further show the ability to target two independent loci in individual cells, exemplified by MYB-GFP and NuMA-Cherry double KI, allowing multicolor labeling of regulatory factors at physiological endogenous levels. Our KI tagging approach allowed us to perform genome-wide TF analysis at increased signal-to-noise ratios, and highlighted previously unidentified MYB target genes and pathways. Overall, we establish a versatile CRISPR-Cas9-based platform, offering attractive opportunities for the dissection of the erythroid differentiation process.
RESUMO
The 2 related basic helix loop helix genes, LYL1 and TAL-1 are active in hematopoietic and endothelial lineages. While Tal-1 is essential for both hematopoietic and vascular development, the role of Lyl1 appears to be distinct as deficient mice are viable and display modest hematopoietic defects. Here, we reveal a role for Lyl1 as a major regulator of adult neovascularization. Tumors implanted into Lyl1-deficient mice showed higher proliferation and angiogenesis, as evidenced by enlarged lumens, reduced pericyte coverage and increased permeability, compared with wild type littermates. Of note, Lyl1-deficient tumor vessels exhibited an up-regulation of Tal-1, the VE-Cadherin target gene, as well as Angiopoietin-2, 3 major actors in angiogenesis. Hematopoietic reconstitution experiments demonstrated that this sustained tumor angiogenesis was of endothelial origin. Moreover, the angiogenic phenotype observed in the absence of Lyl1 function was not tumor-restricted as microvessels forming in Matrigel or originating from aortic explants were also more numerous and larger than their wild-type counterparts. Finally, LYL1 depletion in human endothelial cells revealed that LYL1 controls the expression of molecules involved in the stabilization of vascular structures. Together, our data show a role for LYL1 in the postnatal maturation of newly formed blood vessels.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentais/irrigação sanguínea , Neoplasias Experimentais/metabolismo , Neovascularização Patológica/metabolismo , Angiopoietina-2/biossíntese , Angiopoietina-2/genética , Animais , Antígenos CD/biossíntese , Antígenos CD/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Caderinas/biossíntese , Caderinas/genética , Regulação Neoplásica da Expressão Gênica/genética , Hematopoese/genética , Humanos , Camundongos , Camundongos Mutantes , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Neoplasias Experimentais/genética , Neovascularização Patológica/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Regulação para Cima/genéticaRESUMO
Glioblastomas (GBM) are high-grade brain tumors, containing cells with distinct phenotypes and tumorigenic potentials, notably aggressive and treatment-resistant multipotent glioblastoma stem cells (GSC). The molecular mechanisms controlling GSC plasticity and growth have only partly been elucidated. Contact with endothelial cells and the Notch1 pathway control GSC proliferation and fate. We used three GSC cultures and glioma resections to examine the expression, regulation, and role of two transcription factors, SLUG (SNAI2) and TAL1 (SCL), involved in epithelial to mesenchymal transition (EMT), hematopoiesis, vascular identity, and treatment resistance in various cancers. In vitro, SLUG and a truncated isoform of TAL1 (TAL1-PP22) were strongly upregulated upon Notch1 activation in GSC, together with LMO2, a known cofactor of TAL1, which formed a complex with truncated TAL1. SLUG was also upregulated by TGF-ß1 treatment and by co-culture with endothelial cells. In patient samples, the full-length isoform TAL1-PP42 was expressed in all glioma grades. In contrast, SLUG and truncated TAL1 were preferentially overexpressed in GBMs. SLUG and TAL1 are expressed in the tumor microenvironment by perivascular and endothelial cells, respectively, and to a minor extent, by a fraction of epidermal growth factor receptor (EGFR) -amplified GBM cells. Mechanistically, both SLUG and truncated TAL1 reduced GSC growth after their respective overexpression. Collectively, this study provides new evidence for the role of SLUG and TAL1 in regulating GSC plasticity and growth.
RESUMO
The basic helix-loop-helix TAL-1/SCL essential for hematopoietic development is also required during vascular development for embryonic angiogenesis. We reported that TAL-1 acts positively on postnatal angiogenesis by stimulating endothelial morphogenesis. Here, we investigated the functional consequences of TAL-1 silencing in human primary endothelial cells. We found that TAL-1 knockdown caused the inhibition of in vitro tubulomorphogenesis, which was associated with a dramatic reduction in vascular endothelial cadherin (VE-cadherin) at intercellular junctions. Consistently, silencing of TAL-1 as well as of its cofactors E47 and LMO2 down-regulated VE-cadherin at both the mRNA and the protein level. Endogenous VE-cadherin transcription could be activated in nonendothelial HEK-293 cells by the sole concomitant ectopic expression of TAL-1, E47, and LMO2. Transient transfections in human primary endothelial cells derived from umbilical vein (HUVECs) demonstrated that VE-cadherin promoter activity was dependent on the integrity of a specialized E-box associated with a GATA motif and was maximal with the coexpression of the different components of the TAL-1 complex. Finally, chromatin immunoprecipitation assays showed that TAL-1 and its cofactors occupied the VE-cadherin promoter in HUVECs. Together, these data identify VE-cadherin as a bona fide target gene of the TAL-1 complex in the endothelial lineage, providing a first clue to TAL-1 function in angiogenesis.
Assuntos
Antígenos CD/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Caderinas/biossíntese , Proteínas de Ligação a DNA/fisiologia , Células Endoteliais/metabolismo , Metaloproteínas/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Fatores de Transcrição TCF/fisiologia , Regulação para Cima , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Antígenos CD/genética , Sequência de Bases , Caderinas/genética , Células Cultivadas , Colágeno , Combinação de Medicamentos , Células Endoteliais/fisiologia , Endotélio Vascular/citologia , Humanos , Proteínas com Domínio LIM , Laminina , Dados de Sequência Molecular , Neovascularização Fisiológica , Regiões Promotoras Genéticas , Ligação Proteica , Proteoglicanas , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Proteína 1 Semelhante ao Fator 7 de TranscriçãoRESUMO
OBJECTIVES: Interleukin (IL)-10 has anti-atherogenic properties. However, the molecular mechanisms involved in IL-10 protection against atherosclerosis in vivo remain poorly understood. In this study, we examined the effect of IL-10 cDNA in vivo electrotransfer on diet-induced, endothelial activation. METHODS: C57BL/6J mice were fed an atherogenic diet for 10 days. Expression of VCAM-1 and ICAM-1 was examined in the aortic sinus, a region predisposed to atherogenesis in mice, using immunohistochemistry. NF-kappaB activation was examined using a monoclonal antibody that selectively reacts with the activated form of the p65 subunit. RESULTS: We detected a low basal expression of activated NF-kappaB, VCAM-1 and ICAM-1 in the endothelium of the aortic sinus. Endothelial expression of activated NF-kappaB, VCAM-1 and ICAM-1 was markedly increased after 10 days on the atherogenic diet (p < 0.001). In vivo electrotransfer of a murine IL-10-encoding plasmid completely prevented diet-induced endothelial upregulation of activated NF-kappaB, VCAM-1 and ICAM-1 (p < 0.01). CONCLUSION: In vivo electrotransfer of IL-10 cDNA prevents diet-induced endothelial activation. These results suggest that the protective effects of IL-10 may already occur in the very early stages of atherogenesis.
Assuntos
Dieta Aterogênica , Endotélio/metabolismo , Molécula 1 de Adesão Intercelular/biossíntese , Interleucina-10/metabolismo , NF-kappa B/biossíntese , Seio Aórtico/metabolismo , Molécula 1 de Adesão de Célula Vascular/biossíntese , Animais , DNA Complementar , Eletroporação , Ativação Enzimática , Feminino , Técnicas de Transferência de Genes , Interleucina-10/genética , Camundongos , Camundongos Endogâmicos C57BL , Regulação para CimaRESUMO
Cell invasion targets specific tissues in physiological placental implantation and pathological metastasis, which raises questions about how this process is controlled. We compare dermis and endometrium capacities to support trophoblast invasion, using matching sets of human primary fibroblasts in a coculture assay with human placental explants. Substituting endometrium, the natural trophoblast target, with dermis dramatically reduces trophoblast interstitial invasion. Our data reveal that endometrium expresses a higher rate of the fibronectin (FN) extra type III domain A+ (EDA+) splicing isoform, which displays stronger matrix incorporation capacity. We demonstrate that the high FN content of the endometrium matrix, and not specifically the EDA domain, supports trophoblast invasion by showing that forced incorporation of plasma FN (EDA-) promotes efficient trophoblast invasion. We further show that the serine/arginine-rich protein serine/arginine-rich splicing factor 1 (SRSF1) is more highly expressed in endometrium and, using RNA interference, that it is involved in the higher EDA exon inclusion rate in endometrium. Our data therefore show a mechanism by which tissues can be distinguished, for their capacity to support invasion, by their different rates of EDA inclusion, linked to their SRSF1 protein levels. In the broader context of cancer pathology, the results suggest that SRSF1 might play a central role not only in the tumor cells, but also in the surrounding stroma.
Assuntos
Processamento Alternativo/genética , Fibronectinas/genética , Invasividade Neoplásica/genética , Proteínas Nucleares/genética , Placentação , Proteínas de Ligação a RNA/genética , Aborto Induzido , Endométrio/crescimento & desenvolvimento , Endométrio/metabolismo , Feminino , Fibronectinas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Proteínas Nucleares/biossíntese , Especificidade de Órgãos/genética , Placenta/metabolismo , Gravidez , Cultura Primária de Células , Proteínas de Ligação a RNA/biossíntese , Fatores de Processamento de Serina-Arginina , Trofoblastos/citologia , Trofoblastos/metabolismoRESUMO
The two related basic helix-loop-helix, TAL1 and LYL1, and their cofactor LIM-only-2 protein (LMO2) are present in blood and endothelial cells. While their crucial role in early hematopoiesis is well established, their function in endothelial cells and especially in angiogenesis is less understood. Here, we identified ANGIOPOIETIN-2 (ANG-2), which encodes a major regulator of angiogenesis, as a direct transcriptional target of TAL1, LYL1 and LMO2. Knockdown of any of the three transcription factors in human blood and lymphatic endothelial cells caused ANG-2 mRNA and protein down-regulation. Transient transfections showed that the full activity of the ANG-2 promoter required the integrity of a highly conserved Ebox-GATA composite element. Accordingly, chromatin immunoprecipitation assays demonstrated that TAL1, LYL1, LMO2 and GATA2 occupied this region of ANG-2 promoter in human endothelial cells. Furthermore, we showed that LMO2 played a central role in assembling TAL1-E47, LYL1-LYL1 or/and LYL1-TAL1 dimers with GATA2. The resulting complexes were able to activate endogenous ANG-2 expression in endothelial cells as well as in non-endothelial cells. Finally, we showed that ANG-2 gene activation during angiogenesis concurred with the up-regulation of TAL1 and LMO2. Altogether, we identified ANG-2 as a bona fide target gene of LMO2-complexes with TAL1 and/or LYL1, highlighting a new function of the three hematopoietic factors in the endothelial lineage.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Angiopoietina-2/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteínas com Domínio LIM/fisiologia , Proteínas de Neoplasias/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Angiopoietina-2/metabolismo , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sequência Conservada , Fator de Transcrição GATA2/fisiologia , Técnicas de Silenciamento de Genes , Genes Reporter , Células HEK293 , Humanos , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismo , Luciferases de Vaga-Lume/biossíntese , Luciferases de Vaga-Lume/genética , Microvasos/citologia , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Multimerização Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno/genética , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Transcrição GênicaRESUMO
Dengue virus (DV) is an important re-emerging arthropod-borne virus of global significance. The defining characteristic of DV infection-associated pathology is haemorrhagic fever, which often leads to a fatal shock-like syndrome (DHF/DSS) owing to an increase in vascular endothelial permeability. Here, we show, in a viral dose-dependent manner, that DV-infected immature dendritic cells overproduce soluble gelatinolytic matrix metalloproteinase (MMP)-9-and to a lesser extent MMP-2-which enhances endothelial permeability, but which are reduced by specific inhibitors and a neutralizing anti-MMP-9 antibody. This permeability was associated with a loss of expression of the platelet endothelial adhesion molecule 1 (PECAM-1) and vascular endothelium (VE)-cadherin cell adhesion molecules and redistribution of F-actin fibres. These in vitro observations were confirmed in an in vivo vascular-leakage mouse model. These results provide a molecular basis for DHF/DSS that could be a basis for a general model of haemorrhagic fever-inducing viruses, and identify a new therapeutic approach for the treatment of viral-induced vascular leakage by specifically targeting gelatinolytic metalloproteases.
Assuntos
Permeabilidade Capilar , Células Dendríticas/virologia , Vírus da Dengue , Metaloproteases/metabolismo , Dengue Grave/metabolismo , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Células Dendríticas/imunologia , Vírus da Dengue/imunologia , Endotélio Vascular/imunologia , Endotélio Vascular/virologia , Humanos , Metaloproteases/antagonistas & inibidores , Dengue Grave/complicações , Dengue Grave/imunologia , SíndromeRESUMO
Transfected muscle can be used as a secreting tissue for therapeutic proteins. Skeletal muscle transfection is increased by suitable electric pulse application (electrotransfer). We and others had shown that electrotransfer of interleukin-10 encoding plasmid is an effective strategy in animal models of chronic diseases such as myocarditis, atherosclerosis, or rheumatoid arthritis. In the present work, we have studied murine interleukin-10 production and secretion after i.m. electrotransfer. In immunocompetent mice, serum and muscle mIL-10 levels were enhanced by electrotransfer. Serum mIL-10 concentration reached rapidly a peak level 2 days after electrotransfer. It then decreased to background at day 14. Muscle mIL-10 mRNA and protein remained more stable, being detectable up to 84 days after electrotransfer. A boost reinjection led to similar high level of circulating mIL-10. The fast decrease of serum mIL-10 was not observed in SCID mice.
Assuntos
Técnicas de Transferência de Genes , Interleucina-10/biossíntese , Interleucina-10/genética , Animais , Estimulação Elétrica , Feminino , Interleucina-10/sangue , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Músculo Esquelético/patologia , Músculos/patologia , Plasmídeos/metabolismo , RNA Mensageiro/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
When administered to mice systemically or via the airways, LPS induces bronchoconstriction (BC) and/or bronchopulmonary hyperreactivity (BHR), associated with inflammation. Accordingly, a relationship between inflammation and allergic and nonallergic BHR can be hypothesized. We therefore studied the interference of the anti-inflammatory cytokine murine IL-10 (mIL-10) with LPS-induced lung inflammation, BC, and BHR. mIL-10 was administered directly into the airways by intranasal instillation or generated in vivo after muscle electrotransfer of mIL-10-encoding plasmid. Electrotransfer led to high mIL-10 circulating levels for a longer time than after the injection of recombinant mIL-10 (rmIL-10). rmIL-10 administered intranasally reduced lung inflammation and BHR after LPS administration into airways. It also reduced the ex vivo production of TNF-alpha by LPS-stimulated lung tissue explants. Two days after electrotransfer, mIL-10 blood levels were elevated, but lung inflammation, BC, and BHR persisted unaffected. Blood mIL-10 reaches the airways poorly, which probably accounts for the ineffectiveness of mIL-10-encoding plasmid electrotransfer. When LPS was aerosolized 15 days after electrotransfer, lung inflammation persisted but BHR was significantly reduced, an effect that may be related to the longer exposure of the relevant cells to mIL-10. The dissociation between inflammation and BHR indicates that both are not directly correlated. In conclusion, this study shows that mIL-10 is efficient against BHR when present in the airway compartment. Despite this, the muscle electrotransfer with mIL-10-encoding plasmid showed a protective effect against BHR after a delay of 2 wk that should be further investigated.
Assuntos
Hiper-Reatividade Brônquica/tratamento farmacológico , Terapia Genética , Interleucina-10/genética , Interleucina-10/farmacologia , Lipopolissacarídeos/farmacologia , Administração Intranasal , Animais , Hiper-Reatividade Brônquica/induzido quimicamente , Hiper-Reatividade Brônquica/imunologia , Eletroporação , Feminino , Técnicas In Vitro , Interleucina-10/sangue , Camundongos , Camundongos Endogâmicos C57BL , Nebulizadores e Vaporizadores , Plasmídeos/farmacologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The basic helix-loop-helix tal-1 gene (or scl), known for its fundamental role in embryonic and adult hematopoiesis in vertebrates, is also required for embryonic vascular remodeling. In adults, TAL-1 protein is undetectable in quiescent endothelium but it is present in newly formed vessels including tumoral vasculature, indicating its involvement in angiogenesis. Here, we demonstrate that TAL-1 expression is tightly regulated during in vitro angiogenesis: it is low during the initial step of migration and is upregulated during formation of capillary-like structures. We investigated whether ectopic expression of either wild-type TAL-1 or a dominant-negative mutant lacking the DNA-binding domain (Delta-bas) modulates the activity of human primary endothelial cells in the angiogenic processes of migration, proliferation and cell morphogenesis. Overexpression of either wild-type or Delta-bas TAL-1 affected chemotactic migration of primary endothelial cells without modifying their proliferative properties. Ectopic expression of wild-type TAL-1 accelerated the formation of capillary-like structures in vitro and, in vivo, enhanced vascularisation in mice (Matrigel implants) associated with a general enlargement of capillary lumens. Importantly, transduction of the mutant Delta-bas completely impaired in vitro angiogenesis and strongly inhibited vascularisation in mice. Taken together, our data show that TAL-1 modulates the angiogenic response of endothelial cells by stimulating cell morphogenesis and by influencing their behavior in migration. This study highlights the importance of TAL-1 regulation in postnatal vascular remodeling and provides the first physiological evidence that links TAL-1 activity to endothelial cell morphogenic processes.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Endotélio Vascular/citologia , Endotélio Vascular/crescimento & desenvolvimento , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Divisão Celular , Movimento Celular/fisiologia , Células Cultivadas , Proteínas de Ligação a DNA/química , Endotélio Vascular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Sequências Hélice-Alça-Hélice , Humanos , Técnicas In Vitro , Masculino , Camundongos , Camundongos SCID , Neovascularização Fisiológica , Proteínas Proto-Oncogênicas/química , Interferência de RNA , RNA Interferente Pequeno/genética , Deleção de Sequência , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Fatores de Transcrição/químicaRESUMO
BACKGROUND: Gene therapy is very promising in the treatment of rheumatoid arthritis (RA). Electrotransfer is a recent method reported to enhance in vivo intramuscular DNA transfection. Interleukin-10 (IL-10) has antiinflammatory effects in RA and in collagen-induced arthritis (CIA), a murine model of RA. In order to improve our strategy of gene therapy, we used electrotransfer to enhance penetration into skeletal muscle with CIA of plasmids encoding IL-10. METHODS: CIA was induced in DBA/1 mice by immunization with bovine type II collagen. Injection into the tibial cranial muscle of low-dose (200 ng) pCOR plasmid encoding murine IL-10 (pCOR-CMV-mIL-10) was immediately followed by application of square-wave electric pulses (8 pulses of 200V/cm, 20 ms duration at 2 Hz). Control groups received empty plasmid or saline before electrotransfer. RESULTS: When electrotransfer was performed twice on days 10 and 25 postimmunization, CIA was significantly delayed (P < 0.05) and attenuated (P < 0.001) in groups treated by electrotransfer or pCOR-CMV-mIL-10 plasmid vs. control groups. When electrotransfer of pCOR-CMV-mIL-10 plasmid was performed on days 25 and 40 postimmunization, at disease onset, the clinical severity of CIA was reduced (P < 0.05). All groups which had been electrotransferred early or late by pCOR-CMV-mIL-10 plasmid showed suppression of histological signs of arthritis. CONCLUSIONS: Taken together, these data indicate that administration of an antiinflammatory plasmid-born gene by electrotransfer of naked DNA is effective in vivo in an arthritis model.