Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 7 de 7
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Nat Commun ; 7: 10160, 2016 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-26744078

RESUMO

Previous studies have shown that biological noise may drive dynamic phenotypic mosaicism in isogenic unicellular organisms. However, there is no evidence for a similar mechanism operating in metazoans. Here we show that the endothelial-restricted gene, von Willebrand factor (VWF), is expressed in a mosaic pattern in the capillaries of many vascular beds and in the aorta. In capillaries, the mosaicism is dynamically regulated, with VWF switching between ON and OFF states during the lifetime of the animal. Clonal analysis of cultured endothelial cells reveals that dynamic mosaic heterogeneity is controlled by a low-barrier, noise-sensitive bistable switch that involves random transitions in the DNA methylation status of the VWF promoter. Finally, the hearts of VWF-null mice demonstrate an abnormal endothelial phenotype as well as cardiac dysfunction. Together, these findings suggest a novel stochastic phenotype switching strategy for adaptive homoeostasis in the adult vasculature.


Assuntos
Aorta/metabolismo , Capilares/metabolismo , Metilação de DNA , Células Endoteliais/metabolismo , Mosaicismo , RNA Mensageiro/metabolismo , Fator de von Willebrand/genética , Animais , Imunoprecipitação da Cromatina , Citometria de Fluxo , Imunofluorescência , Expressão Gênica , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Células NIH 3T3 , Fenótipo , Regiões Promotoras Genéticas , Artéria Pulmonar/citologia , Reação em Cadeia da Polimerase em Tempo Real , Fator de von Willebrand/metabolismo
2.
Circ Res ; 115(2): 238-251, 2014 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-24874427

RESUMO

RATIONALE: Forkhead box-O transcription factors (FoxOs) transduce a wide range of extracellular signals, resulting in changes in cell survival, cell cycle progression, and several cell type-specific responses. FoxO1 is expressed in many cell types, including endothelial cells (ECs). Previous studies have shown that Foxo1 knockout in mice results in embryonic lethality at E11 because of impaired vascular development. In contrast, somatic deletion of Foxo1 is associated with hyperproliferation of ECs. Thus, the precise role of FoxO1 in the endothelium remains enigmatic. OBJECTIVE: To determine the effect of endothelial-specific knockout and overexpression of FoxO1 on vascular homeostasis. METHODS AND RESULTS: We show that EC-specific disruption of Foxo1 in mice phenocopies the full knockout. Although endothelial expression of FoxO1 rescued otherwise Foxo1-null animals, overexpression of constitutively active FoxO1 resulted in increased EC size, occlusion of capillaries, elevated peripheral resistance, heart failure, and death. Knockdown of FoxO1 in ECs resulted in marked inhibition of basal and vascular endothelial growth factor-induced Akt-mammalian target of rapamycin complex 1 (mTORC1) signaling. CONCLUSIONS: Our findings suggest that in mice, endothelial expression of FoxO1 is both necessary and sufficient for embryonic development. Moreover, FoxO1-mediated feedback activation of Akt maintains growth factor responsive Akt/mTORC1 activity within a homeostatic range.


Assuntos
Células Endoteliais/metabolismo , Fatores de Transcrição Forkhead/fisiologia , Insuficiência Cardíaca/genética , Complexos Multiproteicos/fisiologia , Neovascularização Fisiológica/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Serina-Treonina Quinases TOR/fisiologia , Animais , Indução Enzimática , Proteína Forkhead Box O1 , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/antagonistas & inibidores , Fatores de Transcrição Forkhead/deficiência , Fatores de Transcrição Forkhead/genética , Insuficiência Cardíaca/fisiopatologia , Homeostase , Células Endoteliais da Veia Umbilical Humana , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Neovascularização Fisiológica/genética , Óxido Nítrico Sintase Tipo III/biossíntese , Óxido Nítrico Sintase Tipo III/genética , Especificidade de Órgãos , RNA Interferente Pequeno/farmacologia , Proteínas Recombinantes de Fusão , Transdução de Sinais/fisiologia , Saco Vitelino/irrigação sanguínea
3.
Blood ; 121(21): 4404-12, 2013 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-23529929

RESUMO

We previously demonstrated that the first intron of the human von Willebrand factor (vWF) is required for gene expression in the endothelium of transgenic mice. Based on this finding, we hypothesized that RNA splicing plays a role in mediating vWF expression in the vasculature. To address this question, we used transient transfection assays in human endothelial cells and megakaryocytes with intron-containing and intronless human vWF promoter-luciferase constructs. Next, we generated knockin mice in which LacZ was targeted to the endogenous mouse vWF locus in the absence or presence of the native first intron or heterologous introns from the human ß-globin, mouse Down syndrome critical region 1, or hagfish coagulation factor X genes. In both the in vitro assays and the knockin mice, the loss of the first intron of vWF resulted in a significant reduction of reporter gene expression in endothelial cells but not megakaryocytes. This effect was rescued to varying degrees by the introduction of a heterologous intron. Intron-mediated enhancement of expression was mediated at a posttranscriptional level. Together, these findings implicate a role for intronic splicing in mediating lineage-specific expression of vWF in the endothelium.


Assuntos
Linhagem da Célula/genética , Endotélio Vascular/fisiologia , Splicing de RNA/genética , Fator de von Willebrand/genética , Animais , Bovinos , Éxons/genética , Técnicas de Introdução de Genes , Hemostasia/fisiologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Íntrons/genética , Óperon Lac , Camundongos , Regiões Promotoras Genéticas/genética , Especificidade da Espécie
4.
Blood ; 117(1): 342-51, 2011 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-20980682

RESUMO

A region of the human von Willebrand factor (VWF) gene between -2812 and the end of the first intron (termed vWF2) was previously shown to direct expression in the endothelium of capillaries and a subset of larger blood vessels in the heart and skeletal muscle. Here, our goal was to delineate the DNA sequences responsible for this effect. A series of constructs containing deletions or mutations of vWF2 coupled to LacZ were targeted to the Hprt locus of mice, and the resulting animals were analyzed for reporter gene expression. The findings demonstrate that DNA sequences between -843 and -620 are necessary for expression in capillary but not large vessel endothelium in heart and skeletal muscle. Further, expression of VWF in capillaries and larger vessels of both tissues required the presence of a native or heterologous intron. In vitro assays implicated a role for ERG-binding ETS motif at -56 in mediating basal expression of VWF. In Hprt-targeted mice, mutation of the ETS consensus motif resulted in loss of LacZ expression in the endothelium of the heart and skeletal muscle. Together, these data indicate that distinct DNA modules regulate vascular bed-specific expression of VWF.


Assuntos
Endotélio Vascular/metabolismo , Regulação da Expressão Gênica , Coração/fisiologia , Músculo Esquelético/metabolismo , Regiões Promotoras Genéticas/genética , Fator de von Willebrand/genética , Animais , Western Blotting , Células Cultivadas , Imunoprecipitação da Cromatina , Endotélio Vascular/citologia , Feminino , Humanos , Hipoxantina Fosforribosiltransferase/genética , Íntrons/genética , Óperon Lac , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição , Regulador Transcricional ERG , Fator de von Willebrand/metabolismo
5.
Blood ; 114(27): 5557-66, 2009 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-19822898

RESUMO

Vascular endothelial growth factor receptor 1 (VEGFR1) is a marker for endothelial-specific gene expression. We previously reported that the human VEGFR1 promoter (between -748 and +284) contains information for expression in the intact endothelium of transgenic mice. The objective of this study was to dissect the cis-regulatory elements underlying VEGFR1 promoter activity in vitro and in vivo. In primary endothelial cells, binding sites for E74-like factor 1 (ELF-1; between -49 and -52), cyclic adenosine monophosphate response element binding (CREB; between -74 and -81), and early growth response factor 1/3 (EGR-1/3; between -16 to -25) were shown to play a positive role in gene transcription, whereas a putative E26 transformation-specificsequence (ETS) motif between -36 and -39 had a net negative effect on promoter activity. When targeted to the Hprt locus of mice, mutations of the ELF-1 binding site and the CRE element reduced promoter activity in the embryonic vasculature and resulted in a virtual loss of expression in adult endothelium. Postnatally, the EGR binding site mutant displayed significantly reduced promoter activity in a subset of vascular beds. In contrast, mutation of the -39 ETS site resulted in increased LacZ staining in multiple vascular beds. Together, these results provide new insights into the transcriptional regulatory mechanisms of VEGFR1.


Assuntos
Proteína de Ligação a CREB/metabolismo , Fatores de Transcrição de Resposta de Crescimento Precoce/metabolismo , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Animais , Sequência de Bases , Sítios de Ligação/genética , Sítios de Ligação/fisiologia , Células Cultivadas , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Proteína 3 de Resposta de Crescimento Precoce/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Feminino , Regulação da Expressão Gênica/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Mutação , Ligação Proteica , Homologia de Sequência do Ácido Nucleico
6.
Blood ; 112(6): 2336-9, 2008 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-18519813

RESUMO

We recently demonstrated that the 3-kb 5'-flanking region of the human ROBO4 gene directs endothelial cell-specific expression in vitro and in vivo. Moreover, a GA-binding protein (GABP)-binding motif at -119 was necessary for mediating promoter activity in vitro. The goal of the present study was to confirm the functional relevance of the -119 GABP-binding site in vivo. To that end, the Hprt locus of mice was targeted with a Robo4-LacZ transgenic cassette in which the GABP site was mutated. In other studies, the GABP mutation was introduced into the endogenous mouse Robo4 locus in which LacZ was knocked-in. Compared with their respective controls, the mutant promoters displayed a significant reduction in activity in embryoid bodies, embryos, and adult animals. Together, these data provide strong support for the role of the GABP-binding motif in mediating Robo4 expression in the intact endothelium.


Assuntos
Endotélio/metabolismo , Fator de Transcrição de Proteínas de Ligação GA/metabolismo , Regiões Promotoras Genéticas , Receptores de Superfície Celular/genética , Animais , Sítios de Ligação/genética , Embrião de Mamíferos , Humanos , Camundongos , Camundongos Transgênicos , Mutação , Neoplasias Experimentais , Distribuição Tecidual , Transplante Heterólogo
7.
Oncogene ; 21(52): 7945-56, 2002 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-12439745

RESUMO

The roles played by the endogenous angiogenesis inhibitor thrombospondin-1 (TSP-1) in the early stages of multi-step carcinogenesis and in the control of hematogenous versus lymphatic metastasis are unknown. To investigate these issues we compared tumor development in normal mice and in transgenic mice with targeted overexpression of TSP-1 in the epidermis following a standard two-step chemical skin carcinogenesis regimen. Overexpression of TSP-1 resulted in delayed and reduced development of premalignant epithelial hyperplasias, but did not inhibit the malignant conversion to squamous cell carcinomas. TSP-1 overexpression also suppressed tumor angiogenesis and distant organ metastasis, but failed to inhibit tumor-associated lymphangiogenesis or lymphatic tumor spread to regional lymph nodes. Concomitant with these results, we found that the endothelial TSP-1 receptor CD36 was mostly absent from cutaneous lymphatic vessels. Our findings indicate the potential use of TSP-1 for the prevention of premalignant stages of tumorigenesis and are likely to have implications for the further development of anti-angiogenic cancer therapies.


Assuntos
Transformação Celular Neoplásica , Metástase Linfática/prevenção & controle , Neovascularização Patológica/prevenção & controle , Trombospondina 1/fisiologia , Animais , Camundongos , Camundongos Transgênicos , Neoplasias Cutâneas/irrigação sanguínea , Neoplasias Cutâneas/patologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...