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1.
Development ; 151(3)2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38223992

RESUMO

The generation of the post-cranial embryonic body relies on the coordinated production of spinal cord neurectoderm and presomitic mesoderm cells from neuromesodermal progenitors (NMPs). This process is orchestrated by pro-neural and pro-mesodermal transcription factors that are co-expressed in NMPs together with Hox genes, which are essential for axial allocation of NMP derivatives. NMPs reside in a posterior growth region, which is marked by the expression of Wnt, FGF and Notch signalling components. Although the importance of Wnt and FGF in influencing the induction and differentiation of NMPs is well established, the precise role of Notch remains unclear. Here, we show that the Wnt/FGF-driven induction of NMPs from human embryonic stem cells (hESCs) relies on Notch signalling. Using hESC-derived NMPs and chick embryo grafting, we demonstrate that Notch directs a pro-mesodermal character at the expense of neural fate. We show that Notch also contributes to activation of HOX gene expression in human NMPs, partly in a non-cell-autonomous manner. Finally, we provide evidence that Notch exerts its effects via the establishment of a negative-feedback loop with FGF signalling.


Assuntos
Padronização Corporal , Genes Homeobox , Animais , Embrião de Galinha , Humanos , Padronização Corporal/genética , Diferenciação Celular/genética , Mesoderma/metabolismo , Medula Espinal , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento
2.
Arterioscler Thromb Vasc Biol ; 43(4): 547-561, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36794585

RESUMO

BACKGROUND: Hemodynamic wall shear stress (WSS) exerted on the endothelium by flowing blood determines the spatial distribution of atherosclerotic lesions. Disturbed flow (DF) with a low WSS magnitude and reversing direction promotes atherosclerosis by regulating endothelial cell (EC) viability and function, whereas un-DF which is unidirectional and of high WSS magnitude is atheroprotective. Here, we study the role of EVA1A (eva-1 homolog A), a lysosome and endoplasmic reticulum-associated protein linked to autophagy and apoptosis, in WSS-regulated EC dysfunction. METHODS: The effect of WSS on EVA1A expression was studied using porcine and mouse aortas and cultured human ECs exposed to flow. EVA1A was silenced in vitro in human ECs and in vivo in zebrafish using siRNA (small interfering RNA) and morpholinos, respectively. RESULTS: EVA1A was induced by proatherogenic DF at both mRNA and protein levels. EVA1A silencing resulted in decreased EC apoptosis, permeability, and expression of inflammatory markers under DF. Assessment of autophagic flux using the autolysosome inhibitor, bafilomycin coupled to the autophagy markers LC3-II (microtubule-associated protein 1 light chain 3-II) and p62, revealed that EVA1A knockdown promotes autophagy when ECs are exposed to DF, but not un-DF . Blocking autophagic flux led to increased EC apoptosis in EVA1A-knockdown cells exposed to DF, suggesting that autophagy mediates the effects of DF on EC dysfunction. Mechanistically, EVA1A expression was regulated by flow direction via TWIST1 (twist basic helix-loop-helix transcription factor 1). In vivo, knockdown of EVA1A orthologue in zebrafish resulted in reduced EC apoptosis, confirming the proapoptotic role of EVA1A in the endothelium. CONCLUSIONS: We identified EVA1A as a novel flow-sensitive gene that mediates the effects of proatherogenic DF on EC dysfunction by regulating autophagy.


Assuntos
Aterosclerose , Peixe-Zebra , Animais , Humanos , Camundongos , Apoptose , Aterosclerose/patologia , Autofagia , Endotélio/metabolismo , Suínos , Peixe-Zebra/genética
3.
J Cell Sci ; 132(11)2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31076511

RESUMO

Endothelial cell (EC) sensing of fluid shear stress direction is a critical determinant of vascular health and disease. Unidirectional flow induces EC alignment and vascular homeostasis, whereas bidirectional flow has pathophysiological effects. ECs express several mechanoreceptors that respond to flow, but the mechanism for sensing shear stress direction is poorly understood. We determined, by using in vitro flow systems and magnetic tweezers, that ß1 integrin is a key sensor of force direction because it is activated by unidirectional, but not bidirectional, shearing forces. ß1 integrin activation by unidirectional force was amplified in ECs that were pre-sheared in the same direction, indicating that alignment and ß1 integrin activity has a feedforward interaction, which is a hallmark of system stability. En face staining and EC-specific genetic deletion studies in the murine aorta revealed that ß1 integrin is activated and is essential for EC alignment at sites of unidirectional flow but is not activated at sites of bidirectional flow. In summary, ß1 integrin sensing of unidirectional force is a key mechanism for decoding blood flow mechanics to promote vascular homeostasis.This article has an associated First Person interview with the first author of the paper.


Assuntos
Aorta/fisiologia , Integrina beta1/metabolismo , Fluxo Sanguíneo Regional/fisiologia , Animais , Linhagem Celular , Feminino , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Integrina beta1/genética , Mecanorreceptores/fisiologia , Camundongos , Camundongos Knockout , Estresse Fisiológico/fisiologia
4.
Cardiovasc Drugs Ther ; 33(2): 231-237, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30809744

RESUMO

BACKGROUND: Endothelial cell (EC) dysfunction (enhanced inflammation, proliferation and permeability) is the initial trigger for atherosclerosis. Atherosclerosis shows preferential development near branches and bends exposed to disturbed blood flow. By contrast, sites that are exposed to non-disturbed blood flow are atheroprotected. Disturbed flow promotes atherosclerosis by promoting EC dysfunction. Blood flow controls EC function through transcriptional and post-transcriptional mechanisms that are incompletely understood. METHODS AND RESULTS: We identified the developmental transcription factors Twist1 and GATA4 as being enriched in EC at disturbed flow, atheroprone regions of the porcine aorta in a microarray study. Further work using the porcine and murine aortae demonstrated that Twist1 and GATA4 expression was enhanced at the atheroprone, disturbed flow sites in vivo. Using controlled in vitro flow systems, the expression of Twist1 and GATA4 was enhanced under disturbed compared to non-disturbed flow in cultured cells. Disturbed flow promoted Twist1 expression through a GATA4-mediated transcriptional mechanism as revealed by a series of in vivo and in vitro studies. GATA4-Twist1 signalling promoted EC proliferation, inflammation, permeability and endothelial-to-mesenchymal transition (EndoMT) under disturbed flow, leading to atherosclerosis development, as shown in a combination of in vitro and in vivo studies using GATA4 and Twist1-specific siRNA and EC-specific GATA4 and Twist1 Knock out (KO) mice. CONCLUSIONS: We revealed that GATA4-Twist1-Snail signalling triggers EC dysfunction and atherosclerosis; this work could lead to the development of novel anti-atherosclerosis therapeutics.


Assuntos
Artérias/metabolismo , Aterosclerose/metabolismo , Endotélio Vascular/metabolismo , Fator de Transcrição GATA4/metabolismo , Mecanotransdução Celular , Proteínas Nucleares/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Animais , Artérias/patologia , Artérias/fisiopatologia , Aterosclerose/patologia , Aterosclerose/fisiopatologia , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Transição Epitelial-Mesenquimal , Humanos , Placa Aterosclerótica , Fluxo Sanguíneo Regional
5.
Development ; 142(21): 3649-60, 2015 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-26534985

RESUMO

NOTCH signalling is an evolutionarily conserved pathway involved in intercellular communication essential for cell fate choices during development. Although dispensable for early aspects of mouse development, canonical RBPJ-dependent NOTCH signalling has been shown to influence lineage commitment during embryonic stem cell (ESC) differentiation. NOTCH activation in ESCs promotes the acquisition of a neural fate, whereas its suppression favours their differentiation into cardiomyocytes. This suggests that NOTCH signalling is implicated in the acquisition of distinct embryonic fates at early stages of mammalian development. In order to investigate in vivo such a role for NOTCH signalling in shaping cell fate specification, we use genetic approaches to constitutively activate the NOTCH pathway in the mouse embryo. Early embryonic development, including the establishment of anterior-posterior polarity, is not perturbed by forced NOTCH activation. By contrast, widespread NOTCH activity in the epiblast triggers dramatic gastrulation defects. These are fully rescued in a RBPJ-deficient background. Epiblast-specific NOTCH activation induces acquisition of neurectoderm identity and disrupts the formation of specific mesodermal precursors including the derivatives of the anterior primitive streak, the mouse organiser. In addition, we show that forced NOTCH activation results in misregulation of NODAL signalling, a major determinant of early embryonic patterning. Our study reveals a previously unidentified role for canonical NOTCH signalling during mammalian gastrulation. It also exemplifies how in vivo studies can shed light on the mechanisms underlying cell fate specification during in vitro directed differentiation.


Assuntos
Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Gastrulação , Receptores Notch/metabolismo , Transdução de Sinais , Animais , Ectoderma/metabolismo , Implantação do Embrião , Camadas Germinativas/metabolismo , Camundongos , Proteína Nodal/metabolismo
6.
Blood ; 128(12): 1567-77, 2016 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-27421959

RESUMO

The first definitive hematopoietic stem cells (dHSCs) in the mouse emerge in the dorsal aorta of the embryonic day (E) 10.5 to 11 aorta-gonad-mesonephros (AGM) region. Notch signaling is essential for early HSC development but is dispensable for the maintenance of adult bone marrow HSCs. How Notch signaling regulates HSC formation in the embryo is poorly understood. We demonstrate here that Notch signaling is active in E10.5 HSC precursors and involves both Notch1 and Notch2 receptors, but is gradually downregulated while they progress toward dHSCs at E11.5. This downregulation is accompanied by gradual functional loss of Notch dependency. Thus, as early as at final steps in the AGM region, HSCs begin acquiring the Notch independency characteristic of adult bone marrow HSCs as part of the maturation program. Our data indicate that fine stage-dependent tuning of Notch signaling may be required for the generation of definitive HSCs from pluripotent cells.


Assuntos
Aorta/embriologia , Embrião de Mamíferos/citologia , Gônadas/embriologia , Células-Tronco Hematopoéticas/citologia , Mesonefro/embriologia , Receptor Notch2/metabolismo , Células Estromais/citologia , Animais , Aorta/metabolismo , Células Cultivadas , Embrião de Mamíferos/metabolismo , Gônadas/metabolismo , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Mesonefro/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transdução de Sinais , Células Estromais/metabolismo
7.
Arterioscler Thromb Vasc Biol ; 37(11): 2087-2101, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28882872

RESUMO

OBJECTIVE: Atherosclerosis develops near branches and bends of arteries that are exposed to low shear stress (mechanical drag). These sites are characterized by excessive endothelial cell (EC) proliferation and inflammation that promote lesion initiation. The transcription factor HIF1α (hypoxia-inducible factor 1α) is canonically activated by hypoxia and has a role in plaque neovascularization. We studied the influence of shear stress on HIF1α activation and the contribution of this noncanonical pathway to lesion initiation. APPROACH AND RESULTS: Quantitative polymerase chain reaction and en face staining revealed that HIF1α was expressed preferentially at low shear stress regions of porcine and murine arteries. Low shear stress induced HIF1α in cultured EC in the presence of atmospheric oxygen. The mechanism involves the transcription factor nuclear factor-κB that induced HIF1α transcripts and induction of the deubiquitinating enzyme Cezanne that stabilized HIF1α protein. Gene silencing revealed that HIF1α enhanced proliferation and inflammatory activation in EC exposed to low shear stress via induction of glycolysis enzymes. We validated this observation by imposing low shear stress in murine carotid arteries (partial ligation) that upregulated the expression of HIF1α, glycolysis enzymes, and inflammatory genes and enhanced EC proliferation. EC-specific genetic deletion of HIF1α in hypercholesterolemic apolipoprotein E-defecient mice reduced inflammation and endothelial proliferation in partially ligated arteries, indicating that HIF1α drives inflammation and vascular dysfunction at low shear stress regions. CONCLUSIONS: Mechanical low shear stress activates HIF1α at atheroprone regions of arteries via nuclear factor-κB and Cezanne. HIF1α promotes atherosclerosis initiation at these sites by inducing excessive EC proliferation and inflammation via the induction of glycolysis enzymes.


Assuntos
Aterosclerose/metabolismo , Células Endoteliais/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inflamação/metabolismo , Mecanotransdução Celular , Placa Aterosclerótica , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/patologia , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Endopeptidases/metabolismo , Células Endoteliais/patologia , Indução Enzimática , Feminino , Predisposição Genética para Doença , Glicólise , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Inflamação/genética , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Camundongos Knockout , NF-kappa B/metabolismo , Oxigênio/metabolismo , Fenótipo , Estabilidade Proteica , Proteólise , Interferência de RNA , Fluxo Sanguíneo Regional , Estresse Mecânico , Sus scrofa , Fatores de Tempo , Transfecção , Ubiquitinação , Regulação para Cima
8.
Arterioscler Thromb Vasc Biol ; 37(1): 130-143, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27834691

RESUMO

OBJECTIVE: Atherosclerosis is initiated at branches and bends of arteries exposed to disturbed blood flow that generates low shear stress. This mechanical environment promotes lesions by inducing endothelial cell (EC) apoptosis and dysfunction via mechanisms that are incompletely understood. Although transcriptome-based studies have identified multiple shear-responsive genes, most of them have an unknown function. To address this, we investigated whether zebrafish embryos can be used for functional screening of mechanosensitive genes that regulate EC apoptosis in mammalian arteries. APPROACH AND RESULTS: First, we demonstrated that flow regulates EC apoptosis in developing zebrafish vasculature. Specifically, suppression of blood flow in zebrafish embryos (by targeting cardiac troponin) enhanced that rate of EC apoptosis (≈10%) compared with controls exposed to flow (≈1%). A panel of candidate regulators of apoptosis were identified by transcriptome profiling of ECs from high and low shear stress regions of the porcine aorta. Genes that displayed the greatest differential expression and possessed 1 to 2 zebrafish orthologues were screened for the regulation of apoptosis in zebrafish vasculature exposed to flow or no-flow conditions using a knockdown approach. A phenotypic change was observed in 4 genes; p53-related protein (PERP) and programmed cell death 2-like protein functioned as positive regulators of apoptosis, whereas angiopoietin-like 4 and cadherin 13 were negative regulators. The regulation of perp, cdh13, angptl4, and pdcd2l by shear stress and the effects of perp and cdh13 on EC apoptosis were confirmed by studies of cultured EC exposed to flow. CONCLUSIONS: We conclude that a zebrafish model of flow manipulation coupled to gene knockdown can be used for functional screening of mechanosensitive genes in vascular ECs, thus providing potential therapeutic targets to prevent or treat endothelial injury at atheroprone sites.


Assuntos
Apoptose , Aterosclerose/genética , Células Endoteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mecanotransdução Celular/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Aterosclerose/metabolismo , Aterosclerose/patologia , Aterosclerose/fisiopatologia , Células Cultivadas , Embrião não Mamífero/irrigação sanguínea , Células Endoteliais/patologia , Feminino , Perfilação da Expressão Gênica/métodos , Técnicas de Silenciamento de Genes , Redes Reguladoras de Genes , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Camundongos , Fenótipo , Interferência de RNA , Fluxo Sanguíneo Regional , Estresse Mecânico , Suínos , Transcriptoma , Transfecção , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo
10.
Nat Commun ; 15(1): 3745, 2024 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-38702304

RESUMO

Early childhood tumours arise from transformed embryonic cells, which often carry large copy number alterations (CNA). However, it remains unclear how CNAs contribute to embryonic tumourigenesis due to a lack of suitable models. Here we employ female human embryonic stem cell (hESC) differentiation and single-cell transcriptome and epigenome analysis to assess the effects of chromosome 17q/1q gains, which are prevalent in the embryonal tumour neuroblastoma (NB). We show that CNAs impair the specification of trunk neural crest (NC) cells and their sympathoadrenal derivatives, the putative cells-of-origin of NB. This effect is exacerbated upon overexpression of MYCN, whose amplification co-occurs with CNAs in NB. Moreover, CNAs potentiate the pro-tumourigenic effects of MYCN and mutant NC cells resemble NB cells in tumours. These changes correlate with a stepwise aberration of developmental transcription factor networks. Together, our results sketch a mechanistic framework for the CNA-driven initiation of embryonal tumours.


Assuntos
Diferenciação Celular , Variações do Número de Cópias de DNA , Proteína Proto-Oncogênica N-Myc , Crista Neural , Neuroblastoma , Humanos , Neuroblastoma/genética , Neuroblastoma/patologia , Crista Neural/metabolismo , Crista Neural/patologia , Feminino , Proteína Proto-Oncogênica N-Myc/genética , Proteína Proto-Oncogênica N-Myc/metabolismo , Aberrações Cromossômicas , Células-Tronco Embrionárias Humanas/metabolismo , Transcriptoma , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica
12.
Vascul Pharmacol ; 150: 107178, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37137436

RESUMO

Flowing blood regulates vascular development, homeostasis and disease by generating wall shear stress which has major effects on endothelial cell (EC) physiology. Low oscillatory shear stress (LOSS) induces a form of cell plasticity called endothelial-to-mesenchymal transition (EndMT). This process has divergent effects; in embryos LOSS-induced EndMT drives the development of atrioventricular valves, whereas in adult arteries it is associated with inflammation and atherosclerosis. The Notch ligand DLL4 is essential for LOSS-dependent valve development; here we investigated whether DLL4 is required for responses to LOSS in adult arteries. Analysis of cultured human coronary artery EC revealed that DLL4 regulates the transcriptome to induce markers of EndMT and inflammation under LOSS conditions. Consistently, genetic deletion of Dll4 from murine EC reduced SNAIL (EndMT marker) and VCAM-1 (inflammation marker) at a LOSS region of the murine aorta. We hypothesized that endothelial Dll4 is pro-atherogenic but this analysis was confounded because endothelial Dll4 negatively regulated plasma cholesterol levels in hyperlipidemic mice. We conclude that endothelial DLL4 is required for LOSS-induction of EndMT and inflammation regulators at atheroprone regions of arteries, and is also a regulator of plasma cholesterol.


Assuntos
Aterosclerose , Vasos Coronários , Células Endoteliais , Animais , Camundongos , Humanos , Células Cultivadas , Vasos Coronários/metabolismo , Vasos Coronários/patologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Transição Epitelial-Mesenquimal , Aterosclerose/metabolismo , Masculino
13.
Elife ; 112022 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-36154671

RESUMO

The neural crest (NC) is an important multipotent embryonic cell population and its impaired specification leads to various developmental defects, often in an anteroposterior (A-P) axial level-specific manner. The mechanisms underlying the correct A-P regionalisation of human NC cells remain elusive. Recent studies have indicated that trunk NC cells, the presumed precursors of childhood tumour neuroblastoma, are derived from neuromesodermal-potent progenitors of the postcranial body. Here we employ human embryonic stem cell differentiation to define how neuromesodermal progenitor (NMP)-derived NC cells acquire a posterior axial identity. We show that TBXT, a pro-mesodermal transcription factor, mediates early posterior NC/spinal cord regionalisation together with WNT signalling effectors. This occurs by TBXT-driven chromatin remodelling via its binding in key enhancers within HOX gene clusters and other posterior regulator-associated loci. This initial posteriorisation event is succeeded by a second phase of trunk HOX gene control that marks the differentiation of NMPs toward their TBXT-negative NC/spinal cord derivatives and relies predominantly on FGF signalling. Our work reveals a previously unknown role of TBXT in influencing posterior NC fate and points to the existence of temporally discrete, cell type-dependent modes of posterior axial identity control.


Assuntos
Mesoderma , Crista Neural , Diferenciação Celular/genética , Humanos , Fatores de Transcrição/metabolismo , Via de Sinalização Wnt
14.
Sci Adv ; 8(35): eabo7958, 2022 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-36044575

RESUMO

Endothelial cell (EC) sensing of disturbed blood flow triggers atherosclerosis, a disease of arteries that causes heart attack and stroke, through poorly defined mechanisms. The Notch pathway plays a central role in blood vessel growth and homeostasis, but its potential role in sensing of disturbed flow has not been previously studied. Here, we show using porcine and murine arteries and cultured human coronary artery EC that disturbed flow activates the JAG1-NOTCH4 signaling pathway. Light-sheet imaging revealed enrichment of JAG1 and NOTCH4 in EC of atherosclerotic plaques, and EC-specific genetic deletion of Jag1 (Jag1ECKO) demonstrated that Jag1 promotes atherosclerosis at sites of disturbed flow. Mechanistically, single-cell RNA sequencing in Jag1ECKO mice demonstrated that Jag1 suppresses subsets of ECs that proliferate and migrate. We conclude that JAG1-NOTCH4 sensing of disturbed flow enhances atherosclerosis susceptibility by regulating EC heterogeneity and that therapeutic targeting of this pathway may treat atherosclerosis.


Assuntos
Aterosclerose , Proteína Jagged-1 , Placa Aterosclerótica , Receptor Notch4 , Animais , Aterosclerose/genética , Aterosclerose/metabolismo , Vasos Coronários/metabolismo , Células Endoteliais/metabolismo , Humanos , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Camundongos , Placa Aterosclerótica/metabolismo , Receptor Notch4/genética , Receptor Notch4/metabolismo , Transdução de Sinais , Suínos
15.
Nat Rev Cardiol ; 17(1): 52-63, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31366922

RESUMO

Flowing blood generates a frictional force called shear stress that has major effects on vascular function. Branches and bends of arteries are exposed to complex blood flow patterns that exert low or low oscillatory shear stress, a mechanical environment that promotes vascular dysfunction and atherosclerosis. Conversely, physiologically high shear stress is protective. Endothelial cells are critical sensors of shear stress but the mechanisms by which they decode complex shear stress environments to regulate physiological and pathophysiological responses remain incompletely understood. Several laboratories have advanced this field by integrating specialized shear-stress models with systems biology approaches, including transcriptome, methylome and proteome profiling and functional screening platforms, for unbiased identification of novel mechanosensitive signalling pathways in arteries. In this Review, we describe these studies, which reveal that shear stress regulates diverse processes and demonstrate that multiple pathways classically known to be involved in embryonic development, such as BMP-TGFß, WNT, Notch, HIF1α, TWIST1 and HOX family genes, are regulated by shear stress in arteries in adults. We propose that mechanical activation of these pathways evolved to orchestrate vascular development but also drives atherosclerosis in low shear stress regions of adult arteries.


Assuntos
Aterosclerose/genética , Endotélio Vascular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mecanotransdução Celular/genética , Animais , Aterosclerose/metabolismo , Aterosclerose/fisiopatologia , Endotélio Vascular/fisiopatologia , Predisposição Genética para Doença , Humanos , Neovascularização Fisiológica/genética , Fenótipo , Fluxo Sanguíneo Regional , Fatores de Risco , Estresse Mecânico , Remodelação Vascular/genética
16.
Cardiovasc Res ; 116(7): 1300-1310, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31504243

RESUMO

AIMS: Atherosclerosis develops near branches and bends of arteries that are exposed to disturbed blood flow which exerts low wall shear stress (WSS). These mechanical conditions alter endothelial cells (EC) by priming them for inflammation and by inducing turnover. Homeobox (Hox) genes are developmental genes involved in the patterning of embryos along their anterior-posterior and proximal-distal axes. Here we identified Hox genes that are regulated by WSS and investigated their functions in adult arteries. METHODS AND RESULTS: EC were isolated from inner (low WSS) and outer (high WSS) regions of the porcine aorta and the expression of Hox genes was analysed by quantitative real-time PCR. Several Hox genes (HoxA10, HoxB4, HoxB7, HoxB9, HoxD8, HoxD9) were significantly enriched at the low WSS compared to the high WSS region. Similarly, studies of cultured human umbilical vein EC (HUVEC) or porcine aortic EC revealed that the expression of multiple Hox genes (HoxA10, HoxB9, HoxD8, HoxD9) was enhanced under low (4 dyn/cm2) compared to high (13 dyn/cm2) WSS conditions. Gene silencing studies identified Hox genes (HoxB9, HoxD8, HoxD9) that are positive regulators of inflammatory molecule expression in EC exposed to low WSS, and others (HoxB9, HoxB7, HoxB4) that regulated EC turnover. We subsequently focused on HoxB9 because it was strongly up-regulated by low WSS and, uniquely, was a driver of both inflammation and proliferation. At a mechanistic level, we demonstrate using cultured EC and murine models that bone morphogenic protein 4 (BMP4) is an upstream regulator of HoxB9 which elicits inflammation via induction of numerous inflammatory mediators including TNF and downstream NF-κB activation. Moreover, the BMP4-HoxB9-TNF pathway was potentiated by hypercholesterolaemic conditions. CONCLUSIONS: Low WSS induces multiple Hox genes that control the activation state and turnover of EC. Notably, low WSS activates a BMP4-HoxB9-TNF signalling pathway to initiate focal arterial inflammation, thereby demonstrating integration of the BMP and Hox systems in vascular pathophysiology.


Assuntos
Aorta Torácica/metabolismo , Doenças da Aorta/metabolismo , Aterosclerose/metabolismo , Proteína Morfogenética Óssea 4/metabolismo , Proteínas de Homeodomínio/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Inflamação/metabolismo , Placa Aterosclerótica , Animais , Aorta Torácica/patologia , Aorta Torácica/fisiopatologia , Doenças da Aorta/genética , Doenças da Aorta/patologia , Doenças da Aorta/fisiopatologia , Aterosclerose/genética , Aterosclerose/patologia , Aterosclerose/fisiopatologia , Proteína Morfogenética Óssea 4/genética , Células Cultivadas , Modelos Animais de Doenças , Proteínas de Homeodomínio/genética , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Inflamação/genética , Inflamação/patologia , Inflamação/fisiopatologia , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Fluxo Sanguíneo Regional , Transdução de Sinais , Estresse Mecânico , Sus scrofa , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
17.
18.
Nat Commun ; 11(1): 214, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31924781

RESUMO

Neutrophils are implicated in the pathogenesis of atherosclerosis but are seldom detected in atherosclerotic plaques. We investigated whether neutrophil-derived microvesicles may influence arterial pathophysiology. Here we report that levels of circulating neutrophil microvesicles are enhanced by exposure to a high fat diet, a known risk factor for atherosclerosis. Neutrophil microvesicles accumulate at disease-prone regions of arteries exposed to disturbed flow patterns, and promote vascular inflammation and atherosclerosis in a murine model. Using cultured endothelial cells exposed to disturbed flow, we demonstrate that neutrophil microvesicles promote inflammatory gene expression by delivering miR-155, enhancing NF-κB activation. Similarly, neutrophil microvesicles increase miR-155 and enhance NF-κB at disease-prone sites of disturbed flow in vivo. Enhancement of atherosclerotic plaque formation and increase in macrophage content by neutrophil microvesicles is dependent on miR-155. We conclude that neutrophils contribute to vascular inflammation and atherogenesis through delivery of microvesicles carrying miR-155 to disease-prone regions.


Assuntos
Aterosclerose/metabolismo , Endotélio/metabolismo , MicroRNAs/metabolismo , Neutrófilos/metabolismo , Animais , Aterosclerose/patologia , Dieta Hiperlipídica , Modelos Animais de Doenças , Células Endoteliais , Endotélio/patologia , Regulação da Expressão Gênica , Humanos , Macrófagos/metabolismo , Camundongos , Camundongos Knockout para ApoE , MicroRNAs/genética , NF-kappa B/metabolismo , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia
19.
Mol Cell Biol ; 26(13): 4769-74, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16782866

RESUMO

The Notch signaling pathway is an evolutionarily conserved signaling system which has been shown to be essential in cell fate specification and in numerous aspects of embryonic development in all metazoans thus far studied. We recently demonstrated that several components of the Notch signaling pathway, including the four Notch receptors and their five ligands known in mammals, are expressed in mouse oocytes, in mouse preimplantation embryos, or both. This suggested a possible implication of the Notch pathway in the first cell fate specification of the dividing mouse embryo, which results in the formation of the blastocyst. To address this issue directly, we generated zygotes in which both the maternal and the zygotic expression of Rbpsuh, a key element of the core Notch signaling pathway, were abrogated. We find that such zygotes give rise to blastocysts which implant and develop normally. Nevertheless, after gastrulation, these embryos die around midgestation, similarly to Rbpsuh-null mutants. This demonstrates that the RBP-Jkappa-dependent pathway, otherwise called the canonical Notch pathway, is dispensable for blastocyst morphogenesis and the establishment of the three germ layers, ectoderm, endoderm, and mesoderm. These results are discussed in the light of recent observations which have challenged this conclusion.


Assuntos
Blastocisto/metabolismo , Implantação do Embrião , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/fisiologia , Receptores Notch/fisiologia , Animais , Blastocisto/citologia , Implantação do Embrião/genética , Feminino , Gástrula/citologia , Gástrula/metabolismo , Deleção de Genes , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Masculino , Camundongos , Camundongos Knockout , Oócitos/metabolismo , Transdução de Sinais , Transcrição Gênica/genética , Zigoto/metabolismo
20.
Mol Cell Biol ; 26(9): 3541-9, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16611995

RESUMO

Notch signaling is an evolutionarily conserved pathway involved in intercellular communication and is essential for proper cell fate choices. Numerous genes participate in the modulation of the Notch signaling pathway activity. Among them, Notchless (Nle) is a direct regulator of the Notch activity identified in Drosophila melanogaster. Here, we characterized the murine ortholog of Nle and demonstrated that it has conserved the ability to modulate Notch signaling. We also generated mice deficient for mouse Nle (mNle) and showed that its disruption resulted in embryonic lethality shortly after implantation. In late mNle(-/-) blastocysts, inner cell mass (ICM) cells died through a caspase 3-dependent apoptotic process. Most deficient embryos exhibited a delay in the temporal down-regulation of Oct4 expression in the trophectoderm (TE). However, mNle-deficient TE was able to induce decidual swelling in vivo and properly differentiated in vitro. Hence, our results indicate that mNle is mainly required in ICM cells, being instrumental for their survival, and raise the possibility that the death of mNle-deficient embryos might result from abnormal Notch signaling during the first steps of development.


Assuntos
Linhagem da Célula/genética , Implantação do Embrião/genética , Embrião de Mamíferos/citologia , Genes Letais , Proteínas de Membrana/fisiologia , Receptores Notch/fisiologia , Sequência de Aminoácidos , Animais , Apoptose , Drosophila melanogaster/genética , Drosophila melanogaster/fisiologia , Embrião não Mamífero/citologia , Feminino , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Mutantes , Dados de Sequência Molecular , Xenopus
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