RESUMO
Endothelial cell responses to fluid shear stress from blood flow are crucial for vascular development, function, and disease. A complex of PECAM-1, VE-cadherin, VEGF receptors (VEGFRs), and Plexin D1 located at cell-cell junctions mediates many of these events. However, available evidence suggests that another mechanosensor upstream of PECAM-1 initiates signaling. Hypothesizing that GPCR and Gα proteins may serve this role, we performed siRNA screening of Gα subunits and found that Gαi2 and Gαq/11 are required for activation of the junctional complex. We then developed a new activation assay, which showed that these G proteins are activated by flow. We next mapped the Gα residues required for activation and developed an affinity purification method that used this information to identify latrophilin-2 (Lphn2/ADGRL2) as the upstream GPCR. Latrophilin-2 is required for all PECAM-1 downstream events tested. In both mice and zebrafish, latrophilin-2 is required for flow-dependent angiogenesis and artery remodeling. Furthermore, endothelial-specific knockout demonstrates that latrophilin plays a role in flow-dependent artery remodeling. Human genetic data reveal a correlation between the latrophilin-2-encoding Adgrl2 gene and cardiovascular disease. Together, these results define a pathway that connects latrophilin-dependent G protein activation to subsequent endothelial signaling, vascular physiology, and disease.
Assuntos
Junções Intercelulares , Mecanotransdução Celular , Receptores Acoplados a Proteínas G , Receptores de Peptídeos , Animais , Humanos , Camundongos , Células Endoteliais/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Junções Intercelulares/metabolismo , Junções Intercelulares/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores de Peptídeos/metabolismo , Receptores de Peptídeos/genética , Estresse Mecânico , Peixe-Zebra/metabolismo , Peixe-Zebra/genéticaRESUMO
The dorsal aorta (DA) is the first major blood vessel to develop in the embryonic cardiovascular system. Its formation is governed by a coordinated process involving the migration, specification, and arrangement of angioblasts into arterial and venous lineages, a process conserved across species. While vascular endothelial growth factor a (VEGF-A) drives DA specification and formation, the kinases involved in this process remain ambiguous. Thus, we investigated the role of protein kinase B, Akt, in zebrafish by generating a quadruple mutant (aktΔ/Δ), where expression and activity of all akt genes-akt 1, 2, 3a, and 3b are strongly decreased. Live imaging of developing aktΔ/Δ DA uncovers early arteriovenous malformations. Single-cell RNA sequencing analysis of aktΔ/Δ endothelial cells corroborates the impairment of arterial, yet not venous, cell specification. Notably, endothelial specific expression of ligand-independent activation of Notch or constitutively active Akt1 were sufficient to reestablish normal arterial specification in aktΔ/Δ. The Akt-loss-of-function mutant unveils that Akt kinase can act upstream of Notch in arterial endothelial cells, and is involved in proper embryonic artery specification. This sheds light on cardiovascular development, revealing a mechanism behind congenital malformations.
RESUMO
Anaplastic lymphoma kinase (Alk) and leucocyte tyrosine kinase (Ltk) were identified as "orphan" receptor tyrosine kinases (RTKs) with oncogenic potential. Recently ALKAL1 and ALKAL2 (also named "augmentor-ß" and "augmentor-α" or "FAM150A" and "FAM150B," respectively) were discovered as physiological ligands of Alk and Ltk. Here, we employ zebrafish as a model system to explore the physiological function and to characterize in vivo links between Alk and Ltk with their ligands. Unlike the two ligands encoded by mammalian genomes, the zebrafish genome contains three genes: aug-α1, aug-α2, and aug-ß Our experiments demonstrate that these ligands play an important role in zebrafish pigment development. Deficiency in aug-α1, aug-α2, and aug-ß results in strong impairment in iridophore patterning of embryonic and adult zebrafish that is phenocopied in zebrafish deficient in Ltk. We show that aug-α1 and aug-α2 are essential for embryonic iridophore development and adult body coloration. In contrast, aug-α2 and aug-ß are essential for iridophore formation in the adult eye. Importantly, these processes are entirely mediated by Ltk and not by Alk. These experiments establish a physiological link between augmentor ligands and Ltk and demonstrate that particular augmentors activate Ltk in a tissue-specific context to induce iridophore differentiation from neural crest-derived cells and pigment progenitor cells.
Assuntos
Receptores Proteína Tirosina Quinases/genética , Peixe-Zebra/genética , Sequência de Aminoácidos , Quinase do Linfoma Anaplásico , Animais , Diferenciação Celular/genética , Olho/metabolismo , Genoma/genética , Ligantes , Crista Neural/fisiologia , Pigmentos da Retina/genética , Células-Tronco/fisiologia , Peixe-Zebra/fisiologiaRESUMO
Vascular endothelial growth factor C (Vegfc) is a secreted protein that guides lymphatic development in vertebrate embryos. However, its role during developmental angiogenesis is not well characterized. Here, we identify a mutation in zebrafish vegfc that severely affects lymphatic development and leads to angiogenesis defects on sensitized genetic backgrounds. The um18 mutation prematurely truncated Vegfc, blocking its secretion and paracrine activity but not its ability to activate its receptor Flt4. When expressed in endothelial cells, vegfc(um18) could not rescue lymphatic defects in mutant embryos, but induced ectopic blood vessel branching. Furthermore, vegfc-deficient endothelial cells did not efficiently contribute to tip cell positions in developing sprouts. Computational modeling together with assessment of endothelial cell dynamics by time-lapse analysis suggested that an autocrine Vegfc/Flt4 loop plays an important role in migratory persistence and filopodia stability during sprouting. Our results suggest that Vegfc acts in two distinct modes during development: as a paracrine factor secreted from arteries to guide closely associated lymphatic vasculature and as an autocrine factor to drive migratory persistence during angiogenesis.
Assuntos
Vasos Sanguíneos/embriologia , Sistema Linfático/embriologia , Fator C de Crescimento do Endotélio Vascular/genética , Fator C de Crescimento do Endotélio Vascular/fisiologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologia , Alelos , Animais , Animais Geneticamente Modificados , Comunicação Autócrina/genética , Comunicação Autócrina/fisiologia , Vasos Sanguíneos/crescimento & desenvolvimento , Movimento Celular/genética , Movimento Celular/fisiologia , Códon sem Sentido/fisiologia , Embrião não Mamífero , Feminino , Sistema Linfático/crescimento & desenvolvimento , Camundongos , Camundongos Knockout , Neovascularização Fisiológica/genética , Neovascularização Fisiológica/fisiologia , Comunicação Parácrina/genética , Comunicação Parácrina/fisiologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Transdução de Sinais/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genéticaRESUMO
Within the circulatory system, blood flow regulates vascular remodelling, stimulates blood stem cell formation, and has a role in the pathology of vascular disease. During vertebrate embryogenesis, vascular patterning is initially guided by conserved genetic pathways that act before circulation. Subsequently, endothelial cells must incorporate the mechanosensory stimulus of blood flow with these early signals to shape the embryonic vascular system. However, few details are known about how these signals are integrated during development. To investigate this process, we focused on the aortic arch (AA) blood vessels, which are known to remodel in response to blood flow. By using two-photon imaging of live zebrafish embryos, we observe that flow is essential for angiogenesis during AA development. We further find that angiogenic sprouting of AA vessels requires a flow-induced genetic pathway in which the mechano-sensitive zinc finger transcription factor klf2a induces expression of an endothelial-specific microRNA, mir-126, to activate Vegf signalling. Taken together, our work describes a novel genetic mechanism in which a microRNA facilitates integration of a physiological stimulus with growth factor signalling in endothelial cells to guide angiogenesis.
Assuntos
Aorta Torácica/embriologia , Hemodinâmica , MicroRNAs/metabolismo , Neovascularização Fisiológica , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Peixe-Zebra/genética , Animais , Células Endoteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , MicroRNAs/genética , Células NIH 3T3 , Fluxo Sanguíneo Regional/fisiologia , Peixe-Zebra/sangue , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
The cilium is a signaling platform of the vertebrate cell. It has a critical role in polycystic kidney disease and nephronophthisis. Cilia have been detected on endothelial cells, but the function of these organelles in the vasculature remains incompletely defined. In this study, using genetic and chemical genetic tools in the model organism zebrafish, we reveal an essential role of cilia in developmental vascular integrity. Embryos expressing mutant intraflagellar transport genes, which are essential and specific for cilia biogenesis, displayed increased risk of developmental intracranial hemorrhage, whereas the morphology of the vasculature remained normal. Moreover, cilia were present on endothelial cells in the developing zebrafish vasculature. We further show that the involvement of cilia in vascular integrity is endothelial autonomous, because endothelial-specific re-expression of intraflagellar transport genes in respective mutants rescued the intracranial hemorrhage phenotype. Finally, whereas inhibition of Hedgehog signaling increased the risk of intracranial hemorrhage in ciliary mutants, activation of the pathway rescued this phenotype. In contrast, embryos expressing an inactivating mutation in pkd2, one of two autosomal dominant cystic kidney disease genes, did not show increased risk of developmental intracranial hemorrhage. These results suggest that Hedgehog signaling is a major mechanism for this novel role of endothelial cilia in establishing vascular integrity.
Assuntos
Cílios/fisiologia , Endotélio Vascular/fisiologia , Proteínas Hedgehog/metabolismo , Hemorragias Intracranianas/etiologia , Animais , Células Endoteliais/citologia , Mecanotransdução Celular , Canais de Cátion TRPP/fisiologia , Peixe-ZebraRESUMO
Vascular smooth muscle cells (VSMCs) envelop vertebrate brain arteries, playing a crucial role in regulating cerebral blood flow and neurovascular coupling. The dedifferentiation of VSMCs is implicated in cerebrovascular diseases and neurodegeneration. Despite its importance, the process of VSMC differentiation on brain arteries during development remains inadequately characterized. Understanding this process could aid in reprogramming and regenerating differentiated VSMCs in cerebrovascular diseases. In this study, we investigated VSMC differentiation on the zebrafish circle of Willis (CoW), comprising major arteries that supply blood to the vertebrate brain. We observed that the arterial expression of CoW endothelial cells (ECs) occurs after their migration from the cranial venous plexus to form CoW arteries. Subsequently, acta2+ VSMCs differentiate from pdgfrb+ mural cell progenitors upon recruitment to CoW arteries. The progression of VSMC differentiation exhibits a spatiotemporal pattern, advancing from anterior to posterior CoW arteries. Analysis of blood flow suggests that earlier VSMC differentiation in anterior CoW arteries correlates with higher red blood cell velocity wall shear stress. Furthermore, pulsatile blood flow is required for differentiation of human brain pdgfrb+ mural cells into VSMCs as well as VSMC differentiation on zebrafish CoW arteries. Consistently, the flow-responsive transcription factor klf2a is activated in ECs of CoW arteries prior to VSMC differentiation, and klf2a knockdown delays VSMC differentiation on anterior CoW arteries. In summary, our findings highlight the role of blood flow activation of endothelial klf2a as a mechanism regulating the initial VSMC differentiation on vertebrate brain arteries.
RESUMO
Vascular smooth muscle cells (VSMCs) envelop vertebrate brain arteries and play a crucial role in regulating cerebral blood flow and neurovascular coupling. The dedifferentiation of VSMCs is implicated in cerebrovascular disease and neurodegeneration. Despite its importance, the process of VSMC differentiation on brain arteries during development remains inadequately characterized. Understanding this process could aid in reprogramming and regenerating dedifferentiated VSMCs in cerebrovascular diseases. In this study, we investigated VSMC differentiation on zebrafish circle of Willis (CoW), comprising major arteries that supply blood to the vertebrate brain. We observed that arterial specification of CoW endothelial cells (ECs) occurs after their migration from cranial venous plexus to form CoW arteries. Subsequently, acta2+ VSMCs differentiate from pdgfrb+ mural cell progenitors after they were recruited to CoW arteries. The progression of VSMC differentiation exhibits a spatiotemporal pattern, advancing from anterior to posterior CoW arteries. Analysis of blood flow suggests that earlier VSMC differentiation in anterior CoW arteries correlates with higher red blood cell velocity and wall shear stress. Furthermore, pulsatile flow induces differentiation of human brain PDGFRB+ mural cells into VSMCs, and blood flow is required for VSMC differentiation on zebrafish CoW arteries. Consistently, flow-responsive transcription factor klf2a is activated in ECs of CoW arteries prior to VSMC differentiation, and klf2a knockdown delays VSMC differentiation on anterior CoW arteries. In summary, our findings highlight blood flow activation of endothelial klf2a as a mechanism regulating initial VSMC differentiation on vertebrate brain arteries.
Assuntos
Diferenciação Celular , Círculo Arterial do Cérebro , Hemodinâmica , Músculo Liso Vascular , Peixe-Zebra , Animais , Círculo Arterial do Cérebro/embriologia , Músculo Liso Vascular/fisiologia , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Humanos , Miócitos de Músculo Liso/fisiologia , Miócitos de Músculo Liso/metabolismo , Células Endoteliais/fisiologia , Células Endoteliais/metabolismoRESUMO
Endothelial cell responses to fluid shear stress from blood flow are crucial for vascular development, function and disease. A complex of PECAM-1, VE-cadherin, VEGF receptors (VEGFRs) and PlexinD1 located at cell-cell junctions mediates many of these events. But available evidence suggests that another mechanosensor upstream of PECAM-1 initiates signaling. Hypothesizing that GPCR and Gα proteins may serve this role, we performed siRNA screening of Gα subunits and found that Gαi2 and Gαq/11 are required for activation of the junctional complex. We then developed a new activation assay, which showed that these G proteins are activated by flow. We next mapped the Gα residues required for activation and developed an affinity purification method that used this information to identify latrophilin-2 (Lphn-2/ADGRL2) as the upstream GPCR. Latrophilin-2 is required for all PECAM-1 downstream events tested. In both mice and zebrafish, latrophilin-2 is required for flow-dependent angiogenesis and artery remodeling. Furthermore, endothelial specific knockout demonstrates that latrophilin plays a role in flow-dependent artery remodeling. Human genetic data reveal a correlation between the latrophilin-2-encoding Adgrl2 gene and cardiovascular disease. Together, these results define a pathway that connects latrophilin-dependent G protein activation to subsequent endothelial signaling, vascular physiology and disease.
RESUMO
OBJECTIVE: The goal of this study was to determine the in vivo functions of the synaptic proteins neurexins and neuroligins in embryonic vascular system development using zebrafish as animal model. METHODS AND RESULTS: In the present study, we show that the knockdown of the α-form of neurexin 1a induces balance defects and reduced locomotory activity, whereas ß-neurexin 1a and neuroligin 1 morphants present defects in sprouting angiogenesis and vascular remodeling, in particular in the caudal plexus and subintestinal vessels. Coinjection of low doses of morpholinos for ß-neurexin 1a and neuroligin 1 together or in combination with morpholinos targeting the -heparin--binding isoforms of vascular endothelial growth factor A (encoded by the VEGFAb gene) recapitulates the observed abnormalities, suggesting synergistic activity of these molecules. Similar coinjection experiments with morpholinos, targeting the enzyme heparan sulfate 6-O-sulfotransferase 2, confirm the presence of a functional correlation between extracellular matrix maturation and ß-neurexin 1a or neuroligin 1. CONCLUSIONS: Our data represent the first in vivo evidence of the role of neurexin and neuroligin in embryonic blood vessel formation and provide insights into their mechanism of action.
Assuntos
Vasos Sanguíneos/embriologia , Moléculas de Adesão Celular Neuronais/fisiologia , Glicoproteínas/fisiologia , Heparina/metabolismo , Neovascularização Fisiológica , Neuropeptídeos/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Peixe-Zebra/embriologia , Animais , Matriz Extracelular/fisiologia , Sulfotransferases/fisiologiaRESUMO
OBJECTIVE: Endothelial sphingosine-1-phosphate (S1P) receptor-1 (S1P(1)) affects different vascular functions, including blood vessel maturation and permeability. Here, we characterized the role of the zS1P(1) ortholog in vascular development in zebrafish. METHODS AND RESULTS: zS1P(1) is expressed in dorsal aorta and posterior cardinal vein of zebrafish embryos at 24 to 30 hours postfertilization. zS1P(1) downregulation by antisense morpholino oligonucleotide injection causes early pericardial edema, lack of blood circulation, alterations of posterior cardinal vein structure, and late generalized edema. Also, zS1P(1) morphants are characterized by downregulation of vascular endothelial cadherin (VE-cadherin) and Eph receptor EphB4a expression and by disorganization of zonula occludens 1 junctions in posterior cardinal vein endothelium, with no alterations of dorsal aorta endothelium. VE-cadherin knockdown results in similar vascular alterations, whereas VE-cadherin overexpression is sufficient to rescue venous vascular integrity defects and EphB4a downregulation in zS1P(1) morphants. Finally, S1P(1) small interfering RNA transfection and the S1P(1) antagonist (R)-3-amino-(3-hexylphenylamino)-4-oxobutylphosphonic acid (W146) cause EPHB4 receptor down-modulation in human umbilical vein endothelial cells and the assembly of zonula occludens 1 intercellular contacts is prevented by the EPHB4 antagonist TNYL-RAW peptide in these cells. CONCLUSIONS: The data demonstrate a nonredundant role of zS1P(1) in the regulation of venous endothelial barrier in zebrafish and identify a S1P(1)/VE-cadherin/EphB4a genetic pathway that controls venous vascular integrity.
Assuntos
Permeabilidade Capilar , Células Endoteliais/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Veias/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Anilidas/farmacologia , Animais , Animais Geneticamente Modificados , Antígenos CD/metabolismo , Células CHO , Caderinas/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , Cricetinae , Cricetulus , Células Endoteliais/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Morfolinos/metabolismo , Oligonucleotídeos Antissenso/metabolismo , Organofosfonatos/farmacologia , Fosfoproteínas/metabolismo , Interferência de RNA , Receptor EphB4/metabolismo , Receptores de Lisoesfingolipídeo/antagonistas & inibidores , Receptores de Lisoesfingolipídeo/genética , Receptores de Esfingosina-1-Fosfato , Junções Íntimas/metabolismo , Transfecção , Veias/efeitos dos fármacos , Veias/embriologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética , Proteína da Zônula de Oclusão-1RESUMO
Definitive haematopoietic stem and progenitor cells (HSPCs) generate erythroid, lymphoid and myeloid lineages. HSPCs are produced in the embryo via transdifferentiation of haemogenic endothelial cells in the aorta-gonad-mesonephros (AGM). HSPCs in the AGM are heterogeneous in differentiation and proliferative output, but how these intrinsic differences are acquired remains unanswered. Here we discovered that loss of microRNA (miR)-128 in zebrafish leads to an expansion of HSPCs in the AGM with different cell cycle states and a skew towards erythroid and lymphoid progenitors. Manipulating miR-128 in differentiating haemogenic endothelial cells, before their transition to HSPCs, recapitulated the lineage skewing in both zebrafish and human pluripotent stem cells. miR-128 promotes Wnt and Notch signalling in the AGM via post-transcriptional repression of the Wnt inhibitor csnk1a1 and the Notch ligand jag1b. De-repression of cskn1a1 resulted in replicative and erythroid-biased HSPCs, whereas de-repression of jag1b resulted in G2/M and lymphoid-biased HSPCs with long-term consequence on the respective blood lineages. We propose that HSPC heterogeneity arises in the AGM endothelium and is programmed in part by Wnt and Notch signalling.
Assuntos
Hemangioblastos , MicroRNAs , Animais , Humanos , Peixe-Zebra/genética , Células-Tronco Hematopoéticas/metabolismo , Diferenciação Celular/genética , Endotélio , MicroRNAs/metabolismo , Hematopoese/genéticaRESUMO
Tissue resident macrophages are largely of embryonic (fetal liver) origin and long-lived, while bone marrow-derived macrophages (BMDM) are recruited following an acute perturbation, such as hypoxia in the setting of myocardial ischemia. Prior transcriptome analyses identified BMDM and fetal liver-derived macrophage (FLDM) differences at the RNA expression level. Posttranscriptional regulation determining mRNA stability and translation rate may override transcriptional signals in response to hypoxia. We profiled differentially regulated BMDM and FLDM transcripts in response to hypoxia at the level of mRNA translation. Using a translating ribosome affinity purification (TRAP) assay and RNA-seq, we identified non-overlapping transcripts with increased translation rate in BMDM (Ly6e, vimentin, PF4) and FLDM (Ccl7, Ccl2) after hypoxia. We further identified hypoxia-induced transcripts within these subsets that are regulated by the RNA-binding protein HuR. These findings define translational differences in macrophage subset gene expression programs, highlighting potential therapeutic targets in ischemic myocardium.
RESUMO
(1) Background: Whole Exome Sequencing of patients with thoracic aortic aneurysm often identifies "Variants of Uncertain Significance" (VUS), leading to uncertainty in clinical management. We assess a novel mechanism for potential routine assessment of these genes in TAA patients. Zebrafish are increasingly used as experimental models of disease. Advantages include low cost, rapid maturation, and physical transparency, permitting direct microscopic assessment. (2) Methods: Zebrafish loss of function mutations were generated using a CRISPRC/CAS9 approach for EMILIN1 and MIB1 genes similar to VUSs identified in clinical testing. Additionally, "positive control" mutants were constructed for known deleterious variants in FBN1 (Marfan's) and COL1A2, COL5A1, COL5A2 (Ehlers-Danlos). Zebrafish embryos were followed to six days post-fertilization. Embryos were studied by brightfield and confocal microscopy to ascertain any vascular, cardiac, and skeletal abnormalities. (3) Results: A dramatic pattern of cardiac, cerebral, aortic, and skeletal abnormalities was identified for the known pathogenic FBN1 and COL1A2, COL5A1, and COL5A2 mutants, as well as for the EMILIN1 and MIB1 mutants of prior unknown significance. Visualized abnormalities included hemorrhage (peri-aortic and cranial), cardiomegaly, reduced diameter of the aorta and intersegmental vessels, lower aortic cell counts, and scoliosis (often extremely severe). (4) Conclusion: This pilot study suggests that candidate genes arising in clinical practice may be rapidly assessed via zebrafish mutants-thus permitting evidence-based decisions about pathogenicity. Thus, years-long delays to clinically demonstrate pathogenicity may be obviated. Zebrafish data would represent only one segment of analysis, which would also include frequency of the variant in the general population, in silico genetic analysis, and degree of preservation in phylogeny.
Assuntos
Aneurisma da Aorta Torácica/patologia , Modelos Animais de Doenças , Embrião não Mamífero/patologia , Mutação com Perda de Função , Fenótipo , Proteínas de Peixe-Zebra/genética , Animais , Aneurisma da Aorta Torácica/genética , Embrião não Mamífero/metabolismo , Humanos , Projetos Piloto , Peixe-ZebraRESUMO
Response to hypoxia is a highly regulated process, but little is known about single-cell responses to hypoxic conditions. Using fluorescent reporters of hypoxia response factor-1α (HIF-1α) activity in various cancer cell lines and patient-derived cancer cells, we show that hypoxic responses in individual cancer cells can be highly dynamic and variable. These responses fall into three classes, including oscillatory activity. We identify a molecular mechanism that can account for all three response classes, implicating reactive-oxygen-species-dependent chaperone-mediated autophagy of HIF-1α in a subset of cells. Furthermore, we show that oscillatory response is modulated by the abundance of extracellular lactate in a quorum-sensing-like mechanism. We show that oscillatory HIF-1α activity rescues hypoxia-mediated inhibition of cell division and causes broad suppression of genes downregulated in cancers and activation of genes upregulated in many cancers, suggesting a mechanism for aggressive growth in a subset of hypoxic tumor cells.
Assuntos
Autofagia Mediada por Chaperonas , Ácido Láctico , Humanos , Ácido Láctico/metabolismo , Linhagem Celular Tumoral , Hipóxia/metabolismo , Proliferação de CélulasRESUMO
The HIV-1 transactivating factor Tat accumulates on the surface of endothelium by interacting with heparan sulfate proteoglycans (HSPGs). Tat also interacts with B-lymphoid Namalwa cells but only when these overexpress HSPGs after syndecan-1 cDNA transfection (SYN-NCs). Accordingly, SYN-NCs, but not mock-transfected cells, adhere to endothelial cells (ECs) when Tat is bound to the surface of either one of the 2 cell types or when SYN-NCs are transfected with a Tat cDNA. Moreover, endogenously produced Tat bound to cell-surface HSPGs mediates cell adhesion of HIV(+) ACH-2 lymphocytes to the endothelium. This heterotypic lymphocyte-EC interaction is prevented by HSPG antagonist or heparinase treatment, but not by integrin antagonists and requires the homodimerization of Tat protein. Tat tethered to the surface of SYN-NCs or of peripheral blood monocytes from healthy donors promotes their transendothelial migration in vitro in response to CXCL12 or CCL5, respectively, and SYN-NC extravasation in vivo in a zebrafish embryo model of inflammation. In conclusion, Tat homodimers bind simultaneously to HSPGs expressed on lymphoid and EC surfaces, leading to HSPG/Tat-Tat/HSPG quaternary complexes that physically link HSPG-bearing lymphoid cells to the endothelium, promoting their extravasation. These data provide new insights about how lymphoid cells extravasate during HIV infection.
Assuntos
Movimento Celular , Endotélio Vascular/metabolismo , Infecções por HIV/metabolismo , HIV-1/metabolismo , Linfócitos/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Animais , Bovinos , Adesão Celular , Linhagem Celular Tumoral , Quimiocina CCL5/farmacologia , Quimiocina CXCL12/farmacologia , Embrião não Mamífero/metabolismo , Infecções por HIV/genética , Proteoglicanas de Heparan Sulfato , Humanos , Multimerização Proteica , Sindecana-1 , Transfecção , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
Protein transport between the trans-Golgi network and endosomes is mediated by transport vesicles formed by the adaptor-protein complex AP-1, consisting of the adaptins γ1, ß1, µ1, σ1. Mammalia express µ1A ubiquitously and isoform µ1B in polarized epithelia. Mouse γ1 or µ1A 'knock out's revealed that AP-1 is indispensable for embryonic development. We isolated µ1A and µ1B from Danio rerio. Analysis of µ1A and µ1B expression revealed tissue-specific expression for either one during embryogenesis and in adult tissues in contrast to their expression in mammalia. µ1B transcript was detected in organs of endodermal derivation and "knock-down" experiments gave rise to embryos defective in formation of intestine, liver, and pronephric ducts. Development ceased at 7-8 dpf. µ1B is not expressed in murine liver, indicating loss of µ1B expression and establishment of alternative sorting mechanisms during mammalian development.
Assuntos
Complexo 1 de Proteínas Adaptadoras/metabolismo , Subunidades mu do Complexo de Proteínas Adaptadoras/metabolismo , Morfogênese/fisiologia , Isoformas de Proteínas/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/crescimento & desenvolvimento , Complexo 1 de Proteínas Adaptadoras/genética , Subunidades mu do Complexo de Proteínas Adaptadoras/classificação , Subunidades mu do Complexo de Proteínas Adaptadoras/genética , Sequência de Aminoácidos , Animais , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Genoma , Humanos , Camundongos , Dados de Sequência Molecular , Fenótipo , Filogenia , Isoformas de Proteínas/classificação , Isoformas de Proteínas/genética , Alinhamento de Sequência , Distribuição Tecidual , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/classificação , Proteínas de Peixe-Zebra/genéticaRESUMO
Intracranial aneurysm (IA) rupture leads to subarachnoid hemorrhage, a sudden-onset disease that often causes death or severe disability. Although genome-wide association studies have identified common genetic variants that increase IA risk moderately, the contribution of variants with large effect remains poorly defined. Using whole-exome sequencing, we identified significant enrichment of rare, deleterious mutations in PPIL4, encoding peptidyl-prolyl cis-trans isomerase-like 4, in both familial and index IA cases. Ppil4 depletion in vertebrate models causes intracerebral hemorrhage, defects in cerebrovascular morphology and impaired Wnt signaling. Wild-type, but not IA-mutant, PPIL4 potentiates Wnt signaling by binding JMJD6, a known angiogenesis regulator and Wnt activator. These findings identify a novel PPIL4-dependent Wnt signaling mechanism involved in brain-specific angiogenesis and maintenance of cerebrovascular integrity and implicate PPIL4 gene mutations in the pathogenesis of IA.
Assuntos
Encéfalo/irrigação sanguínea , Ciclofilinas/genética , Aneurisma Intracraniano/genética , Neovascularização Patológica/genética , Proteínas de Ligação a RNA/genética , Ciclofilinas/fisiologia , Humanos , Mutação , Proteínas de Ligação a RNA/fisiologia , Sequenciamento do Exoma , Via de Sinalização Wnt/fisiologiaRESUMO
Fibroblast growth factor-2 (FGF2) plays a major role in angiogenesis. The pattern recognition receptor long-pentraxin 3 (PTX3) inhibits the angiogenic activity of FGF2. To identify novel FGF2-antagonistic peptide(s), four acetylated (Ac) synthetic peptides overlapping the FGF2-binding region PTX3-(97-110) were assessed for their FGF2-binding capacity. Among them, the shortest pentapeptide Ac-ARPCA-NH(2) (PTX3-[100-104]) inhibits the interaction of FGF2 with PTX3 immobilized to a BIAcore sensorchip and suppresses FGF2-dependent proliferation in endothelial cells, without affecting the activity of unrelated mitogens. Also, Ac-ARPCA-NH(2) inhibits angiogenesis triggered by FGF2 or by tumorigenic FGF2-overexpressing murine endothelial cells in chick and zebrafish embryos, respectively. Accordingly, the peptide hampers the binding of FGF2 to Chinese Hamster ovary cells overexpressing the tyrosine-kinase FGF receptor-1 (FGFR1) and to recombinant FGFR1 immobilized to a BIAcore sensorchip without affecting heparin interaction. In all the assays the mutated Ac-ARPSA-NH(2) peptide was ineffective. In keeping with the observation that hydrophobic interactions dominate the interface between FGF2 and the FGF-binding domain of the Ig-like loop D2 of FGFR1, amino acid substitutions in Ac-ARPCA-NH(2) and saturation transfer difference-nuclear magnetic resonance analysis of its mode of interaction with FGF2 implicate the hydrophobic methyl groups of the pentapeptide in FGF2 binding. These results will provide the basis for the design of novel PTX3-derived anti-angiogenic FGF2 antagonists.