RESUMEN
Dendritic spines are essential for synaptic function because they constitute the postsynaptic compartment of the neurons that receives the most excitatory input. The extracellularly shorter variant of the presynaptic cell adhesion molecules neurexins, ß-neurexin, has been implicated in various aspects of synaptic function, including neurotransmitter release. However, its role in developing or stabilizing dendritic spines as fundamental computational units of excitatory synapses has remained unclear. Here, we show through morphological analysis that the deletion of ß-neurexins in hippocampal neurons in vitro and in hippocampal tissue in vivo affects presynaptic dense-core vesicles, as hypothesized earlier, and, unexpectedly, alters the postsynaptic spine structure. Specifically, we observed that the absence of ß-neurexins led to an increase in filopodial-like protrusions in vitro and more mature mushroom-type spines in the CA1 region of adult knockout mice. In addition, the deletion of ß-neurexins caused alterations in the spine head dimension and an increase in spines with perforations of their postsynaptic density but no changes in the overall number of spines or synapses. Our results indicate that presynaptic ß-neurexins play a role across the synaptic cleft, possibly by aligning with postsynaptic binding partners and glutamate receptors via transsynaptic columns.
Asunto(s)
Espinas Dendríticas , Neurexinas , Ratones , Animales , Espinas Dendríticas/metabolismo , Sinapsis/metabolismo , Neuronas/metabolismo , Transmisión Sináptica/fisiología , Hipocampo/metabolismo , Ratones NoqueadosRESUMEN
Junctional adhesion molecule (JAM)-A is a cell adhesion receptor localized at epithelial cell-cell contacts with enrichment at the tight junctions. Its role during cell-cell contact formation and epithelial barrier formation has intensively been studied. In contrast, its role during collective cell migration is largely unexplored. Here, we show that JAM-A regulates collective cell migration of polarized epithelial cells. Depletion of JAM-A in MDCK cells enhances the motility of singly migrating cells but reduces cell motility of cells embedded in a collective by impairing the dynamics of cryptic lamellipodia formation. This activity of JAM-A is observed in cells grown on laminin and collagen-I but not on fibronectin or vitronectin. Accordingly, we find that JAM-A exists in a complex with the laminin- and collagen-I-binding α3ß1 integrin. We also find that JAM-A interacts with tetraspanins CD151 and CD9, which both interact with α3ß1 integrin and regulate α3ß1 integrin activity in different contexts. Mapping experiments indicate that JAM-A associates with α3ß1 integrin and tetraspanins CD151 and CD9 through its extracellular domain. Similar to depletion of JAM-A, depletion of either α3ß1 integrin or tetraspanins CD151 and CD9 in MDCK cells slows down collective cell migration. Our findings suggest that JAM-A exists with α3ß1 integrin and tetraspanins CD151 and CD9 in a functional complex to regulate collective cell migration of polarized epithelial cells.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Integrina alfa3beta1/metabolismo , Tetraspanina 24/metabolismo , Tetraspanina 29/metabolismo , Animales , Moléculas de Adhesión Celular/antagonistas & inhibidores , Moléculas de Adhesión Celular/genética , Línea Celular , Movimiento Celular/efectos de los fármacos , Perros , Doxorrubicina/farmacología , Humanos , Molécula A de Adhesión de Unión/antagonistas & inhibidores , Molécula A de Adhesión de Unión/genética , Células de Riñón Canino Madin Darby , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/metabolismoRESUMEN
The neuropilin-1 (NRP1)-MET signaling axis regulates the motility of individual endothelial cells (ECs). It is unknown how this signaling pathway affects the endothelial barrier in coherent ECs forming a tight monolayer. We hypothesized that it is involved both in modulation of the endothelial barrier and in EC activation. To investigate the role of NRP1-MET signaling in inflammatory processes (e.g., systemic inflammatory response syndrome [SIRS] or snakebite-induced SIRS-like conditions), we employed the C-type lectin-related protein rhodocetin-αß (RCαß) as a specific trigger of this signal axis in ECs in vitro. In coherent HUVECs, RCαß reinforced the actin cytoskeleton and increased cell stiffness, thus favoring vascular endothelial cadherin-mediated transmission of intercellular forces. Increased cell stiffness was associated with enhanced activation of RhoA and nuclear translocation of NF-κB. Simultaneously, RCαß-triggered signaling via the NRP1-MET axis increased EC monolayer permeability, induced transcription of proinflammatory genes such as ICAM-1 and, consequently, leukocyte tethering. The RCαß-induced transcriptome differed from that induced by hepatocyte growth factor, although in both cases the same tyrosine kinase, MET, was involved. This was due to RCαß-mediated recruitment of the MET coreceptor NRP1 and additional Rho-mediated activation of the actomyosin system. RCαß induced similar transcriptional and cellular changes if external shear forces were applied. These data highlight the modulatory role of NRP1 as MET coreceptor, and they explain how some snake venoms induce SIRS-like conditions. Additionally, this study demonstrates that inflammatory activation of coherent ECs is triggered by converging signals that are induced by NRP1-MET signaling and influenced by intercellular forces.
Asunto(s)
Células Endoteliales de la Vena Umbilical Humana/inmunología , Inflamación/inmunología , Neuropilina-1/inmunología , Proteínas Proto-Oncogénicas c-met/inmunología , Transducción de Señal/inmunología , Células Cultivadas , HumanosRESUMEN
Blood vessels are covered with endothelial cells on their inner surfaces, forming a selective and semipermeable barrier between the blood and the underlying tissue. Many pathological processes, such as inflammation or cancer metastasis, are accompanied by an increased vascular permeability. Progress in live cell imaging techniques has recently revealed that the structure of endothelial cell contacts is constantly reorganized and that endothelial junctions display high heterogeneities at a subcellular level even within one cell. Although it is assumed that this dynamic remodeling is associated with a local change in endothelial barrier function, a direct proof is missing mainly because of a lack of appropriate experimental techniques. Here, we describe a new assay to dynamically measure local endothelial barrier function with a lateral resolution of â¼15 µm and a temporal resolution of 1 min. In this setup, fluorescence-labeled molecules are added to the apical compartment of an endothelial monolayer, and the penetration of molecules from the apical to the basal compartment is recorded by total internal reflection fluorescence microscopy utilizing the generated evanescent field. With this technique, we found a remarkable heterogeneity in the local permeability for albumin within confluent endothelial cell layers. In regions with low permeability, stimulation with the proinflammatory agent histamine results in a transient increase in paracellular permeability. The effect showed a high variability along the contact of one individual cell, indicating a local regulation of endothelial barrier function. In regions with high basal permeability, histamine had no obvious effect. In contrast, the barrier-enhancing drug forskolin reduces the permeability for albumin and dextran uniformly along the cell junctions. Because this new approach can be readily combined with other live cell imaging techniques, it will contribute to a better understanding of the mechanisms underlying subcellular junctional reorganization during wound healing, inflammation, and angiogenesis.
Asunto(s)
Células Endoteliales de la Vena Umbilical Humana/metabolismo , Microscopía/métodos , Supervivencia Celular/efectos de los fármacos , Colforsina/farmacología , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Procesamiento de Imagen Asistido por Computador , Cinética , Permeabilidad/efectos de los fármacosRESUMEN
Endothelial cells of the vascular system are dynamic cells whose molecular adaptability is decisive for the adjustment of homeostasis and organ perfusion. Advanced microscopic techniques, automation processing, and image analysis software was shown to improve the understanding of vascular biology. In this work, we describe advanced methods that allow investigating the dynamics of endothelial cell contacts. The development of viral vectors has contributed significantly to the genetic manipulation of endothelial cells. We used the Gibson assembly as a quick and cheap cloning system for introducing sequences into the lentiviral-based pFUGW vector. Furthermore, classical fluorescence tags such as mCherry and EGFP were compared with self-labeling tags such as Halo and SNAP for their suitability to study junction dynamics in cultured endothelium, and found the self-labeling tags as useful tools. Using such combinations, we found maintained cell junction integrity during shear stress-induced junction remodeling using VE-cadherin-EGFP. Remodeling was accompanied by VE-cadherin plaque formation, indicating that this process is mediated by the for-mation of the actin-driven junction-associated intermittent lamellipodia, JAIL. The combined methods including the Gibson assembly, lentiviral mediated gene transfer, spinning disk-based live cell imaging, and software for quantification allow analyses of the endothelial cell junction dynamics under static and under shear stress conditions.
Asunto(s)
Clonación Molecular/métodos , Células Endoteliales/fisiología , Células Endoteliales/ultraestructura , Colorantes Fluorescentes , Uniones Intercelulares/fisiología , Animales , Anticuerpos , Anticuerpos Monoclonales , Cadherinas/análisis , Cadherinas/genética , Expresión Génica , Vectores Genéticos , Cabras/inmunología , Proteínas Fluorescentes Verdes/genética , Células Endoteliales de la Vena Umbilical Humana , Humanos , Immunoblotting , Uniones Intercelulares/química , Ratones , Conejos/inmunología , beta Catenina/análisis , gamma Catenina/análisisRESUMEN
RATIONALE: Angiogenesis and vessel integrity depend on the adhesion of endothelial cells (ECs) to the extracellular matrix and to adjacent ECs. The focal adhesion protein α-parvin (α-pv) is essential for vascular development. However, the role of α-pv in ECs in vivo is not known. OBJECTIVE: To determine the function of α-pv in ECs during vascular development in vivo and the underlying mechanisms. METHODS AND RESULTS: We deleted the α-pv gene specifically in ECs of mice to study its role in angiogenesis and vascular development. Here, we show that endothelial-specific deletion of α-pv in mice results in late embryonic lethality associated with hemorrhages and reduced vascular density. Postnatal-induced EC-specific deletion of α-pv leads to retinal hypovascularization because of reduced vessel sprouting and excessive vessel regression. In the absence of α-pv, blood vessels display impaired VE-cadherin junction morphology. In vitro, α-pv-deficient ECs show reduced stable adherens junctions, decreased monolayer formation, and impaired motility, associated with reduced formation of integrin-mediated cell-extracellular matrix adhesion structures and an altered actin cytoskeleton. CONCLUSIONS: Endothelial α-pv is essential for vessel sprouting and for vessel stability.
Asunto(s)
Uniones Adherentes/ultraestructura , Vasos Sanguíneos/embriología , Células Endoteliales/citología , Endotelio Vascular/fisiología , Proteínas de Microfilamentos/fisiología , Neovascularización Fisiológica/fisiología , Uniones Adherentes/fisiología , Animales , Antígenos CD/análisis , Vasos Sanguíneos/crecimiento & desarrollo , Cadherinas/análisis , Movimiento Celular , Forma de la Célula , Células Cultivadas , Citoesqueleto/ultraestructura , Células Endoteliales/metabolismo , Endotelio Vascular/ultraestructura , Matriz Extracelular/ultraestructura , Femenino , Genes Letales , Células Endoteliales de la Vena Umbilical Humana , Masculino , Ratones , Ratones Transgénicos , Proteínas de Microfilamentos/deficiencia , Proteínas de Microfilamentos/genética , Neovascularización Fisiológica/genética , Seudópodos/fisiología , Seudópodos/ultraestructura , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Vasos Retinianos/patologíaRESUMEN
Endothelial junctions are dynamic structures organized by multi-protein complexes that control monolayer integrity, homeostasis, inflammation, cell migration and angiogenesis. Newly developed methods for both the genetic manipulation of endothelium and microscopy permit time-lapse recordings of fluorescent proteins over long periods of time. Quantitative data analyses require automated methods. We developed a software package, the CellBorderTracker, allowing quantitative analysis of fluorescent-tagged cell junction protein dynamics in time-lapse sequences. The CellBorderTracker consists of the CellBorderExtractor that segments cells and identifies cell boundaries and mapping tools for data extraction. The tool is illustrated by analyzing fluorescent-tagged VE-cadherin the backbone of adherence junctions in endothelium. VE-cadherin displays high dynamics that is forced by junction-associated intermittent lamellipodia (JAIL) that are actin driven and WASP/ARP2/3 complex controlled. The manual segmentation and the automatic one agree to 90 %, a value that indicates high reliability. Based on segmentations, different maps were generated allowing more detailed data extraction. This includes the quantification of protein distribution pattern, the generation of regions of interest, junction displacements, cell shape changes, migration velocities and the visualization of junction dynamics over many hours. Furthermore, we demonstrate an advanced kymograph, the J-kymograph that steadily follows irregular cell junction dynamics in time-lapse sequences for individual junctions at the subcellular level. By using the CellBorderTracker, we demonstrate that VE-cadherin dynamics is quickly arrested upon thrombin stimulation, a phenomenon that was largely due to transient inhibition of JAIL and display a very heterogeneous subcellular and divers VE-cadherin dynamics during intercellular gap formation and resealing.
Asunto(s)
Cadherinas/análisis , Endotelio Vascular/citología , Uniones Intercelulares/metabolismo , Programas Informáticos , Animales , Cadherinas/metabolismo , Células Cultivadas , Drosophila , Endotelio Vascular/metabolismo , Fluorescencia , Técnica del Anticuerpo Fluorescente , Humanos , Uniones Intercelulares/químicaRESUMEN
Endothelial cells line the inner surface of all blood vessels and constitute a selective barrier between blood and tissue. Permeation of solutes across the endothelial cell monolayer occurs either paracellularly through specialized endothelial cell-cell junctions or transcellularly via special transport mechanisms including transcytosis, via the formation of transcellular channels, or by cell membrane transport proteins. Several in vitro assays have been developed in the past few decades to analyze the molecular mechanisms of transendothelial permeability. Measurement of the electrical resistance of the cell monolayer has proven to be particularly suitable for analyzing paracellular barrier function with high-time resolution over long time periods. We review the various permeability assays and focus on the electrical impedance analysis of endothelial cell monolayers. We also address current progress in the development of techniques used to investigate endothelial permeability with high-lateral resolution and under mechanical loads.
Asunto(s)
Barrera Hematoencefálica/fisiología , Células Endoteliales/fisiología , Transporte Biológico , Barrera Hematoencefálica/metabolismo , Permeabilidad de la Membrana Celular , Células Cultivadas , Impedancia Eléctrica , Células Endoteliales/metabolismo , HumanosRESUMEN
The vascular endothelium is a cellular interface between the blood and the interstitial space of tissue, which controls the exchange of fluid, solutes and cells by both transcellular and paracellular means. To accomplish the demands on barrier function, the regulation of the endothelium requires quick and adaptive mechanisms. This is, among others, accomplished by actin dynamics that interdependently interact with both the VE-cadherin/catenin complex, the main components of the adherens type junctions in endothelium and the membrane cytoskeleton. Actin filaments in endothelium are components of super-structured protein assemblies that control a variety of dynamic processes such as endo- and exocytosis, shape change, cell-substrate along with cell-cell adhesion and cell motion. In endothelium, actin filaments are components of: (1) contractile actin bundles appearing as stress fibers and junction-associated circumferential actin filaments, (2) actin networks accompanied by endocytotic ruffles, lamellipodia at leading edges of migrating cells and junction-associated intermittent lamellipodia (JAIL) that dynamically maintain junction integrity, (3) cortical actin and (4) the membrane cytoskeleton. All these structures, most probably interact with cell junctions and cell-substrate adhesion sites. Due to the rapid growth in information, we aim to provide a bird's eye view focusing on actin filaments in endothelium and its functional relevance for entire cell and junction integrity, rather than discussing the detailed molecular mechanism for control of actin dynamics.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Uniones Adherentes/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , Animales , Antígenos CD/metabolismo , Cadherinas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Endotelio Vascular/metabolismo , HumanosRESUMEN
The vascular endothelium provides the critical barrier during hematogenous spreading of bacteria, a phenomenon that might contribute to severe diseases in humans including endocarditis and sepsis as known from infections by Staphylococcus aureus. Here we aimed to uncover early responses of the endothelium to S. aureus infection with respect to (a) inflammatory reactions such as paracellular endothelial barrier function and expression of cell adhesion molecule-1 (ICAM-1) and (b) translocation through the endothelium. After infection of the cultured endothelium with 22 different clinical isolates of S. aureus and two well-characterized lab strains a diverse and strain-specific change in para- and transcellular endothelial barrier function was observed. Bayesian data analysis revealed positive correlation of paracellular barrier function decrease followed by expression of ICAM-1 while these parameters negatively correlated with transcellular bacterial translocation. Translocating bacteria largely blocked TNFα-induced ICAM-1 expression indicating an active anti-inflammatory effect mediated by those strains probably due to intracellularly released virulence factors. Furthermore, the underlying background of barrier function decrease was investigated in more detail using two well-characterized lab strains, ls 8325-4 and ls 6850 and respective mutants. Barrier function decrease was found to be independent of early cell death and early release of virulence factors into the medium, but require internalization of live bacteria. The data show for the first time that endothelial cells respond diversely to infection with different strains of S. aureus and that translocating strains downregulate inflammatory response of the endothelium. Furthermore, data indicate that S. aureus-mediated activation of the endothelium reduces bacterial translocation.
Asunto(s)
Traslocación Bacteriana , Permeabilidad Capilar , Células Endoteliales/microbiología , Células Endoteliales/fisiología , Interacciones Huésped-Patógeno , Staphylococcus aureus/fisiología , Células Cultivadas , Expresión Génica , Humanos , Molécula 1 de Adhesión Intercelular/biosíntesisRESUMEN
Ebola virus (EBOV), an enveloped, single-stranded, negative-sense RNA virus, causes severe hemorrhagic fever in humans and nonhuman primates. The EBOV glycoprotein (GP) gene encodes the nonstructural soluble glycoprotein (sGP) but also produces the transmembrane glycoprotein (GP1,2) through transcriptional editing. A third GP gene product, a small soluble glycoprotein (ssGP), has long been postulated to be produced also as a result of transcriptional editing. To identify and characterize the expression of this new EBOV protein, we first analyzed the relative ratio of GP gene-derived transcripts produced during infection in vitro (in Vero E6 cells or Huh7 cells) and in vivo (in mice). The average percentages of transcripts encoding sGP, GP1,2, and ssGP were approximately 70, 25, and 5%, respectively, indicating that ssGP transcripts are indeed produced via transcriptional editing. N-terminal sequence similarity with sGP, the absence of distinguishing antibodies, and the abundance of sGP made it difficult to identify ssGP through conventional methodology. Optimized 2-dimensional (2D) gel electrophoresis analyses finally verified the expression and secretion of ssGP in tissue culture during EBOV infection. Biochemical analysis of recombinant ssGP characterized this protein as a disulfide-linked homodimer that was exclusively N glycosylated. In conclusion, we have identified and characterized a new EBOV nonstructural glycoprotein, which is expressed as a result of transcriptional editing of the GP gene. While ssGP appears to share similar structural properties with sGP, it does not appear to have the same anti-inflammatory function on endothelial cells as sGP.
Asunto(s)
Ebolavirus/genética , Regulación Viral de la Expresión Génica , Glicoproteínas/biosíntesis , Glicoproteínas de Membrana/biosíntesis , Edición de ARN , Proteínas de la Matriz Viral/biosíntesis , Proteínas no Estructurales Virales/biosíntesis , Animales , Línea Celular , Electroforesis en Gel Bidimensional , Perfilación de la Expresión Génica , Glicoproteínas/genética , Humanos , Glicoproteínas de Membrana/genética , Ratones , Proteínas de la Matriz Viral/genética , Proteínas no Estructurales Virales/genéticaRESUMEN
Vascular endothelial cell (EC) junctions are key structures controlling tissue homeostasis in physiology. In the last three decades, excellent studies have addressed many aspects of this complex and highly dynamic regulation, including cell signaling, remodeling processes of the proteins of tight junctions, adherens junctions, and gap junctions, the cytoskeleton, and post-transcriptional modifications, transcriptional activation, and gene silencing. In this dynamic process, vascular endothelial cadherin (VE-cadherin) provides the core structure of EC junctions mediating the physical adhesion of cells as well as the control of barrier function and monolayer integrity via remodeling processes, regulation of protein expression and post-translational modifications. In recent years, research teams have documented locally restricted dynamics of EC junctions in which actin-driven protrusions in plasma membranes play a central role. In this regard, our research group showed that the dynamics of VE-cadherin is driven by small (1-5 µm) actin-mediated protrusions in plasma membranes that, due to this specific function, were named "junction-associated intermittent lamellipodia" (JAIL). JAIL form at overlapping, adjacent cells, and exactly at this site new VE-cadherin interactions occur, leading to new VE-cadherin adhesion sites, a process that restores weak or lost VE-cadherin adhesion. Mechanistically, JAIL formation occurs locally restricted (1-5 µm) and underlies autoregulation in which the local VE-cadherin concentration is an important parameter. A decrease in the local concentration of VE-cadherin stimulates JAIL formation, whereas an increase in the concentration of VE-cadherin blocks it. JAIL mediated VE-cadherin remodeling at the subjunctional level have been shown to be of crucial importance in angiogenesis, wound healing, and changes in permeability during inflammation. The concept of subjunctional regulation of EC junctions is strongly supported by permeability assays, which can be employed to quantify actin-driven subjunctional changes. In this brief review, we summarize and discuss the current knowledge and concepts of subjunctional regulation in the endothelium.
RESUMEN
Nicotine adenine dinucleotide phosphate (NADPH) oxidase (Nox) complexes are the main sources of reactive oxygen species (ROS) formation in the vessel wall. We have used DNA microarray, real-time PCR and Western blot to demonstrate that the subunit Nox4 is the major Nox isoform in primary human endothelial cells; we also found high levels of NADPH oxidase subunit p22(phox) expression. Nox4 was localized by laser scanning confocal microscopy within the cytoplasm of endothelial cells. Endothelial Nox4 overexpression enhanced superoxide anion formation and phosphorylation of p38 MAPK. Nox4 down-regulation by shRNA has in contrast to TGF-beta no effect on p38 MAPK phosphorylation. We conclude that Nox4 is the major Nox isoform in human endothelial cells, and forms an active complex with p22(phox). The Nox4-containing complex mediates formation of reactive oxygen species and p38 MAPK activation. This is a novel mechanism of redox-sensitive signaling in human endothelial cells.
Asunto(s)
Células Endoteliales/enzimología , Endotelio Vascular/enzimología , NADPH Oxidasas/biosíntesis , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Células Cultivadas , Humanos , Isoenzimas/metabolismo , NADPH Oxidasa 4 , Análisis de Secuencia por Matrices de Oligonucleótidos , Transducción de SeñalRESUMEN
Fluid shear stress stimulates endothelial nitric oxide synthase (eNOS) activation and nitric oxide (NO) production through multiple kinases, including protein kinase A (PKA), AMP-activated protein kinase (AMPK), AKT and Ca2+/calmodulin-dependent protein kinase II (CaMKII). Membrane-associated guanylate kinase (MAGUK) with inverted domain structure-1 (MAGI1) is an adaptor protein that stabilizes epithelial and endothelial cell-cell contacts. The aim of this study was to assess the unknown role of endothelial cell MAGI1 in response to fluid shear stress. We show constitutive expression and co-localization of MAGI1 with vascular endothelial cadherin (VE-cadherin) in endothelial cells at cellular junctions under static and laminar flow conditions. Fluid shear stress increases MAGI1 expression. MAGI1 silencing perturbed flow-dependent responses, specifically, Krüppel-like factor 4 (KLF4) expression, endothelial cell alignment, eNOS phosphorylation and NO production. MAGI1 overexpression had opposite effects and induced phosphorylation of PKA, AMPK, and CAMKII. Pharmacological inhibition of PKA and AMPK prevented MAGI1-mediated eNOS phosphorylation. Consistently, MAGI1 silencing and PKA inhibition suppressed the flow-induced NO production. Endothelial cell-specific transgenic expression of MAGI1 induced PKA and eNOS phosphorylation in vivo and increased NO production ex vivo in isolated endothelial cells. In conclusion, we have identified endothelial cell MAGI1 as a previously unrecognized mediator of fluid shear stress-induced and PKA/AMPK dependent eNOS activation and NO production.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Moléculas de Adhesión Celular/fisiología , Células Endoteliales/metabolismo , Guanilato-Quinasas/fisiología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico/metabolismo , Resistencia al Corte , Estrés Mecánico , Animales , Antígenos CD/metabolismo , Cadherinas/metabolismo , Células Endoteliales/citología , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Factor 4 Similar a Kruppel , Ratones , Ratones Transgénicos , Transducción de SeñalRESUMEN
Actin-binding proteins are essential for linear and branched actin filament dynamics that control shape change, cell migration, and cell junction remodeling in vascular endothelium (endothelial cells [ECs]). The epithelial protein lost in neoplasm (EPLIN) is an actin-binding protein, expressed as EPLIN-α and EPLIN-ß by alternative promoters; however, the isoform-specific functions are not yet understood. Aortic compared to cava vein ECs and shear stress-exposed cultured ECs express increased EPLIN-ß levels that stabilize stress fibers. In contrast, EPLIN-α expression is increased in growing and migrating ECs, is targeted to membrane protrusions, and terminates their growth via interaction with the Arp2/3 complex. The data indicate that EPLIN-α controls protrusion dynamics while EPLIN-ß has an actin filament stabilizing role, which is consistent with FRAP analyses demonstrating a lower EPLIN-ß turnover rate compared to EPLIN-α. Together, EPLIN isoforms differentially control actin dynamics in ECs, essential in shear stress responses, cell migration, and barrier function.
Asunto(s)
Actinas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Endotelio Vascular/metabolismo , Animales , Aorta/citología , Aorta/metabolismo , Proliferación Celular , Proteínas del Citoesqueleto/genética , Endotelio Vascular/citología , Femenino , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Células MCF-7 , Masculino , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fibras de Estrés/metabolismoRESUMEN
Endothelial cells are constantly exposed to high or low shear stress in arteries and veins by the flowing blood. Angiopoietin-2 (Ang-2) is acting as a critical regulator of vessel maturation and endothelial cell quiescence. In this study, flow-dependent regulation of Ang-2 was analyzed in vitro and in vivo. Ang-2 mRNA, protein expression and release was upregulated by 24 h of low (1 dyne/cm(2)), but downregulated by high flow (30 dyne/cm(2)) in human endothelial cells. Increased endothelial NO synthase expression and NO formation was not affecting regulation of Ang-2 by low or high flow. Low and high flow increased VEGF-A expression. Inhibition of VEGFR-2 prevented upregulation of Ang-2 by low flow, but not downregulation of Ang-2 by high flow. Furthermore, upregulation of Ang-2 by VEGF was reduced by application of high flow. Forkhead box O (FOXO) transcription factor FOXO1 has been shown to regulate Ang-2 expression in endothelial cells. FOXO1 binding activity was reduced by high flow. Nuclear localization of transcription factor FOXO1 was not changed by low flow, but reduced by high flow. In vivo, Ang-2 was higher expressed in veins compared to arteries. Arterial ligation augmented Ang-2 expression in distal arterial low flow areas. Our results support a VEGF-dependent induction of Ang-2 in low flow areas, and FOXO1-dependent downregulation of Ang-2 in high flow areas. These data suggest a new mechanism of flow-dependent regulation of vessel stability and differentiation.
Asunto(s)
Angiopoyetina 2/metabolismo , Endotelio Vascular/metabolismo , Arteria Femoral/fisiología , Regulación de la Expresión Génica , Angiopoyetina 1/farmacología , Animales , Benzoquinonas/farmacología , Separación Celular/métodos , Células Cultivadas , Cromonas/farmacología , Medio de Cultivo Libre de Suero , Dactinomicina/farmacología , Endotelio Vascular/efectos de los fármacos , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/análisis , Factores de Transcripción Forkhead/metabolismo , Humanos , Lactamas Macrocíclicas/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Morfolinas/farmacología , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/análisis , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , Nitritos/análisis , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/análisis , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/análisis , Receptor TIE-2/metabolismo , Flujo Sanguíneo Regional , Rifabutina/análogos & derivados , Transducción de Señal/efectos de los fármacos , Estrés Mecánico , Triazenos/farmacología , Venas Umbilicales/citología , Factor A de Crecimiento Endotelial Vascular/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
OBJECTIVE: Flow-induced conversion of endothelial cells into an elongated arterial phenotype requires a coordinated regulation of cell junctions. Here we investigated the effect of acute and chronic flow on junction regulation. METHODS AND RESULTS: Using an extended experimental setup that allows analyses of endothelial barrier function under flow conditions, we found a flow-induced upregulation of the transendothelial electrical resistance within minutes. This was accompanied by an increase in actin filaments along the junctions and vascular endothelial (VE)-cadherin clustering, which was identified at nanoscale resolution by stimulated emission depletion microscopy. In addition, a transient tyrosine phosphorylation of VE-cadherin and catenins occurred within minutes following the onset of flow. VE-cadherin and actin distribution were maintained under chronic flow over 24 h and associated with the upregulation of VE-cadherin and alpha-catenin expression, thus compensating for the cell elongation-mediated increase in cell border length. Importantly, all observed effects were rac1 dependent as verified by the inhibitory effect of dominant negative N17rac1. CONCLUSION: These results show that flow-induced conversion of endothelial cells into an arterial phenotype occurs while intercellular junctions remain intact. The data place rac1 in a central multimodal regulatory position that might be important in the development of vascular diseases, such as arteriosclerosis.
Asunto(s)
Células Endoteliales/metabolismo , Uniones Intercelulares/metabolismo , Actinas/metabolismo , Antígenos CD/metabolismo , Arterias , Cadherinas/metabolismo , Adhesión Celular , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular , Células Cultivadas , Electrofisiología , Humanos , Microscopía Electrónica , Microscopía Fluorescente , Fenotipo , Fosforilación , Flujo Sanguíneo Regional , Estrés Mecánico , Venas , Proteína de Unión al GTP rac1/metabolismo , Proteínas de Unión al GTP rho/metabolismoRESUMEN
VEGFR-2/Notch signalling regulates angiogenesis in part by driving the remodelling of endothelial cell junctions and by inducing cell migration. Here, we show that VEGF-induced polarized cell elongation increases cell perimeter and decreases the relative VE-cadherin concentration at junctions, triggering polarized formation of actin-driven junction-associated intermittent lamellipodia (JAIL) under control of the WASP/WAVE/ARP2/3 complex. JAIL allow formation of new VE-cadherin adhesion sites that are critical for cell migration and monolayer integrity. Whereas at the leading edge of the cell, large JAIL drive cell migration with supportive contraction, lateral junctions show small JAIL that allow relative cell movement. VEGFR-2 activation initiates cell elongation through dephosphorylation of junctional myosin light chain II, which leads to a local loss of tension to induce JAIL-mediated junctional remodelling. These events require both microtubules and polarized Rac activity. Together, we propose a model where polarized JAIL formation drives directed cell migration and junctional remodelling during sprouting angiogenesis.
Asunto(s)
Actinas/metabolismo , Antígenos CD/metabolismo , Cadherinas/metabolismo , Movimiento Celular/fisiología , Polaridad Celular/fisiología , Células Endoteliales/metabolismo , Uniones Intercelulares/metabolismo , Neovascularización Fisiológica/fisiología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteína 2 Relacionada con la Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Proteína 3 Relacionada con la Actina/metabolismo , Actinas/efectos de los fármacos , Antígenos CD/efectos de los fármacos , Cadherinas/efectos de los fármacos , Miosinas Cardíacas/metabolismo , Adhesión Celular , Movimiento Celular/efectos de los fármacos , Polaridad Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Endotelio Vascular , Células Endoteliales de la Vena Umbilical Humana , Humanos , Uniones Intercelulares/efectos de los fármacos , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Modelos Cardiovasculares , Cadenas Ligeras de Miosina/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Seudópodos/efectos de los fármacos , Seudópodos/metabolismo , Seudópodos/fisiología , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Remodelación Vascular , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismo , Proteínas de Unión al GTP rac/metabolismoRESUMEN
Intercellular junctions of the vascular endothelium are dynamic structures that display a high degree of plasticity, which is required to contribute to their regulation of many physiological and pathological processes including monolayer integrity, barrier function, wound healing and angiogenesis. Vascular endothelial cadherin (VE-cadherin) is connected via catenins to the actin cytoskeleton, both of which are key structures in endothelial junction regulation, and thus are the focus of much investigation. Fluorescence-based live cell imaging is the method of choice to study dynamic remodeling in living cells. Although these methods have been successfully applied to many cell types, investigations of endothelial junction dynamics were for a long time limited as they are largely resistant to transfection using many classical protocols. Application of virus-based gene transduction techniques, together with advanced microscopy, now allows both sufficient expression of fluorescence tagged junction-localized proteins in the endothelium and time-lapse recording over long periods. Using highly spatiotemporally resolved fluorescence microscopy it turned out that endothelial junctions display extensive junction heterogeneity at the subcellular level; a fact that largely limits automated quantification by available software. Recent work describes open software tools to quantitatively analyze large amounts of fluorescence-based image data in either single or confluent epithelial and endothelial cells. Based on quantitative VE-cadherin and actin dynamics novel key players, mechanisms and concepts have been suggested that control endothelial junction dynamics. Here we aim to summarize the recent developments in the field.
RESUMEN
Endothelial barrier function depends on the integrity of intercellular adherens junctions controlled by the association of VE-cadherin/catenin complex with cortical actin filaments. Both tyrosine phosphorylation/dephosphorylation of junctional proteins and actin reorganization mediated by rho-GTPases regulate barrier function but the relationship between these regulatory mechanisms is unclear. Here we studied the effects of factors increasing protein tyrosine phosphorylation, pervanadate (PV) and VEGF, on distribution of VE-cadherin, F-actin polymerization and transendothelial electrical resistance (TER) in human umbilical vein endothelial cells (HUVECs). Changes in protein tyrosine phosphorylation of cytoplasmic and junctional proteins, as well as the activity of rho-GTPase racl, were also measured. We report for the first time that PV and VEGF induced a rapid transient increase in endothelial barrier function accompanied by rac1 activation, a differentiated tyrosine phosphorylation of the VE-cadherin/catenin complex, recruitment of actin filament to cell junctions and ruffle formation. A sustained decrease in endothelial barrier function was observed at later times of PV and VEGF treatment. Expression of dominant negative rac1, N17rac1 abolished the barrier-enhancing effects of PV and VEGF, while the sustained decrease in barrier function was unaffected. These observations bring into focus early short-term effects of protein tyrosine phosphorylation in cells, often overshadowed by more pronounced and long-lasting later effects and may play an important role in the regulation of endothelial barrier function.