RESUMO
Endothelial cell-cell adhesive junctions are formed by transmembrane adhesive proteins linked to a complex cytoskeletal network. These structures are important not only for maintaining adhesion between endothelial cells and, as a consequence, for the control of vascular permeability, but also for intracellular signalling properties. The establishment of intercellular junctions might affect the endothelial functional phenotype by the downregulation or upregulation of endothelial-specific activities.
Assuntos
Endotélio/fisiologia , Junções Intercelulares/fisiologia , Animais , Adesão Celular , Endotélio/ultraestrutura , Humanos , Transdução de Sinais/fisiologiaRESUMO
In the past, year targeted null mutation studies have further supported the concept that endothelial cell-matrix and cell-cell adhesion is involved in the formation and maintenance of the network of branched tubes within the vascular tree. In addition, recent results derived from the closely related experimental system of branching tubulogenesis in epithelial cells may provide an appealing model for endothelial biology.
Assuntos
Adesão Celular , Endotélio Vascular/metabolismo , Integrinas/fisiologia , Morfogênese/fisiologia , Neovascularização Fisiológica , Transativadores , Animais , Caderinas/fisiologia , Proteínas de Ligação ao Cálcio , Proteínas do Citoesqueleto/fisiologia , Desmoplaquinas , Dimerização , Matriz Extracelular/metabolismo , Marcação de Genes , Proteínas de Homeodomínio/fisiologia , Humanos , Integrinas/deficiência , Integrinas/genética , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Knockout , Mutação , Fosfolipídeos/metabolismo , Coelhos , Receptores Notch , Proteínas Serrate-Jagged , alfa Catenina , beta CateninaRESUMO
The present study was designed to define molecules and structures involved in the interaction of natural killer (NK) cells with the vascular endothelium in vitro. Resting and interleukin 2 (IL-2)-activated NK cells were studied for their capacity to adhere to resting and IL-1-treated human umbilical vein endothelial cells (EC). In the absence of stimuli, NK cells showed appreciable adhesion to EC, with levels of binding intermediate between polymorphs and monocytes. The binding ability was increased by pretreatment of NK cells with IL-2. Using the appropriate monoclonal antibody, the beta 2 leukocyte integrin CD18/CD11a was identified as the major adhesion pathway of NK cells to unstimulated EC. Activation of EC with IL-1 increased the binding of NK cells. In addition to the CD18-CD11a/intercellular adhesion molecule pathway, the interaction of resting or IL-2-activated NK cells to IL-1-activated EC involved the VLA-4 (alpha 4 beta 1)-vascular cell adhesion molecule 1 receptor/counter-receptor pair. No evidence for appreciable involvement of endothelial-leukocyte adhesion molecule was obtained. Often, NK cells interacted either with the culture substrate or with the EC surface via dot-shaped adhesion structures (podosomes) protruding from the ventral surface and consisting of a core of F-actin surrounded by a ring of vinculin and talin. The identification of molecules and microanatomical structures involved in the interaction of NK cells with EC may provide a better understanding of the regulation of NK cell recruitment from blood, their extravasation, and their migration to tissues.
Assuntos
Moléculas de Adesão Celular/fisiologia , Endotélio Vascular/citologia , Cadeias alfa de Integrinas , Células Matadoras Naturais/citologia , Organelas/fisiologia , Anticorpos Monoclonais/imunologia , Antígenos CD18 , Adesão Celular , Citometria de Fluxo , Imunofluorescência , Humanos , Interleucina-1/farmacologia , Interleucina-2/farmacologia , Cinética , Ativação Linfocitária/imunologia , Antígeno-1 Associado à Função Linfocitária/fisiologia , Receptores de Adesão de Leucócito/fisiologia , Receptores de Antígeno muito Tardio/fisiologiaRESUMO
The mechanisms that govern leukocyte transmigration through the endothelium are not yet fully defined. Junctional adhesion molecule (JAM) is a newly cloned member of the immunoglobulin superfamily which is selectively concentrated at tight junctions of endothelial and epithelial cells. A blocking monoclonal antibody (BV11 mAb) directed to JAM was able to inhibit monocyte transmigration through endothelial cells in in vitro and in vivo chemotaxis assays. In this study, we report that BV11 administration was able to attenuate cytokine-induced meningitis in mice. The intravenous injection of BV11 mAb significantly inhibited leukocyte accumulation in the cerebrospinal fluid and infiltration in the brain parenchyma. Blood-brain barrier permeability was also reduced by the mAb. We conclude that JAM may be a new target in limiting the inflammatory response that accompanies meningitis.
Assuntos
Anticorpos Monoclonais/farmacologia , Moléculas de Adesão Celular/imunologia , Quimiotaxia/imunologia , Leucócitos/imunologia , Meningite/imunologia , Animais , Barreira Hematoencefálica/imunologia , Encéfalo/imunologia , Citocinas/farmacologia , Modelos Animais de Doenças , Eosinófilos/metabolismo , Imunofluorescência , Inflamação/imunologia , Interleucina-1/farmacologia , Moléculas de Adesão Juncional , Meningite/líquido cefalorraquidiano , Camundongos , Microscopia de Fluorescência , Monócitos/metabolismo , Fatores de Tempo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The two major cadherins of endothelial cells are neural (N)-cadherin and vascular endothelial (VE)- cadherin. Despite similar level of protein expression only VE-cadherin is located at cell-cell contacts, whereas N-cadherin is distributed over the whole cell membrane. Cotransfection of VE-cadherin and N-cadherin in CHO cells resulted in the same distribution as that observed in endothelial cells indicating that the behavior of the two cadherins was not cell specific but related to their structural characteristics. Similar amounts of alpha- and beta-catenins and plakoglobin were associated to VE- and N-cadherins, whereas p120 was higher in the VE-cadherin complex. The presence of VE-cadherin did not affect N-cadherin homotypic adhesive properties or its capacity to localize at junctions when cotransfectants were cocultured with cells transfected with N-cadherin only. To define the molecular domain responsible for the VE-cadherin-dominant activity we prepared a chimeric construct formed by VE-cadherin extracellular region linked to N-cadherin intracellular domain. The chimera lost the capacity to exclude N-cadherin from junctions indicating that the extracellular domain of VE-cadherin alone is not sufficient for the preferential localization of the molecule at the junctions. A truncated mutant of VE-cadherin retaining the full extracellular domain and a short cytoplasmic tail (Arg621-Pro702) lacking the catenin-binding region was able to exclude N-cadherin from junctions. This indicates that the Arg621-Pro702 sequence in the VE-cadherin cytoplasmic tail is required for N-cadherin exclusion from junctions. Competition between cadherins for their clustering at intercellular junctions in the same cell has never been described before. We speculate that, in the endothelium, VE- and N-cadherin play different roles; whereas VE-cadherin mostly promotes the homotypic interaction between endothelial cells, N-cadherin may be responsible for the anchorage of the endothelium to other surrounding cell types expressing N-cadherin such as vascular smooth muscle cells or pericytes.
Assuntos
Caderinas/análise , Endotélio Vascular/química , Junções Intercelulares/química , Transativadores , Animais , Células CHO/química , Caderinas/biossíntese , Caderinas/química , Adesão Celular/fisiologia , Cricetinae , Proteínas do Citoesqueleto/análise , Proteínas do Citoesqueleto/metabolismo , Endotélio Vascular/citologia , Humanos , Fosforilação , Estrutura Terciária de Proteína , Transfecção , Tirosina/metabolismo , Veias Umbilicais/citologia , alfa Catenina , beta CateninaRESUMO
It has been recently proposed that adhesion of polymorphonuclear cells (PMNs) to human umbilical vein endothelial cells leads to the disorganization of the vascular endothelial cadherin-dependent endothelial adherens junctions. Combined immunofluorescence and biochemical data suggested that after adhesion of PMNs to the endothelial cell surface, beta-catenin, as well as plakoglobin was lost from the cadherin/catenin complex and from total cell lysates. In this study we present data that strongly suggest that the adhesion-dependent disappearance of endothelial catenins is not mediated by a leukocyte to endothelium signaling event, but is due to the activity of a neutrophil protease that is released upon detergent lysis of the cells.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Endopeptidases/sangue , Endotélio Vascular/metabolismo , Neutrófilos/enzimologia , Transativadores , Caderinas/metabolismo , Adesão Celular , Células Cultivadas , Desmoplaquinas , Técnica Indireta de Fluorescência para Anticorpo , Humanos , beta Catenina , gama CateninaRESUMO
This paper shows that, in confluent human umbilical vein endothelial cell (EC) monolayers, the integrin heterodimers alpha 2 beta 1 and alpha 5 beta 1, but not other members of the beta 1 subfamily, are located at cell-cell contact borders and not at cellular free edges. Also the alpha v chain, but not its most common partner beta 3, that is widely expressed in EC cell-matrix junctions, is found at cell-cell borders. In EC monolayers, the putative ligands of alpha 2 beta 1 and alpha 5 beta 1 receptors, i.e., laminin, collagen type IV, and fibronectin, are also organized in strands corresponding to cell-cell borders. The location of the above integrin receptors is not an artifact of in vitro culture since it has been noted also in explanted islets of the native umbilical vein endothelium. The integrins alpha 2 beta 1 and alpha 5 beta 1 play a role in the maintenance of endothelial monolayer continuity in vitro. Indeed, specific antibodies to alpha 2 beta 1, alpha 5 beta 1, and the synthetic peptide GRGDSP alter its continuity without any initial cell detachment. Moreover, antibodies to alpha 5 beta 1 increase the permeation of macromolecules across confluent EC monolayers. In contrast beta 3 antibodies were ineffective. It is suggested that the relocation of integrins to cell-cell borders is a feature of cells programmed to form polarized monolayers since integrins have a different distribution in nonpolar confluent dermal fibroblasts. The conclusion is that some members of the integrin superfamily collaborate with other intercellular molecules to form lateral junctions and to control both the monolayer integrity and the permeability properties of the vascular endothelial lining. This also suggest that integrins are adhesion molecules provided with a unique biochemical adaptability to different biological functions.
Assuntos
Endotélio Vascular/citologia , Integrinas/fisiologia , Actinas/análise , Sequência de Aminoácidos , Complexo Antígeno-Anticorpo , Permeabilidade da Membrana Celular , Células Cultivadas , Proteínas do Citoesqueleto/análise , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/imunologia , Imunofluorescência , Humanos , Integrinas/análise , Substâncias Macromoleculares , Dados de Sequência Molecular , Oligopeptídeos/farmacologia , Veias Umbilicais , VinculinaRESUMO
Human umbilical vein endothelial cells (ECs) adhere in vitro to proteins of the extracellular matrix including fibronectin (fn) and vitronectin (vn). Specific receptors for fn and vn have been previously characterized. These receptors belong to a family of membrane glycoproteins characterized (a) by being a transmembrane complex of two noncovalently linked subunits and (b) by recognizing the tripeptide Arg-Gly-Asp on their respective ligands. In this paper we investigated how vn and fn control the organization of their respective receptors over the surface of ECs. It was found that the clustering of individual receptors and the organization thereafter of focal contacts occurred only when ECs were exposed to the specific ligand and did not occur on the opposite ligand. The shape of receptor clusters was slightly different and a colocalization of the two receptors was found when ECs were cultured on a mixed matrix of fn plus vn. Adhesion was selectively inhibited by vn or fn receptor antibodies on their respective substrates. The clustering of both receptors preceded the association of vinculin with focal contacts and stress fiber formation. Also, the vn receptor, in the absence of associated fn receptor, was capable of inducing the organization of the membrane-microfilament interaction complex. Overall, these results indicate that individual matrix ligands induce only the clustering of their respective membrane receptors. The clustering of only one receptor is capable of supporting the subsequent formation of focal contacts and the local assembly of related cytoskeletal proteins.
Assuntos
Proteínas Sanguíneas/fisiologia , Endotélio Vascular/metabolismo , Fibronectinas/fisiologia , Glicoproteínas/fisiologia , Receptores Imunológicos/metabolismo , Receptores de Peptídeos , Adesão Celular , Células Cultivadas , Imunofluorescência , Humanos , Imunoensaio , Ligantes , Proteínas Musculares/análise , Proteínas Musculares/metabolismo , Testes de Precipitina , Receptores de Fibronectina , Receptores Imunológicos/análise , Receptores de Vitronectina , Vinculina , VitronectinaRESUMO
The integrity of the endothelial layer, which lines the entire cavity of the vascular system, depends on tight adhesion of the cells to the underlying basement membrane as well as to each other. It has been previously shown that such interactions occur via membrane receptors that determine the specificity, topology, and mechanical properties of the surface adhesion. Cell-cell junctions between endothelial cells, in culture and in situ, involve both Ca(2+)-dependent and -independent mechanisms that are mediated by distinct adhesion molecules. Ca(2+)-dependent cell-cell adhesion occurs mostly via members of the cadherin family, which locally anchor the microfilament system to the plasma membrane, in adherens junctions. Ca(2+)-independent adhesions were reported to mainly involve members of the Ig superfamily. In this study, we performed three-dimensional microscopic analysis of the relative subcellular distributions of these two endothelial intercellular adhesion systems. We show that cadherins are located at adjacent (usually more apical), yet clearly distinct domains of the lateral plasma membrane, compared to PECAM-1. Moreover, cadherins were first organized in adherens junctions within 2 h after seeding of endothelial cells, forming multiple lateral patches which developed into an extensive belt-like structure over a period of 24 h. PECAM-1 became associated with surface adhesions significantly later and became progressively associated with the cadherin-containing adhesions. Cadherins and PECAM-1 also differed in their detergent extractability, reflecting differences in their mode of association with the cytoskeleton. Moreover, the two adhesion systems could be differentially modulated since short treatment with the Ca2+ chelator EGTA, disrupted the cadherin junctions leaving PECAM-1 apparently intact. These results confirm that endothelial cells possess distinct intercellular contact mechanisms that differ in their spatial and temporal organization as well as in their functional properties.
Assuntos
Antígenos de Diferenciação Mielomonocítica/isolamento & purificação , Caderinas/isolamento & purificação , Moléculas de Adesão Celular/isolamento & purificação , Adesão Celular/fisiologia , Endotélio Vascular/ultraestrutura , Junções Intercelulares/química , Adesão Celular/efeitos dos fármacos , Compartimento Celular , Citoplasma/química , Citoesqueleto/química , Ácido Egtázico/farmacologia , Imunofluorescência , Humanos , Junções Intercelulares/ultraestrutura , Microscopia Imunoeletrônica , Placenta , Molécula-1 de Adesão Celular Endotelial a Plaquetas , Fatores de Tempo , Veias UmbilicaisRESUMO
When cultivated on substrates that prevent cell adhesion (the polymer polyhydroxyethylmethacrylate, bovine serum albumin, and Teflon), human endothelial cells (EC) rapidly lost viability with a half-life of approximately 10 h. Dying EC showed the morphological and biochemical characteristics of apoptosis. The apoptotic process of suspended EC was delayed by the protein synthesis inhibitor cycloheximide. To obtain information as to the mechanism involved in the apoptosis of suspended EC, we investigated whether adhesion to matrix proteins or integrin occupancy in EC retaining a round shape may affect EC suicide. EC bound to low coating concentration of either fibronectin or vitronectin, retaining a round shape and failing to organize actin microfilaments, underwent to rapid cell death; by contrast, cells on high substrate concentrations became flattened, showed actin microfilament organization, and retained viability. Addition of saturating amounts of soluble vitronectin to suspended round-shaped EC did not reduce the process of apoptosis. Finally, when suspended EC bound Gly-Arg-Gly-Asp-Ser-coated microbeads (approximately 10 microbeads/cell), yet retaining a round shape, the apoptotic process was not affected. Oncogene-transformed EC in suspension were less susceptible to cell death and apoptosis than normal EC. Overall, these data indicate that cell attachment to matrix or integrin binding per se is not sufficient for maintaining cell viability, and that cells need to undergo some minimal degree of shape change to survive. Modulation of interaction with the extracellular matrix can, therefore, be an important target for the control of angiogenesis.
Assuntos
Apoptose , Endotélio Vascular/citologia , Matriz Extracelular/metabolismo , Sequência de Aminoácidos , Adesão Celular , Movimento Celular , Tamanho Celular , Sobrevivência Celular , Transformação Celular Neoplásica , Células Cultivadas , Cicloeximida/farmacologia , Fibronectinas/metabolismo , Glicoproteínas/metabolismo , Humanos , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , VitronectinaRESUMO
mAbs were raised in mice against cultured human endothelial cells (EC) and screened by indirect immunofluorescence for their ability to stain intercellular contacts. One mAb denoted 7B4 was identified which, out of many cultured cell types, specifically decorated cultured human EC. The antigen recognized by mAb 7B4 is bound at the appositional surfaces of cultured EC only as they become confluent and is stably expressed at intercellular boundaries of confluent monolayers. EC recognition specificity was maintained when the antibody was assayed by immuno-histochemistry in tissue sections of many normal and malignant tissues and in blood vessels of different size and type. The antigen recognized by 7B4 was enriched at EC intercellular boundaries similarly in vitro and in situ. In vitro, addition of mAb 7B4 to confluent EC increased permeation of macromolecules across monolayers even without any obvious changes of cell morphology. In addition, when EC permeability was increased by agents such as thrombin, elastase, and TNF/gamma IFN, its distribution pattern at intercellular contact rims was severely altered. mAb 7B4 immunoprecipitated a major protein of 140 kD from metabolically and surface-labeled cultured EC extracts which appeared to be an integral membrane glycoprotein. On the basis of its distribution in cultured cells and in tissues in situ, 7B4 antigen is distinct from other described EC proteins enriched at intercellular contacts. NH2-terminal sequencing of the antigen, immunopurified from human placenta, and sequencing of peptides from tryptic peptide maps revealed identity to the cDNA deduced sequence of a recently identified new member of the cadherin family (Suzuki, S., K. Sano, and H. Tanihara. 1991. Cell Regul. 2:261-270.) These data indicate that 7B4 antigen is an endothelial-specific cadherin that plays a role in the organization of lateral endothelial junctions and in the control of permeability properties of vascular endothelium.
Assuntos
Caderinas/análise , Endotélio Vascular/química , Junções Intercelulares/química , Sequência de Aminoácidos , Anticorpos Monoclonais , Antígenos de Diferenciação Mielomonocítica/análise , Biomarcadores , Caderinas/química , Adesão Celular/fisiologia , Moléculas de Adesão Celular/análise , Células Cultivadas , Cromatografia de Afinidade , Endotélio Vascular/metabolismo , Imunofluorescência , Humanos , Dados de Sequência Molecular , Mapeamento de Peptídeos , Molécula-1 de Adesão Celular Endotelial a PlaquetasRESUMO
Human umbilical vein endothelial cells (ECs) have been shown to attach to a substratum of fibrinogen (fg). Later, ECs undergo spreading, organization of thick microfilament bundles of the stress fiber type, and formation of focal contacts (adhesion plaques) that correspond to accumulation of vinculin at the cytoplasmic aspect of the ventral membrane. The rate of attachment to fg and the type of spreading is virtually identical to that obtained on substrata coated with fibronectin (FN). Antibodies to fg, but not to FN, prevent EC adhesion to fg; conversely, antibodies to FN, but not to fg, prevent adhesion of ECs to a FN-coated substratum. The removal of residual FN contamination from fg preparations by means of DEAE-cellulose chromatography does not result in any difference in EC adhesion on fg. Moreover, pretreatment of cells with inhibitors of synthesis and release of proteins does not impair their adhesion capacity on an fg-coated substratum. In contrast, human arterial smooth muscle cells do not adhere and spread on fg substrata but do so on FN. The synthetic peptides (Gly-Arg-Gly-Asp[GRGD] and Gly-Arg-Gly-Asp-Ser-Pro[GRGDSP]) containing the tripeptide Arg-Gly-Asp (RGD), originally found to be responsible for the cell binding activity of FN, have been found to inhibit EC spreading and the redistribution of their cytoskeleton, including the formation of stress fibers and the localization of vinculin either on fg or on FN. Conversely, the synthetic peptide Arg-Gly-Gly (RGG) was completely uneffective in inhibiting the adhesion and the sequence of events leading to spreading and cytoskeletal organization. These results indicate that ECs, but not smooth muscle cells, specifically adhere and spread on an fg substratum and this occurs by recognition mechanisms similar to those reported for FN.
Assuntos
Citoesqueleto de Actina/ultraestrutura , Adesão Celular , Citoesqueleto/ultraestrutura , Endotélio/citologia , Fibrinogênio/fisiologia , Aprotinina/farmacologia , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Endotélio/efeitos dos fármacos , Endotélio/ultraestrutura , Feminino , Hirudinas/farmacologia , Humanos , Biossíntese de Proteínas , Veias Umbilicais/citologia , Veias Umbilicais/ultraestruturaRESUMO
Von Willebrand factor (vWF) is a constitutive and specific component of endothelial cell (EC) matrix. In this paper we show that, in vitro, vWF can induce EC adhesion and promote organization of microfilaments and adhesion plaques. In contrast, human vascular smooth muscle cells and MG63 osteosarcoma cells did not adhere and spread on vWF. Using antibodies to the beta chains of fibronectin (beta 1) and vitronectin (beta 3) receptors it was found that ECs adherent to vWF show clustering of both receptors. The beta 1 receptor antibodies are arranged along stress fibers at sites of extracellular matrix contact while the beta 3 receptor antibodies were sharply confined at adhesion plaques. ECs release and organize endogenous fibronectin early during adhesion to vWF. Upon blocking protein synthesis and secretion, ECs can equally adhere and spread on vWF but, while the beta 3 receptors are regularly organized, the beta 1 receptors remain diffuse. This suggests that the organization of the beta 1 receptors depend on the release of fibronectin and/or other matrix proteins operated by the same cell. Antibodies to the beta 3 receptors fully block EC adhesion to vWF and detach ECs seeded on this substratum. In contrast, antibodies to the beta 1 receptors are poorly active. Overall these results fit with an accessory role of beta 1 receptors and indicate a leading role for the beta 3 receptors in EC interaction with vWF. To identify the EC binding domain on vWF we used monoclonal antibodies produced against a peptide representing the residues Glu1737-Ser1750 of the mature vWF and thought to be important in mediating its binding to the platelet receptor glycoprotein IIb-IIIa. We found that the antibody that recognizes the residues 1,744-1,746, containing the Arg-Gly-Asp sequence, completely inhibit EC adhesion to vWF whereas a second antibody recognizing the adjacent residues 1,740-1,742 (Arg-Gly-Asp-free) is inactive. Both antibodies do not interfere with EC adhesion to vitronectin. This defines the molecular domain on vWF that is specifically recognized by ECs and reaffirms the direct role of the Arg-Gly-Asp sequence as the integrin receptor recognition site also in the vWF molecule.
Assuntos
Adesão Celular , Endotélio Vascular/citologia , Receptores de Superfície Celular/fisiologia , Fator de von Willebrand/fisiologia , Citoesqueleto de Actina/ultraestrutura , Sequência de Aminoácidos , Sítios de Ligação , Emetina/farmacologia , Humanos , Técnicas In Vitro , Monensin/farmacologia , Relação Estrutura-AtividadeRESUMO
Thrombin, in addition to its central role in hemostasis, possesses diverse cellular bioregulatory functions implicated in wound healing, inflammation, and atherosclerosis. In the present study we demonstrate that thrombin molecules modified either at the procoagulant or catalytic sites induce endothelial cell (EC) adhesion, spreading, and cytoskeletal reorganization. The most potent adhesive thrombin analogue (NO2-alpha-thrombin) was obtained by nitration of tyrosine residues. The cell adhesion promoting activity of NO2-alpha-thrombin was blocked upon the formation of thrombin-antithrombin III (ATIII) complexes and by antiprothrombin antibodies, but was unaffected by hirudin. Arg-Gly-Asp-containing peptides, fully inhibited EC adhesion to NO2-alpha-thrombin, while synthetic peptides corresponding to thrombin "Loop B" mitogenic site and the thrombin-derived chemotactic fragment "CB67-129", were uneffective. Immunofluorescence studies indicated that EC adhesion to NO2-alpha-thrombin was followed by cell spreading, actin microfilament assembly, and formation of focal contacts. By the use of specific antibodies, the vitronectin (vn) receptor (alpha v beta 3) was found to be localized in clusters upon cell adhesion to NO2-alpha-thrombin. An anti alpha v beta 3 antibody blocked EC adhesion and spreading while antifibronectin (fn) receptor (alpha 5 beta 1) antibodies were uneffective. While native thrombin exhibited a very low cell attachment activity, thrombin that was incubated at 37 degrees C before coating of plastic surfaces induced EC attachment and spreading. We propose that under certain conditions the naturally hindered RGD domain within thrombin is exposed for interaction with alpha v beta 3 on EC. This in turn promotes cell adhesion, spreading, and reorganization of cytoskeletal elements, which may altogether contribute to repair mechanisms in the disturbed vessel wall. This study defines a new biological role of thrombin and characterizes a new recognition mechanism on EC for this molecule.
Assuntos
Adesão Celular , Endotélio Vascular/citologia , Oligopeptídeos/metabolismo , Trombina/metabolismo , Sequência de Aminoácidos , Animais , Aorta , Bovinos , Células Cultivadas , Endotélio Vascular/metabolismo , Imunofluorescência , Humanos , Dados de Sequência Molecular , Trombina/antagonistas & inibidores , Trombina/químicaRESUMO
Integrins from the very late activation antigen (VLA) subfamily are involved in cellular attachment to extracellular matrix (ECM) proteins and in intercellular adhesions. It is known that the interaction of integrin proteins with their ligands can be regulated during cellular activation. We have investigated the regulation of different VLA-mediated adhesive interactions through the common beta 1 chain. We have found that certain anti-beta 1 antibodies strongly enhance binding of myelomonocytic U-937 cells to fibronectin. This beta 1-mediated regulatory effect involved both VLA-4 and VLA-5 fibronectin receptors. Moreover, anti-beta 1 mAb also induced VLA-4-mediated binding to a recombinant soluble form of its endothelial cell ligand VCAM-1. Non-activated peripheral blood T lymphocytes, unable to mediate VLA-4 interactions with fibronectin or VCAM-1, acquired the ability to bind these ligands in the presence of anti-beta 1 mAb. The anti-beta 1-mediated changes in the affinities of beta 1 integrin for their ligands were comparable to those triggered by different lymphocyte activation agents such as anti-CD3 mAb or phorbol ester. Adhesion of melanoma cells to other ECM proteins such as laminin or collagen as well as that of alpha 2-transfected K-562 cells to collagen, was also strongly enhanced by anti-beta 1 mAb. These beta 1-mediated regulatory effects on different VLA-ligand interactions do not involve changes in cell surface membrane expression of different VLA heterodimers. The anti-beta 1-mediated functional effects required an active metabolism, cytoskeleton integrity and the existence of physiological levels of intracellular calcium as well as a functional Na+/H+ antiporter. Beta 1 antibodies not only increased cell attachment but also promoted spreading and cytoplasmic extension of endothelial cells on plates coated with either fibronectin, collagen, or laminin as well as induced the rapid appearance of microspikes in U-937 cells on fibronectin. Moreover, both beta 1 integrin and the cytoskeletal protein talin colocalized in the anti-beta 1 induced microspikes. These results emphasize the central role of the common beta 1 chain in regulating different adhesive functions mediated by VLA integrins as well as cellular morphology.
Assuntos
Antígenos de Superfície/metabolismo , Adesão Celular/fisiologia , Integrinas/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Antígenos de Superfície/imunologia , Antiporters , Proteínas de Transporte/metabolismo , Moléculas de Adesão Celular/metabolismo , Comunicação Celular/fisiologia , Colágeno/metabolismo , Citoesqueleto/ultraestrutura , Fibronectinas/metabolismo , Integrinas/imunologia , Ligantes , Morfogênese/fisiologia , Linfócitos T/fisiologia , Células Tumorais Cultivadas , Regulação para Cima , Molécula 1 de Adesão de Célula VascularRESUMO
Polymorphonuclear leukocytes (PMN) infiltration into tissues is frequently accompanied by increase in vascular permeability. This suggests that PMN adhesion and transmigration could trigger modifications in the architecture of endothelial cell-to-cell junctions. In the present paper, using indirect immunofluorescence, we found that PMN adhesion to tumor necrosis factor-activated endothelial cells (EC) induced the disappearance from endothelial cell-to-cell contacts of adherens junction (AJ) components: vascular endothelial (VE)-cadherin, alpha-catenin, beta-catenin, and plakoglobin. Immunoprecipitation and Western blot analysis of the VE-cadherin/catenin complex showed that the amount of beta-catenin and plakoglobin was markedly reduced from the complex and from total cell extracts. In contrast, VE-cadherin and alpha-catenin were only partially affected. Disorganization of endothelial AJ by PMN was not accompanied by EC retraction or injury and was specific for VE-cadherin/catenin complex, since platelet/endothelial cell adhesion molecule 1 (PECAM-1) distribution at cellular contacts was unchanged. PMN adhesion to EC seems to be a prerequisite for VE-cadherin/catenin complex disorganization. This phenomenon could be fully inhibited by blocking PMN adhesion with an anti-integrin beta 2 mAb, while it could be reproduced by any condition that induced increase of PMN adhesion, such as addition of PMA or an anti-beta 2-activating mAb. The effect on endothelial AJ was specific for PMN since adherent activated lymphocytes did not induce similar changes. High concentrations of protease inhibitors and oxygen metabolite scavengers were unable to prevent AJ disorganization mediated by PMN. PMN adhesion to EC was accompanied by increase in EC permeability in vitro. This effect was dependent on PMN adhesion, was not mediated by proteases and oxygen-reactive metabolites, and could be reproduced by EC treatment with EGTA. Finally, immunohistochemical analysis showed that VE-cadherin distribution was affected by PMN adhesion to the vessel wall in vivo too. This work suggests that PMN adhesion could trigger intracellular signals in EC that possibly regulate VE-cadherin /catenin complex disorganization. This effect could increase EC permeability and facilitate PMN transmigration during the acute inflammatory reaction.
Assuntos
Adesão Celular , Endotélio Vascular/fisiologia , Junções Intercelulares/fisiologia , Neutrófilos/fisiologia , Transativadores , Western Blotting , Caderinas/análise , Caderinas/fisiologia , Células Cultivadas , Proteínas do Citoesqueleto/análise , Proteínas do Citoesqueleto/fisiologia , Desmoplaquinas , Endopeptidases/metabolismo , Endotélio Vascular/ultraestrutura , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Junções Intercelulares/ultraestrutura , Neutrófilos/ultraestrutura , Veias Umbilicais , alfa Catenina , beta Catenina , gama CateninaRESUMO
In this paper we report that the assembly of interendothelial junctions containing the cell type-specific vascular endothelial cadherin (VE-cadherin or cadherin-5) is a dynamic process which is affected by the functional state of the cells. Immunofluorescence double labeling of endothelial cells (EC) cultures indicated that VE-cadherin, alpha-catenin, and beta-catenin colocalized in areas of cell to cell contact both in sparse and confluent EC monolayers. In contrast, plakoglobin became associated with cell-cell junctions only in tightly confluent cells concomitantly with an increase in its protein and mRNA levels. Furthermore, the amount of plakoglobin coimmunoprecipitated with VE-cadherin, increased in closely packed monolayers. Artificial wounding of confluent EC monolayers resulted in a major reorganization of VE-cadherin, alpha-catenin, beta-catenin, and plakoglobin. All these proteins decreased in intensity at the boundaries of EC migrating into the lesion. In contrast, EC located immediately behind the migrating front retained junctional VE-cadherin, alpha-catenin, and beta-catenin while plakoglobin was absent from these sites. In line with this observation, the amount of plakoglobin coimmunoprecipitated with VE-cadherin decreased in migrating EC. These data suggest that VE-cadherin, alpha-catenin, and beta-catenin are already associated with each other at early stages of intercellular adhesion and become readily organized at nascant cell contacts. Plakoglobin, on the other hand, associates with junctions only when cells approach confluence. When cells migrate, this order is reversed, namely, plakoglobin dissociates first and, then, VE-cadherin, alpha-catenin, and beta-catenin disassemble from the junctions. The late association of plakoglobin with junctions suggests that while VE-cadherin/alpha-catenin/beta-catenin complex can function as an early recognition mechanism between EC, the formation of mature, cytoskeleton-bound junctions requires plakoglobin synthesis and organization.
Assuntos
Caderinas/análise , Proteínas do Citoesqueleto/análise , Endotélio Vascular/ultraestrutura , Junções Intercelulares/ultraestrutura , Transativadores , Northern Blotting , Western Blotting , Caderinas/biossíntese , Moléculas de Adesão Celular/análise , Células Cultivadas , Proteínas do Citoesqueleto/biossíntese , Desmoplaquinas , Eletroforese em Gel de Poliacrilamida , Endotélio Vascular/fisiologia , Imunofluorescência , Humanos , Junções Intercelulares/fisiologia , Cinética , Peso Molecular , Fatores de Tempo , Veias Umbilicais , alfa Catenina , beta Catenina , gama CateninaRESUMO
Tight junctions are the most apical components of endothelial and epithelial intercellular cleft. In the endothelium these structures play an important role in the control of paracellular permeability to circulating cells and solutes. The only known integral membrane protein localized at sites of membrane-membrane interaction of tight junctions is occludin, which is linked inside the cells to a complex network of cytoskeletal and signaling proteins. We report here the identification of a novel protein (junctional adhesion molecule [JAM]) that is selectively concentrated at intercellular junctions of endothelial and epithelial cells of different origins. Confocal and immunoelectron microscopy shows that JAM codistributes with tight junction components at the apical region of the intercellular cleft. A cDNA clone encoding JAM defines a novel immunoglobulin gene superfamily member that consists of two V-type Ig domains. An mAb directed to JAM (BV11) was found to inhibit spontaneous and chemokine-induced monocyte transmigration through an endothelial cell monolayer in vitro. Systemic treatment of mice with BV11 mAb blocked monocyte infiltration upon chemokine administration in subcutaneous air pouches. Thus, JAM is a new component of endothelial and epithelial junctions that play a role in regulating monocyte transmigration.
Assuntos
Antígenos/fisiologia , Moléculas de Adesão Celular/fisiologia , Movimento Celular , Imunoglobulinas/fisiologia , Monócitos/fisiologia , Junções Íntimas/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Antígenos/genética , Antígenos/metabolismo , Sequência de Bases , Células COS , Adesão Celular , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Clonagem Molecular , DNA Complementar , Endotélio/citologia , Células Epiteliais/metabolismo , Imunoglobulinas/genética , Imunoglobulinas/metabolismo , Moléculas de Adesão Juncional , Masculino , Camundongos , Dados de Sequência Molecular , Ratos , Ratos Endogâmicos Lew , Pele/imunologia , TransfecçãoRESUMO
Supernatants from cultures of human monocytes that had been stimulated with endotoxin or silica induced the synthesis of prostacyclin in endothelial and smooth muscle cells. The lymphokine mediating these effects on the cells of the blood vessel wall was identified as interleukin-1; interferons and interleukin-2 were inactive. Interleukin-1-induced prostacyclin synthesis represents a new aspect of the interaction between the immune system (as well as other tissues) and the vessel wall and may serve as a basis for the development of new strategies in antithrombotic therapy.
Assuntos
Vasos Sanguíneos/metabolismo , Endotélio/metabolismo , Epoprostenol/biossíntese , Interleucina-1/farmacologia , Músculo Liso/metabolismo , Células Cultivadas , Humanos , Interferons/farmacologia , Interleucina-2/farmacologia , Monócitos/fisiologia , Fatores de TempoRESUMO
Selective pharmacological inhibition of thromboxane A2 synthesis did not prevent arachidonate-induced aggregation of human platelets in vitro. Prevention was instead achieved by a combination of thromboxane A2 inhibitors with low concentrations of aspirin. The latter partially reduced the proaggregatory cyclooxygenase products that accumulated when thromboxane A2 synthesis was blocked. The aspirin concentrations did not affect per se either platelet aggregation or prostacyclin synthesis in cultured human endothelial cells. The combination of thromboxane synthetase inhibitors with low doses of aspirin may offer greater antithrombotic potential than either drug alone.