RESUMEN
The homeobox containing gene HoxB7 is functionally associated with melanoma growth promotion through the direct transactivation of bFGF. Accordingly, the introduction of HoxB7 in the breast cancer line SkBr3 (SkBr3/B7), strongly increases its tumorigenic properties. Here we show that in SkBr3/B7 cells, HoxB7 regulates the expression of TALE Hox cofactors by increasing Pbx2 and Prep1 and decreasing Pbx1. The functional requirement of Hox cofactors in the oncogenic activity of HoxB7 was proven with a dominant-negative Pbx1 mutant, Pbx1NT, which sequesters Prep1 in the cytoplasm. The less aggressive phenotype of the SkBr3/B7/PbxNT cells, evaluated in vitro as well as in vivo, correlated well with increased apoptosis, decreased cycling and up-regulation of p16 and p53. Tumor cell-type specific functional effects of Pbx1NT were observed, possibly related to the presence of different Hox genes in melanoma or breast adenocarcinoma DNA-protein ternary complexes.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas de Homeodominio/metabolismo , Proteínas Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Factores de Transcripción/metabolismo , Animales , Apoptosis , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Proteínas de Homeodominio/análisis , Proteínas de Homeodominio/antagonistas & inhibidores , Proteínas de Homeodominio/genética , Humanos , Ratones , Ratones Desnudos , Mutación , Proteínas Oncogénicas/antagonistas & inhibidores , Factor de Transcripción 1 de la Leucemia de Células Pre-B , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , ARN Mensajero/metabolismo , Factores de Transcripción/genéticaRESUMEN
The function of urokinase and its receptor is essential for cell migration in pathological conditions, as shown by the analysis of knockout mice phenotypes. How a protease of a fibrinolytic pathway can induce migration is not understood and no link between this protease and migration-promoting G protein-coupled receptors has been described. We now show that FPRL1/LXA4R, a G protein-coupled receptor for a number of polypeptides and for the endogenous lipoxin A4 (LXA4), is the link between urokinase-type plasminogen activator (uPA) and migration as it directly interacts with an activated, soluble, cleaved form of uPA receptor (uPAR) (D2D3(88-274)) to induce chemotaxis. In this article we show that (i) both uPAR and FPRL1/LXA4R are necessary for the chemotactic activity of uPA whereas FPRL1/LXA4R is sufficient to mediate D2D3(88-274)-induced cell migration. (ii) Inhibition or desensitization of FPRL1/LXA4R by antibodies or specific ligands specifically prevents chemotaxis induced by D2D3(88-274) in THP-1 cells and human peripheral blood monocytes. (iii) Desensitization of FPRL1/LXA4R prevents the activation of tyrosine kinase Hck induced by D2D3(88-274). (iv) D2D3(88-274) directly binds to FPRL1/LXA4R and is competed by two specific FPRL1/LXA4R agonists, the synthetic MMK-1 peptide and a stable analog of LXA4. Thus, a naturally produced cleaved form of uPAR is a unique endogenous chemotactic agonist for FPRL1/LXA4R receptor and its activity can be antagonized by specific ligands. These results provide the first direct link, to our knowledge, between the fibrinolytic machinery and the inflammatory response, demonstrating that uPA-derived peptide fragments can activate a specific chemotactic receptor.
Asunto(s)
Lipoxinas , Receptores de Superficie Celular/metabolismo , Receptores Inmunológicos/fisiología , Receptores de Lipoxina , Receptores de Péptidos/fisiología , Animales , Línea Celular , Quimiotaxis de Leucocito/efectos de los fármacos , Quimiotaxis de Leucocito/fisiología , Proteínas de Unión al GTP/metabolismo , Humanos , Ácidos Hidroxieicosatetraenoicos/metabolismo , Leucemia Basofílica Aguda , Monocitos/fisiología , N-Formilmetionina Leucil-Fenilalanina/farmacología , Péptidos/farmacología , Activadores Plasminogénicos/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Ratas , Receptores de Formil Péptido , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Células Tumorales Cultivadas , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
PAI-1 (plasminogen activator inhibitor-1) binds the urokinase-type plasminogen activator (uPA) and causes its degradation via its receptor uPAR and low-density lipoprotein receptor-related protein (LRP). While both uPA and PAI-1 are chemoattractants, we find that a preformed uPA-PAI-1 complex has no chemotactic activity and that PAI-1 inhibits uPA-induced chemotaxis. The inhibitory effect of PAI-1 on uPA-dependent chemotaxis is reversed when uPAR internalization is inhibited by the 39 kDa receptor-associated protein or by anti-LRP antibodies. Under the same conditions, the uPA-PAI-1 complex is turned into a chemoattractant causing cytoskeleton reorganization and extracellular-regulated kinase/mitogen-activated protein kinases activation. Thus, uPAR internalization by PAI-1 regulates cell migration.
Asunto(s)
Quimiotaxis , Inhibidor 1 de Activador Plasminogénico/metabolismo , Receptores de Superficie Celular/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Transporte Activo de Núcleo Celular , Animales , Movimiento Celular , Células Cultivadas , Citoesqueleto/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Ligandos , Sistema de Señalización de MAP Quinasas , Ratones , Microscopía Fluorescente , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Músculo Liso/citología , Unión Proteica , Ratas , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Proteínas Recombinantes/metabolismoRESUMEN
Vitronectin (VN) and pro-urokinase (pro-uPA) stimulated migration of rat smooth muscle cells in a dose-dependent and additive way, and induced motile-type changes in cell morphology together with a complete reorganization of the actin filaments and of the microtubules. All these effects were inhibited by pertussis toxin, or by antibodies directed against the urokinase receptor (uPAR) or against the VN receptor alpha(v)beta(3) suggesting that an association between the two receptors is required to mediate both signals. Investigation of the signaling pathways showed that increasing the intracellular cAMP resulted in a selective inhibition of VN-induced cell migration. On the other hand, PD 98059, an inhibitor of MEK, differentially inhibited the pro-uPA- but not the VN-induced cell migration. Phosphorylation and nuclear translocation of Erk by pro-uPA was directly observed. We conclude that the signaling pathways of pro-uPA and VN must be at least in part different.
Asunto(s)
Quimiotaxis/fisiología , Receptores de Superficie Celular/fisiología , Receptores de Vitronectina/fisiología , Transducción de Señal/fisiología , Activador de Plasminógeno de Tipo Uroquinasa/fisiología , Vitronectina/fisiología , Animales , Anticuerpos/inmunología , Anticuerpos/farmacología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Tamaño de la Célula/efectos de los fármacos , Tamaño de la Célula/fisiología , Quimiotaxis/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Citoesqueleto/fisiología , Activación Enzimática , Sistema de Señalización de MAP Quinasas/fisiología , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Músculo Liso/citología , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Toxina del Pertussis , Ratas , Receptor Cross-Talk/fisiología , Receptores de Superficie Celular/inmunología , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Receptores de Vitronectina/inmunología , Proteínas Recombinantes/farmacología , Activador de Plasminógeno de Tipo Uroquinasa/farmacología , Factores de Virulencia de Bordetella/farmacología , Vitronectina/farmacologíaRESUMEN
HMG1 (high mobility group 1) is a ubiquitous and abundant chromatin component. However, HMG1 can be secreted by activated macrophages and monocytes, and can act as a mediator of inflammation and endotoxic lethality. Here we document a role of extracellular HMG1 in cell migration. HMG1 (and its individual DNA-binding domains) stimulated migration of rat smooth muscle cells in chemotaxis, chemokinesis, and wound healing assays. HMG1 induced rapid and transient changes of cell shape, and actin cytoskeleton reorganization leading to an elongated polarized morphology typical of motile cells. These effects were inhibited by antibodies directed against the receptor of advanced glycation endproducts, indicating that the receptor of advanced glycation endproducts is the receptor mediating the HMG1-dependent migratory responses. Pertussis toxin and the mitogen-activated protein kinase kinase inhibitor PD98059 also blocked HMG1-induced rat smooth muscle cell migration, suggesting that a G(i/o) protein and mitogen-activated protein kinases are required for the HMG1 signaling pathway. We also show that HMG1 can be released by damage or necrosis of a variety of cell types, including endothelial cells. Thus, HMG1 has all the hallmarks of a molecule that can promote atherosclerosis and restenosis after vascular damage.
Asunto(s)
Tamaño de la Célula/fisiología , Factores Quimiotácticos/metabolismo , Quimiotaxis/fisiología , Citoesqueleto/metabolismo , Proteínas del Grupo de Alta Movilidad/metabolismo , Músculo Liso Vascular/citología , Proteínas Nucleares/metabolismo , Animales , Células Cultivadas , Factores Quimiotácticos/genética , Factores Quimiotácticos/farmacología , Quimiotaxis/efectos de los fármacos , Medio de Cultivo Libre de Suero , Citoesqueleto/efectos de los fármacos , Endotelio Vascular/química , Endotelio Vascular/citología , Endotelio Vascular/ultraestructura , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Productos Finales de Glicación Avanzada/metabolismo , Proteínas del Grupo de Alta Movilidad/genética , Humanos , Microscopía Fluorescente , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/ultraestructura , Proteínas Nucleares/genética , Toxina del Pertussis , Unión Proteica , Ratas , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Transducción de Señal/fisiología , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
We recently described a subset of peripheral CD14+CD34+ cells able to migrate across endothelial cell monolayers and differentiate into immunostimulatory dendritic cells (DC). In this paper we show that immature DC derived from CD14+CD34+ precursors are also capable of reverse transendothelial migration and extracellular matrix (ECM) invasion using the urokinase plasminogen activator receptor (uPAR). We found that these cells respond to macrophage-inflammatory protein (MIP)-1alpha, enhancing their ability to invade ECM and supporting the idea that immature DC are selectively recruited at the site of inflammation to expand the pool of APCs. Interestingly, MIP-1alpha was also capable of preventing the decreased matrix invasion observed by blocking uPAR, suggesting that the uPA/uPAR system and MIP-1alpha cooperate in driving immature DC migration through the subendothelial matrix. Upon exposure to maturating stimuli, such as TNF-alpha, CD14+CD34+-derived DC enhance their APC function and decrease the capacity of invading ECM; these changes are accompanied by altered expression and function of uPAR. Moreover, mature DC shift their sensitivity from MIP-1alpha to MIP-3beta, enhancing their transendothelial migration capability in response to the latter chemokine. Our data support the hypothesis that bloodborne DC can move through ECM toward the site of pathogen entry where they differentiate into fully mature APCs with their motility and function regulated by microenvironmental stimuli, including MIP-1alpha, MIP-3beta, and TNF-alpha.
Asunto(s)
Antígenos CD34/biosíntesis , Células Dendríticas/enzimología , Regulación hacia Abajo/inmunología , Receptores de Lipopolisacáridos/biosíntesis , Receptores de Superficie Celular/metabolismo , Células Madre/enzimología , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Diferenciación Celular/inmunología , Movimiento Celular/inmunología , Células Cultivadas , Quimiocina CCL19 , Quimiocina CCL3 , Quimiocina CCL4 , Quimiocina CCL5/fisiología , Quimiocinas CC/fisiología , Células Dendríticas/citología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Endotelio Vascular/citología , Activación Enzimática/inmunología , Matriz Extracelular/enzimología , Matriz Extracelular/inmunología , Humanos , Proteínas Inflamatorias de Macrófagos/fisiología , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/fisiología , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Células Madre/citología , Células Madre/inmunología , Células Madre/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/antagonistas & inhibidores , Activador de Plasminógeno de Tipo Uroquinasa/fisiologíaRESUMEN
The catalytically inactive precursor of urokinase-type plasminogen activator (pro-u-PA) induced a chemotactic response in rat smooth muscle cells (RSMC) through binding to the membrane receptor of urokinase (u-PA receptor [u-PAR]). A soluble form of u-PAR activated by chymotrypsin cleavage as well as a peptide located between domain 1 and 2 of u-PAR reproduced the effect of pro-u-PA on cell migration. The chemotactic pro-u-PA effect correlates with a dramatic reorganization of actin cytoskeleton, of adhesion plaques, and with major cell shape changes in RSMC. Pro-u-PA induced a decrease in stress fiber content, membrane ruffling, actin ring formation, and disruption leading to the characteristic elongated cell shape of motile cells with an actin semi-ring located close to the leading edge of cells. u-PAR effects on both chemotaxis and cytoskeleton were sensitive to pertussis toxin and, hence, possibly require G proteins. u-PAR effects are accompanied by a relocation of u-PAR, vitronectin receptor (VNR) alphavbeta3, beta1 integrin subunit, and Src tyrosine kinase to the leading membrane of migrating cells. In conclusion, our data show that pro-u-PA, via binding to u-PAR, controls a signaling pathway, regulated by tyrosine kinases and possibly G proteins, leading to cell cytoskeleton reorganization and cell migration.
Asunto(s)
Quimiotaxis/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Precursores Enzimáticos/farmacología , Músculo Liso/efectos de los fármacos , Toxina del Pertussis , Receptores de Superficie Celular/fisiología , Transducción de Señal/efectos de los fármacos , Activador de Plasminógeno de Tipo Uroquinasa/farmacología , Factores de Virulencia de Bordetella/farmacología , Familia-src Quinasas/fisiología , Actinas/análisis , Animales , Tamaño de la Célula/efectos de los fármacos , Células Cultivadas , Citoesqueleto/ultraestructura , Proteínas de Unión al GTP/metabolismo , Ratones , Microscopía Fluorescente , Músculo Liso/citología , Músculo Liso/ultraestructura , Fragmentos de Péptidos/farmacología , Conformación Proteica , Proteínas Proto-Oncogénicas pp60(c-src)/fisiología , Ratas , Receptores de Superficie Celular/química , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Receptores de Vitronectina/metabolismo , Proteínas Recombinantes/farmacologíaRESUMEN
The binding of the urokinase plasminogen activator (uPA) to its receptor (uPAR) regulates cell adhesion, surface proteolysis, chemotaxis and cell extravasation in a number of experimental systems. Recent evidences have suggested that uPAR can by itself mediate chemotaxis of human monocytes and cause profound changes in cytoskeletal organization indicating that this receptor has the properties of a cell-surface regulated chemokine. Indeed, it is likely that upon binding to uPA, uPAR undergoes a conformational change that uncovers a new epitope located in the linker region between domain 1 and 2 of the receptor and is endowed with a potent chemotactic activity. This conformational change can be mimicked in vitro by enzymatic processing of a recombinant receptor. We have shown that chymotrypsin cleaves uPAR between domain 1 and 2 in an area that can be also cleaved by uPA at high efficiency and generate a receptor that can mediate monocytes migration independently of uPA binding. This mechanism is pertussis-toxin sensitive and involves activation of tyrosine kinases and cytoskeletal reorganization events in vitro. These studies indicate that in addition to its receptor function, upon binding to uPA, uPAR becomes a pleiotropic ligand for other still to be identified surface molecules.
Asunto(s)
Receptores de Superficie Celular , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Comunicación Autocrina , Quimiocinas/química , Quimiocinas/genética , Quimiocinas/metabolismo , Humanos , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Transducción de SeñalRESUMEN
The role of urokinase-type plasminogen activator (uPA) and its receptor (uPAR/CD87) in cell migration and invasion is well substantiated. Recently, uPA has been shown to be essential in cell migration, since uPA-/- mice are greatly impaired in inflammatory cell recruitment. We have shown previously that the uPA-induced chemotaxis requires interaction with and modification of uPAR/CD87, which is the true chemoattracting molecule acting through an unidentified cell surface component which mediates this cell surface chemokine activity. By expressing and testing several uPAR/CD87 variants, we have located and functionally characterized a potent uPAR/CD87 epitope that mimics the effects of the uPA-uPAR interaction. The chemotactic activity lies in the region linking domains 1 and 2, the only protease-sensitive region of uPAR/CD87, efficiently cleaved by uPA at physiological concentrations. Synthetic peptides carrying this epitope promote chemotaxis and activate p56/p59(hck) tyrosine kinase. Both chemotaxis and kinase activation are pertussis toxin sensitive, involving a Gi/o protein in the pathway.
Asunto(s)
Quimiotaxis/fisiología , Proteínas de Unión al GTP/metabolismo , Receptores de Superficie Celular/química , Receptores de Superficie Celular/fisiología , Activador de Plasminógeno de Tipo Uroquinasa/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Quimiocinas/fisiología , Quimiotaxis/efectos de los fármacos , Cartilla de ADN , Humanos , Inflamación , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Toxina del Pertussis , Reacción en Cadena de la Polimerasa , Receptores de Superficie Celular/biosíntesis , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Activador de Plasminógeno de Tipo Uroquinasa/biosíntesis , Activador de Plasminógeno de Tipo Uroquinasa/deficiencia , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
Physiological concentrations of urokinase plasminogen activator (uPA) stimulated a chemotactic response in human monocytic THP-1 through binding to the urokinase receptor (uPAR). The effect did not require the protease moiety of uPA, as stimulation was achieved also with the N-terminal fragment (ATF), while the 33 kDa low molecular weight uPA was ineffective. Co-immunoprecipitation experiments showed association of uPAR with intracellular kinase(s), as demonstrated by in vitro kinase assays. Use of specific antibodies identified p56/p59hck as a kinase associated with uPAR in THP-1 cell extracts. Upon addition of ATF, p56/p59hck activity was stimulated within 2 min and returned to normal after 30 min. Since uPAR lacks an intracellular domain capable of interacting with intracellular kinase, activation of p56/p59hck must require a transmembrane adaptor. Evidence for this was strongly supported by the finding that a soluble form of uPAR (suPAR) was capable of inducing chemotaxis not only in THP-1 cells but also in cells lacking endogenous uPAR (IC50, 5 pM). However, activity of suPAR require chymotrypsin cleavage between the N-terminal domain D1 and D2 + D3. Chymotrypsin-cleaved suPAR also induced activation of p56/p59hck in THP-1 cells, with a time course comparable with ATF. Our data show that uPA-induced signal transduction takes place via uPAR, involves activation of intracellular tyrosine kinase(s) and requires an as yet undefined adaptor capable of connecting the extracellular ligand binding uPAR to intracellular transducer(s).
Asunto(s)
Quimiotaxis de Leucocito/fisiología , Receptores de Superficie Celular/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Animales , Anticuerpos Bloqueadores/farmacología , Anticuerpos Monoclonales/farmacología , Línea Celular , Quimiotaxis de Leucocito/efectos de los fármacos , Cricetinae , Endopeptidasas/metabolismo , Activación Enzimática , Humanos , Técnicas In Vitro , Ratones , Monocitos/fisiología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-hck , Agregación de Receptores , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Transducción de Señal , Activador de Plasminógeno de Tipo Uroquinasa/química , Activador de Plasminógeno de Tipo Uroquinasa/farmacologíaRESUMEN
The biosynthesis and the surface localization of the urokinase plasminogen activator receptor (uPAR) were analysed in MDCK epithelial cells and in unpolarized fibroblasts. No differences were observed with respect to rate of synthesis, nature of precursors and time of surface appearance. uPAR was localized particularly at the focal and cell-cell contacts when expressed in fibroblasts. On the contrary, in MDCK cells uPAR was found mostly on the apical surface; in agreement with its localization, down-regulation of uPAR by the uPA-PAI-1 complex was observed only from the apical membrane.
Asunto(s)
Membrana Celular/química , Polaridad Celular , Receptores de Superficie Celular/análisis , Receptores de Superficie Celular/biosíntesis , Células 3T3 , Animales , Adhesión Celular , Línea Celular , Perros , Regulación hacia Abajo , Células Epiteliales , Glicosilfosfatidilinositoles , Humanos , Riñón/citología , Células L , Ratones , Peso Molecular , Receptores de Superficie Celular/genética , Receptores del Activador de Plasminógeno Tipo Uroquinasa , TransfecciónRESUMEN
Two monoclonal antibodies (mAb), MEC 7.46 (IgG1) and MEC 13.3 (IgG2a) that specifically recognize mouse endothelial cells (EC) of blood vessels, were produced immunizing a Lewis rat with a polyoma middle T transformed EC line. Antibodies were screened by enzyme-linked immunosorbent assay (ELISA) and by immunofluorescence on different cultured cell lines and by immunoperoxidase staining on frozen sections of various mouse normal and inflammatory tissues. Both mAbs reacted with eight transformed endothelial lines tested in vitro, but were consistently negative on various cell lines of different histological origin. Reactivity was not altered by preexposure of the cell lines to IL-1. Microscopic immunofluorescence analysis showed that the MEC mAbs localized at the cell-cell contacts in EC. Immunohistochemical staining of various mouse tissue was always restricted to the EC of all blood vessels of the organ considered. Staining of the endothelial lining of blood vessels was greater at cell-to-cell contacts. Weak reactivity was detected in bone marrow and spleen megakaryocytes. This picture was not altered in inflamed and tumor tissues. In the developing mouse embryo, MEC 13.3 specifically stained proliferating and sprouting endothelium in all organs and tissues examined. Both MEC 7.46 and MEC 13.3 mAbs were able to precipitate a molecule with an apparent molecular mass of 130 kDa from endothelioma lysates. The protein was synthesized by the cells and exposed on the cell surface. Immunodepletion analysis indicated that MEC 13.3 recognized a molecule related to the murine from of PECAM or CD31. We believe that these mAbs are promising tools for the identification of murine EC and for studying their ontogenesis and functions.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Endotelio Vascular/inmunología , Animales , Antígenos de Diferenciación Mielomonocítica/metabolismo , Moléculas de Adhesión Celular/metabolismo , Línea Celular , Endotelio Vascular/embriología , Endotelio Vascular/crecimiento & desarrollo , Femenino , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Molécula-1 de Adhesión Celular Endotelial de Plaqueta , Ratas , Ratas Endogámicas LewRESUMEN
Freshly cultured vascular endothelial cells express the CD34 antigen in a diffuse cell surface pattern with some concentration on microvilli. Expression is downregulated with proliferation in continuous culture and undetectable after nine population doublings but can be maintained by restraining cell proliferation and promoting cell contact. Expression of CD34 at the antigen and mRNA levels on early passage cells is rapidly downregulated by interleukin-1 beta (IL-1 beta), interferon-gamma (INF-gamma), and tumor necrosis factor-alpha (TNF-alpha) under conditions in which these ligands upregulate the adhesion molecules: endothelial leukocyte adhesion molecule 1 (ELAM-1) and intracellular adhesion molecule 1 (ICAM-1). This reciprocal pattern of expression and the topographic distribution of CD34 molecules on the lumenal interdigitated microprocesses of adjacent endothelial cells in vivo suggest that CD34 might have a negative modulating role on adhesion functions of endothelia.
Asunto(s)
Antígenos CD/metabolismo , Moléculas de Adhesión Celular/metabolismo , Endotelio Vascular/inmunología , Antígenos CD/genética , Antígenos CD34 , Adhesión Celular , División Celular , Membrana Celular/inmunología , Membrana Celular/metabolismo , Selectina E , Endotelio Vascular/metabolismo , Endotelio Vascular/ultraestructura , Expresión Génica , Humanos , Interferón gamma/farmacología , Interleucina-1/farmacología , Microscopía Electrónica de Rastreo , Microvellosidades/inmunología , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
In order to select a candidate segmented polyurethane (SPU) elastomer for the preparation of cardio-vascular prostheses, a series of biological tests (namely haemolysis, aPTT and PT coagulation tests, cytotoxicity, human endothelial cells seeding) was carried out on five commercially available biomedical polyurethanes. The tests were performed on solvent cast samples, from THF (Cardiothane 51, Pellethane 2363 80A, Estane 5714 F1, and Estane 58810), or DMAC (Biomer). All the materials were sterilized by gamma-irradiation before being tested. From the results obtained all the polyurethanes used in this study were shown to be devoid of toxicity towards blood (as proved by haemolysis and coagulation time tests) or blood cells (as proved by cytotoxicity and cell adhesion assays). A clear difference among the tested copolymers didn't stand out under our test conditions, although Cardiothane, possibly due to its physico-chemical characteristics, was less effective in promoting endothelial cell adhesion.
Asunto(s)
Prótesis Vascular , Prótesis Valvulares Cardíacas , Poliuretanos/toxicidad , Animales , Bovinos , Adhesión Celular , Células Cultivadas , Pruebas Inmunológicas de Citotoxicidad , Endotelio , Hemólisis , Humanos , Ensayo de Materiales , Tiempo de Tromboplastina Parcial , Tiempo de ProtrombinaRESUMEN
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.
Asunto(s)
Cadherinas/análisis , Endotelio Vascular/química , Uniones Intercelulares/química , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Antígenos de Diferenciación Mielomonocítica/análisis , Biomarcadores , Cadherinas/química , Adhesión Celular/fisiología , Moléculas de Adhesión Celular/análisis , Células Cultivadas , Cromatografía de Afinidad , Endotelio Vascular/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Datos de Secuencia Molecular , Mapeo Peptídico , Molécula-1 de Adhesión Celular Endotelial de PlaquetaRESUMEN
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.