RESUMEN
Interleukin (IL)-13 is a key mediator of tissue fibrosis caused by T helper cell type 2 inflammation. We hypothesized that the fibrogenic effects of IL-13 are mediated by transforming growth factor (TGF)-beta. To test this hypothesis we compared the regulation of TGF-beta in lungs from wild-type mice and CC10-IL-13 mice in which IL-13 overexpression causes pulmonary fibrosis. IL-13 selectively stimulated TGF-beta(1) production in transgenic animals and macrophages were the major site of TGF-beta(1) production and deposition in these tissues. IL-13 also activated TGF-beta(1) in vivo. This activation was associated with decreased levels of mRNA encoding latent TGF-beta-binding protein-1 and increased mRNA encoding urinary plasminogen activator, matrix metalloproteinase (MMP)-9, and CD44. TGF-beta(1) activation was abrogated by the plasmin/serine protease antagonist aprotinin. It was also decreased in progeny of crosses of CC10-IL-13 mice and MMP-9 null mice but was not altered in crosses with CD44 null animals. IL-13-induced fibrosis was also significantly ameliorated by treatment with the TGF-beta antagonist soluble TGFbetaR-Fc (sTGFbetaR-Fc). These studies demonstrate that IL-13 is a potent stimulator and activator of TGF-beta(1) in vivo. They also demonstrate that this activation is mediated by a plasmin/serine protease- and MMP-9-dependent and CD44-independent mechanism(s) and that the fibrogenic effects of IL-13 are mediated, in great extent, by this TGF-beta pathway.
Asunto(s)
Interleucina-13/inmunología , Fibrosis Pulmonar/inmunología , Factor de Crecimiento Transformador beta/inmunología , Animales , Receptores de Hialuranos/fisiología , Interleucina-13/genética , Metaloproteinasa 9 de la Matriz/fisiología , Ratones , Ratones Noqueados , Ratones Transgénicos , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta1 , Activador de Plasminógeno de Tipo Uroquinasa/fisiologíaRESUMEN
Kidney fibrosis is the hallmark of most types of progressive kidney disease, including the genetic disorder Alport's syndrome. We undertook gene expression analysis in Alport's syndrome mouse kidneys using microchip arrays to characterize the development of fibrosis. In addition to matrix and matrix-remodeling genes, consistent with interstitial fibrosis, macrophage-related genes show elevated expression levels in Alport's syndrome kidneys. Immunohistochemical analysis of kidney sections illustrated that macrophages as well as myofibroblasts accumulate in the tubular interstitium. Deletion of alpha(1) integrin results in decreased accumulation of both myofibroblasts and macrophages in the tubular interstitium in Alport's syndrome mice and delays disease progression. Transforming growth factor beta antagonism, although reducing interstitial fibrosis, does not limit macrophage accumulation in the tubular interstitium and disease progression. In this study, we identified previously overlooked inflammatory events that occur in the tubulointerstitial region. We propose that in addition to the previously suggested role for the alpha(1)beta(1) integrin in mesangial expansion and abnormal laminin deposition, this integrin may be critical for monocyte accumulation that, in turn, may lead directly to renal failure. Our gene expression and immunohistochemical data indicate that macrophage accumulation is dependent on alpha(1) integrin expression on the macrophage cell surface and that anti-alpha(1) integrin strategies may be employed as therapeutics in the treatment of chronic inflammatory and fibrotic diseases.
Asunto(s)
Antígenos CD/fisiología , Riñón/patología , Animales , Antígenos CD/genética , Mapeo Cromosómico , Modelos Animales de Enfermedad , Regulación hacia Abajo , Fibrosis , Inmunohistoquímica , Inflamación/genética , Integrina alfa1 , Riñón/metabolismo , Laminina/metabolismo , Ratones , Nefritis Hereditaria/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Complementario/metabolismo , ARN Mensajero/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factores de Tiempo , Factor de Crecimiento Transformador beta/metabolismo , Regulación hacia ArribaRESUMEN
We have shown that the integrin alphavbeta6 activates latent TGF-beta in the lungs and skin. We show here that mice lacking this integrin are completely protected from pulmonary edema in a model of bleomycin-induced acute lung injury (ALI). Pharmacologic inhibition of TGF-beta also protected wild-type mice from pulmonary edema induced by bleomycin or Escherichia coli endotoxin. TGF-beta directly increased alveolar epithelial permeability in vitro by a mechanism that involved depletion of intracellular glutathione. These data suggest that integrin-mediated local activation of TGF-beta is critical to the development of pulmonary edema in ALI and that blocking TGF-beta or its activation could be effective treatments for this currently untreatable disorder.
Asunto(s)
Antígenos de Neoplasias , Síndrome de Dificultad Respiratoria/etiología , Factor de Crecimiento Transformador beta/fisiología , Animales , Bleomicina , Barrera Alveolocapilar/fisiología , Células Cultivadas , Endotoxinas , Glutatión/metabolismo , Integrinas/genética , Ratones , Ratones Noqueados , Proteínas Serina-Treonina Quinasas , Alveolos Pulmonares/metabolismo , Edema Pulmonar/etiología , Edema Pulmonar/patología , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/administración & dosificación , Síndrome de Dificultad Respiratoria/metabolismo , Síndrome de Dificultad Respiratoria/patología , Factor de Crecimiento Transformador beta/antagonistas & inhibidoresRESUMEN
Integrins are a family of transmembrane glycoproteins that can interact with components of the extracellular matrix. The alpha4beta1 and alpha4beta7 integrins are heterodimeric leukocyte cell surface molecules critical to their cell and matrix adhesive interactions. Evidence for a central role for the alpha4 integrins in leukocyte pathophysiology in the lung is well documented. In this study, we tested the hypothesis that neutralizing antibody for integrin alpha4 (PS2) may reduce bleomycin (BL)-induced lung fibrosis in vivo. Male C57BL/6 mice were injected intratracheally with saline (SA) or BL (0.08 U/mouse) followed by intraperitoneal injection of SA, isotype control antibody (1E6), or PS2 (100 microg) three times a week. Twenty-one days after the intratracheal instillation, mice were killed for bronchoalveolar lavage (BAL), biochemical, histopathological, and immunohistological analyses. Treatment with PS2 significantly reduced BL-induced increases in lung lipid peroxidation and hydroxyproline content. Lung histopathology also showed reduced fibrotic lesions in the BL-treated lungs by treatment with PS2. BL-treated mouse lungs also showed induction of cells with the myofibroblast phenotype, as indicated by the increased expression of alpha-smooth muscle actin (alphaSMA), whereas treatment with PS2 minimized the BL-induced alphaSMA expression. Furthermore, treatment with PS2 reduced the BL-induced increase in the BAL total cell number, and attenuated the BL-induced increase in the BAL protein level. It is concluded that integrin alpha4 may play an important role in BL-induced pulmonary fibrosis, and the use of anti-alpha4 antibody offers therapeutic antifibrotic potential in vivo.
Asunto(s)
Antígenos CD/inmunología , Antígenos CD/metabolismo , Fibrosis Pulmonar/prevención & control , Actinas/análisis , Animales , Antibacterianos , Anticuerpos/inmunología , Bleomicina , Líquido del Lavado Bronquioalveolar/citología , Diferenciación Celular , Hidroxiprolina/metabolismo , Inmunohistoquímica , Integrina alfa4 , Peroxidación de Lípido , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Liso/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/enzimología , Fibrosis Pulmonar/patologíaRESUMEN
Alport syndrome is a genetic disorder resulting from mutations in type IV collagen genes. The defect results in pathological changes in kidney glomerular and inner-ear basement membranes. In the kidney, progressive glomerulonephritis culminates in tubulointerstitial fibrosis and death. Using gene knockout-mouse models, we demonstrate that two different pathways, one mediated by transforming growth factor (TGF)-beta1 and the other by integrin alpha1beta1, affect Alport glomerular pathogenesis in distinct ways. In Alport mice that are also null for integrin alpha1 expression, expansion of the mesangial matrix and podocyte foot process effacement are attenuated. The novel observation of nonnative laminin isoforms (laminin-2 and/or laminin-4) accumulating in the glomerular basement membrane of Alport mice is markedly reduced in the double knockouts. The second pathway, mediated by TGF-beta1, was blocked using a soluble fusion protein comprising the extracellular domain of the TGF-beta1 type II receptor. This inhibitor prevents focal thickening of the glomerular basement membrane, but does not prevent effacement of the podocyte foot processes. If both integrin alpha1beta1 and TGF-beta1 pathways are functionally inhibited, glomerular foot process and glomerular basement membrane morphology are primarily restored and renal function is markedly improved. These data suggest that integrin alpha1beta1 and TGF-beta1 may provide useful targets for a dual therapy aimed at slowing disease progression in Alport glomerulonephritis.
Asunto(s)
Integrinas/fisiología , Glomérulos Renales/fisiopatología , Nefritis Hereditaria/tratamiento farmacológico , Nefritis Hereditaria/etiología , Factor de Crecimiento Transformador beta/fisiología , Animales , Membrana Basal/patología , Progresión de la Enfermedad , Fragmentos Fc de Inmunoglobulinas/genética , Integrina alfa1beta1 , Integrinas/genética , Glomérulos Renales/patología , Ratones , Ratones Noqueados/genética , Microscopía Electrónica , Microscopía Electrónica de Rastreo , Nefritis Hereditaria/patología , Receptores de Factores de Crecimiento Transformadores beta/genética , Proteínas Recombinantes de Fusión/uso terapéutico , Factor de Crecimiento Transformador beta1RESUMEN
BACKGROUND & AIMS: Transforming growth factor (TGF)-beta has been implicated in many fibrotic conditions. However, its mechanistic role in radiation toxicity is equivocal despite compelling correlative evidence. This study assessed whether in vivo administration of a soluble TGF-beta type II receptor (TbetaR-II) protein ameliorates intestinal radiation injury (radiation enteropathy). METHODS: A recombinant fusion protein, consisting of the extracellular portion of mouse TbetaR-II and the Fc portion of mouse immunoglobulin (Ig) G, was produced. A 5-cm segment of mouse ileum was exposed to 19 Gy x-radiation. TbetaR-II:Fc fusion protein (1 mg/kg every other day) or mouse IgG was administered from 2 days before to 6 weeks after irradiation. Radiation injury was assessed at 6 weeks using quantitative histology, morphometry, and immunohistochemistry. Collagen was measured colorimetrically, and TGF-beta1 messenger RNA was assessed with fluorogenic probe reverse-transcription polymerase chain reaction. RESULTS: Compared with IgG controls, TbetaR-II:Fc-treated mice exhibited less structural injury, preservation of mucosal surface area, and less intestinal wall fibrosis. Intestinal TGF-beta1 messenger RNA increased in TbetaR-II:Fc-treated mice, whereas TGF-beta immunoreactivity decreased. TbetaR-II:Fc treatment increased crypt cell proliferation but otherwise did not affect unirradiated intestine. CONCLUSIONS: Long-term modulation of TGF-beta with a TbetaR-II:Fc fusion protein is feasible and ameliorates radiation enteropathy. These data confirm the putative role of TGF-beta in intestinal radiation fibrosis.
Asunto(s)
Enfermedades Intestinales/tratamiento farmacológico , Traumatismos por Radiación/tratamiento farmacológico , Receptores de Factores de Crecimiento Transformadores beta/genética , Proteínas Recombinantes de Fusión/uso terapéutico , Animales , Células CHO , Colágeno/metabolismo , Cricetinae , Íleon/efectos de los fármacos , Íleon/metabolismo , Íleon/patología , Íleon/efectos de la radiación , Fragmentos Fc de Inmunoglobulinas/genética , Inmunoglobulina G/genética , Enfermedades Intestinales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , ARN Mensajero/metabolismo , Traumatismos por Radiación/patología , Receptores de Factores de Crecimiento Transformadores beta/química , Solubilidad , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta1RESUMEN
Adhesive interactions play an important role in inflammation by promoting leukocyte attachment and extravasation from the vasculature into the peripheral tissues. However, the importance of adhesion molecules within the extracellular matrix-rich environment of peripheral tissues, in which cells must migrate and be activated, has not been well explored. We investigated the role of the major collagen-binding integrins, alpha1beta1 and alpha2beta1, in several in vivo models of inflammation. mAb's against murine alpha1 and alpha2 were found to significantly inhibit effector phase inflammatory responses in animal models of delayed-type hypersensitivity (DTH), contact hypersensitivity (CHS), and arthritis. Mice that were alpha1-deficient also showed decreased inflammatory responses in the CHS and arthritis models when compared with wild-type mice. Decreased leukocyte infiltration and edema formation accompanied inhibition of antigen-specific models of inflammation, as nonspecific inflammation induced by croton oil was not inhibited. This study demonstrates the importance in vivo of alpha1beta1 and alpha2beta1, the collagen-binding integrins, in inflammatory diseases. The study also extends the role of integrins in inflammation beyond leukocyte attachment and extravasation at the vascular endothelial interface, revealing the extracellular matrix environment of peripheral tissues as a new point of intervention for adhesion-based therapies.
Asunto(s)
Artritis/prevención & control , Adhesión Celular/fisiología , Colágeno/metabolismo , Dermatitis Alérgica por Contacto/prevención & control , Hipersensibilidad Tardía/prevención & control , Integrinas/fisiología , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Artritis/inmunología , Artritis/patología , Colágeno/toxicidad , Dermatitis Alérgica por Contacto/inmunología , Dermatitis Alérgica por Contacto/patología , Dermatitis Irritante/inmunología , Dermatitis Irritante/patología , Dermatitis Irritante/prevención & control , Edema/etiología , Edema/prevención & control , Femenino , Hipersensibilidad Tardía/inmunología , Hipersensibilidad Tardía/patología , Integrina alfa1beta1 , Integrinas/inmunología , Leucocitos/patología , Lipopolisacáridos/toxicidad , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Receptores de ColágenoRESUMEN
Rats injected in the hind paw with a mixture of Mycobacterium butirricum emulsified in mineral oil (FA) developed a severe polyarthritis that shared some immunological features with human rheumatoid arthritis. After this local administration, rats developed a secondary lesion (edema) in the contralateral paw, which is a hallmark of immune system activation. In vivo intravenous treatment with a monoclonal anti-very late antigen (VLA)-1 antibody (HA31/8) significantly reduced the edema formation in the contralateral paw. T cells isolated from contralateral paw draining lymph nodes of FA rats treated with HA31/8 showed a reduced cell proliferation in vitro, after stimulation with concanavalin A. Furthermore FACS analysis showed that the reduction in proliferation was concomitant to a reduction in the number of T cells positive to surface IL-2 receptor expression. Our data indicate that after in vivo treatment with a monoclonal anti-very late antigen-1 antibody, there is a beneficial effect on the development of the secondary lesion, which correlates to the reduced ability of T cells to proliferate in vitro as well as to a reduced surface expression of IL-2 receptor. The association of this antibody to other drugs interfering at other levels in rheumatoid arthritis may open a new therapeutic window.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Artritis Infecciosa/terapia , Integrinas/inmunología , Linfocitos T/inmunología , Animales , Artritis Infecciosa/sangre , Artritis Infecciosa/inmunología , Artritis Infecciosa/patología , Separación Celular , Citometría de Flujo , Humanos , Integrina alfa1beta1 , Interleucina-1/sangre , Masculino , Mycobacterium/patogenicidad , Ratas , Ratas Endogámicas Lew , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Central to immune and inflammatory responses are the integrin-mediated adhesive interactions of cells with their extracellular matrix (ECM)-rich environment. Using a comprehensive and quantitative mRNA profiling technique, we analyzed the effect of ECM-induced attachment on monocyte gene expression, its regulation by growth factors, and the integrin specificity of this event. Adhesion of cells to fibronectin resulted in increased expression of a large number of genes, which was strongly potentiated by the presence of growth factors. Adhesion activated both the NF-kappaB and Jak/STAT pathways of gene transcription and increased expression of genes involved in inflammatory and immune responses, revealing the importance of ECM-integrin interactions in these processes.
Asunto(s)
Matriz Extracelular/metabolismo , Integrinas/metabolismo , Monocitos/metabolismo , Adhesión Celular/fisiología , Línea Celular , Proteínas de Unión al ADN/metabolismo , Fibronectinas/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Sustancias de Crecimiento/metabolismo , Humanos , Janus Quinasa 1 , FN-kappa B/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Factor de Transcripción STAT1 , Transducción de Señal , Transactivadores/metabolismoRESUMEN
BACKGROUND: Transforming growth factor beta (TGF-beta) is a key mediator of collagen synthesis in the development of lung fibrosis. It has previously been shown that the administration of TGF-beta antibody and TGF-beta binding proteoglycan, decorin, reduced bleomycin (BL) induced lung fibrosis in animals. The present study was carried out to investigate whether intratracheal instillation of TGF-beta soluble receptor (TR) would minimise the BL induced lung fibrosis in hamsters. METHODS: The effect of a recombinant TR (TGFbetaRII) on the lung collagen accumulation was evaluated in a BL hamster model of pulmonary fibrosis. Animals were divided into four groups and intratracheally injected with saline or BL at 6.5 U/4 ml/kg followed by intratracheal instillation of phosphate buffered saline (PBS) or 4 nmol TR in 0.3 ml twice a week. Twenty days after the first intratracheal instillation the hamsters were killed for bronchoalveolar lavage (BAL) fluid, biochemical, and histopathological analyses. RESULTS: Treatment of hamsters with TR after intratracheal instillation of BL significantly reduced BL induced lung fibrosis as shown by decreases in the lung hydroxyproline level and prolyl hydroxylase activity, although they were still significantly higher than those of the saline control. Histopathological examination showed a considerable decrease in BL induced fibrotic lesions by TR treatment. However, TR did not prevent the BL induced increases in total cells and protein in the BAL fluid. CONCLUSIONS: These results suggest that TR has antifibrotic potential in vivo and may be useful in the treatment of fibrotic diseases where increased TGF-beta is associated with excess collagen accumulation.
Asunto(s)
Antibióticos Antineoplásicos/efectos adversos , Bleomicina/efectos adversos , Fibrosis Pulmonar/prevención & control , Receptores de Factores de Crecimiento Transformadores beta/uso terapéutico , Animales , Líquido del Lavado Bronquioalveolar , División Celular/efectos de los fármacos , Colágeno , Cricetinae , Evaluación Preclínica de Medicamentos , Hidroxiprolina/farmacología , Masculino , Mesocricetus , Oxigenasas de Función Mixta/farmacología , Fibrosis Pulmonar/inducido químicamente , Receptores de Factores de Crecimiento Transformadores beta/administración & dosificaciónRESUMEN
Recent structural and functional analyses of alpha integrin subunit I domains implicate a region in cation and ligand binding referred to as the metal ion-dependent adhesion site (MIDAS). Although the molecular interactions between Mn2+ and Mg2+ and the MIDAS region have been defined by crystallographic analyses, the role of cation in I domain function is not well understood. Recombinant alpha 1 beta 1 integrin I domain (alpha1-I domain) binds collagen in a cation-dependent manner. We have generated and characterized a panel of antibodies directed against the alpha1-I domain, and selected one (AJH10) that blocks alpha 1 beta 1 integrin function for further study. The epitope of AJH10 was localized within the loop between the alpha 3 and alpha 4 helices which contributes one of the metal coordination sites of the MIDAS structure. Kinetic analyses of antibody binding to the I domain demonstrate that divalent cation is required to stabilize the epitope. Denaturation experiments demonstrate that cation has a dramatic effect on the stabilization of the I domain structure. Mn2+ shifts the point at which the I domain denatures from 3.4 to 6.3 M urea in the presence of the denaturant, and from 49.5 to 58.6 degrees C following thermal denaturation. The structural stability provided to the alpha1-I domain by divalent cations may contribute to augmented ligand binding that occurs in the presence of these cations.
Asunto(s)
Cationes Bivalentes/química , Integrinas/química , Magnesio/fisiología , Manganeso/fisiología , Fragmentos de Péptidos/química , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacología , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Colágeno/metabolismo , Epítopos/química , Epítopos/metabolismo , Femenino , Humanos , Integrina alfa1beta1 , Integrinas/antagonistas & inhibidores , Integrinas/genética , Integrinas/inmunología , Magnesio/química , Manganeso/química , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Unión Proteica/genética , Unión Proteica/inmunología , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
The alpha1beta1 integrin is a major cell surface receptor for collagen. Ligand binding is mediated, in part, through a 200 amino acid inserted 'I'-domain contained in the extracellular part of the integrin alpha chain. Integrin I-domains contain a divalent cation binding (MIDAS) site and require cations to interact with integrin ligands. We have determined the crystal structure of recombinant I-domain from the rat alpha1beta1 integrin at 2.2 A resolution in the absence of divalent cations. The alpha1 I-domain adopts the dinucleotide binding fold that is characteristic of all I-domain structures that have been solved to date and has a structure very similar to that of the closely related alpha2beta1 I-domain which also mediates collagen binding. A unique feature of the alpha1 I-domain crystal structure is that the MIDAS site is occupied by an arginine side chain from another I-domain molecule in the crystal, in place of a metal ion. This interaction supports a proposed model for ligand-induced displacement of metal ions. Circular dichroism spectra determined in the presence of Ca2+, Mg2+ and Mn2+ indicate that no changes in the structure of the I-domain occur upon metal ion binding in solution. Metal ion binding induces small changes in UV absorption spectra, indicating a change in the polarity of the MIDAS site environment.
Asunto(s)
Integrinas/química , Animales , Sitios de Unión , Cationes Bivalentes , Gráficos por Computador , Simulación por Computador , Cristalografía por Rayos X , Dimerización , Integrina alfa1beta1 , Modelos Moleculares , Conformación Proteica , Estructura Secundaria de Proteína , Ratas , Proteínas Recombinantes/químicaRESUMEN
Using the rat balloon catheter denudation model, we examined the role of transforming growth factor-beta (TGF-beta) isoforms in vascular repair processes. By en face in situ hybridization, proliferating and quiescent smooth muscle cells in denuded vessels expressed high levels of mRNA for TGF-beta1, TGF-beta2, TGF-beta3, and lower levels of TGF-beta receptor II (TGF-betaRII) mRNA. Compared with normal endothelium, TGF-beta1 and TGF-beta2, as well as TGF-betaRII, mRNA were upregulated in endothelium at the wound edge. Injected recombinant soluble TGF-betaRII (TGF-betaR:Fc) localized preferentially to the adventitia and developing neointima in the injured carotid artery, causing a reduction in intimal lesion formation (up to 65%) and an increase in lumen area (up to 88%). The gain in lumen area was largely due to inhibition of negative remodeling, which coincided with reduced adventitial fibrosis and collagen deposition. Four days after injury, TGF-betaR:Fc treatment almost completely inhibited the induction of smooth muscle alpha-actin expression in adventitial cells. In the vessel wall, TGF-betaR:Fc caused a marked reduction in mRNA levels for collagens type I and III. TGF-betaR:Fc had no effect on endothelial proliferation as determined by reendothelialization of the denuded rat aorta. Together, these findings identify the TGF-beta isoforms as major factors mediating adventitial fibrosis and negative remodeling after vascular injury, a major cause of restenosis after angioplasty.
Asunto(s)
Endotelio Vascular/lesiones , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Actinas/metabolismo , Angioplastia de Balón/efectos adversos , Animales , Aorta/citología , Arterias Carótidas/química , Arterias Carótidas/patología , Traumatismos de las Arterias Carótidas , Diferenciación Celular/fisiología , División Celular/fisiología , Colágeno/metabolismo , Endotelio Vascular/química , Endotelio Vascular/patología , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Fibroblastos/química , Fibroblastos/citología , Fibroblastos/enzimología , Fibrosis , Expresión Génica/fisiología , Hiperplasia , Hibridación in Situ , Ligandos , Proteínas Serina-Treonina Quinasas , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Solubilidad , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Túnica Íntima/química , Túnica Íntima/citología , Túnica Íntima/enzimologíaRESUMEN
Many extracellular matrix proteins contain the tripeptide sequence arginine-glycine-aspartate (RGD). This RGD motif is recognized by integrins, a family of adhesion receptors present on vascular smooth muscle cells. In the present study, we examined the ability of different RGD-containing peptides to affect the contraction of rat aortic rings in response to different agonists. We found that the peptide RGDS inhibited angiotensin-induced contraction in a dose dependent manner. In contrast, the peptides RGDW and RGES had no effect on angiotensin-induced contractility. We show that function-blocking antibodies to the integrins alphavbeta3 and alpha5beta1 also inhibit angiotensin-induced contraction. These effects were observed in the absence of an intact endothelium. In contrast, neither an antibody directed against the beta1 subunit nor the peptide RGDS had an effect on phenylephrine or 5-hydroxytryptamine-induced contraction. These data suggest that interactions of vascular smooth muscle with components of the surrounding extracellular matrix may influence the response of smooth muscle to agonists.
Asunto(s)
Angiotensina II/antagonistas & inhibidores , Aorta/efectos de los fármacos , Integrinas/metabolismo , Contracción Muscular/efectos de los fármacos , Oligopéptidos/farmacología , Angiotensina II/farmacología , Animales , Anticuerpos/farmacología , Matriz Extracelular/química , Músculo Liso Vascular/efectos de los fármacos , Fenilefrina/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de Fibronectina/metabolismo , Receptores de Vitronectina/metabolismo , Serotonina/farmacologíaRESUMEN
Integrins are a major class of cell surface receptors involved in cell-cell and cell-matrix adhesion and communication. Ha2/11 is a function-blocking anti-rat beta 1 integrin hamster IgM that should be a useful reagent for understanding beta 1 integrin function. We demonstrate that Ha2/11 cross reacts with human, Xenopus, and Drosophila beta 1 integrins, and use phage display to map the epitope for Ha2/11 to residues within the sequence LRSGEPQTF which lies 18 amino acids proximal to the putative I domain in beta 1 integrins. Monoclonal antibody mapping experiments, mutational analyses, and direct binding assays have implicated integrin I domains in both cation and ligand binding. Our data therefore suggest that Ha2/11 blocks beta 1 integrin function by interfering with I domain-mediated ligand binding.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Colifagos/genética , Epítopos/química , Inmunoglobulina M/farmacología , Integrina beta1/inmunología , Fragmentos de Péptidos/química , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Reacciones Antígeno-Anticuerpo , Línea Celular , Pollos/inmunología , Secuencia de Consenso , Cricetinae , Reacciones Cruzadas , Drosophila melanogaster/citología , Drosophila melanogaster/inmunología , Ensayo de Inmunoadsorción Enzimática , Epítopos/metabolismo , Citometría de Flujo , Humanos , Inmunoglobulina M/inmunología , Inmunoglobulina M/metabolismo , Técnicas de Inmunoadsorción , Riñón/citología , Ligandos , Ratones , Músculo Liso Vascular/citología , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/metabolismo , Unión Proteica , Ratas , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Xenopus laevis/inmunologíaRESUMEN
Many cell-surface and extracellular matrix proteins contain multiple modular domains known as fibronectin type III (FNIII) repeats. Cells adhere to the extracellular matrix proteins fibronectin and tenascin in part by the interaction of certain integrins with the Arg-Gly-Asp (RGD) sequence, displayed on specific FNIII repeats. We have found that, after experimental activation of beta1 integrins, a number of cell types adhere and spread on FNIII repeats lacking RGD, derived from extracellular matrix proteins and cytokine receptors. Interaction between individual FNIII domains and beta1 integrins mediates focal adhesion kinase phosphorylation and subsequent stress fiber and focal contact formation. These data suggest that many FNIII-containing proteins may bind and signal through activated beta1 integrins, dramatically expanding the potential for integrin-dependent intercellular and cell-matrix communication.
Asunto(s)
Adhesión Celular/fisiología , Fibronectinas/química , Integrina beta1/fisiología , Oligopéptidos/fisiología , Transducción de Señal/fisiología , Animales , Sitios de Unión , Calcio/metabolismo , Moléculas de Adhesión Celular/metabolismo , Comunicación Celular/fisiología , Proteínas de la Matriz Extracelular/química , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Manganeso/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Ratas , Receptores de Citocinas/químicaRESUMEN
Alpha 1 beta 1 heterodimer is a member of the integrin receptor superfamily that has been described to be involved in cell-matrix binding through its interaction with collagens, fibronectin and laminin. The alpha 1 integrin belongs to a subset of I-domain containing integrins that includes alpha M, alpha L, alpha X and alpha 2. In this study we describe an anti-alpha 1 mAb (FB12) that recognizes an epitope located in the human alpha 1 I-domain, since the mAb can bind to human, but not to rat, recombinant I-domain GST fusion protein. FB12 mAb efficiently and specifically inhibits the binding of activated human lymphocytes to laminin, collagen and fibronectin. These data support the notion that the alpha 1 I-domain itself has an important role in receptor-ligand binding. In particular, we show that alpha 1 integrin-dependent lymphocyte adhesion to fibronectin is I-domain mediated, at variance with the RGD-dependent adhesion which seems to be mediated by the beta 1 rather than the alpha 1 integrin chain. Lastly, the overexpression of the alpha 1-integrin by stromal cells and blood vessels of solid tumors may suggest a role for this integrin in tumor biology.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos CD/inmunología , Integrinas/inmunología , Animales , Especificidad de Anticuerpos , Antígenos CD/genética , Colágeno/metabolismo , Fibronectinas/metabolismo , Células HeLa/inmunología , Humanos , Inmunohistoquímica , Integrina alfa1 , Integrinas/genética , Células Asesinas Naturales/inmunología , Laminina/metabolismo , Ratones , Ratones Endogámicos BALB C , Neoplasias/inmunología , Reacción en Cadena de la Polimerasa , RatasRESUMEN
Remodeling of the extracellular matrix by activated mesenchymal cells (myofibroblasts) is a critical aspect of wound repair in all adult organs. Collagen-dependent gel contraction, a process requiring integrin function, is an established in vitro assay thought to mimic in vivo matrix remodeling. Numerous data have implicated the alpha2beta1 integrin in various cell types as the primary collagen receptor responsible for collagen gel contraction. However, evidence from the literature suggests that the major collagen binding integrin expressed on mesenchymally derived cells in situ is the alpha1beta1 integrin, not the alpha2beta1 integrin. In this report, we use a rat vascular injury model to illustrate that the alpha1beta1 integrin is the major collagen receptor expressed on vascular smooth muscle cells after injury. Using two smooth muscle cell lines, expressing either the alpha1beta1 integrin alone or both the alpha1beta1 and alpha2beta1 integrins, along with Chinese hamster ovary cells transfected with the alpha1 integrin, we demonstrate that alpha1beta1 supports not only collagen-dependent adhesion and migration, but also gel contraction. These data suggest that in vivo the alpha1beta1 integrin is a critical collagen receptor on mesenchymally derived cells potentially involved in matrix remodeling after injury.
Asunto(s)
Arteria Carótida Común/fisiología , Colágeno/metabolismo , Integrinas/biosíntesis , Músculo Liso Vascular/fisiología , Túnica Íntima/fisiología , Cicatrización de Heridas , Animales , Antígenos CD/biosíntesis , Aorta/lesiones , Aorta/fisiología , Células CHO , Traumatismos de las Arterias Carótidas , Adhesión Celular , Línea Celular , Movimiento Celular , Cricetinae , Matriz Extracelular/fisiología , Humanos , Inmunohistoquímica , Hibridación in Situ , Integrina alfa1 , Integrina alfa1beta1 , Masculino , Arteria Pulmonar/fisiología , Ratas , Ratas Sprague-Dawley , TransfecciónRESUMEN
We have expressed Drosophila position-specific (PS) integrins on the surfaces of Schneider S2 cells and tested for adhesion and spreading on various matrix molecules. We report that PS1 integrin is a laminin receptor and that PS1 and PS2 integrins promote cell spreading on two different Drosophila extracellular matrix molecules, laminin and tiggrin, respectively. The differing ligand specificities of these two integrins, combined with data on the in vivo expression patterns of the integrins and their ligands, lead to a model for the structure of integrin-dependent attachments in the pupal wings and embryonic muscles of Drosophila.
Asunto(s)
Adhesión Celular , Drosophila melanogaster/citología , Integrinas/metabolismo , Laminina/metabolismo , Receptores de Laminina/metabolismo , Animales , Línea Celular , Ligandos , Músculos/citología , Proteínas Recombinantes , Alas de Animales/citologíaRESUMEN
The major advance during the past year was the identification of ligands for two of the previously known position-specific integrins in Drosophila. At the same time, two new Drosophila integrin subunits (one alpha and one beta) were discovered, and significant progress was made on developmental genetic analyses of integrin functions, shedding light on the roles of integrins in Drosophila development.