RESUMEN
FGFs and their high-affinity receptors (FGFRs) play key roles in development, tissue repair, and disease. Because FGFRs bind overlapping sets of ligands, their individual functions cannot be determined using ligand stimulation. Here, we generated a light-activated FGFR2 variant (OptoR2) to selectively activate signaling by the major FGFR in keratinocytes. Illumination of OptoR2-expressing HEK 293T cells activated FGFR signaling with remarkable temporal precision and promoted cell migration and proliferation. In murine and human keratinocytes, OptoR2 activation rapidly induced the classical FGFR signaling pathways and expression of FGF target genes. Surprisingly, multi-level counter-regulation occurred in keratinocytes in vitro and in transgenic mice in vivo, including OptoR2 down-regulation and loss of responsiveness to light activation. These results demonstrate unexpected cell type-specific limitations of optogenetic FGFRs in long-term in vitro and in vivo settings and highlight the complex consequences of transferring optogenetic cell signaling tools into their relevant cellular contexts.
Asunto(s)
Queratinocitos/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Animales , Femenino , Factores de Crecimiento de Fibroblastos/metabolismo , Factores de Crecimiento de Fibroblastos/fisiología , Células HEK293 , Humanos , Queratinocitos/fisiología , Ligandos , Luz , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/genética , Receptores de Factores de Crecimiento de Fibroblastos/fisiología , Transducción de SeñalRESUMEN
Fibroblast growth factors (FGFs) play key roles in the pathogenesis of different human diseases, but the cross-talk between FGFs and other cytokines remains largely unexplored. We identified an unexpected antagonistic effect of FGFs on the interferon (IFN) signaling pathway. Genetic or pharmacological inhibition of FGF receptor signaling in keratinocytes promoted the expression of interferon-stimulated genes (ISG) and proteins in vitro and in vivo. Conversely, FGF7 or FGF10 treatment of keratinocytes suppressed ISG expression under homeostatic conditions and in response to IFN or poly(I:C) treatment. FGF-mediated ISG suppression was independent of IFN receptors, occurred at the transcriptional level, and required FGF receptor kinase and proteasomal activity. It is not restricted to keratinocytes and functionally relevant, since FGFs promoted the replication of herpes simplex virus I (HSV-1), lymphocytic choriomeningitis virus, and Zika virus. Most importantly, inhibition of FGFR signaling blocked HSV-1 replication in cultured human keratinocytes and in mice. These results suggest the use of FGFR kinase inhibitors for the treatment of viral infections.
Asunto(s)
Infección por el Virus Zika , Virus Zika , Animales , Factores de Crecimiento de Fibroblastos , Humanos , Interferones , Ratones , Receptores de Factores de Crecimiento de Fibroblastos , Transducción de Señal , Replicación ViralRESUMEN
Tissue injury initiates a complex repair process, which in some organisms can lead to the complete regeneration of a tissue. In mammals, however, the repair of most organs is imperfect and results in scar formation. Both regeneration and repair are orchestrated by a highly coordinated interplay of different growth factors and cytokines. Among the key players are the fibroblast growth factors (FGFs), which control the migration, proliferation, differentiation and survival of different cell types. In addition, FGFs influence the expression of other factors involved in the regenerative response. Here, we summarize current knowledge on the roles of endogenous FGFs in regeneration and repair in different organisms and in different tissues and organs. Gaining a better understanding of these FGF activities is important for appropriate modulation of FGF signaling after injury to prevent impaired healing and to promote organ regeneration in humans.
Asunto(s)
Factores de Crecimiento de Fibroblastos/metabolismo , Regeneración/fisiología , Cicatrización de Heridas , Animales , Humanos , Modelos Biológicos , Especificidad de Órganos , Transducción de SeñalRESUMEN
Primary myelofibrosis (PMF) is a Philadelphia-negative (Ph-) myeloproliferative disorder, showing abnormal CD34+ progenitor cell trafficking, splenomegaly, marrow fibrosis leading to extensive extramedullary haematopoiesis, and abnormal neoangiogenesis in either the bone marrow or the spleen. Monocytes expressing the angiopoietin-2 receptor (Tie2) have been shown to support abnormal angiogenic processes in solid tumors through a paracrine action that takes place in proximity to the vessels. In this study we investigated the frequency of Tie2 expressing monocytes in the spleen tissue samples of patients with PMF, and healthy subjects (CTRLs), and evaluated their possible role in favouring spleen angiogenesis. We show by confocal microscopy that in the spleen tissue of patients with PMF, but not of CTRLs, the most of the CD14+ cells are Tie2+ and are close to vessels; by flow cytometry, we found that Tie2 expressing monocytes were Tie2+CD14lowCD16brightCDL62-CCR2- (TEMs) and their frequency was higher (p = 0.008) in spleen tissue-derived mononuclear cells (MNCs) of patients with PMF than in spleen tissue-derived MNCs from CTRLs undergoing splenectomy for abdominal trauma. By in vitro angiogenesis assay we evidenced that conditioned medium of immunomagnetically selected spleen tissue derived CD14+ cells of patients with PMF induced a denser tube like net than that of CTRLs; in addition, CD14+Tie2+ cells sorted from spleen tissue derived single cell suspension of patients with PMF show a higher expression of genes involved in angiogenesis than that found in CTRLs. Our results document the enrichment of Tie2+ monocytes expressing angiogenic genes in the spleen of patients with PMF, suggesting a role for these cells in starting/maintaining the pathological angiogenesis in this organ.
Asunto(s)
Monocitos/metabolismo , Neovascularización Patológica/metabolismo , Mielofibrosis Primaria/metabolismo , Receptor TIE-2/metabolismo , Bazo/metabolismo , Anciano , Estudios de Casos y Controles , Diferenciación Celular , Femenino , Humanos , Masculino , Persona de Mediana Edad , Monocitos/patología , Mielofibrosis Primaria/patología , Mielofibrosis Primaria/cirugía , Bazo/patología , EsplenectomíaRESUMEN
Cerebral cavernous malformations (CCMs) are vascular malformations located within the central nervous system often resulting in cerebral hemorrhage. Pharmacological treatment is needed, since current therapy is limited to neurosurgery. Familial CCM is caused by loss-of-function mutations in any of Ccm1, Ccm2, and Ccm3 genes. CCM cavernomas are lined by endothelial cells (ECs) undergoing endothelial-to-mesenchymal transition (EndMT). This switch in phenotype is due to the activation of the transforming growth factor beta/bone morphogenetic protein (TGFß/BMP) signaling. However, the mechanism linking Ccm gene inactivation and TGFß/BMP-dependent EndMT remains undefined. Here, we report that Ccm1 ablation leads to the activation of a MEKK3-MEK5-ERK5-MEF2 signaling axis that induces a strong increase in Kruppel-like factor 4 (KLF4) in ECs in vivo. KLF4 transcriptional activity is responsible for the EndMT occurring in CCM1-null ECs. KLF4 promotes TGFß/BMP signaling through the production of BMP6. Importantly, in endothelial-specific Ccm1 and Klf4 double knockout mice, we observe a strong reduction in the development of CCM and mouse mortality. Our data unveil KLF4 as a therapeutic target for CCM.
Asunto(s)
Hemangioma Cavernoso del Sistema Nervioso Central/genética , Hemangioma Cavernoso del Sistema Nervioso Central/patología , Factores de Transcripción de Tipo Kruppel/metabolismo , Animales , Proteína Morfogenética Ósea 6/antagonistas & inhibidores , Proteína Morfogenética Ósea 6/genética , Proteína Morfogenética Ósea 6/metabolismo , Proliferación Celular , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Células Endoteliales/citología , Células Endoteliales/metabolismo , Células HEK293 , Hemangioma Cavernoso del Sistema Nervioso Central/metabolismo , Humanos , Proteína KRIT1 , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/antagonistas & inhibidores , Factores de Transcripción de Tipo Kruppel/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/antagonistas & inhibidores , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteína Quinasa 7 Activada por Mitógenos/metabolismo , Mutación , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Interferencia de ARN , Transducción de Señal , Proteína Smad1/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Chronic skin inflammation resulting from a defective epidermal barrier is a hallmark of atopic dermatitis (AD). We previously demonstrated that mice lacking FGF receptors 1 and 2 in keratinocytes (K5-R1/R2 mice) develop an AD-like chronic dermatitis as a result of an impaired epidermal barrier. Here, we show that γδ T cells, which rapidly respond to various insults, accumulate in the epidermis of K5-R1/R2 mice before the development of histological abnormalities. Their number and activation further increase as the phenotype progresses, most likely as a consequence of increased expression of Il-2 and Il-7 and the stress-induced proteins Rae-1, H60c, Mult1, PlexinB2, and Skint1. To determine the role of γδ T cells in the skin phenotype, we generated quadruple mutant K5-R1/-R2 mice lacking γδ T cells. Surprisingly, loss of γδ T cells did not or only marginally affect keratinocyte proliferation, epidermal thickness, epidermal barrier function, and accumulation and activation of different immune cells in the skin of K5-R1/R2 mice, possibly due to partial compensation by αß T cells. These results demonstrate that γδ T cells do not contribute to the development or maintenance of chronic inflammation in response to a defect in the epidermal barrier.
Asunto(s)
Dermatitis/inmunología , Queratinocitos/inmunología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/deficiencia , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/deficiencia , Receptores de Antígenos de Linfocitos T gamma-delta/deficiencia , Subgrupos de Linfocitos T/inmunología , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Proliferación Celular , Enfermedad Crónica , Dermatitis/genética , Dermatitis/patología , Modelos Animales de Enfermedad , Epidermis/inmunología , Epidermis/patología , Regulación de la Expresión Génica , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Inmunoglobulinas/genética , Inmunoglobulinas/inmunología , Interleucina-2/genética , Interleucina-2/inmunología , Interleucina-7/genética , Interleucina-7/inmunología , Queratinocitos/patología , Activación de Linfocitos , Depleción Linfocítica , Proteínas de la Membrana , Ratones , Ratones Transgénicos , Antígenos de Histocompatibilidad Menor/genética , Antígenos de Histocompatibilidad Menor/inmunología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/inmunología , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas Asociadas a Matriz Nuclear/inmunología , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/inmunología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/inmunología , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Transducción de Señal , Subgrupos de Linfocitos T/patologíaRESUMEN
Cerebral cavernous malformation (CCM) is a disease of the central nervous system causing hemorrhage-prone multiple lumen vascular malformations and very severe neurological consequences. At present, the only recommended treatment of CCM is surgical. Because surgery is often not applicable, pharmacological treatment would be highly desirable. We describe here a murine model of the disease that develops after endothelial-cell-selective ablation of the CCM3 gene. We report an early, cell-autonomous, Wnt-receptor-independent stimulation of ß-catenin transcription activity in CCM3-deficient endothelial cells both in vitro and in vivo and a triggering of a ß-catenin-driven transcription program that leads to endothelial-to-mesenchymal transition. TGF-ß/BMP signaling is then required for the progression of the disease. We also found that the anti-inflammatory drugs sulindac sulfide and sulindac sulfone, which attenuate ß-catenin transcription activity, reduce vascular malformations in endothelial CCM3-deficient mice. This study opens previously unidentified perspectives for an effective pharmacological therapy of intracranial vascular cavernomas.
Asunto(s)
Neoplasias del Sistema Nervioso Central/tratamiento farmacológico , Hemangioma Cavernoso del Sistema Nervioso Central/tratamiento farmacológico , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Sulindac/análogos & derivados , Animales , Antiinflamatorios no Esteroideos/farmacología , Proteínas Reguladoras de la Apoptosis , Neoplasias del Sistema Nervioso Central/genética , Neoplasias del Sistema Nervioso Central/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Hemangioma Cavernoso del Sistema Nervioso Central/genética , Hemangioma Cavernoso del Sistema Nervioso Central/metabolismo , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones Noqueados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Sulindac/farmacología , Factor de Crecimiento Transformador beta/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
While tumor blood vessels share many characteristics with normal vasculature, they also exhibit morphological and functional aberrancies. For example, the neural adhesion molecule L1, which mediates neurite outgrowth, fasciculation, and pathfinding, is expressed on tumor vasculature. Here, using an orthotopic mouse model of pancreatic carcinoma, we evaluated L1 functionality in cancer vessels. Tumor-bearing mice specifically lacking L1 in endothelial cells or treated with anti-L1 antibodies exhibited decreased angiogenesis and improved vascular stabilization, leading to reduced tumor growth and metastasis. In line with these dramatic effects of L1 on tumor vasculature, the ectopic expression of L1 in cultured endothelial cells (ECs) promoted phenotypical and functional alterations, including proliferation, migration, tubulogenesis, enhanced vascular permeability, and endothelial-to-mesenchymal transition. L1 induced global changes in the EC transcriptome, altering several regulatory networks that underlie endothelial pathophysiology, including JAK/STAT-mediated pathways. In particular, L1 induced IL-6-mediated STAT3 phosphorylation, and inhibition of the IL-6/JAK/STAT signaling axis prevented L1-induced EC proliferation and migration. Evaluation of patient samples revealed that, compared with that in noncancerous tissue, L1 expression is specifically enhanced in blood vessels of human pancreatic carcinomas and in vessels of other tumor types. Together, these data indicate that endothelial L1 orchestrates multiple cancer vessel functions and represents a potential target for tumor vascular-specific therapies.
Asunto(s)
Células Endoteliales/citología , Endotelio Vascular/patología , Neoplasias/irrigación sanguínea , Neovascularización Patológica , Molécula L1 de Adhesión de Célula Nerviosa/fisiología , Animales , Vasos Sanguíneos , Permeabilidad Capilar , Carcinoma/metabolismo , Movimiento Celular , Proliferación Celular , Femenino , Hemangioma/metabolismo , Humanos , Interleucina-6/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Ratones Transgénicos , Metástasis de la Neoplasia , Molécula L1 de Adhesión de Célula Nerviosa/genética , Neoplasias Pancreáticas/metabolismo , Permeabilidad , Fenotipo , Interferencia de ARN , Receptor TIE-2/genética , Factor de Transcripción STAT3/metabolismoRESUMEN
Reproducing the characteristics and the functional responses of the blood-brain barrier (BBB) in vitro represents an important task for the research community, and would be a critical biotechnological breakthrough. Pharmaceutical and biotechnology industries provide strong demand for inexpensive and easy-to-handle in vitro BBB models to screen novel drug candidates. Recently, it was shown that canonical Wnt signaling is responsible for the induction of the BBB properties in the neonatal brain microvasculature in vivo. In the present study, following on from earlier observations, we have developed a novel model of the BBB in vitro that may be suitable for large scale screening assays. This model is based on immortalized endothelial cell lines derived from murine and human brain, with no need for co-culture with astrocytes. To maintain the BBB endothelial cell properties, the cell lines are cultured in the presence of Wnt3a or drugs that stabilize ß-catenin, or they are infected with a transcriptionally active form of ß-catenin. Upon these treatments, the cell lines maintain expression of BBB-specific markers, which results in elevated transendothelial electrical resistance and reduced cell permeability. Importantly, these properties are retained for several passages in culture, and they can be reproduced and maintained in different laboratories over time. We conclude that the brain-derived endothelial cell lines that we have investigated gain their specialized characteristics upon activation of the canonical Wnt pathway. This model may be thus suitable to test the BBB permeability to chemicals or large molecular weight proteins, transmigration of inflammatory cells, treatments with cytokines, and genetic manipulation.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Encéfalo/citología , Encéfalo/metabolismo , Células Endoteliales/metabolismo , Proteínas Wnt/metabolismo , Animales , Ratones , Microscopía Fluorescente , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , beta Catenina/metabolismoRESUMEN
Cerebral cavernous malformation (CCM) is a vascular dysplasia, mainly localized within the brain and affecting up to 0.5% of the human population. CCM lesions are formed by enlarged and irregular blood vessels that often result in cerebral haemorrhages. CCM is caused by loss-of-function mutations in one of three genes, namely CCM1 (also known as KRIT1), CCM2 (OSM) and CCM3 (PDCD10), and occurs in both sporadic and familial forms. Recent studies have investigated the cause of vascular dysplasia and fragility in CCM, but the in vivo functions of this ternary complex remain unclear. Postnatal deletion of any of the three Ccm genes in mouse endothelium results in a severe phenotype, characterized by multiple brain vascular malformations that are markedly similar to human CCM lesions. Endothelial-to-mesenchymal transition (EndMT) has been described in different pathologies, and it is defined as the acquisition of mesenchymal- and stem-cell-like characteristics by the endothelium. Here we show that endothelial-specific disruption of the Ccm1 gene in mice induces EndMT, which contributes to the development of vascular malformations. EndMT in CCM1-ablated endothelial cells is mediated by the upregulation of endogenous BMP6 that, in turn, activates the transforming growth factor-ß (TGF-ß) and bone morphogenetic protein (BMP) signalling pathway. Inhibitors of the TGF-ß and BMP pathway prevent EndMT both in vitro and in vivo and reduce the number and size of vascular lesions in CCM1-deficient mice. Thus, increased TGF-ß and BMP signalling, and the consequent EndMT of CCM1-null endothelial cells, are crucial events in the onset and progression of CCM disease. These studies offer novel therapeutic opportunities for this severe, and so far incurable, pathology.
Asunto(s)
Progresión de la Enfermedad , Transición Epitelial-Mesenquimal , Hemangioma Cavernoso del Sistema Nervioso Central/patología , Animales , Proteína Morfogenética Ósea 6/antagonistas & inhibidores , Proteína Morfogenética Ósea 6/metabolismo , Proteína Morfogenética Ósea 6/farmacología , Modelos Animales de Enfermedad , Transición Epitelial-Mesenquimal/efectos de los fármacos , Transición Epitelial-Mesenquimal/genética , Hemangioma Cavernoso del Sistema Nervioso Central/genética , Humanos , Proteína KRIT1 , Ratones , Proteínas Asociadas a Microtúbulos/deficiencia , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/metabolismo , Regulación hacia ArribaRESUMEN
Endothelial adherens junctions maintain vascular integrity. Arteries and veins differ in their permeability but whether organization and strength of their adherens junctions vary has not been demonstrated in vivo. Here we report that vascular endothelial cadherin, an endothelial specific adhesion protein located at adherens junctions, is phosphorylated in Y658 and Y685 in vivo in veins but not in arteries under resting conditions. This difference is due to shear stress-induced junctional Src activation in veins. Phosphorylated vascular endothelial-cadherin is internalized and ubiquitinated in response to permeability-increasing agents such as bradykinin and histamine. Inhibition of Src blocks vascular endothelial cadherin phosphorylation and bradykinin-induced permeability. Point mutation of Y658F and Y685F prevents vascular endothelial cadherin internalization, ubiquitination and an increase in permeability by bradykinin in vitro. Thus, phosphorylation of vascular endothelial cadherin contributes to a dynamic state of adherens junctions, but is not sufficient to increase vascular permeability in the absence of inflammatory agents.
Asunto(s)
Antígenos CD/metabolismo , Cadherinas/metabolismo , Permeabilidad Capilar , Hemodinámica , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos/efectos de los fármacos , Antígenos CD/inmunología , Arterias/efectos de los fármacos , Arterias/fisiología , Bradiquinina/farmacología , Cadherinas/inmunología , Permeabilidad Capilar/efectos de los fármacos , Endocitosis/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Hemodinámica/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Fosforilación/efectos de los fármacos , Estrés Mecánico , Ubiquitinación/efectos de los fármacos , Venas/efectos de los fármacos , Venas/fisiología , Familia-src Quinasas/metabolismoRESUMEN
Dendritic cells (DCs) flexibly adapt to different microenvironments by using diverse migration strategies that are ultimately dependent on the dynamics and structural organization of the actin cytoskeleton. Here, we have shown that DCs require the actin capping activity of the signaling adaptor Eps8 to polarize and to form elongated migratory protrusions. DCs from Eps8-deficient mice are impaired in directional and chemotactic migration in 3D in vitro and are delayed in reaching the draining lymph node (DLN) in vivo after inflammatory challenge. Hence, Eps8-deficient mice are unable to mount a contact hypersensitivity response. We have also shown that the DC migratory defect is cell autonomous and that Eps8 is required for the proper architectural organization of the actin meshwork and dynamics of cell protrusions. Yet, Eps8 is not necessary for antigen uptake, processing, and presentation. Thus, we have identified Eps8 as a unique actin capping protein specifically required for DC migration.
Asunto(s)
Proteínas de Capping de la Actina/inmunología , Proteínas Adaptadoras Transductoras de Señales/inmunología , Proteínas del Citoesqueleto/inmunología , Células Dendríticas/inmunología , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Presentación de Antígeno , Movimiento Celular/inmunología , Proliferación Celular , Células Cultivadas , Proteínas del Citoesqueleto/deficiencia , Proteínas del Citoesqueleto/genética , Dermatitis por Contacto/inmunología , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Linfocitos T/inmunologíaRESUMEN
Cerebral cavernous malformations (CCM) are vascular malformations of the central nervous system (CNS) that lead to cerebral hemorrhages. Familial CCM occurs as an autosomal dominant condition caused by loss-of-function mutations in one of the three CCM genes. Constitutive or tissue-specific ablation of any of the Ccm genes in mice previously established the crucial role of Ccm gene expression in endothelial cells for proper angiogenesis. However, embryonic lethality precluded the development of relevant CCM mouse models. Here, we show that endothelial-specific Ccm2 deletion at postnatal day 1 (P1) in mice results in vascular lesions mimicking human CCM lesions. Consistent with CCM1/3 involvement in the same human disease, deletion of Ccm1/3 at P1 in mice results in similar CCM lesions. The lesions are located in the cerebellum and the retina, two organs undergoing intense postnatal angiogenesis. Despite a pan-endothelial Ccm2 deletion, CCM lesions are restricted to the venous bed. Notably, the consequences of Ccm2 loss depend on the developmental timing of Ccm2 ablation. This work provides a highly penetrant and relevant CCM mouse model.
Asunto(s)
Proteínas Portadoras/genética , Malformaciones Vasculares del Sistema Nervioso Central/metabolismo , Hemorragia Cerebral/metabolismo , Endotelio Vascular/metabolismo , Proteínas de Microfilamentos/genética , Animales , Proteínas Portadoras/metabolismo , Malformaciones Vasculares del Sistema Nervioso Central/genética , Malformaciones Vasculares del Sistema Nervioso Central/patología , Malformaciones Vasculares del Sistema Nervioso Central/fisiopatología , Cerebelo/irrigación sanguínea , Cerebelo/metabolismo , Cerebelo/patología , Hemorragia Cerebral/genética , Hemorragia Cerebral/patología , Hemorragia Cerebral/fisiopatología , Modelos Animales de Enfermedad , Endotelio Vascular/patología , Eliminación de Gen , Humanos , Ratones , Ratones Transgénicos , Proteínas de Microfilamentos/metabolismo , Retina/metabolismo , Retina/patología , Retina/fisiopatologíaRESUMEN
Epithelial ovarian carcinoma (EOC) is an aggressive neoplasm, which mainly disseminates to organs of the peritoneal cavity, an event mediated by molecular mechanisms that remain elusive. Here, we investigated the expression and functional role of neural cell adhesion molecule (NCAM), a cell surface glycoprotein involved in brain development and plasticity, in EOC. NCAM is absent from normal ovarian epithelium but becomes highly expressed in a subset of human EOC, in which NCAM expression is associated with high tumour grade, suggesting a causal role in cancer aggressiveness. We demonstrate that NCAM stimulates EOC cell migration and invasion in vitro and promotes metastatic dissemination in mice. This pro-malignant function of NCAM is mediated by its interaction with fibroblast growth factor receptor (FGFR). Indeed, not only FGFR signalling is required for NCAM-induced EOC cell motility, but targeting the NCAM/FGFR interplay with a monoclonal antibody abolishes the metastatic dissemination of EOC in mice. Our results point to NCAM-mediated stimulation of FGFR as a novel mechanism underlying EOC malignancy and indicate that this interplay may represent a valuable therapeutic target.
Asunto(s)
Carcinoma/patología , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neoplasias Ováricas/patología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal , Animales , Carcinoma/secundario , Movimiento Celular , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunohistoquímica , Ratones , Invasividad Neoplásica , Metástasis de la Neoplasia/patología , Neoplasias Ováricas/secundarioRESUMEN
Little is known about the molecular mechanisms that regulate the organization of vascular lumen. In this paper we show that lumen formation correlates with endothelial polarization. Adherens junctions (AJs) and VE-cadherin (VEC, encoded by CDH5) are required for endothelial apicobasal polarity in vitro and during embryonic development. Silencing of CDH5 gene expression leads to abrogation of endothelial polarity accompanied by strong alterations in lumenal structure. VEC co-distributes with members of the Par polarity complex (Par3 and PKCzeta) and is needed for activation of PKCzeta. CCM1 is encoded by the CCM1 gene, which is mutated in 60% of patients affected by cerebral cavernous malformation (CCM). The protein interacts with VEC and directs AJ organization and AJ association with the polarity complex, both in cell-culture models and in human CCM1 lesions. Both VEC and CCM1 control Rap1 concentration at cell-cell junctions. We propose that VEC, CCM1 and Rap1 form a signaling complex. In the absence of any of these proteins, AJs are dismantled, cell polarity is lost and vascular lumenal structure is severely altered.
Asunto(s)
Neoplasias Encefálicas/genética , Células Endoteliales/metabolismo , Hemangioma Cavernoso del Sistema Nervioso Central/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Neovascularización Fisiológica , Proteínas Proto-Oncogénicas/metabolismo , Uniones Adherentes/metabolismo , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Neoplasias Encefálicas/patología , Cadherinas/genética , Cadherinas/metabolismo , Línea Celular , Polaridad Celular/genética , Células Endoteliales/patología , Predisposición Genética a la Enfermedad , Hemangioma Cavernoso del Sistema Nervioso Central/patología , Humanos , Proteína KRIT1 , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/genética , Complejos Multiproteicos/metabolismo , Polimorfismo Genético , Unión Proteica/genética , Proteínas Proto-Oncogénicas/genética , ARN Interferente Pequeño/genética , Transducción de Señal , Proteínas de Unión al GTP rap1/genética , Proteínas de Unión al GTP rap1/metabolismoRESUMEN
Junctional adhesion molecule-A (JAM-A)-null dendritic cells (DCs) are more motile and effective than their wild-type counterpart in promoting contact hypersensitivity reaction. Here, we show that the growth and aggressiveness of pancreatic islet cell carcinoma induced by SV40 T antigen expression in beta cells (Rip1Tag2 mice) are significantly reduced in JAM-A-null mice. Because these tumor cells do not express JAM-A, we focused on changes in stroma reactivity. In the absence of JAM-A, tumors showed a small but significant reduction in angiogenesis and a marked increase in the immune reaction with enhanced infiltration of DCs (CD11c+ and MHC-II+) and CD4+ and CD8+ lymphocytes. In contrast, phagocyte number was not affected. DC capacity to produce cytokines was not significantly altered, but transmigration through JAM-A-null endothelial cells was increased as compared with JAM-A-positive endothelium. On adoptive transfer, JAM-A(-/-) DCs were recruited to tumors at slightly but significantly higher rate than JAM-A(+/+) DCs. Ablation of CD4+ and CD8+ cells with specific antibodies abrogated the inhibitory effect of JAM-A deletion on tumor growth and angiogenesis. These findings support the idea that, in the Rip1Tag2 tumor model, abrogation of JAM-A reduces cancer development by increasing antitumor immune response.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Carcinoma de Células de los Islotes Pancreáticos/inmunología , Moléculas de Adhesión Celular/deficiencia , Células Dendríticas/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Neoplasias Pancreáticas/inmunología , Receptores de Superficie Celular/deficiencia , Animales , Carcinoma de Células de los Islotes Pancreáticos/genética , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/inmunología , Femenino , Masculino , Ratones , Ratones Noqueados , Neoplasias Pancreáticas/genética , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunologíaRESUMEN
Cell adhesion molecules (CAMs) are cell surface glycoproteins that mediate the physical interactions between adjacent cells and between cells and the surrounding extracellular matrix. CAMs belong to different protein families, depending on their structural and functional properties. Furthermore, the expression of certain CAMs under physiological conditions is restricted to specific cell types. Besides playing a key homeostatic role in maintaining the architecture of quiescent tissues, CAMs have also to adapt to the microenvironmental changes that occur during certain physiological and pathological processes. This is best exemplified by cancer vascularization, where the expression and function of vascular CAMs are dynamically regulated in response to tissue alterations induced by tumor growth as well as by changes in the surrounding stroma. This enables endothelial cells (ECs) to leave the quiescent state and re-enter the angiogenic cascade. The latter is a multistep process carried out by different types of specialized ECs. This review describes the actual or supposed function of the various CAM subsets in the sequential series of events that underlie vascular changes during tumor angiogenesis. Notably, elucidating the mechanism of action of endothelial CAMs in cancer vasculature is expected to open new therapeutic avenues aimed at interfering with tumor growth and dissemination.
Asunto(s)
Moléculas de Adhesión Celular/fisiología , Adhesión Celular/fisiología , Neoplasias/irrigación sanguínea , Neovascularización Patológica/metabolismo , HumanosRESUMEN
The adhesion molecule L1, which is extensively characterized in the nervous system, is also expressed in dendritic cells (DCs), but its function there has remained elusive. To address this issue, we ablated L1 expression in DCs of conditional knockout mice. L1-deficient DCs were impaired in adhesion to and transmigration through monolayers of either lymphatic or blood vessel endothelial cells, implicating L1 in transendothelial migration of DCs. In agreement with these findings, L1 was expressed in cutaneous DCs that migrated to draining lymph nodes, and its ablation reduced DC trafficking in vivo. Within the skin, L1 was found in Langerhans cells but not in dermal DCs, and L1 deficiency impaired Langerhans cell migration. Under inflammatory conditions, L1 also became expressed in vascular endothelium and enhanced transmigration of DCs, likely through L1 homophilic interactions. Our results implicate L1 in the regulation of DC trafficking and shed light on novel mechanisms underlying transendothelial migration of DCs. These observations might offer novel therapeutic perspectives for the treatment of certain immunological disorders.