RESUMO
In human glioblastoma, the blood-brain barrier (BBB) is disturbed. According to our concept, the glio-vascular relationships and thus the control of the BBB are essentially dependent on the polarity of astroglial cells. This polarity is characterized by the uneven distribution of the water channel protein aquaporin-4 (AQP4), dystroglycan and other molecules. Recently, we were able to show that the extracellular matrix component agrin is important for the construction and localization of the so-called orthogonal arrays of particles (OAPs), which consist in AQP4. Here, combining freeze-fracture electron microscopy, immunohistochemistry and Western blotting, we describe alterations of expression and distribution of AQP4, dystroglycan, agrin and the matrix metalloproteinases (MMP) 2, 3 and 9 in human primary glioblastomas (eight primary tumours, six recurrent tumours). Increase of MMP3- and MMP2/9 immunoreactivities went along with loss of agrin and dystroglycan respectively. On the protein level, AQP4 expression was increased in glioblastoma compared to control tissue. This was not accompanied by an increase of OAPs, suggesting that AQP4 can also occur without forming OAPs. The results underline our concept of the loss of glioma cell polarity as one of the factors responsible for the disturbance of the neurovascular unit and as an explanation for the formation of edemas in the glioblastoma.
Assuntos
Agrina/metabolismo , Aquaporina 4/metabolismo , Neoplasias Encefálicas/metabolismo , Encéfalo/metabolismo , Distroglicanas/metabolismo , Glioblastoma/metabolismo , Metaloproteinases da Matriz/metabolismo , Barreira Hematoencefálica/metabolismo , Polaridade Celular , Matriz Extracelular/metabolismo , Técnica de Fratura por Congelamento , Humanos , Imuno-Histoquímica , Microscopia EletrônicaRESUMO
Leukocyte extravasation depends on various adhesion receptors at endothelial cell contacts. Here we have analyzed how mouse CD99 and CD99L2 cooperate with PECAM-1. We found that antibodies against mouse CD99 and PECAM-1 trap neutrophils between endothelial cells in in vitro transmigration assays. A sequential function, as has been suggested for human PECAM-1 and CD99, could not be demonstrated. In contrast to these in vitro results, blocking CD99 or CD99L2 or gene disruption of PECAM-1 trapped neutrophils in vivo between endothelial cells and the underlying basement membrane as revealed by electron microscopy and by 3-dimensional confocal fluorescence microscopy in the inflamed cremaster tissue. Leukocyte extravasation was inhibited in interleukin-1beta-inflamed peritoneum and in the cremaster by PECAM-1 gene disruption and was further attenuated by blocking antibodies against CD99 and CD99L2. In addition, CD99 and CD99L2 were required for leukocyte extravasation in the cremaster after stimulation with tumor necrosis factor-alpha, where the need for PECAM-1 is known to be bypassed. We conclude that CD99 and CD99L2 act independently of PECAM-1 in leukocyte extravasation and cooperate in an independent way to help neutrophils overcome the endothelial basement membrane.
Assuntos
Antígenos CD/fisiologia , Endotélio Vascular/metabolismo , Leucócitos/imunologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/fisiologia , Antígeno 12E7 , Animais , Membrana Basal/imunologia , Membrana Basal/metabolismo , Adesão Celular , Movimento Celular , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/imunologia , Imunofluorescência , Humanos , Inflamação , Leucócitos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/imunologia , Neutrófilos/metabolismo , Peritônio/imunologiaRESUMO
Mutant ataxin-3 is aberrantly folded and proteolytically cleaved in spinocerebellar ataxia type 3. The C-terminal region of the protein includes a polyglutamine stretch that is expanded in spinocerebellar ataxia type 3. Here, we report on the analysis of an ataxin-3 mutant mouse that has been obtained by gene trap integration. The ataxin-3 fusion protein encompasses 259 N-terminal amino acids including the Josephin domain and an ubiquitin-interacting motif but lacks the C-terminus with the polyglutamine stretch, the valosin-containing protein binding region and part of the ubiquitin-interacting motif 2. Homozygous ataxin-3 mutant mice were viable and showed no apparent anatomical defects at birth. However, at the age of 9 months, homozygous and heterozygous mutant mice revealed significantly altered behaviour and progressing deficits of motor coordination followed by premature death at â¼12 months. At this time, prominent extranuclear protein aggregates and neuronal cell death was found in mutant mice. This was associated with disturbances of the endoplasmic reticulum-mediated unfolded protein response, consistent with the normal role of ataxin-3 in endoplasmic reticulum homeostasis. Thus, the ataxin-3 gene trap model provides evidence for a contribution of the non-polyglutamine containing ataxin-3 N-terminus, which mimics a calpain fragment that has been observed in spinocerebellar ataxia type 3. Consistent with the disease in humans, gene trap mice develop cytoplasmic inclusion bodies and implicate impaired unfolded protein response in the pathogenesis of spinocerebellar ataxia type 3.
Assuntos
Encéfalo/ultraestrutura , Retículo Endoplasmático/patologia , Corpos de Inclusão/patologia , Doença de Machado-Joseph/etiologia , Doença de Machado-Joseph/patologia , Proteínas Nucleares/química , Ribossomos/patologia , Fatores de Transcrição/química , Análise de Variância , Animais , Ataxina-3 , Peso Corporal/genética , Encéfalo/patologia , Células Cultivadas , Modelos Animais de Doenças , Embrião de Mamíferos , Retículo Endoplasmático/genética , Retículo Endoplasmático/ultraestrutura , Fibroblastos/efeitos dos fármacos , Fibroblastos/ultraestrutura , Regulação da Expressão Gênica/genética , Marcação In Situ das Extremidades Cortadas/métodos , Corpos de Inclusão/genética , Corpos de Inclusão/ultraestrutura , Locomoção/genética , Doença de Machado-Joseph/genética , Doença de Machado-Joseph/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Imunoeletrônica/métodos , Modelos Biológicos , Atividade Motora/genética , Mutação/genética , Proteínas Nucleares/genética , Fragmentos de Peptídeos/genética , Desempenho Psicomotor/fisiologia , Ribossomos/genética , Ribossomos/ultraestrutura , Fatores de Transcrição/genética , beta-Galactosidase/metabolismoRESUMO
The dystrophin-dystroglycan complex (DDC) is a molecular array of proteins in muscle and brain cells. The central component of the DDC is dystroglycan, which comprises α- and ß-subunits. α-Dystroglycan (α-DG) binds to extracellular matrix components such as agrin, whereas ß-dystroglycan (ß-DG) is a membrane-spanning protein linking α-DG to the cytoskeleton and other intracellular components such as α-syntrophin. In astrocytes, α-syntrophin binds to the water channel protein aquaporin-4 (AQP4). Recently, it has been shown that AQP4 expression is unaltered in agrin-knockout mice, but that formation of orthogonal arrays of particles (OAPs), consisting of AQP4, is abnormal. As the brain-selective deletion of the DG gene causes a disorganization of the astroglial endfeet, we investigated whether DG deletion has an impact on AQP4. Western blotting revealed reduced AQP4 in the parenchymal but not in the superficial compartment of the astrocyte-conditioned DG-knockout mouse brain. Accordingly, immunohistochemical stainings of AQP4 revealed a selective loss of AQP4 in perivascular but not in superficial astroglial endfeet. In both superficial and perivascular endfeet of the DG-knockout brain, we observed a loss of OAPs. We conclude that in the absence of DG the majority of superficial AQP4 molecules did not form OAPs, and that expression of AQP4 in perivascular endfeet is compromised. However, the decreased number of perivascular AQP4 molecules obviously did form a few OAPs, even in the absence of DG.
Assuntos
Astrócitos/fisiologia , Membrana Celular/fisiologia , Distroglicanas/fisiologia , Animais , Aquaporina 4/metabolismo , Aquaporina 4/fisiologia , Astrócitos/metabolismo , Astrócitos/ultraestrutura , Barreira Hematoencefálica/fisiologia , Barreira Hematoencefálica/ultraestrutura , Encéfalo/metabolismo , Encéfalo/fisiologia , Encéfalo/ultraestrutura , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Distroglicanas/genética , Técnica de Fratura por Congelamento/métodos , Camundongos , Camundongos KnockoutRESUMO
The blood-brain barrier (BBB) does not exclusively refer to brain endothelial cells, which are the site of the barrier proper. In the past few years, it has become increasingly clear that BBB endothelial cells depend considerably on the brain microenvironment to a degree exceeding the environmental influence in other organs. The concept of the BBB has been continuously developed over the decades, culminating now in the recognition that endothelial cell function in the brain is not limited to simply mediating energy and oxygen transfer between blood and neural tissue. Endothelial cells are rather "Janus-headed beings" that are active partners of both luminal molecules and cells, as well as subendothelial cells such as pericytes, astrocytes, and neurons. In this overview, the authors present and discuss both the role of astroglial cells in managing the BBB and aspects of pathological alterations in the brain as far as the BBB is involved. After a brief introduction of the BBB that describes the structure and function of the brain capillary endothelial cells, the authors report on both the water channel protein aquaporin-4 (AQP4) in astrocytes and the extracellular matrix between astrocytes/pericytes and endothelial cells. The AQP4 has an important impact on the homeostasis in the brain parenchyma; however, the mechanistic cascade from the composition of the astrocyte membrane to the maintenance of BBB properties in the endothelial cells, including their tight junction formation, is still completely unknown.
Assuntos
Agrina/metabolismo , Aquaporina 4/metabolismo , Astrócitos/metabolismo , Barreira Hematoencefálica/citologia , Polaridade Celular/fisiologia , Animais , Barreira Hematoencefálica/fisiologia , Humanos , Modelos BiológicosRESUMO
We present and discuss the role of endothelial and astroglial cells in managing the blood-brain barrier (BBB) and aspects of pathological alterations in the BBB. The impact of astrocytes, pericytes, and perivascular cells on the induction and maintenance of the gliovascular unit is largely unidentified so far. An understanding of the signaling pathways that lie between these cell types and the endothelium and that possibly are mediated by components of the basal lamina is just beginning to emerge. The metabolism for the maintenance of the endothelial barrier is intimately linked to and dependent on the microenvironment of the brain parenchyma. We report the structure and function of the endothelial cells of brain capillaries by describing structures involved in the regulation of permeability, including transporter systems, caveolae, and tight junctions. There is increasing evidence that caveolae are not only vehicles for endo- and transcytosis, but also important regulators of tight-junction-based permeability. Tight junctions separate the luminal from the abluminal membrane domains of the endothelial cell ("fence function") and control the paracellular pathway ("gate function") thus representing the most significant structure of the BBB. In addition, the extracellular matrix between astrocytes/pericytes and endothelial cells contains numerous molecules with inherent signaling properties that have to be considered if we are to improve our knowledge of the complex and closely regulated BBB.
Assuntos
Astrócitos/metabolismo , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Células Endoteliais/metabolismo , Pericitos/metabolismo , Transdução de Sinais/fisiologia , Animais , Astrócitos/citologia , Barreira Hematoencefálica/citologia , Encéfalo/irrigação sanguínea , Encéfalo/citologia , Capilares/citologia , Capilares/metabolismo , Permeabilidade Capilar/fisiologia , Endocitose/fisiologia , Células Endoteliais/citologia , Matriz Extracelular/metabolismo , Humanos , Pericitos/citologia , Junções Íntimas/metabolismoRESUMO
In multiple sclerosis (MS), and its animal model experimental autoimmune encephalomyelitis (EAE), dysfunction of the blood-brain barrier (BBB) leads to edema formation within the central nervous system. The molecular mechanisms of edema formation in EAE/MS are poorly understood. We hypothesized that edema formation is due to imbalanced water transport across the BBB caused by a disturbed crosstalk between BBB endothelium and astrocytes. Here, we demonstrate at the light microscopic and ultrastructural level, the loss of polarized localization of the water channel protein aquaporin-4 (AQP4) in astrocytic endfeet surrounding microvessels during EAE. AQP4 was found to be redistributed over the entire astrocytic cell surface and lost its arrangement in orthogonal arrays of intramembranous particles as seen in the freeze-fracture replica. In addition, immunostaining for the astrocytic extracellular matrix receptor beta-dystroglycan disappeared from astroglial membranes in the vicinity of inflammatory cuffs, whereas immunostaining for the dystroglycan ligands agrin and laminin in the perivascular basement membrane remained unchanged. Our data suggest that during EAE, loss of beta-dystroglycan-mediated astrocyte foot process anchoring to the basement membrane leads to loss of polarized AQP4 localization in astrocytic endfeet, and thus to edema formation in EAE.
Assuntos
Astrócitos/patologia , Barreira Hematoencefálica/patologia , Encefalomielite Autoimune Experimental/patologia , Agrina/metabolismo , Animais , Aquaporina 4/metabolismo , Astrócitos/metabolismo , Barreira Hematoencefálica/metabolismo , Edema Encefálico/etiologia , Edema Encefálico/metabolismo , Polaridade Celular , Distroglicanas/metabolismo , Encefalomielite Autoimune Experimental/complicações , Encefalomielite Autoimune Experimental/metabolismo , Feminino , Imuno-Histoquímica , Camundongos , Microscopia Confocal , Microscopia Eletrônica de TransmissãoRESUMO
The aim of this article is to describe alterations of the blood-brain barrier (BBB) in gliomas. The main clinical problem of human gliomas is the edematous swelling and the dramatic increase of intracerebral pressure, also compromising healthy areas of the brain. According to our concept, one of the main reasons on the cellular level for these clinical problems is the loss or reduction of astroglial polarity. Astroglial polarity means the specific accumulation of potassium and water channels in the superficial and perivascular astroglial endfeet membranes. The most important water channel in the CNS is the astroglial water channel protein aquaporin-4 (AQP4) which is arranged in a morphologically spectacular way, the so-called orthogonal arrays of particles (OAPs) to be observed in freeze-fracture replicas. In brain tumors, but also under conditions of trauma or inflammation, these OAPs are redistributed to membrane domains apart from endfeet areas. Probably, this dislocation might be due to the degradation of the proteoglycan agrin by the matrix metalloproteinase 3 (MMP3). Agrin binds to the dystrophin-dystroglycan-complex (DDC), which in turn is connected to AQP4. As a consequence, agrin loss may lead to a redistribution of AQP4 and a compromised directionality of water transport out of the cell, finally to cytotoxic edema. This in turn is hypothesized to lead to a breakdown of the BBB characterized by disturbed tight junctions, and thus to the development of vasogenic edema. However, the mechanism how the loss of polarity is related to the disturbance of microvascular tight junctions is completely unknown so far.
Assuntos
Aquaporina 4/metabolismo , Barreira Hematoencefálica/metabolismo , Edema Encefálico/metabolismo , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Desequilíbrio Hidroeletrolítico/metabolismo , Agrina/química , Agrina/metabolismo , Aquaporina 4/química , Astrócitos/metabolismo , Astrócitos/patologia , Barreira Hematoencefálica/patologia , Edema Encefálico/complicações , Edema Encefálico/patologia , Neoplasias Encefálicas/complicações , Neoplasias Encefálicas/patologia , Polaridade Celular , Distroglicanas/química , Distroglicanas/metabolismo , Distrofina/química , Distrofina/metabolismo , Glioblastoma/complicações , Glioblastoma/patologia , Humanos , Pressão Intracraniana , Metaloproteinase 3 da Matriz/química , Metaloproteinase 3 da Matriz/metabolismo , Conformação Proteica , Desequilíbrio Hidroeletrolítico/complicações , Desequilíbrio Hidroeletrolítico/patologiaRESUMO
Aquaporin-4 (AQP4), the main water channel of the brain, is highly expressed in animal glioma and human glioblastoma in situ. In contrast, most cultivated glioma cell lines don't express AQP4, and primary cell cultures of human glioblastoma lose it during the first passages. Accordingly, in C6 cells and RG2 cells, two glioma cell lines of the rat, and in SMA mouse glioma cell lines, we found no AQP4 expression. We confirmed an AQP4 loss in primary human glioblastoma cell cultures after a few passages. RG-2 glioma cells if grafted into the brain developed AQP4 expression. This led us consider the possibility of AQP4 expression depends on brain microenvironment. In previous studies, we observed that the typical morphological conformation of AQP4 as orthogonal arrays of particles (OAP) depended on the extracellular matrix component agrin. In this study, we showed for the first time implanted AQP4 negative glioma cells in animal brain or flank to express AQP4 specifically in the intracerebral gliomas but neither in the extracranial nor in the flank gliomas. AQP4 expression in intracerebral gliomas went along with an OAP loss, compared to normal brain tissue. AQP4 staining in vivo normally is polarized in the astrocytic endfoot membranes at the glia limitans superficialis and perivascularis, but in C6 and RG2 tumors the AQP4 staining is redistributed over the whole glioma cell as in human glioblastoma. In contrast, primary rat or mouse astrocytes in culture did not lose their ability to express AQP4, and they were able to form few OAPs.
Assuntos
Aquaporina 4/biossíntese , Neoplasias Encefálicas/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioma/metabolismo , Proteínas de Neoplasias/biossíntese , Neoplasias Experimentais/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Microambiente Tumoral , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Glioma/patologia , Humanos , Camundongos , Transplante de Neoplasias , Neoplasias Experimentais/patologia , Ratos , Ratos Wistar , Transplante HomólogoRESUMO
Orthogonal arrays or assemblies of intramembranous particles (OAPs) are structures in the membrane of diverse cells which were initially discovered by means of the freeze-fracturing technique. This technique, developed in the 1960s, was important for the acceptance of the fluid mosaic model of the biological membrane. OAPs were first described in liver cells, and then in parietal cells of the stomach, and most importantly, in the astrocytes of the brain. Since the discovery of the structure of OAPs and the identification of OAPs as the morphological equivalent of the water channel protein aquaporin-4 (AQP4) in the 1990s, a plethora of morphological work on OAPs in different cells was published. Now, we feel a need to balance new and old data on OAPs and AQP4 to elucidate the interrelationship of both structures and molecules. In this review, the identity of OAPs as AQP4-based structures in a diversity of cells will be described. At the same time, arguments are offered that under pathological or experimental circumstances, AQP4 can also be expressed in a non-OAP form. Thus, we attempt to project classical work on OAPs onto the molecular biology of AQP4. In particular, astrocytes and glioma cells will play the major part in this review, not only due to our own work but also due to the fact that most studies on structure and function of AQP4 were done in the nervous system.
Assuntos
Aquaporina 4/metabolismo , Aquaporina 4/ultraestrutura , Astrócitos/ultraestrutura , Membrana Celular/ultraestrutura , Agrina/metabolismo , Animais , Aquaporina 4/genética , Astrócitos/metabolismo , Barreira Hematoencefálica/metabolismo , Neoplasias Encefálicas/metabolismo , Membrana Celular/metabolismo , Sistema Nervoso Central/citologia , Sistema Nervoso Central/metabolismo , Plexo Corióideo/citologia , Distroglicanas/metabolismo , Distrofina/metabolismo , Técnica de Fratura por Congelamento , Glioma/metabolismo , Humanos , Inflamação/metabolismo , Modelos Moleculares , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/ultraestrutura , Estrutura Quaternária de Proteína , Distribuição Tecidual , Água/metabolismoRESUMO
The olfactory ensheathing (glial) cells (OECs) have been identified to be useful candidate cells to support regeneration after being transplanted into injured fiber tracts of the central nervous system. We investigated by means of immunocytochemistry and freeze-fracturing the morphology and molecular composition of OEC tight junctions in the rat olfactory system. In addition, we tested the hypothesis whether tight junctions and orthogonal arrays of particles (OAPs) which contain the water channel protein aquaporin-4 (AQP4), are mutually exclusive as suggested in previous studies. In OECs, we found neither OAPs nor AQP4, but tight junctions immunoreactive for ZO-1, occludin, and claudin-5, but immunonegative for ZO-2 and claudin-3. To shed more light on the function of OEC tight junctions, we tested the permeability and tight junction composition of blood vessels and fila olfactoria. We found them both, permeable for infused lanthanum nitrate, and to be immunopositive for ZO-1 and claudin-5. The tight junctions of the OECs are discussed to be responsible for micro-compartmentalization within the olfactory fiber tract providing a benefit for axonal growth.
Assuntos
Endotélio/metabolismo , Cavidade Nasal/metabolismo , Neuroglia/metabolismo , Junções Íntimas/metabolismo , Animais , Aquaporina 4/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , Permeabilidade Capilar/fisiologia , Claudina-5 , Endotélio/ultraestrutura , Técnica de Fratura por Congelamento , Lantânio/farmacologia , Masculino , Proteínas de Membrana/metabolismo , Microscopia Eletrônica de Transmissão , Cavidade Nasal/ultraestrutura , Mucosa Nasal/metabolismo , Mucosa Nasal/ultraestrutura , Neuroglia/ultraestrutura , Ocludina , Fosfoproteínas/metabolismo , Ratos , Ratos Wistar , Junções Íntimas/ultraestrutura , Proteína da Zônula de Oclusão-1RESUMO
CD99 is a long-known leukocyte antigen that does not belong to any of the known protein families. It was recently found on endothelial cells, where it mediates transendothelial migration of human monocytes and lymphocyte recruitment into inflamed skin in the mouse. Here, we show that CD99L2, a recently cloned, widely expressed antigen of unknown function with moderate sequence homology to CD99, is expressed on mouse leukocytes and endothelial cells. Using antibodies, we found that CD99L2 and CD99 are involved in transendothelial migration of neutrophils in vitro and in the recruitment of neutrophils into inflamed peritoneum. Intravital and electron microscopy of cremaster venules revealed that blocking CD99L2 inhibited leukocyte transmigration through the vessel wall (diapedesis) at the level of the perivascular basement membrane. We were surprised to find that, in contrast to CD99, CD99L2 was not relevant for the extravasation of lymphocytes into inflamed tissue. Although each protein promoted cell aggregation of transfected cells, endothelial CD99 and CD99L2 participated in neutrophil extravasation independent of these proteins on neutrophils. Our results establish CD99L2 as a new endothelial surface protein involved in neutrophil extravasation. In addition, this is the first evidence for a role of CD99 and CD99L2 in the process of leukocyte diapedesis in vivo.
Assuntos
Antígenos CD/fisiologia , Movimento Celular , Células Endoteliais/química , Neutrófilos/fisiologia , Antígeno 12E7 , Animais , Antígenos CD/análise , Adesão Celular , Células Cultivadas , Endotélio Vascular , Inflamação/patologia , Linfócitos/fisiologia , Camundongos , Vênulas/citologiaRESUMO
A cell polarity complex consisting of partitioning defective 3 (PAR-3), atypical protein kinase C (aPKC) and PAR-6 has a central role in the development of cell polarity in epithelial cells. In vertebrate epithelial cells, this complex localizes to tight junctions. Here, we provide evidence for the existence of a distinct PAR protein complex in endothelial cells. Both PAR-3 and PAR-6 associate directly with the adherens junction protein vascular endothelial cadherin (VE-cadherin). This association is direct and mediated through non-overlapping domains in VE-cadherin. PAR-3 and PAR-6 are recruited independently to cell-cell contacts. Surprisingly, the VE-cadherin-associated PAR protein complex lacks aPKC. Ectopic expression of VE-cadherin in epithelial cells affects tight junction formation. Our findings suggest that in endothelial cells, another PAR protein complex exists that localizes to adherens junctions and does not promote cellular polarization through aPKC activity. They also point to a direct role of a cadherin in the regulation of cell polarity in vertebrates.
Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células Endoteliais/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Células CHO , Comunicação Celular , Linhagem Celular , Cricetinae , Cães , Células HeLa , Humanos , Proteína Quinase C/metabolismo , Junções Íntimas/metabolismo , Junções Íntimas/patologia , TransfecçãoRESUMO
VE-PTP, a receptor-type phosphotyrosine phosphatase, associates with the tyrosine kinase receptor Tie-2 and VE-cadherin and enhances the adhesive function of the latter. Here, VE-PTP was found to be restricted to endothelial cells, with a preference for arterial endothelium. Mutant mice expressing a truncated, secreted form of VE-PTP lacking the cytoplasmic and transmembrane domains and the most membrane-proximal extracellular fibronectin type III repeat, showed severe vascular malformations causing lethality at 10 days of gestation. Although blood vessels were initially formed, the intraembryonic vascular system soon deteriorated. Blood vessels in the yolk sac developed into dramatically enlarged cavities. In explant cultures of mutant allantoides, endothelial cells were found next to vessel structures growing as cell layers. No signs for enhanced endothelial apoptosis or proliferation were observed. Thus, the activity of VE-PTP is not required for the initial formation of blood vessels, yet it is essential for their maintenance and remodeling.
Assuntos
Vasos Sanguíneos/embriologia , Células Endoteliais/enzimologia , Neovascularização Fisiológica/fisiologia , Proteínas Tirosina Fosfatases/metabolismo , Saco Vitelino/irrigação sanguínea , Sequência de Aminoácidos/genética , Animais , Antígenos CD , Apoptose/genética , Vasos Sanguíneos/anormalidades , Caderinas/metabolismo , Proliferação de Células , Perda do Embrião/genética , Camundongos , Camundongos Mutantes , Estrutura Terciária de Proteína/genética , Proteínas Tirosina Fosfatases/genética , Receptor TIE-2/metabolismo , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores , Deleção de Sequência/genética , Saco Vitelino/anormalidadesRESUMO
Diapedesis of leukocytes across endothelial barriers is generally believed to require the opening of endothelial tight junctions. At the blood-brain barrier (BBB), endothelial cells are interconnected by complex tight junctions. Here, we show by serial section conventional electron microscopy that during experimental autoimmune encephalomyelitis mononuclear cells traverse cerebral microvessels by a transcellular pathway, leaving the endothelial tight junctions intact. Cerebral endothelial cells were found to form filopodia-like membrane protrusions on their luminal aspect, thus embracing the mononuclear cells and forming cup-like structures, and eventually pores, through which the traversing cell could reach the abluminal side. At the abluminal side endothelial cell protrusions surrounding a migrating inflammatory cell were found to be progressively lined with basal lamina, suggesting a change from luminal to abluminal membrane characteristics of endothelial cell membranes during inflammatory cell diapedesis. Morphological evidence for the involvement of tight junctions in the diapedesis of mononuclear cells across the inflamed BBB could not be obtained in any case. Taken together, the presence of morphologically intact tight junctions and our novel finding of the presence of a basal lamina on both sides of abluminal endothelial cell protrusions surrounding migrating inflammatory cells suggests that during experimental autoimmune encephalomyelitis diapedesis of mononuclear cells occurs via a transendothelial process.
Assuntos
Encefalomielite Autoimune Experimental/patologia , Células Endoteliais/patologia , Endotélio Vascular/patologia , Leucócitos Mononucleares/patologia , Animais , Modelos Animais de Doenças , Células Endoteliais/ultraestrutura , Endotélio Vascular/ultraestrutura , Feminino , Leucócitos Mononucleares/ultraestrutura , Camundongos , Camundongos Endogâmicos , Microscopia Eletrônica de Transmissão/métodos , Junções Íntimas/ultraestruturaRESUMO
The coxsackievirus-adenovirus receptor (CAR) is a cell contact protein on various cell types with unknown physiological function. It belongs to a subfamily of the immunoglobulin-superfamily of which some members are junctional adhesion molecules on epithelial and/or endothelial cells. CAR is dominantly expressed in the hearts and brains of mice until the newborne phase after which it becomes mainly restricted to various epithelial cells. To understand more about the physiological function of CAR, we have generated CAR-deficient mice by gene targeting. We found that these mice die between E11.5 and E13.5 of embryonal development. Ultrastructural analysis of cardiomyocytes revealed that the density of myofibrils was reduced and that their orientation and bundling was disorganized. In addition, mitochondria were enlarged and glycogen storage strongly enriched. In line with these defects, we observed pericardial edema formation as a clear sign of insufficient heart function. Developmental abnormalities likely to be secondary effects of gene ablation were the persistent singular cardial atrio-ventricular canal and dilatations of larger blood vessels such as the cardinal veins. The secondary nature of these defects was supported by the fact that CAR was not expressed on vascular cells or on cells of the vascular wall. No obvious signs for alterations of the histological organization of the placenta were observed. We conclude that CAR is required for embryonal heart development, most likely due to its function during the organization of myofibrils in cardiomyocytes.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Receptores Virais/fisiologia , Animais , Doenças Cardiovasculares/patologia , Linhagem Celular , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/ultraestrutura , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células Endoteliais/ultraestrutura , Biblioteca Genômica , Coração/crescimento & desenvolvimento , Camundongos , Miócitos Cardíacos/patologia , Miócitos Cardíacos/ultraestrutura , Miofibrilas/patologia , Miofibrilas/ultraestrutura , Receptores Virais/deficiência , Receptores Virais/genéticaRESUMO
In the central nervous system (CNS) complex endothelial tight junctions (TJs) form a restrictive paracellular diffusion barrier, the blood-brain barrier (BBB). During inflammation, BBB properties are frequently lost, resulting in brain edema. To investigate whether BBB leakiness correlates with molecular changes at BBB TJs, we performed immunofluorescence stainings for TJ molecules in a mouse model of experimental autoimmune encephalomyelitis (EAE) and in human tissue with glioblastoma multiforme (GBM). In TJs of healthy CNS vessels in both mouse and man we detected occludin, ZO-1, claudin-5 and claudin-3. In EAE brain and spinal cord sections we observed the selective loss of claudin-3 immunostaining from TJs of venules surrounded by inflammatory cuffs, whereas the localization of the other TJ proteins remained unchanged. In addition, selective loss of claudin-3 immunostaining was also observed in altered cerebral microvessels of human GBM. Our data demonstrate the selective loss of claudin-3 from BBB TJs under pathological conditions such as EAE or GBM when the integrity of the BBB is compromised, and therefore suggest that claudin-3 is a central component determining the integrity of BBB TJs in vivo.
Assuntos
Barreira Hematoencefálica , Encefalomielite Autoimune Experimental/metabolismo , Glioblastoma/metabolismo , Proteínas de Membrana/metabolismo , Junções Íntimas/metabolismo , Animais , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Claudina-3 , Claudina-5 , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/patologia , Feminino , Glioblastoma/patologia , Humanos , Imuno-Histoquímica/métodos , Camundongos , Camundongos Endogâmicos , Ocludina , Fosfoproteínas/metabolismo , Junções Íntimas/patologia , Proteína da Zônula de Oclusão-1RESUMO
Searching for cell surface proteins expressed at interendothelial cell contacts, we have raised monoclonal antibodies against intact mouse endothelial cells. We obtained two monoclonal antibodies, 1G8 and 4C10, that stain endothelial cell contacts and recognize a protein of 55 kDa. Purification and identification by mass spectrometry of this protein revealed that it contains two extracellular Ig domains, reminiscent of the JAM family, but a much longer 120-amino acid cytoplasmic domain. The antigen is exclusively expressed on endothelial cells of various organs as was analyzed by immunohistochemistry. Immunogold labeling of ultrathin sections of brain as well as skeletal muscle revealed that the antigen strictly colocalizes in capillaries with the tight junction markers occludin, claudin-5, and ZO-1. Upon transfection into MDCK cells, the antigen was restricted to the most apical tip of the lateral cell surface, where it colocalized with ZO-1 but not with beta-catenin. In contrast to JAM-1, however, the 1G8 antigen does not associate with the PDZ domain proteins ZO-1, AF-6, or ASIP/PAR-3, despite the presence of a PDZ-binding motif. The 1G8 antigen was not detected on peripheral blood mouse leukocytes, whereas similar to JAM-1 it was strongly expressed on platelets and megakaryocytes. The 1G8 antigen supports homophilic interactions on transfected Chinese hamster ovary cells. Based on the similarity to the JAM molecules, it is plausible that the 1G8 antigen might be involved in interendothelial cell adhesion.
Assuntos
Plaquetas/metabolismo , Moléculas de Adesão Celular/fisiologia , Endotélio Vascular/metabolismo , Megacariócitos/metabolismo , Proteínas de Membrana/metabolismo , Junções Íntimas/fisiologia , Animais , Anticorpos , Anticorpos Monoclonais , Encéfalo/metabolismo , Moléculas de Adesão Celular/análise , Espectrometria de Massas , Megacariócitos/ultraestrutura , Proteínas de Membrana/sangue , Proteínas de Membrana/genética , Camundongos , Microscopia Imunoeletrônica , Músculo Esquelético/metabolismo , Coelhos , Ratos , Proteínas Recombinantes de Fusão/metabolismoRESUMO
In the central nervous system (CNS) complex endothelial tight junctions (TJs) form a restrictive paracellular diffusion barrier, the blood-brain barrier (BBB). Pathogenic changes within the CNS are frequently accompanied by the loss of BBB properties, resulting in brain edema. In order to investigate whether BBB leakiness can be monitored by a loss of TJ proteins from cellular borders, we used an in vitro BBB model where brain endothelial cells in co-culture with astrocytes form a tight permeability barrier for 3H-inulin and 14C-sucrose. Removal of astrocytes from the co-culture resulted in an increased permeability to small tracers across the brain endothelial cell monolayer and an opening of the TJs to horseradish peroxidase as detected by electron microscopy. Strikingly, opening of the endothelial TJs was not accompanied by any visible change in the molecular composition of endothelial TJs as junctional localization of the TJ-associated proteins claudin-3, claudin-5, occludin, ZO-1 or ZO-2 or the adherens junction-associated proteins beta-catenin or p120cas did not change. Thus, opening of BBB TJs is not readily accompanied by the complete loss of the junctional localization of TJ proteins.