Karen Wolburg-Buchholz

Localization of claudin-3 in tight junctions of the blood-brain barrier is selectively lost during experimental autoimmune encephalomyelitis and human glioblastoma multiforme.

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.

Brain endothelial cells and the glio-vascular complex

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.

Diapedesis of mononuclear cells across cerebral venules during experimental autoimmune encephalomyelitis leaves tight junctions intact

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.

Involvement of the choroid plexus in central nervous system inflammation

During inflammatory conditions in the central nervous system (CNS), immune cells immigrate into the CNS and can be detected in the CNS parenchyma and in the cerebrospinal fluid (CSF). The most comprehensively investigated model for CNS inflammation is experimental autoimmune encephalomyelitis (EAE), which is considered the prototype model for the human disease multiple sclerosis (MS). In EAE autoagressive CD4+, T cells gain access to the CNS and initiate the molecular and cellular events leading to edema, inflammation, and demyelination in the CNS. The endothelial blood‐brain barrier (BBB) has been considered the obvious place of entry for the circulating immune cells into the CNS. A role of the choroid plexus in the pathogenesis of EAE or MS, i.e., as an alternative entry site for circulating lymphocytes directly into the CSF, has not been seriously considered before. However, during EAE, we observed massive ultrastructural changes within the choroid plexus, which are different from changes observed during hypoxia. Using immunohistochemistry and in situ hybridization, we observed expression of VCAM‐1 and ICAM‐1 in the choroid plexus and demonstrated their upregulation and also de novo expression of MAdCAM‐1 during EAE. Ultrastructural studies revealed polar localization of ICAM‐1, VCAM‐1, and MAdCAM‐1 on the apical surface of choroid plexus epithelial cells and their complete absence on the fenestrated endothelial cells within the choroid plexus parenchyme. Furthermore, ICAM‐1, VCAM‐1, and MAdCAM‐1 expressed in choroid plexus epithelium mediated binding of lymphocytes via their known ligands. In vitro, choroid plexus epithelial cells can be induced to express ICAM‐1, VCAM‐1, MAdCAM‐1, and, additionally, MHC class I and II molecules on their surface. Taken together, our observations imply a previously unappreciated function of the choroid plexus in the immunosurveillance of the CNS. Microsc. Res. Tech. 52:112–129, 2001.

Astrocyte mediated modulation of blood-brain barrier permeability does not correlate with a loss of tight junction proteins from the cellular contacts

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 β-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.

Claudin-1, claudin-2 and claudin-11 are present in tight junctions of choroid plexus epithelium of the mouse.

The choroid plexus epithelium forms the blood-cerebrospinal fluid (CSF) barrier and is responsible for the secretion of the CSF from the blood. The morphological correlate of the blood-CSF barrier are the tight junctions of choroid plexus epithelium. By freeze-fracture electron microscopy it has been demonstrated that choroid plexus epithelial tight junctions form parallel strands resembling those of Sertoli cells building the blood-testis barrier and those of the myelin sheaths of oligodendrocytes. As the oligodendrocyte specific protein/claudin-11 has been shown to be the central mediator of parallel-array tight junctions in Sertoli cells and myelin sheaths in mice, we asked whether claudin-11 is present in the tight junctions of choroid plexus epithelial cells of the mouse. Here, we present the first direct evidence that claudin-11 besides claudin-1 and -2, occludin and the zonula occludens protein ZO-1 is present in choroid plexus epithelial tight junctions. During inflammation in the central nervous system such as experimental autoimmune encephalomyelitis, the molecular composition of choroid plexus epithelial tight junctions does not change considerably. Their unique molecular composition, with claudin-11 accompanied by claudin-1 and claudin-2 points to a unique regulatory mechanism of the blood-CSF-barrier function.

The disturbed blood-brain barrier in human glioblastoma.

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.

Coxsackievirus-adenovirus receptor (CAR) is essential for early embryonic cardiac development.

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.

Agrin, Aquaporin-4, and Astrocyte Polarity as an Important Feature of the Blood-Brain Barrier

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.

Loss of astrocyte polarity marks blood–brain barrier impairment during experimental autoimmune encephalomyelitis

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 β-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 β-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.