Hannelore Bauer

In search of the astrocytic factor(s) modulating blood-brain barrier functions in brain capillary endothelial cells in vitro.

Summary1. The blood–brain barrier (BBB) is formed by brain capillary endothelial cells (ECs). There are various cell types, in particular astrocytes, but also pericytes and neurons, located in close vicinity to the capillary ECs which may influence formation and function of the BBB. Based on this consideration, this paper discusses various aspects of the influence of the surrounding cells on brain capillary ECs with special focus on the role of astrocytes.2. Based on the morphology of the BBB, important aspects of brain EC functions are summarized, such as transport functions and maintenance of low paracellular permeability. Moreover, various facets are discussed with respect to the influence of astrocytes, pericytes, microglia, and neurons on the BBB. Data on the role of glial cells in the ontogenesis of the BBB are presented subsequently. The knowledge on this subject is far from being complete, however, these data imply that the neural/neuronal environment rather than glial cells may be of importance in the maturation of the barrier.3. The role of glial cells in the induction and maintenance of the BBB is discussed under physiological as well as pathological conditions. Although the literature presents manifold evidence for a great variety of effects induced by astroglia, there are also many controversies, which may result from different cellular models and experimental conditions used in the respective studies. Numerous factors secreted by astrocytes have been shown to induce a BBB phenotype. On the molecular level, increased expression of barrier-relevant proteins (e.g., tight junction proteins) is documented in the presence of astrocyte-derived factors, and many studies demonstrate the improvement of physiological parameters, such as increased transendothelial resistance and decreased paracellular permeability, in different in vitro models of the BBB. Moreover, one has to take into account that the interaction of brain ECs and astrocytes is bi-directional, and that the other cell types surrounding the brain microvasculature also contribute to BBB function or dysfunction, respectively.4. In conclusion, it is expected that the present and future research focused on molecular mechanisms and signaling pathways will produce new and exciting insights into the complex network of BBB regulation: the cornerstone is laid.

The tight junction-specific protein occludin is a functional target of the E3 ubiquitin-protein ligase itch.

Tight junctions create a highly selective diffusion barrier between epithelial and endothelial cells by preventing the free passage of molecules and ions across the paracellular pathway. Although the regulation of this barrier is still enigmatic, there is evidence that junctional transmembrane proteins are critically involved. Recent evidence confirms the notion thatoccludin, a four-pass integral plasma-membrane protein, is a functional component of the paracellular barrier. The overall hydrophilicity of occludin predicts two extracellular loops bounded by NH2- and COOH-terminal cytoplasmic domains. To date, the binding of the COOH terminus of occludin to intracellular proteins is well documented, but information concerning the function of the cytoplasmic NH2 terminus is still lacking. Using yeast two-hybrid screening we have identified a novel interaction between occludin and the E3 ubiquitin-protein ligase Itch, a member of the HECT domain-containing ubiquitin-protein ligases. We have found that the NH2-terminal portion of occludin binds specifically to a multidomain of Itch, consisting of four WW motifs. This interaction has been confirmed by our results from in vivoand in vitro co-immunoprecipitation experiments. In addition, we provide evidence that Itch is specifically involved in the ubiquitination of occludin in vivo, and that the degradation of occludin is sensitive to proteasome inhibition.

Neural Induction of the Blood-Brain Barrier: Still an Enigma

Abstract1. The study of the blood–brain barrier and its various realms offers a myriad of opportunities for scientific exploration. This review focuses on two of these areas in particular: the induction of the blood–brain barrier and the molecular mechanisms underlying this developmental process.2. The creation of the blood–brain barrier is considered a specific step in the differentiation of cerebral capillary endothelial cells, resulting in a number of biochemical and functional alterations. Although the specific endothelial properties which maintain the homeostasis in the central nervous system necessary for neuronal function have been well described, the inductive mechanisms which trigger blood–brain barrier establishment in capillary endothelial cells are unknown.3. The timetable of blood–brain barrier formation is still a matter of debate, caused largely by the use of varying experimental systems and by the general difficulty of quantitatively measuring the degree of blood–brain barrier “tightness.” However, there is a general consensus that a gradual formation of the blood–brain barrier starts shortly after intraneural neovascularization and that the neural microenvironment (neurons and/or astrocytes) plays a key role in inducing blood–brain barrier function in capillary endothelial cells. This view stems from numerous in vitro experiments using mostly cocultures of capillary endothelial cells and astrocytes and assays for easily measurable blood–brain barrier markers. In vivo, there are great difficulties in proving the inductive influence of the neuronal environment. Also dealt with in this article are brain tumors, the least understood in vivo systems, and the induction or noninduction of barrier function in the newly established tumor vascularization.4. Finally, this review tries to elucidate the question concerning the nature of the inductive signal eliciting blood–brain barrier formation in the cerebral microvasculature.

The dual role of zonula occludens (ZO) proteins.

ZO (zonula occludens) proteins are scaffolding proteins providing the structural basis for the assembly of multiprotein complexes at the cytoplasmic surface of intercellular junctions. In addition, they provide a link between the integral membrane proteins and the filamentous cytoskeleton. ZO proteins belong to the large family of membrane-associated guanylate kinase (MAGUK)-like proteins comprising a number of subfamilies based on domain content and sequence similarity. Besides their structural function at cell-cell contacts, ZO proteins appear to participate in the regulation of cell growth and proliferation. Detailed molecular studies have shown that ZO proteins exhibit conserved functional nuclear localization and nuclear export motifs within their amino acid sequence. Further, ZO proteins interact with dual residency proteins localizing to the plasma membrane and the nucleus. Although the nuclear targeting of ZO proteins has well been described, many questions concerning the biological significance of this process have remained open. This review focuses on the dual role of ZO proteins, being indispensable structural components at the junctional site and functioning in signal transduction pathways related to gene expression and cell behavior.

Effect of oxidative stress on the junctional proteins of cultured cerebral endothelial cells

Summary1. There is increasing evidence that the cerebral endothelium and the blood–brain barrier (BBB) plays an important role in the oxidative stress-induced brain damage. The aim of the present study was to investigate the role of interendothelial junctional proteins in the BBB permeability increase induced by oxidative stress.2. For the experiments, we have used cultured cerebral endothelial cells exposed to hypoxia/reoxygenation or treated with the redox cycling quinone 2,3-Dimethoxy-1,4-naphthoquinone (DMNQ) in the presence or absence of glucose. The expression of junctional proteins and activation of mitogen activated protein kinases (MAPK) was followed by Western-blotting, the interaction of junctional proteins was investigated using coimmunoprecipitation.3. Oxidative stress induces a downregulation of the tight junction protein occludin expression which is more pronounced in the absence of glucose. Furthermore, oxidative stress leads to disruption of the cadherin-β-catenin complex and an activation of extracellular signal-regulated kinase (ERK1/2), which is more intense in the absence of glucose.4. We have shown that one of the causes of the BBB breakdown is probably the structural alteration of the junctional complex caused by oxidative stress, a process in which ERK1/2 may play an important role.

Transmigration of Melanoma Cells through the Blood-Brain Barrier: Role of Endothelial Tight Junctions and Melanoma-Released Serine Proteases

Malignant melanoma represents the third common cause of brain metastasis, having the highest propensity to metastasize to the brain of all primary neoplasms in adults. Since the central nervous system lacks a lymphatic system, the only possibility for melanoma cells to reach the brain is via the blood stream and the blood-brain barrier. Despite the great clinical importance, mechanisms of transmigration of melanoma cells through the blood-brain barrier are incompletely understood. In order to investigate this question we have used an in vitro experimental setup based on the culture of cerebral endothelial cells (CECs) and the A2058 and B16/F10 melanoma cell lines, respectively. Melanoma cells were able to adhere to confluent brain endothelial cells, a process followed by elimination of protrusions and transmigration from the luminal to the basolateral side of the endothelial monolayers. The transmigration process of certain cells was accelerated when they were able to use the routes preformed by previously transmigrated melanoma cells. After migrating through the endothelial monolayer several melanoma cells continued their movement beneath the endothelial cell layer. Melanoma cells coming in contact with brain endothelial cells disrupted the tight and adherens junctions of CECs and used (at least partially) the paracellular transmigration pathway. During this process melanoma cells produced and released large amounts of proteolytic enzymes, mainly gelatinolytic serine proteases, including seprase. The serine protease inhibitor Pefabloc® was able to decrease to 44–55% the number of melanoma cells migrating through CECs. Our results suggest that release of serine proteases by melanoma cells and disintegration of the interendothelial junctional complex are main steps in the formation of brain metastases in malignant melanoma.

“You Shall Not Pass”—tight junctions of the blood brain barrier

The structure and function of the barrier layers restricting the free diffusion of substances between the central nervous system (brain and spinal cord) and the systemic circulation is of great medical interest as various pathological conditions often lead to their impairment. Excessive leakage of blood-borne molecules into the parenchyma and the concomitant fluctuations in the microenvironment following a transient breakdown of the blood-brain barrier (BBB) during ischemic/hypoxic conditions or because of an autoimmune disease are detrimental to the physiological functioning of nervous tissue. On the other hand, the treatment of neurological disorders is often hampered as only minimal amounts of therapeutic agents are able to penetrate a fully functional BBB or blood cerebrospinal fluid barrier. An in-depth understanding of the molecular machinery governing the establishment and maintenance of these barriers is necessary to develop rational strategies allowing a controlled delivery of appropriate drugs to the CNS. At the basis of such tissue barriers are intimate cell-cell contacts (zonulae occludentes, tight junctions) which are present in all polarized epithelia and endothelia. By creating a paracellular diffusion constraint TJs enable the vectorial transport across cell monolayers. More recent findings indicate that functional barriers are already established during development, protecting the fetal brain. As an understanding of the biogenesis of TJs might reveal the underlying mechanisms of barrier formation during ontogenic development numerous in vitro systems have been developed to study the assembly and disassembly of TJs. In addition, monitoring the stage-specific expression of TJ-associated proteins during development has brought much insight into the “developmental tightening” of tissue barriers. Over the last two decades a detailed molecular map of transmembrane and cytoplasmic TJ-proteins has been identified. These proteins not only form a cell-cell adhesion structure, but integrate various signaling pathways, thereby directly or indirectly impacting upon processes such as cell-cell adhesion, cytoskeletal rearrangement, and transcriptional control. This review will provide a brief overview on the establishment of the BBB during embryonic development in mammals and a detailed description of the ultrastructure, biogenesis, and molecular composition of epithelial and endothelial TJs will be given.

Perivascular cells of the supraspinatus tendon express both tendon- and stem cell-related markers

Tendons and ligaments are often affected by mechanical injuries or chronic impairment but other than muscle or bone they possess a low healing capacity. So far, little is known about regeneration of tendons and the role of tendon precursor cells in that process. We hypothesize that perivascular cells of tendon capillaries are progenitors for functional tendon cells and are characterized by expression of marker genes and proteins typical for mesenchymal stem cells and functional tendon cells. Immunohistochemical characterization of biopsies derived from intact human supraspinatus tendons was performed. From these biopsies perivascular cells were isolated, cultured, and characterized using RT-PCR and Western blotting. We have shown for the first time that perivascular cells within tendon tissue express both tendon- and stem/precursor cell-like characteristics. These findings were confirmed by results from in vitro studies focusing on cultured perivascular cells isolated from human supraspinatus tendon biopsies. The results suggest that the perivascular niche may be considered a source for tendon precursor cells. This study provides further information about the molecular nature and localization of tendon precursor cells, which is the basis for developing novel strategies towards tendon healing and facilitated regeneration.

Cytotoxic and genotoxic effects of 4-hydroxynonenal in cerebral endothelial cells

Oxygen free radicals are produced in the central nervous system (CNS) as a consequence of normal physiological metabolic reactions of neuronal cells, but there is evidence accumulating that they are also implicated in the processes leading to a number of pathological changes in the brain. A general mechanism whereby oxygen free radicals induce tissue damage is lipid peroxidation (LPO), which generates a large variety of water-soluble carbonyl compounds. Due to their high reactivity, we focused our investigations on 4-hydroxyalkenals, in particular on 4-hydroxynonenal (HNE), the major 4-hydroxyalkenal. Two phenotypes of cerebral endothelial cells (cECs) were treated with various concentrations of 4-hydroxynonenal and the cyto- and genotoxic effects studied. The cytogenetic endpoints determined were chromosomal aberrations and the induction of micronuclei. Three hours of incubation with HNE induced significantly elevated levels of chromosomal aberrations at concentrations > or = 1 microM and micronuclei at concentrations > or = 10 microM in both cEC phenotypes, compared to the controls. Cytotoxicity was observed at a concentration of 50 microM HNE and was significantly higher in the elongated and spindle-shaped cEC phenotype (type II) than in the epithelial cEC phenotype (type I). The results indicate that cECs are affected by HNE even at low concentrations with minor differences between the two cEC phenotypes.

Comparison of gene expression of extracellular matrix molecules in brain microvascular endothelial cells and astrocytes

By use of random-primed cDNA probes the expression of extracellular matrix molecules in cerebral microvascular endothelial cells (cEC) and in astrocytes from mouse brain was examined. Two phenotypically different batches of cloned cEC were used. Expression of major adhesive ECM molecules, constituting the endothelial basement membrane (i.e., fibronectin, laminin A, B and collagen IV) and of other attachment factors, such as SPARC (osteonectin), tenascin and thrombospondin 1, was examined. We have demonstrated that cEC of different morphology display variations in the expression of fibronectin (FN), thrombospondin 1 (TSP1) and collagen IV (C IV). Astrocytes were shown to contain FN, TSP1, TN and SPARC mRNA. Unexpectedly, SPARC mRNA could not be detected in any of the capillary endothelial cells examined. Therefore, we suggest that astrocytes are likely to be involved in endothelial differentiation and function in the central nervous system via ECM molecule secretion.