Lipedema: The Use of Cultured Adipocytes for Identification of Diagnostic Markers.

BACKGROUND: Lipedema, diagnosed most often in women, is a progressive disease characterized by the disproportionate and symmetrical distribution of adipose tissue, primarily in the extremities. Although numerous results from in vitro and in vivo studies have been published, many questions regarding the pathology and genetic background of lipedema remain unanswered. METHODS: In this study, adipose tissue-derived stromal/stem cells were isolated from lipoaspirates derived from nonobese and obese donors with or without lipedema. Growth and morphology, metabolic activity, differentiation potential, and gene expression were evaluated using quantification of lipid accumulation, metabolic activity assay, live-cell imaging, reverse transcription polymerase chain reaction, quantitative polymerase chain reaction, and immunocytochemical staining. RESULTS: The adipogenic potential of lipedema and nonlipedema adipose tissue-derived stromal/stem cells did not rise in parallel with the donors' body mass index and did not differ significantly between groups. However, in vitro differentiated adipocytes from nonobese lipedema donors showed significant upregulation of adipogenic gene expression compared with nonobese controls. All other genes tested were expressed equally in lipedema and nonlipedema adipocytes. The adiponectin/leptin ratio was significantly reduced in adipocytes from obese lipedema donors compared with their nonobese lipedema counterparts. Increased stress fiber-integrated smooth muscle actin was visible in lipedema adipocytes compared with nonlipedema controls and appeared enhanced in adipocytes from obese lipedema donors. CONCLUSIONS: Not only lipedema per se but also body mass index of donors affect adipogenic gene expression substantially in vitro. The significantly reduced adiponectin/leptin ratio and the increased occurrence of myofibroblast-like cells in obese lipedema adipocyte cultures underscores the importance of attention to the co-occurrence of lipedema and obesity. These are important findings toward accurate diagnosis of lipedema. CLINICAL RELEVANCE STATEMENT: Our study highlights not only the difficulty in lipedema diagnostics but also the tremendous need for further studies on lipedema tissue. Although lipedema might seem to be an underestimated field in plastic and reconstructive surgery, the power it holds to provide better treatment to future patients can not be promoted enough.

Lipedema Research-Quo Vadis?

When studying the current literature, one might get the impression that lipedema is a "modern" disease, with increasing incidence and augmenting prevalence throughout Western countries during the last decade. However, a quick look into older textbooks shows that disproportionate accumulation of fat in female bodies has long been known without being recognized as an independent disease. Nevertheless, it was not until 1940 that Allen and Hines described a "syndrome characterized by fat legs and orthostatic edema" in a seminal publication. The mere awareness that people who have lipedema are not just overweight but suffer from a yet poorly defined pathological condition, may be considered a decisive leap forward in the understanding of lipedema. A number of comprehensive publications have since dealt with the clinical presentation of lipedema and have provided the first clues towards the potential pathological mechanisms underlying its initiation and progression. Nevertheless, despite all effort that has been undertaken to unravel lipedema pathology, many questions have remained unanswered. What can be deduced with certainty from all experimental and medical evidence available so far is that lipedema is neither a cosmetic problem nor is it a problem of lifestyle but should be accepted as a serious disease with yet undetermined genetic background, which makes women's lives unbearable from both a physical and psychological point of view. To date, results from clinical inspections have led to the categorization of various types and stages of lipedema, describing how the extremities are affected and evaluating its progression, as demonstrated by skin alterations, adipose tissue volume increase and physical and everyday-behavioral impediments. There is accumulating evidence showing that advanced stages of lipedema are usually accompanied by excessive weight or obesity. Thus, it is not unreasonable to assume that the progression of lipedema is largely driven by weight gain and the pathological alterations associated with it. Similarly, secondary lymphedema is frequently found in lipedema patients at advanced stages. Needless to say, both conditions considerably blur the clinical presentation of lipedema, making diagnosis difficult and scientific research challenging. The present literature review will focus on lipedema research, based on evidence fromex vivo and in vitro data, which has accumulated throughout the last few decades. We will also open the discussion as to whether the currently used categorization of lipedema stages is still sufficient and up-to-date for the accurate description of this enigmatic disease, whose name, strangely enough, does not match its pathologic correlate.

The Blood-Brain Barrier and Its Intercellular Junctions in Age-Related Brain Disorders.

With age, our cognitive skills and abilities decline. Maybe starting as an annoyance, this decline can become a major impediment to normal daily life. Recent research shows that the neurodegenerative disorders responsible for age associated cognitive dysfunction are mechanistically linked to the state of the microvasculature in the brain. When the microvasculature does not function properly, ischemia, hypoxia, oxidative stress and related pathologic processes ensue, further damaging vascular and neural function. One of the most important and specialized functions of the brain microvasculature is the blood-brain barrier (BBB), which controls the movement of molecules between blood circulation and the brain parenchyma. In this review, we are focusing on tight junctions (TJs), the multiprotein complexes that play an important role in establishing and maintaining barrier function. After a short introduction of the cell types that modulate barrier function via intercellular communication, we examine how age, age related pathologies and the aging of the immune system affects TJs. Then, we review how the TJs are affected in age associated neurodegenerative disorders: Alzheimer's disease and Parkinson's disease. Lastly, we summarize the TJ aspects of Huntington's disease and schizophrenia. Barrier dysfunction appears to be a common denominator in neurological disorders, warranting detailed research into the molecular mechanisms behind it. Learning the commonalities and differences in the pathomechanism of the BBB injury in different neurological disorders will predictably lead to development of new therapeutics that improve our life as we age.

Adipose Stem Cells from Lipedema and Control Adipose Tissue Respond Differently to Adipogenic Stimulation In Vitro.

BACKGROUND: Lipedema is characterized by localized accumulation of fat in the extremities, which is typically unresponsive to dietary regimens or physical activity. Although the disease is well described and has a high incidence, little is known regarding the molecular and cellular mechanisms underlying its pathogenesis. The aim of this study was to investigate the pathophysiology of lipedema adipose cells in vitro. METHODS: Adipose-derived stem cells were isolated from lipoaspirates derived from lipedema and nonlipedema patients undergoing tumescent liposuction. In vitro differentiation studies were performed for up to 14 days using adipogenic or regular culture medium. Supernatants and cell lysates were tested for adiponectin, leptin, insulin-like growth factor-1, aromatase (CYP19A1), and interleukin-8 content at days 7 and 14, using enzyme-linked immunosorbent assays. Adipogenesis was evaluated by visualizing and measuring cytoplasmic lipid accumulation. RESULTS: Lipedema adipose-derived stem cells showed impeded adipogenesis already at early stages of in vitro differentiation. Concomitant with a strongly reduced cytoplasmic lipid accumulation, significantly lower amounts of adiponectin and leptin were detectable in supernatants from lipedema adipose-derived stem cells and adipocytes compared with control cells. In addition, lipedema and nonlipedema cells differed in their expression of insulin-like growth factor-1, aromatase (CYP19A1), and interleukin-8 and in their proliferative activity. CONCLUSIONS: The authors' findings indicate that in vitro adipogenesis of lipedema adipose-derived stem cells is severely hampered compared with nonlipedema adipose-derived stem cells. Lipedema adipose cells differ not only in their lipid storage capacity but also in their adipokine expression pattern. This might serve as a valuable marker for diagnosis of lipedema, probably from an early stage on.

Tight Junctions of the Blood-Brain Barrier - A Molecular Gatekeeper.

A tight regulation of the neuroparenchymal microenvironment is imperative for proper neurological function. The flux of blood-borne ions and solutes is restricted by specialized tissue barriers and of the three main central nervous system barriers, the brain endothelium constituting the blood-brain barrier represents the major interface between blood and brain. At the basis of the bloodbrain barrier are, next to an elaborate transporting machinery, tight junctions which create not only a paracellular diffusion constraint but also enable vectorial transport across the endothelial monolayer. Generally, tight junctions not only represent a cellcell adhesion structure, but integrate various signaling pathways via large multiprotein complexes, thereby impacting upon processes such as cell proliferation, cytoskeletal rearrangement, and transcriptional control. This review provides an overview of tight junction morphology and discusses our current understanding of the molecular composition of endothelial tight junctions at the blood-brain barrier.

Pharmaceutical Targeting of the Brain.

Besides being indispensable for the protection and nutrition of the central nervous system (CNS), blood-brain barrier (BBB)-forming cerebral endothelial cells (CECs) have a major role in hampering drugs to reach therapeutically relevant concentrations in the brain. In this respect, the most important defense systems of CECs are tight junctions (TJs) sealing the paracellular way of transport, efflux pumps (ABC transporters) and metabolic enzymes. Here we review current strategies aiming at overcoming the BBB with the purpose of effectively delivering drugs to the CNS. Besides chemical modification of drug candidates to improve CNS availability, the main strategies include: bypassing the BBB (intracranial or nasal routes), reversible opening of TJs (using hyperosmotic mannitol, ultrasounds, peptides and other physical methods or chemical agents), vector-mediated drug delivery systems (nanocarriers, exploitation of receptor- and carrier-mediated transport) and inhibition of efflux transporters. We discuss the main advantages, disadvantages and clinical relevance of each strategy. Special emphasis will be given to the description of the chemical characteristics of nanoparticles (lipidic, polymeric, inorganic, etc.) and the main strategies of targeting them to the CNS.

"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.

Cellular specificity of the blood-CSF barrier for albumin transfer across the choroid plexus epithelium.

To maintain the precise internal milieu of the mammalian central nervous system, well-controlled transfer of molecules from periphery into brain is required. Recently the soluble and cell-surface albumin-binding glycoprotein SPARC (secreted protein acidic and rich in cysteine) has been implicated in albumin transport into developing brain, however the exact mechanism remains unknown. We postulate that SPARC is a docking site for albumin, mediating its uptake and transfer by choroid plexus epithelial cells from blood into cerebrospinal fluid (CSF). We used in vivo physiological measurements of transfer of endogenous (mouse) and exogenous (human) albumins, in situ Proximity Ligation Assay (in situ PLA), and qRT-PCR experiments to examine the cellular mechanism mediating protein transfer across the blood-CSF interface. We report that at all developmental stages mouse albumin and SPARC gave positive signals with in situ PLAs in plasma, CSF and within individual plexus cells suggesting a possible molecular interaction. In contrast, in situ PLA experiments in brain sections from mice injected with human albumin showed positive signals for human albumin in the vascular compartment that were only rarely identifiable within choroid plexus cells and only at older ages. Concentrations of both endogenous mouse albumin and exogenous (intraperitoneally injected) human albumin were estimated in plasma and CSF and expressed as CSF/plasma concentration ratios. Human albumin was not transferred through the mouse blood-CSF barrier to the same extent as endogenous mouse albumin, confirming results from in situ PLA. During postnatal development Sparc gene expression was higher in early postnatal ages than in the adult and changed in response to altered levels of albumin in blood plasma in a differential and developmentally regulated manner. Here we propose a possible cellular route and mechanism by which albumin is transferred from blood into CSF across a sub-population of specialised choroid plexus epithelial cells.

Role of Rho/ROCK signaling in the interaction of melanoma cells with the blood-brain barrier.

We have investigated the role of the Rho/ROCK signaling pathway in the interaction of metastatic melanoma cells with the brain endothelium. ROCK inhibition induced a shift of melanoma cells to the mesenchymal phenotype, increased the number of melanoma cells attached to the brain endothelium, and strengthened the adhesion force between melanoma and endothelial cells. Inhibition of ROCK raised the number of melanoma cells migrating through the brain endothelial monolayer and promoted the formation of parenchymal brain metastases in vivo. We have shown that inhibition of the Rho/ROCK pathway in melanoma, but not in brain endothelial cells, is responsible for this phenomenon. Our results indicate that the mesenchymal type of tumor cell movement is primordial in the transmigration of melanoma cells through the blood-brain barrier.

Mechanisms that determine the internal environment of the developing brain: a transcriptomic, functional and ultrastructural approach.

We provide comprehensive identification of embryonic (E15) and adult rat lateral ventricular choroid plexus transcriptome, with focus on junction-associated proteins, ionic influx transporters and channels. Additionally, these data are related to new structural and previously published permeability studies. Results reveal that most genes associated with intercellular junctions are expressed at similar levels at both ages. In total, 32 molecules known to be associated with brain barrier interfaces were identified. Nine claudins showed unaltered expression, while two claudins (6 and 8) were expressed at higher levels in the embryo. Expression levels for most cytoplasmic/regulatory adaptors (10 of 12) were similar at the two ages. A few junctional genes displayed lower expression in embryos, including 5 claudins, occludin and one junctional adhesion molecule. Three gap junction genes were enriched in the embryo. The functional effectiveness of these junctions was assessed using blood-delivered water-soluble tracers at both the light and electron microscopic level: embryo and adult junctions halted movement of both 286Da and 3kDa molecules into the cerebrospinal fluid (CSF). The molecular identities of many ion channel and transporter genes previously reported as important for CSF formation and secretion in the adult were demonstrated in the embryonic choroid plexus (and validated with immunohistochemistry of protein products), but with some major age-related differences in expression. In addition, a large number of previously unidentified ion channel and transporter genes were identified for the first time in plexus epithelium. These results, in addition to data obtained from electron microscopical and physiological permeability experiments in immature brains, indicate that exchange between blood and CSF is mainly transcellular, as well-formed tight junctions restrict movement of small water-soluble molecules from early in development. These data strongly indicate the brain develops within a well-protected internal environment and the exchange between the blood, brain and CSF is transcellular and not through incomplete barriers.

Bupivacaine induces short-term alterations and impairment in rat tendons.

BACKGROUND: Toxicity of the local anesthetic bupivacaine (BV) has been a matter of debate across medical fields. Numerous in vitro studies demonstrate considerable toxicity of BV on various cell types. PURPOSE: This study addresses the question of how tendon tissue responds to BV in vivo and in vitro. STUDY DESIGN: Controlled laboratory study. METHODS: In vitro studies on cultured rat Achilles tendon-derived cells were performed with cell viability assays and cleaved caspase 3 immunocytochemistry. Quantitative reverse transcription-polymerase chain reaction, Western blotting, gelatin zymography, and a biomechanical testing routine were applied on rat Achilles tendons at 1 and 4 weeks after a single unilateral peritendinous injection of 0.5% BV. The BV-mediated cell death in tendons was estimated with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and immunohistochemical detection of cleaved caspase 3. RESULTS: Treatment of rat tendon-derived cells with 0.5% bupivacaine for 10 minutes had detrimental effects on cell viability, which can be reduced by N-acetyl-L-cysteine or reduction of extracellular calcium. In vivo, single peritendinous injections of BV caused apoptosis in endotenon cells and an increase of pro-matrix metalloproteinase-9 after 6 hours. The collagen ratio shifted toward collagen type III after 6 hours and 2 days; scleraxis messenger RNA (mRNA) expression was reduced by 87%. Maximum tensile load was reduced by 17.6% after 1 week. CONCLUSION: Bupivacaine exerts a severe, reactive oxygen species-mediated effect on tendon cell viability in vitro in a time- and dose-dependent manner, depending on extracellular calcium concentration. Culture conditions need to be taken into account when in vitro data are translated into the in vivo situation. In vivo, administration of BV elicits a marked but temporary functional damage. CLINICAL RELEVANCE: Local anesthetics cause short-term alterations in rat tendons, which, if occurring in humans to a similar extent, may be relevant regarding decreased biomechanical properties and increased vulnerability to tendon overload or injury.

Beyond cell-cell adhesion: Emerging roles of the tight junction scaffold ZO-2.

Zonula occludens proteins (ZO-1, ZO-2, ZO-3), which belong to the family of membrane-associated guanylate kinase (MAGUK) homologs, serve as molecular hubs for the assembly of multi-protein networks at the cytoplasmic surface of intercellular contacts in epithelial and endothelial cells. These multi-PDZ proteins exert crucial functions in the structural organization of intercellular contacts and in transducing intracellular signals from the plasma membrane to the nucleus. The junctional MAGUK protein ZO-2 not only associates with the C-terminal PDZ-binding motif of various transmembrane junctional proteins but also transiently targets to the nucleus and interacts with a number of nuclear proteins, thereby modulating gene expression and cell proliferation. Recent evidence suggests that ZO-2 is also involved in stress response and cytoprotective mechanisms, which further highlights the multi-faceted nature of this PDZ domain-containing protein. This review focuses on ZO-2 acting as a molecular scaffold at the cytoplasmic aspect of tight junctions and within the nucleus and discusses additional aspects of its cellular activities. The multitude of proteins interacting with ZO-2 and the heterogeneity of proteins either influencing or being influenced by ZO-2 suggests an exceptional functional capacity of this protein far beyond merely serving as a structural component of cellular junctions.

Brain pericyte plasticity as a potential drug target in CNS repair.

Brain pericytes (BrPCs) are essential cellular components of the central nervous system neurovascular unit involved in the regulation of blood flow, blood-brain barrier function, as well as in the stabilization of the vessel architecture. More recently, it became evident that BrPCs, besides their regulatory activities in brain vessel function and homeostasis, have pleiotropic functions in the adult CNS ranging from stromal and regeneration promoting activities to stem cell properties. This special characteristic confers BrPC cell plasticity, being able to display features of other cells within the organism. BrPCs might also be causally involved in certain brain diseases. Due to these properties BrPCs might be potential drug targets for future therapies of neurological disorders. This review summarizes BrPC properties, disorders in which this cell type might be involved, and provides suggestions for future therapeutic developments targeting BrPCs.

Molecular characterisation of transport mechanisms at the developing mouse blood-CSF interface: a transcriptome approach.

Exchange mechanisms across the blood-cerebrospinal fluid (CSF) barrier in the choroid plexuses within the cerebral ventricles control access of molecules to the central nervous system, especially in early development when the brain is poorly vascularised. However, little is known about their molecular or developmental characteristics. We examined the transcriptome of lateral ventricular choroid plexus in embryonic day 15 (E15) and adult mice. Numerous genes identified in the adult were expressed at similar levels at E15, indicating substantial plexus maturity early in development. Some genes coding for key functions (intercellular/tight junctions, influx/efflux transporters) changed expression during development and their expression patterns are discussed in the context of available physiological/permeability results in the developing brain. Three genes: Secreted protein acidic and rich in cysteine (Sparc), Glycophorin A (Gypa) and C (Gypc), were identified as those whose gene products are candidates to target plasma proteins to choroid plexus cells. These were investigated using quantitative- and single-cell-PCR on plexus epithelial cells that were albumin- or total plasma protein-immunopositive. Results showed a significant degree of concordance between plasma protein/albumin immunoreactivity and expression of the putative transporters. Immunohistochemistry identified SPARC and GYPA in choroid plexus epithelial cells in the embryo with a subcellular distribution that was consistent with transport of albumin from blood to cerebrospinal fluid. In adult plexus this pattern of immunostaining was absent. We propose a model of the cellular mechanism in which SPARC and GYPA, together with identified vesicle-associated membrane proteins (VAMPs) may act as receptors/transporters in developmentally regulated transfer of plasma proteins at the blood-CSF interface.

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.

Oxidative stress and blood-brain barrier dysfunction under particular consideration of matrix metalloproteinases.

A cell's "redox" (oxidation and reduction) state is determined by the sum of all redox processes yielding reactive oxygen species (ROS), reactive nitrogen species (RNS), and other reactive intermediates. Low amounts of ROS/RNS are generated by different mechanisms in every cell and are important regulatory mediators in many signaling processes (redox signaling). When the physiological balance between the generation and elimination of ROS/RNS is disrupted, oxidative/nitrosative stress with persistent oxidative damage of the organism occurs. Oxidative stress has been suggested to act as initiator and/or mediator of many human diseases. The cerebral vasculature is particularly susceptible to oxidative stress, which is critical since cerebral endothelial cells play a major role in the creation and maintenance of the blood-brain barrier (BBB). This article will only contain a focused introduction on the biochemical background of redox signaling, since this has been reported already in a series of excellent recent reviews. The goal of this work is to increase the understanding of basic mechanisms underlying ROS/RNS-induced BBB disruption, with a focus on the role of matrix metalloproteinases, which, after all, appear to be a key mediator in the initiation and progression of BBB damage elicited by oxidative stress.

Hyperosmotic stress induces Axl activation and cleavage in cerebral endothelial cells.

Because of the relative impermeability of the blood-brain barrier (BBB), many drugs are unable to reach the CNS in therapeutically relevant concentration. One method to deliver drugs to the CNS is the osmotic opening of the BBB using mannitol. Hyperosmotic mannitol induces a strong phosphorylation on tyrosine residues in a broad spectrum of proteins in cerebral endothelial cells, the principal components of the BBB. Previously, we have shown that among targets of tyrosine phosphorylation are beta-catenin, extracellular signal-regulated kinase 1/2 and the non-receptor tyrosine kinase Src. The aim of this study was to identify new signalling pathways activated by hypertonicity in cerebral endothelial cells. Using an antibody array and immunoprecipitation we identified the receptor tyrosine kinase Axl to become tyrosine phosphorylated in response to hyperosmotic mannitol. Besides activation, Axl was also cleaved in response to osmotic stress. Degradation of Axl proved to be metalloproteinase- and proteasome-dependent and resulted in 50-55 kDa C-terminal products which remained phosphorylated even after degradation. Specific knockdown of Axl increased the rate of apoptosis in hyperosmotic mannitol-treated cells; therefore, we assume that activation of Axl may be a protective mechanism against hypertonicity-induced apoptosis. Our results identify Axl as an important element of osmotic stress-induced signalling.

Nuclear Zonula occludens-2 alters gene expression and junctional stability in epithelial and endothelial cells.

Zonula occludens proteins (ZOPs) are essential scaffold proteins involved in the organization of epithelial and endothelial intercellular junctions. Based on their molecular domain architecture, they are members of the large family of membrane-associated guanylate kinase-like (MAGUK) proteins. As all other MAGUKs, ZOPs contain a core of several PDZ, an src homology-3, and a guanylate kinase-like domain, indicating that these proteins may serve both structural and signaling functions. In addition, ZOPs exhibit some unique motifs not shared by other MAGUKs, i.e., several nuclear localization (NLS) and nuclear export signals (NES), allowing these proteins to shuttle between the cytoplasm and the nucleus. However, the stimuli leading to the nuclear accumulation of ZOPs and the resulting physiological consequences remain poorly defined. We have previously reported the direct binding of nuclear ZO-2 to scaffold attachment factor B, a heterogeneous nuclear ribonucleoprotein involved in chromatin organization and the transcriptional control of eukaryotic genes. We now report that the nuclear accumulation of ZO-2 leads to an increase in the expression of the M2 type of pyruvate kinase (M2-PK) in epithelial and endothelial cells. Further, the proliferative activity was increased, while the intercellular junctional stability of Madin-Darby canine kidney cells was reduced. Our data provide evidence to suggest that ZO-2 exerts a junction-unrelated function that further supports the notion of a general "dual" role of junctional MAGUKs, being an indispensable structural component at cell-cell junctions and a nuclear factor influencing gene expression and cell behavior.

Neuronal stem cells in adults.

Neuronal stem cells are like other tissue-specific stem cells, undifferentiated cells which can proliferate and may give rise to glia and neurons. They are present in mammalians throughout the entire life and are supposed to play an important role in renewal of neurons. However, little is known about the origin, phenotypic expression and function of neuronal stem cells in the adult brain. In the present review the occurrence and origin of neuronal stem cells as well as specific markers, which allow their identification in the brain is being described. Finally the role of these cells in the adult brain and their potential use in neuropathy is discussed.