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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.

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.

Aquaporins (AQPs) are important for ocular homeostasis and function. While AQP expression has been investigated in ocular tissues of human, mouse, rat and dog, comprehensive data in rabbits are missing. As rabbits are frequently used model organisms in ophthalmic research, the aim of this study was to analyze mRNA expression and to localize AQPs in the rabbit eye. The results were compared with the data published for other species. In cross sections of New Zealand White rabbit eyes AQP0 to AQP5 were labeled by immunohistology and analyzed by confocal microscopy. Immunohistological findings were compared to mRNA expression levels, which were analyzed by quantitative reverse transcription real time polymerase chain reaction (qRT-PCR). The primers used were homologous against conserved regions of AQPs. In the rabbit eye, AQP0 protein expression was restricted to the lens, while AQP1 was present in the cornea, the chamber angle, the iris, the ciliary body, the retina and, to a lower extent, in optic nerve vessels. AQP3 and AQP5 showed immunopositivity in the cornea. AQP3 was also present in the conjunctiva, which could not be confirmed for AQP5. However, at a low level AQP5 was also traceable in the lens. AQP4 protein was detected in the ciliary non-pigmented epithelium (NPE), the retina, optic nerve astrocytes and extraocular muscle fibers. For most tissues the qRT-PCR data confirmed the immunohistology results and vice versa. Although species differences exist, the AQP protein expression pattern in the rabbit eye shows that, especially in the anterior section, the AQP distribution is very similar to human, mouse, rat and dog. Depending on the ocular regions investigated in rabbit, different protein and mRNA expression results were obtained. This might be caused by complex gene regulatory mechanisms, post-translational protein modifications or technical limitations. However, in conclusion the data suggest that the rabbit is a useful in-vivo model to study AQP function and the effects of direct and indirect intervention strategies to investigate e. g. mechanisms for intraocular pressure modulation or cornea transparency regulation.

Despite significant advancements in bone tissue-engineering applications, the clinical impact of bone marrow stromal cells (BMSCs) for the treatment of large osseous defects remains limited. Therefore, other cell sources are under investigation for their osteogenic potential to repair bone. In this study, tendon-derived stromal cells (TDSCs) were evaluated in comparison to BMSCs to support the functional repair of a 5 mm critical-sized, segmental defect in the rat femur. Analysis of the trilineage differentiation capacity of TDSCs and BMSCs cultured on collagen sponges revealed impaired osteogenic differentiation and mineral deposition of TDSCs in vitro, whereas chondrogenic and adipogenic differentiation was evident for both cell types. Radiographic assessment demonstrated that neither cell type significantly improved the healing rate of a challenging 5 mm segmental femoral defect. Transplanted TDSCs and BMSCs both led to the formation of only small amounts of bone in the defect area, and histological evaluation revealed non-mineralized, collagen-rich scar tissue to be present within the defect area. Newly formed lamellar bone was restricted to the defect margins, resulting in closure of the medullary cavity. Interestingly, in comparison to BMSCs, significantly more TDSC-derived cells were present at the osteotomy gap up to 8 weeks after transplantation and were also found to be located within newly formed lamellar bone, suggesting their capacity to directly contribute to de novo bone formation. To our knowledge, this is the first study investigating the in vivo capacity of TDSCs to regenerate a critical-sized defect in the rat femur. Copyright © 2015 John Wiley & Sons, Ltd.

The presence of neural stem/progenitor cells (NSPCs) in specific areas of the central nervous system (CNS) supports tissue maintenance as well as regeneration. The subependymal zone (SEZ), located at the lateral ventricle's wall, represents a niche for NSPCs and in response to stroke or demyelination becomes activated with progenitors migrating towards the lesion and differentiating into neurons and glia. The mechanisms that underlie this phenomenon remain largely unknown. The vascular niche and in particular blood-derived elements such as platelets, has been shown to contribute to CNS regeneration in different pathological conditions. Indeed, intracerebroventricularly administrated platelet lysate (PL) stimulates angiogenesis, neurogenesis and neuroprotection in the damaged CNS. Here, we explored the presence of platelets in the activated SEZ after a focal demyelinating lesion in the corpus callosum of mice and we studied the effects of PL on proliferating SEZ-derived NSPCs in vitro. We showed that the lesion-induced increase in the size of the SEZ and in the number of proliferating SEZ-resident NSPCs correlates with the accumulation of platelets specifically along the activated SEZ vasculature. Expanding on this finding, we demonstrated that exposure of NSPCs to PL in vitro led to increased numbers of cells by enhanced cell survival and reduced apoptosis without differences in proliferation and in the differentiation potential of NSPCs. Finally, we demonstrate that the accumulation of platelets within the SEZ is spatially correlated with reduced numbers of apoptotic cells when compared to other periventricular areas. In conclusion, our results show that platelet-derived compounds specifically promote SEZ-derived NSPC survival and suggest that platelets might contribute to the enlargement of the pool of SEZ NSPCs that are available for CNS repair in response to injury.

Chronic and acute tendinopathies are difficult to treat and tendon healing is generally a very slow and incomplete process and our general understanding of tendon biology and regeneration lags behind that of muscle or bone. Although still largely unexplored, several studies suggest a positive effect of nutritional interventions on tendon health and repair. With this study, we aim to reveal effects of a high-glucose diet on tendon neoformation in a non-diabetic rat model of Achilles tenotomy. After surgery animals received either a high-glucose diet or a control diet for 2 and 4 weeks, respectively. Compared to the control group, tendon repair tissue thickness and stiffness were increased in the high-glucose group after 2 weeks and gait pattern was altered after 1 and 2 weeks. Cell proliferation was up to 3-fold higher and the expression of the chondrogenic marker genes Sox9, Col2a1, Acan and Comp was significantly increased 2 and 4 weeks post-surgery. Further, a moderate increase in cartilage-like areas within the repair tissue was evident after 4 weeks of a high-glucose diet regimen. In summary, we propose that a high-glucose diet significantly affects tendon healing after injury in non-diabetic rats, potentially driving chondrogenic degeneration.

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.

Acute and chronic tendinopathies remain clinically challenging and tendons are predisposed to degeneration or injury with age. Despite the high prevalence of tendon disease in the elderly, our current understanding of the mechanisms underlying the age-dependent deterioration of tendon function remains very limited. Here, we show that Secreted protein acidic and rich in cysteine (Sparc) expression significantly decreases in healthy-aged mouse Achilles tendons. Loss of Sparc results in tendon collagen fibrillogenesis defects and Sparc−/− tendons are less able to withstand force in comparison with their respective wild type counterparts. On the cellular level, Sparc-null and healthy-aged tendon-derived cells exhibited a more contracted phenotype and an altered actin cytoskeleton. Additionally, an elevated expression of the adipogenic marker genes PPARγ and Cebpα with a concomitant increase in lipid deposits in aged and Sparc−/− tendons was observed. In summary, we propose that Sparc levels in tendons are critical for proper collagen fibril maturation and its age-related decrease, together with a change in ECM properties favors lipid accretion in tendons.

Brain diseases are one of the most prevalent groups of diseases in Europe with estimated annual costs amounting to euro386 billion. Data collected by the WHO suggest that brain diseases are responsible for 35% of Europe's total disease burden. In the treatment of neurological disease, the blood brain barrier (BBB) still represents an obstacle for the delivery of drugs to the brain and thus a major challenge for the development of therapeutic regimens. Understanding the molecular basis and functioning of the BBB in health and disease, including transport mechanisms across the BBB, therefore holds significant potential for future strategies to prevent and ameliorate neurological disease. Recent research indicates that some neurological disorders have a developmental etiologic component. The major goal of the NEUROBID project is thus to understand the molecular mechanisms and function of the BBB in health and disease both in the developing brain and the adult central nervous system. With an interdisciplinary consortium from the fields of developmental neurobiology and BBB research, NEUROBID aims to (i) understand the involvement of normal and disturbed BBB function in normal and abnormal brain development and (ii) to develop novel strategies for drug delivery to the brain. Unique transport mechanisms across the BBB will be used to target potential therapeutic macromolecular and cellular agents specifically to the brain barriers and transport them into the brain. The main target disorders of NEUROBID are non-inherited neurodevelopmental disorders arising from perinatal adverse exposure, such as cerebral palsy, and classic adult neurological disorders such as multiple sclerosis and stroke. In the long term, NEUROBID hopes to pave the way for new treatment strategies and thus reduce the economic and social burden of neurological disease.

Diabetes mellitus is a risk factor for various types of tendon disorders. The mechanisms underlying diabetes associated tendinopathies remain unclear, but typically, systemic factors related to high blood glucose levels are thought to be causally involved. We hypothesize that tendon immanent cells might be directly involved in diabetic tendinopathy. We therefore analyzed human and rat tendons by immunohistochemistry, laser capture microdissection, and single cell PCR for pancreatic β-cell associated markers. Moreover, we examined the short term effects of a single injection of streptozotocin, a toxin for GLUT2 expressing cells, in rats on insulin expression of tendon cells, and on the biomechanical properties of Achilles tendons. Tendon cells, both in the perivascular area and in the dense collagenous tissue express insulin and Glut2 on both protein and mRNA levels. In addition, glucagon and PDX-1 are present in tendon cells. Intraperitoneal injection of streptozotocin caused a loss of insulin and insulin mRNA in rat Achilles tendons after only 5 days, accompanied by a 40% reduction of mechanical strength. In summary, a so far unrecognized, extrapancreatic, insulin-producing cell type, possibly playing a major role in the pathophysiology of diabetic tendinopathy is described. In view of these data, novel strategies in tendon repair may be considered. The potential of the described cells as a tool for treating diabetes needs to be addressed by further studies.

Inhibition of Hedgehog (HH)/GLI signalling in cancer is a promising therapeutic approach. Interactions between HH/GLI and other oncogenic pathways affect the strength and tumourigenicity of HH/GLI. Cooperation of HH/GLI with epidermal growth factor receptor (EGFR) signalling promotes transformation and cancer cell proliferation in vitro. However, the in vivo relevance of HH-EGFR signal integration and the critical downstream mediators are largely undefined. In this report we show that genetic and pharmacologic inhibition of EGFR signalling reduces tumour growth in mouse models of HH/GLI driven basal cell carcinoma (BCC). We describe HH-EGFR cooperation response genes including SOX2, SOX9, JUN, CXCR4 and FGF19 that are synergistically activated by HH-EGFR signal integration and required for in vivo growth of BCC cells and tumour-initiating pancreatic cancer cells. The data validate EGFR signalling as drug target in HH/GLI driven cancers and shed light on the molecular processes controlled by HH-EGFR signal cooperation, providing new therapeutic strategies based on combined targeting of HH-EGFR signalling and selected downstream target genes.

Neural stem and progenitor cells serve as a reservoir for new neurons in the adult brain throughout lifetime. One of the critical steps determining the net production of new neurons is neural progenitor proliferation, which needs to be tightly controlled. Since inflammation has detrimental effects on neurogenesis and the 5-lipoxygenase/leukotriene pathway is involved in inflammatory processes, we investigated the effects of leukotrienes and montelukast, a small molecule inhibitor of the leukotriene receptors CysLT(1)R and GPR17, on neural stem and progenitor cell proliferation. We demonstrate expression of the leukotriene receptor GPR17 by neural progenitors and by neural stem cells. Stimulation with excess amounts of leukotrienes did not affect progenitor proliferation, whereas blockade of GPR17 with montelukast strongly elevated neural stem and progenitor proliferation, while maintaining their differentiation fate and potential. This effect was associated with increased ERK1/2 phosphorylation suggesting an involvement of the EGF signaling cascade. Based on our results, montelukast and the inhibition of the 5-LOX pathway might be potent candidates for future therapies employing neurogenesis to promote structural and functional improvement in neurodegeneration, neuropsychiatric disease and ageing.

Epithelial and endothelial tissue barriers are based on tight intercellular contacts (Tight Junctions, TJs) between neighbouring cells. TJs are multimeric complexes, located at the most apical border of the lateral membrane. So far, a plethora of proteins locating at tight intercellular contacts have been discovered, the role of which has just partly been unraveled. Yet, there is convincing evidence that many TJ proteins exert a dual role: They act as structural components at the junctional site and they are involved in signalling pathways leading to alterations of gene expression and cell behaviour (migration, proliferation). This review will shortly summarize the classical functions of TJs and TJ-related proteins and will introduce a new category, termed the "non-classical" functions of junctional proteins. A particular focus will be directed towards the nuclear targeting of junctional proteins and the downstream effects elicited by their intranuclear activities.

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.

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.

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.

Despite significant advancements in bone tissue-engineering applications, the clinical impact of bone marrow stromal cells (BMSCs) for the treatment of large osseous defects remains limited. Therefore, other cell sources are under investigation for their osteogenic potential to repair bone. In this study, tendon-derived stromal cells (TDSCs) were evaluated in comparison to BMSCs to support the functional repair of a 5 mm critical-sized, segmental defect in the rat femur. Analysis of the trilineage differentiation capacity of TDSCs and BMSCs cultured on collagen sponges revealed impaired osteogenic differentiation and mineral deposition of TDSCs in vitro, whereas chondrogenic and adipogenic differentiation was evident for both cell types. Radiographic assessment demonstrated that neither cell type significantly improved the healing rate of a challenging 5 mm segmental femoral defect. Transplanted TDSCs and BMSCs both led to the formation of only small amounts of bone in the defect area, and histological evaluation revealed non-mineralized, collagen-rich scar tissue to be present within the defect area. Newly formed lamellar bone was restricted to the defect margins, resulting in closure of the medullary cavity. Interestingly, in comparison to BMSCs, significantly more TDSC-derived cells were present at the osteotomy gap up to 8 weeks after transplantation and were also found to be located within newly formed lamellar bone, suggesting their capacity to directly contribute to de novo bone formation. To our knowledge, this is the first study investigating the in vivo capacity of TDSCs to regenerate a critical-sized defect in the rat femur. Copyright © 2015 John WileySons, Ltd.

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 bloodCSF 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 bloodCSF 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. HEALTH-F2-2009-241778

Evidence is accumulating that capillary endothelial cells (cEC) and astrocytes play a pivotal role in neuroprotection, in particular with respect to counteract oxidative injury. Furthermore, differences among both cell types in response to oxidative stress have been shown and astrocytes seem to be more tolerant in terms of cytotoxicity, however, no reports exist on oxidative stress mediated genotoxicity in astrocytes. We investigated genotoxic and cytotoxic effects of oxidative stress in astrocytes and cECs induced by hypoxia/reoxygenation or by the redox cycling quinone DMNQ. Additionally, the dependence of these effects on glucose availabilty was also studied. On exposure to Hy/Re or 10 μM DMNQ for 24 hr, the frequency of micronucleated and apoptotic cells was significantly increasing, however, astrocytes proved to be more resistant to apoptosis induction, in particular on use of DMNQ. In astrocytes, the low background rates of necrotic cells were not affected and a significant necrosis induction was only detectable in cECs exposed to DMNQ for 24 hr. Short-term exposure to DMNQ (1 hr) had no effect in astrocytes but exerted significant geno- and cytotoxicity in cECs. Increasing the glucose concentration markedly reduced oxidative stress mediated geno- and cytotoxicity in astrocytes. Surprisingly, glucose deprivation (aglycemia) suppressed DMNQ induced micronucleus formation in astrocytes without affecting the frequency of apoptotic cells. Our results indicate that astrocytes are more resistant to oxidative stress than cECs, in particular regarding the potential to counteract genotoxicity as well as apoptosis induction mediated by a short term oxidative insult. © 2006 Wiley-Liss, Inc.

Infections by mouse hepatitis viruses result in disease of the liver, the gastrointestinal tract, respiratory tract, and the central nervous system. Coronaviruses related to mouse hepatitis virus express a hemagglutinin-esterase surface glycoprotein, which specifically hydrolyses either 5-N-acetyl-4-O-acetyl neuraminic acid (Neu4,5Ac(2)) or 5-N-acetyl-9-O-acetyl neuraminic acid (Neu5,9Ac(2)). Moreover, these sialic acids represent potential cellular receptor determinants for murine coronaviruses. Until now, the distribution of these sialic acids in mouse brain was not thoroughly investigated. Particularly Neu4,5Ac(2) was not yet found in mouse brain. Using a sensitive method of gas chromatography coupled to mass spectrometry in the electron impact mode of ionization this manuscript demonstrates the occurrence of 13 different sialic acids varying in their alkyl and acyl substituents in mouse tissues including 5-N-acetyl-4-O-acetyl-9-O-lactyl-neuraminic acid (Neu4,5Ac(2)9Lt), 5-N-acetyl-9-O-lactyl-neuraminic acid (Neu5Ac9Lt), 5-N-acetyl-8-O-methyl-neuraminic acid (Neu5Ac8Me) and the 1,7-lactone (Neu5Ac1,7L) of neuraminic acid. Neu4,5Ac(2), relatively abundant in the gut, was present as a minor compound in all tissues, including liver, olfactory lobe, telencephalon, metencephalon and hippocampus. Neu5,9Ac(2) was also found in these tissues, except in the liver. It is suggested that these sialic acids represent the endogenous substrate and receptor determinants for murine coronaviruses.

Careful examination of relevant literature shows that many of the most cherished concepts of the blood-brain barrier are incorrect. These include an almost mythological belief in its immaturity that is unfortunately often equated with absence or at least leakiness in the embryo and fetus. The original concept of a blood-brain barrier is often attributed to Ehrlich; however, he did not accept that permeability of cerebral vessels was different from other organs. Goldmann is often credited with the first experiments showing dye (trypan blue) exclusion from the brain when injected systemically, but not when injected directly into it. Rarely cited are earlier experiments of Bouffard and of Franke who showed methylene blue and trypan red stained all tissues except the brain. The term "blood-brain barrier" "Blut-Hirnschranke" is often attributed to Lewandowsky, but it does not appear in his papers. The first person to use this term seems to be Stern in the early 1920s. Studies in embryos by Stern and colleagues, Weed and Wislocki showed results similar to those in adult animals. These were well-conducted experiments made a century ago, thus the persistence of a belief in barrier immaturity is puzzling. As discussed in this review, evidence for this belief, is of poor experimental quality, often misinterpreted and often not properly cited. The functional state of blood-brain barrier mechanisms in the fetus is an important biological phenomenon with implications for normal brain development. It is also important for clinicians to have proper evidence on which to advise pregnant women who may need to take medications for serious medical conditions. Beliefs in immaturity of the blood-brain barrier have held the field back for decades. Their history illustrates the importance of taking account of all the evidence and assessing its quality, rather than selecting papers that supports a preconceived notion or intuitive belief. This review attempts to right the wrongs. Based on careful translation of original papers, some published a century ago, as well as providing discussion of studies claiming to show barrier immaturity, we hope that readers will have evidence on which to base their own conclusions.

Mannitol, which is a cell-impermeable and nontoxic polyalcohol, has been shown to be a useful tool for reversible opening of the blood-brain barrier (BBB). Despite successful clinical trials, the molecular mechanism of the mannitol-induced changes in cerebral endothelial cells (CECs) are poorly understood. For our experiments, we used CECs in culture, which were treated with different, clinically relevant concentrations of mannitol. We found that mannitol induced a rapid, concentration-dependent, and reversible tyrosine phosphorylation of a broad range of proteins between 50 and 190 kDa. One of the targets of tyrosine phosphorylation turned out to be the adherens junction protein beta-catenin. Phosphorylation of beta-catenin on tyrosine residues caused its subcellular redistribution and its dissociation from cadherin and alpha-catenin as shown by coimmunoprecipitation studies. All these effects could be inhibited by the Src kinase inhibitor PP-1 but not by the Erk inhibitor U0126, the Rho kinase inhibitor Y27632, or the calcium channel blocker verapamil. Because beta-catenin is a key component of the junctional complex, its Src-mediated phpsphorylation may play an important role in the mannitol induced reversible opening of the BBB.

Zonula occludens proteins (ZOPs), currently comprising ZO-1, ZO-2, and ZO-3, belong to the family of membrane-associated guanylate kinase homologue (MAGUK) proteins that are involved in the organization of epithelial and endothelial intercellular junctions. ZOPs bind to the cytoplasmic C termini of junctional transmembrane proteins linking them to the actin cytoskeleton. They are characterized by several conserved modules, including three PDZ domains, one SH3 domain, and a guanylate kinase-like domain, elements indicating that ZOPs may serve multiple purposes. Interestingly, ZOPs contain some unique motifs not shared by other MAGUK family members, including nuclear localization and nuclear export signals and a leucine zipper-like sequence. Their potential involvement in cell growth and proliferation has been suggested earlier based on the observation that the N-terminal half of ZOPs displays significant similarity to the product of the Drosophila tumor suppressor gene lethal(1)discs-large (dlg). The nuclear targeting of ZOPs in subconfluent epithelial cell cultures is well documented, although the action of the junctional MAGUKs in the nucleus has remained elusive. Here we show for the first time that nuclear ZO-2 directly interacts with the DNA-binding protein scaffold attachment factor-B (SAF-B). Our results from two-hybrid assays and in vivo co-immunoprecipitation studies provide evidence to suggest that ZO-2 associates with the C-terminal portion of SAF-B via its PDZ-1 domain. We further demonstrate that enhanced green fluorescent protein (EGFP)- and DsRed-tagged ZO-2 and SAF-B fusion proteins partially co-localize in nuclei of transfected epithelial cells. As shown by laser confocal microscopy and epifluorescent analysis, nuclear ZO-2 is present in epithelial and endothelial cells, particularly in response to environmental stress conditions. Interestingly, no association of SAF-B with ZO-1 was found, which supports the notion that junctional MAGUKs serve nonredundant functions.

Impaired function of the brain vasculature might contribute to the development of HIV-associated dementia. For example, injury or dysfunction of brain microvascular endothelial cells (BMEC) can lead to the breakdown of the blood-brain barrier (BBB) and thus allow accelerated entry of the HIV-1 virus into the CNS. Mechanisms of injury to BMEC during HIV-1 infection are not fully understood, but the viral gene product Tat may be, at least in part, responsible for this effect. Tat can be released from infected perivascular macrophages in the CNS of patients with AIDS, and thus BMEC can be directly exposed to high concentrations of this protein. To study oxidative and inflammatory mechanisms associated with Tat-induced toxicity, BMEC were exposed to increasing doses of Tat1-72, and markers of oxidative stress, as well as redox-responsive transcription factors such as nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1), were measured. Tat1-72 treatment markedly increased cellular oxidative stress, decreased levels of intracellular glutathione and activated DNA binding activity and transactivation of NF-kappaB and AP-1. To determine if Tat1-72 can stimulate inflammatory responses in brain endothelium in vivo, expression of monocyte chemoattractant protein-1 (MCP-1), an NF-kappaB and AP-1-dependent chemokine, was studied in brain tissue in mice injected with Tat1-72 into the right hippocampus. Tat1-72 markedly elevated the MCP-1 mRNA levels in brain tissue. In addition, a double immunohistochemistry study revealed that MCP-1 protein was markedly overexpressed on brain vascular endothelium. These data indicate that Tat1-72 can induce redox-related inflammatory responses both in in vitro and in vivo environments. These changes can directly lead to disruption of the BBB. Thus, Tat can play an important role in the development of detrimental vascular changes in the brains of HIV-infected patients.

Tears of the anterior cruciate ligament (ACL) are very frequent injuries, particularly in young and active people. Arthroscopic reconstruction using tendon auto- or allograft represents the gold-standard for the management of ACL tears. Interestingly, the ACL has the potential to heal upon intensive non-surgical rehabilitation procedures. Several biological factors influence this healing process as local intraligamentous cytokines and mainly cell repair mechanisms controlled by stem cells or progenitor cells. Understanding the mechanisms of this regeneration process and the cells involved may pave the way for novel, less invasive and biology-based strategies for ACL repair. This review aims to focus on the current knowledge on the mechanisms of ACL healing, the nature and potential of ligament derived stem/progenitor cells as well as on the potential and the limitations of using mesenchymal stem cells (MSCs) for treating injured ACL.

1. 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.

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 concentrationsor = 1 microM and micronuclei at concentrationsor = 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.

A full-length cDNA encoding the porcine monocyte chemoattractant protein-1 (pMCPC-1) was isolated from growth-stimulated porcine cerebral capillary endothelial cells (cEC); the pMCP-1 cDNA showed 89% identity to human MCP-1 and was isolated by use of subtractive hybridization and differential screening of two phenotypically different sub-populations of cloned cEC. pMCP-1 was abundantly expressed in cEC grown in the presence of FCS, ECGF and heparin whereas lower expression was observed in cEC kept in FCS-supplemented medium only. As shown by Northern blot analysis, no pMCP-1 transcripts were present in total RNA derived from freshly isolated brain capillaries, large brain vessels or whole brain homogenate. MCP/JE expression was also demonstrated in ECGF/heparin-treated murine cEC. Astrocytes and smooth muscle cells grown in FCS-supplemented medium did not show MCP-1 expression. Treatment of porcine cEC with TNF-alpha increased pMCP-1 mRNA levels in a dose-dependent manner. These data further support the notion that cerebral capillary endothelial cells actively participate in processes of CNS host defense.

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.

In the brain, the microvascular system is composed of endothelial cells surrounded by a layer of pericytes. The lack of smooth muscle cells in this tissue suggests that any contractile function must be performed by one or both of these cell types. The present study was undertaken in order to identify cells in terminal blood vessels that contain smooth muscle-like contractile machinery. Endothelial cells were reactive with antibodies against smooth muscle myosin but showed no other smooth muscle-related features. In contrast, pericytes of intact microvessels showed a pattern of protein expression similar to that of smooth muscle cells. Pericytes also behaved in tissue culture like cultured smooth muscle cells, with regard to the changes in expression of smooth muscle-related proteins. These data confirm the close relationship between smooth muscle cells and pericytes, and point to their contractile function in the brain microvessels.

Brain pericytes (BrPCs) are essential cellular components of the central nervous system neuromuscularunit involved in the regulation of blood flow, blood–brain barrier function, as well as in the stabilizationof the vessel architecture. More recently, it became evident that BrPCs, besides their regulatory activitiesin brain vessel function and homeostasis, have pleiotropic functions in the adult CNS ranging fromstromal and regeneration promoting activities to stem cell properties. This special characteristic confersBrPC cell plasticity, being able to display features of other cells within the organism. BrPCs might also becausally involved in certain brain diseases. Due to these properties BrPCs might be potential drug targetsfor future therapies of neurological disorders. This review summarizes BrPC properties, disorders inwhich this cell type might be involved, and provides suggestions for future therapeutic developmentstargeting BrPCs.

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.

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. Antioxid. Redox Signal. 15, 1305–1323.

Alarin is a 25 amino acid peptide that belongs to the galanin peptide family. It is derived from the galanin-like peptide gene by a splice variant, which excludes exon 3. Alarin was first identified in gangliocytes of neuroblastic tumors and later shown to have a vasoactive function in the skin. Recently, alarin was demonstrated to stimulate food intake as well as the hypothalamic–pituitary–gonadal axis in rodents, suggesting that it might be a neuromodulatory peptide in the brain. However, the individual neurons in the central nervous system that express alarin have not been identified. Here, we determined the distribution of alarin-like immunoreactivity (alarin-LI) in the adult murine brain. The specificity of the antibody against alarin was demonstrated by the absence of labeling after pre-absorption of the antiserum with synthetic alarin peptide and in transgenic mouse brains lacking neurons expressing the GALP gene. Alarin-LI was observed in different areas of the murine brain. A high intensity of alarin-LI was detected in the accessory olfactory bulb, the medial preoptic area, the amygdala, different nuclei of the hypothalamus such as the arcuate nucleus and the ventromedial hypothalamic nucleus, the trigeminal complex, the locus coeruleus, the ventral chochlear nucleus, the facial nucleus, and the epithelial layer of the plexus choroideus. The distinct expression pattern of alarin in the adult mouse brain suggests potential functions in reproduction and metabolism.

5-Bromo-2′-deoxyuridin (BrdU) is frequently used in anaylsis of neural stem cell biology, in particular to label and to fate-map dividing cells. However, up to now, only a few studies have addressed the question as to whether BrdU labeling per se affects the cells to be investigated. Here, we focused on the potential impact of BrdU on neurosphere cultures derived from the adult rat brain and on proliferation of progenitors in vivo. In vitro, neurospheres were pulsed for 48 h with BrdU, and cell proliferation, cell cycle, differentiation, survival and adhesion properties were subsequently analyzed. BrdU inhibited the expansion of neural progenitors as assessed by MTS assay and increased the fraction of cells in the G0/G1-phase of the cell cycle. Moreover, BrdU increased cell death and dose-dependently induced adherence of NPCs. Cell adherence was accompanied by a reduced amount of active matrix-metalloproteinase-2 (MMP-2). Furthermore, BrdU repressed neuronal and oligodendroglial differentiation, whereas astroglial fate was not affected. In contrast to the in vitro situation, BrdU apparently did not influence endogenous proliferation of NPCs or neurogenesis in concentrations that are typically used for labeling of neural progenitors in vivo. Our results reveal so far uncharacterized effects of BrdU on adult NPCs. We conclude that, because of its ubiquitous use in stem cell biology, any potential effect of BrdU of NPCs has to be scrutinized prior to interpretation of data.

Vascularization and expression of blood-brain barrier (bbb)-associated morphological characteristics during the embryonic development of the mouse central nervous system (CNS) was studied by ultrastructural analysis. At embryonic day 9 (E9) capillaries were only found in the perineural mesenchymal tissue. These capillaries showed fenestrations, and pericyte like cells (PC) were found joined to the vessel walls. Around E10 endothelial cells (EC) together with PC started invading the intraneural section. At this stage, the immigrating endothelial cells lost their fenestrations and exhibited numerous, partly extended junctional complexes, which appeared 'tight' in some places. A first intraneural anastomotic plexus was observed at E10, as evidenced by the presence of blood cells in all capillary lumens. While the number of junctional complexes remained constant in intraneural capillaries, the frequency of pinocytotic vesicles decreased significantly from E10 to E17. These findings indicate that from the first day of intraneural vascularization onwards, the morphological properties of the bbb are present in the early embryonic mouse cerebral cortex.

Introduction We have previously shown that human and rat tendon cells produce insulin and secrete it upon glucose stimulation. Moreover, the level of tendon insulin production is affected by the amount of glucose taken up by nutrition [Lehner, 2012]. We now hypothesise that nutritional glucose affects tendon healing in a rat model. Methods In 60 female Lewis rats full thickness defects were created in one Achilles tendon and left unsutured. The rats were randomly assigned to three groups, one was fed a high glucose diet, one a diet with low glucose/high fat and one a control diet, for 2 weeks each. Before surgery, one and two weeks after, gait analysis was performed using a Noldus TM catwalk system. After two weeks the animals were sacrificed and tendon size was measured and tendons were biomechanically tested an evaluated by various histological methods. Results Gait Analysis revealed a significant difference between the three groups one week after surgery. The intermediate toe spread (the range between second and fourth toe, a measure for the load on the limb) of the high glucose group is significantly increased one week p.o. (0,49 cm ± 0,07; n = 20; p Measurement of length and thickness of the newly formed tissue revealed a significant (p Biomechanical testing revealed no significant difference between the groups in maximum tensile load, however, the new fibrous tissue from the glucose group is significantly (p Discussion Newly formed tendon tissue quality is affected by nutritional glucose. This finding is relevant for understanding diabetes related tendinopathy. Nutritional parameters may account for the interindividual variation of tendon quality and regeneration. The underlying molecular mechanisms will be examined. Reference Lehner et al . Horm Metab Res. 2012;44:506–510

Background Rotator cuff tears are a common cause of shoulder pain and impairment. Subacromial glucocorticoid injections are widely used for treatment of epiphenomenons of chronic impingement syndrome with the possible side effects of tendon rupture and impaired tendon healing. Methods Using qRT-PCR, western blot, immunoflourescence, and measurement of 3H-thymidine uptake we investigated the effects of the crystalline glucocorticoid triamcinolone acetonide (TAA) when added to the culture medium of isolated human rotator cuff tendon cells. Results After 2 weeks of incubation, the cells had lost their fibroblastic appearance and parallel orientation, which is characteristic of cellular degeneration in vivo. Moreover, expression and secretion of collagen I was strongly reduced, and there was a decrease in proliferation rate. Cell migration was blocked and the rate of expression of the matrix metalloproteinases MMP2, MMP8, MMP9, and MMP13 was reduced, but expression of TIMP1 (a tissue inhibitor of MMPs) was upregulated, indicating a reduction in the cellular capacity for tendon repair. In addition, changes in cellular differentiation were observed: the number of adipocytes increased and levels of the protein Sox9—a marker of differentiating and mature chondrocytes—were elevated in triamcinolone acetonide treated cells. Interpretation These results may indicate that the use of TAA is one reason for weaker mechanical tendon properties and for the high rate of re-rupture after supraspinatus tendon repair.

The blood-brain barrier (BBB) in mammals is created and maintained by cerebral endothelial cells (cEC) that express specialized functional properties, including intercellular tight junctions, absence of fenestrae and specific membrane transport systems. It has been proposed that the differentiation of these characteristics, acquired during brain development, is controlled by the neural environment. Co-culture experiments of cloned cEC with astroglial cells, C6 glioma cells and cortical neurons, with plasma membranes or conditioned media of these cells, were used to study induction of some BBB characteristics in vitro. Activities of Na+,K(+)-ATPase and gamma-glutamyl transpeptidase (GGTP), an enzyme responsible for amino acid transport across the BBB, were taken as parameters for BBB function. Co-culture of cEC with C6 glioma cells caused a two-fold increase in GGTP activity and this activity was likewise amplified by incubation with plasma membrane fractions derived from C6 glioma cells, embryonic brain cells and cortical neurons; conditioned media (soluble factors) had no effect. Na+,K(+)-ATPase activity, estimated from the ouabain inhibitable fraction of 86Rb uptake, was increased by about 90% in cEC incubated with C6 glioma plasma membranes. We propose from these data that both neurons and glial cells confer BBB characteristics on cEC via cell-cell contact.