The blood-testis barrier in Aphanius dispar (Teleostei)

Summary Thin sections of normal testes from the cyprinodont Aphanius dispar were studied by electron microscopy after intravascular injection of live specimens with horseradish peroxidase. The intercellular space in the spermatogenic cysts is marked differently by the tracer according to the degree of differentiation of the germ cells. Spermatogonia and gonocytes undergoing meiosis are surrounded by a dark band of the marker. This band gradually disappears during spermiogenesis. In cysts containing ripe spermatozoa, the marker penetrates a short distance between the bases of adjoining Sertoli cells bordering the cysts, but is arrested by tight junctional complexes near the lumina of the cysts. The tight junctions between the Sertoli cells provide a permeability barrier between the vascular spaces of the stroma and the lumina of ripe cysts.Abbreviations BM basement membrane - BTB blood-testis barrier - HRP horseradish peroxidase This research was supported by a grant from the National Council for Research and Development, Israel, and the GKSS Geesthacht-Tesperhude, Federal Republic of Germany     

Differentiating embryonic neural progenitor cells induce blood-brain barrier properties

The blood-brain barrier (BBB) is a multifunctional endothelial interface separating the bloodstream from the brain interior. Although the mature BBB is well characterized, the embryonic development of this complex system remains poorly understood. Embryonic neural progenitor cells (NPC) are a potential inductive cell type populating the developing brain, and their ability to influence BBB properties was therefore examined. When puromycin-purified brain microvascular endothelial cells (BMEC) were co-cultured with embryonic NPC in a two-compartment Transwell system, the BMEC exhibited enhanced barrier properties in the form of increased transendothelial electrical resistance (TEER) and decreased permeability to the small molecule tracer, sodium fluorescein. These changes required the presence of NPC in the early stages of differentiation and were accompanied by alterations in the fidelity of BMEC tight junctions as indicated by occludin, claudin 5, and zonula occluden-1 redistribution at cell-cell borders. In contrast to the findings with NPC, post-natal astrocytes elicited a delayed, but longer duration response in BMEC TEER. BMEC co-culture also suppressed neuronal differentiation of NPC indicating a reciprocal signaling between the two cell populations. This study demonstrates that NPC-BMEC interactions are prevalent and for the first time demonstrates that NPC are capable of inducing BBB properties.     

Blood-brain barrier in acute liver failure

Brain edema remains a challenging obstacle in the management of acute liver failure (ALF). Cytotoxic mechanisms associated with brain edema have been well recognized, but evidence for vasogenic mechanisms in the pathogenesis of brain edema in ALF has been lacking. Recent reports have not only shown a role of matrix metalloproteinase-9 in the pathogenesis of brain edema in experimental ALF but have also found significant alterations in the tight junction elements including occludin and claudin-5, suggesting a vasogenic injury in the blood-brain barrier (BBB) integrity. This article reviews and explores the role of the paracellular tight junction proteins in the increased selective BBB permeability that leads to brain edema in ALF.     

A pump-free tricellular blood–brain barrier on-a-chip model to understand barrier property and evaluate drug response

Disruption of the blood–brain barrier (BBB) leads to various neurovascular diseases. Development of therapeutics required to cross the BBB is difficult due to a lack of relevant in vitro models. We have developed a three-dimensional (3D) microfluidic BBB chip (BBBC) to study cell interactions in the brain microvasculature and to test drug candidates of neurovascular diseases. We isolated primary brain microvascular endothelial cells (ECs), pericytes, and astrocytes from neonatal rats and cocultured them in the BBBC. To mimic the 3D in vivo BBB structure, we used type I collagen hydrogel to pattern the microchannel via viscous finger patterning technique to create a matrix. ECs, astrocytes, and pericytes were cocultured in the collagen matrix. The fluid flow in the BBBC was controlled by a pump-free strategy utilizing gravity as driving force and resistance in a paper-based flow resistor. The primary cells cultured in the BBBC expressed high levels of junction proteins and formed a tight endothelial barrier layer. Addition of tumor necrosis factor alpha to recapitulate neuroinflammatory conditions compromised the BBB functionality. To mitigate the neuroinflammatory stimulus, we treated the BBB model with the glucocorticoid drug dexamethasone, and observed protection of the BBB. This BBBC represents a new simple, cost-effective, and scalable in vitro platform for validating therapeutic drugs targeting neuroinflammatory conditions.     

Molecular make-up of the glomerular filtration barrier

The glomerular filtration barrier is composed of glomerular endothelial cells, the glomerulus basement membrane and the podocyte cell layer. The filtration barrier is a target of injury in several systemic and renal diseases, and this often leads to progressive renal disease and kidney failure. Therefore, it is essential to understand the molecular biology of the glomerulus. During the last two decades, a lot of new information about molecular components of the glomerulus filtration barrier has been generated. Many of the key discoveries have been obtained through studies on the genetic background of inherited glomerular diseases. These studies have emphasized the role of podocytes in the filtration barrier function. During the last decade, the use of knockout mouse technology has become more available and given important new insights into the functional significance of glomerular components. Large-scale approaches, such as microarray profiling, have also given data about molecules involved in the biology and pathology of the glomerulus. In the coming decade, the use of global expression profiling platforms, transgenic mouse lines, and other in vivo gene delivery methods will rapidly expand our understanding of biology and pathology of the glomerular filtration barrier, and hopefully expose novel target molecules for therapy in progressive renal diseases.     

Blood-brain barrier modeling with co-cultured neural progenitor cell-derived astrocytes and neurons

In vitro blood-brain barrier (BBB) models often consist of brain microvascular endothelial cells (BMECs) that are co-cultured with other cells of the neurovascular unit, such as astrocytes and neurons, to enhance BBB properties. Obtaining primary astrocytes and neurons for co-culture models can be laborious, while yield and heterogeneity of primary isolations can also be limiting. Neural progenitor cells (NPCs), because of their self-renewal capacity and ability to reproducibly differentiate into tunable mixtures of neurons and astrocytes, represent a facile, readily scalable alternative. To this end, differentiated rat NPCs were co-cultured with rat BMECs and shown to induce BBB properties such as elevated trans-endothelial electrical resistance, improved tight junction continuity, polarized p-glycoprotein efflux, and low passive permeability at levels indistinguishable from those induced by primary rat astrocyte co-culture. An NPC differentiation time of 12 days, with the presence of 10% fetal bovine serum, was found to be crucial for generating NPC-derived progeny capable of inducing the optimal response. This approach could also be extended to human NPC-derived astrocytes and neurons which similarly regulated BBB induction. The distribution of rat or human NPC-derived progeny under these conditions was found to be a roughly 3 : 1 mixture of astrocytes to neurons with varying degrees of cellular maturity. BMEC gene expression analysis was conducted using a BBB gene panel, and it was determined that 23 of 26 genes were similarly regulated by either differentiated rat NPC or rat astrocyte co-culture while three genes were differentially altered by the rat NPC-derived progeny. Taken together, these results demonstrate that NPCs are an attractive alternative to primary neural cells for use in BBB co-culture models.     

Apelin-13 passes through the ADMA-damaged endothelial barrier and acts on vascular smooth muscle cells

Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, is associated with vascular dysfunction. The polypeptide apelin mediates two major actions on blood vessels. However, their combined effects on vascular function are not fully understood. The present study aimed to determine the effect of apelin-13 on myosin light chain (MLC) phosphorylation in vascular smooth muscle cells (VSMCs) under ADMA-induced endothelial leakage conditions. To assess the increased permeability induced by ADMA, human umbilical vein endothelium cells (HUVECs) were plated in transwell dishes. The FITC-dextran flux and FITC-apelin-13 flux through the endothelial monolayer were measured. To examine the effect of leakage of apelin-13 on MLC phosphorylation in HUVSMCs, transwell dishes were used to establish a coculture system with HUVECs in upper chambers and HUVSMCs in lower chambers. Western blot was performed to assess the phospho-MLC levels. ADMA increased endothelial permeability in a concentration- and time-dependent manner, accompanied by actin stress fiber assembly and intercellular gap formation. When HUVECs were treated with ADMA, the permeability to both macromolecular dextran and micromolecular apelin-13 increased significantly. Both p38 MAPK inhibitor and NADPH oxidase inhibitor could prevent HUVECs from the increased permeability, and the changes of cytoskeleton and intercellular junction, which were induced by ADMA. Apelin-13 passed through the ADMA-stimulated endothelial monolayer and increased the expression of phospho-MLC in VSMCs. These results suggest that ADMA increases endothelial permeability, which may involve the p38 MAPK and NADPH oxidase pathway. Apelin-13 can pass through the damaged endothelial barrier, and acts directly on VSMCs to increase MLC phosphorylation.     

Staphylococcal enterotoxin B suppresses Alix and compromises intestinal epithelial barrier functions

Background

The epithelial barrier dysfunction plays a critical role in the pathogenesis of a broad array of immune diseases. Alix protein is involved in the endolysosome system. This study aims to elucidate the role of Alix in the maintenance of epithelial barrier function.

Results

The results showed that Alix was detected in T84 cells at both mRNA and protein levels. Exposure to Staphylococcal enterotoxin B (SEB) markedly suppressed the expression of Alix in T84 cells, which could be blocked by knocking down the Toll like receptor 2. The exposure to SEB did not affect the TER, but markedly increased the permeability of T84 monolayers to OVA; the OVA passing through T84 monolayers still preserved the antigenicity manifesting inducing antigen specific T cells proliferation.

Conclusions

Alix protein plays a critical role in the maintenance of the barrier function of T84 monolayers.     

Accelerating whole-cell biocatalysis by reducing outer membrane permeability barrier

Whole-cell biocatalysts are preferred in many biocatalysis applications. However, due to permeability barriers imposed by cell envelopes, whole-cell catalyzed reactions are reportedly 10-100-fold slower than reactions catalyzed by free enzymes. In this study, we accelerated whole-cell biocatalysis by reducing the membrane permeability barrier using molecular engineering approaches. Escherichia coli cells with genetically altered outer membrane structures were used. Specifically, a lipopolysaccarides mutant SM101 and a Braun's lipoprotein mutant E609L were used along with two model substrates that differ substantially in size and hydrophobicity, nitrocefin, and a tetrapeptide N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. The reduction of the outer membrane permeability by genetic methods led to significant increases (up to 380%) in reaction rates of whole-cell catalyzed reactions. The magnitude of increase in biocatalysis rates was dependent on the substrates and on the nature of mutations introduced in the outer membrane structure. Notably, mutations in outer membrane can render the outer membrane completely permeable to one substrate, a barrierless condition that maximizes the reaction rate. The impact of the mutations introduced on the permeability barrier of the membranes was compared to the impact of polymixin B nonapeptide, a known potent permeabilizer acting on lipopolysaccharides. Our results suggest that genetic modifications to enhance the permeability of hydrophilic molecules should target the Lipid A region. However, strategies other than reduction of Lipid A synthesis should be considered. As we have demonstrated with tetrapeptide, membrane engineering can be much more effective in reducing a permeability barrier than are exogenous permeabilizers. This work, to our knowledge, is the first use of a molecular membrane engineering approach to address substrate permeability limitations encountered in biocatalysis applications.     

Role of lactadherin in intestinal barrier integrity in experimental neonatal necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is one of the most widespread and devastating gastrointestinal diseases in neonates. Destruction of the intestinal barrier is the main underlying cause of NEC. The aim of this study was to determine the role of lactadherin in preventing NEC in a neonatal rat model and investigate the molecular mechanism of lactadherin-mediated protection of the intestinal barrier. Neonatal rats were divided into three groups: dam feeding (DF), NEC (NEC), and NEC supplemented with 10 μg/(g·day) recombinant human lactadherin (NEC+L). Intestinal permeability, tissue damage, and cell junction protein expression and localization were evaluated. We found that lactadherin reduced weight loss caused by NEC, reduced the incidence of NEC from 100% to 46.7%, and reduced the mean histological score for tissue damage to 1.40 compared with 2.53 in the NEC group. Intestinal permeability of lactadherin-treated rats was significantly reduced when compared with that of the NEC group. In addition, the expression levels of JAM-A, claudin 3, and E-calcium in the ileum of NEC group animals increased compared with those in the ileum of DF group animals, and these levels decreased in the NEC+L group. Lactadherin changed the localization of claudin 3, occludin, and E-cadherin in epithelial cells. The mechanism underlying lactadherin-mediated protection of the intestinal barrier might be restoring the correct expression levels and localization of tight junction and adherent junction proteins. These findings suggest a new candidate agent for the prevention of NEC in newborns.     

Blood-brain barrier transport of pramipexole, a dopamine D2 agonist

The blood-brain barrier (BBB) transport of pramipexole, a potent dopamine receptor agonist with high efficacy for Parkinson's disease, was mainly characterized using immortalized rat brain capillary endothelial cells (RBEC)1 as an in vitro BBB model. [(14)C]Pramipexole uptake by RBEC1 was dependent on temperature and pH, but not sodium ion concentration or membrane potential. The uptake was inhibited by several organic cations including pyrilamine. Mutual inhibition was observed between pramipexole and pyrilamine. In addition, [(14)C]pramipexole uptake was stimulated by preloading unlabeled pramipexole. RT-PCR analysis for organic cation transporters (rOCT1-3, rOCTN1-2) in RBEC1 was performed. The mRNA level of rOCTN2 was the highest, followed by rOCTN1, while expression of rOCT1, rOCT2 and rOCT3 was negligible. The brain uptake of [(14)C]pramipexole, which was measured by the in situ rat brain perfusion technique, was significantly inhibited by unlabeled pramipexole. These results suggest that pramipexole is, at least in part, transported across the BBB by an organic cation-sensitive transporter. The pramipexole transport in RBEC1 was pH-dependent, but sodium- and membrane potential-independent.     

Ginsenoside Re improves skin barrier function in HaCaT keratinocytes under normal growth conditions

Ginsenoside Re (Re), a major ginsenoside of ginseng, enhanced the cornified cell envelope (CE) formation in HaCaT keratinocytes under normal conditions. In HaCaT keratinocytes, Re was also able to upregulate filaggrin protein and caspase-14 activity in a concentration-dependent manner. These findings reasonably imply that Re possesses a desirable property of improving skin barrier function.     

P-glycoprotein in blood-brain barrier endothelial cells: interaction and oligomerization with caveolins

P-glycoprotein (P-gp), an adenosine triphosphate (ATP)-binding cassette transporter which acts as a drug efflux pump, is highly expressed at the blood-brain barrier (BBB) where it plays an important role in brain protection. Recently, P-gp has been reported to be located in the caveolae of multidrug-resistant cells. In this study, we investigated the localization and the activity of P-gp in the caveolae of endothelial cells of the BBB. We used an in vitro model of the BBB which is formed by co-culture of bovine brain capillary endothelial cells (BBCEC) with astrocytes. Caveolar microdomains isolated from BBCEC are enriched in P-gp, cholesterol, caveolin-1, and caveolin-2. Moreover, P-gp interacts with caveolin-1 and caveolin-2; together, they form a high molecular mass complex. P-gp in isolated caveolae is able to bind its substrates, and the caveolae-disrupting agents filipin III and nystatin decrease P-gp transport activity. In addition, mutations in the caveolin-binding motif present in P-gp reduced the interaction of P-gp with caveolin-1 and increased the transport activity of P-gp. Thus, P-gp expressed at the BBB is mainly localized in caveolae and its activity may be modulated by interaction with caveolin-1.     

A fence barrier method of leading edge cell capture for explorative biochemical research

The scratch or wound-healing assay is used ubiquitously for investigating re-epithelialisation and has already revealed the importance of cells comprising the leading edge of healing epithelial wounds. However it is currently limited to studying the effect of known biochemical agents on the tissue of choice. Here we present an adaptation that extends the utility of this model to encompass the collection of cells from the leading edge of migrating epithelial sheets making available explorative biochemical analyses. The method is scalable and does not require expensive apparatus, making it suitable for large and small laboratories alike. We detail the application of our method and exemplify proof of principle data derived from primary human keratinocyte cultures.     

Disruption of blood-testis barrier dynamics in ether-lipid-deficient mice

One of the major roles of Sertoli cells is to establish the blood-testis (Sertoli cell) barrier (BTB), which is permanently assembled and disassembled to accommodate the translocation of leptotene spermatocytes from the basal into the adluminal compartment of the seminiferous epithelium and to guarantee completion of meiosis and spermiogenesis. Recently, we have demonstrated spermatogenesis to be arrested before spermatid elongation in Gnpat-null mice with selective deficiency of ether lipids (ELs) whose functions are poorly understood. In this study, we have focused on the spatio-temporal expression of several BTB tight-junctional proteins in the first wave of spermatogenesis to obtain insights into the physiological role of ELs during BTB establishment and dynamics. Our data confirm the transient existence of Russell’s intermediate or translocation compartment delineated by two separate claudin-3-positive luminal and basal tight junctions and reveal that EL deficiency blocks BTB remodeling. This block is associated with (1) downregulation and mistargeting of claudin-3 and (2) impaired BTB disassembly resulting in deficient sealing of the intermediate compartment as shown by increased BTB permeability to biotin. These results suggest that ELs are essential for cyclic BTB dynamics ensuring the sluice mechanism for leptotene translocation into the adluminal compartment. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was supported by the German Research Foundation (grants Go 432/2-1, Ju 166/3-1, and Sa 172/1-1).     

Simultaneous cell death and desquamation of the embryonic diffusion barrier during epidermal development

The periderm is an epithelial layer covering the emerging epidermis in early embryogenesis of vertebrates. In the chicken embryo, an additional cellular layer, the subperiderm, occurs at later embryonic stages underneath the periderm. The questions arose what is the function of both epithelial layers and, as they are transitory structures, by which mechanism are they removed. By immunocytochemistry, the tight junction (TJ) proteins occludin and claudin-1 were localized in the periderm and in the subperiderm, and sites of close contact between adjacent cells were detected by electron microscopy. Using horseradish peroxidase (HRP) as tracer, these contacts were identified as tight junctions involved in the formation of the embryonic diffusion barrier. This barrier was lost by desquamation at the end of the embryonic period, when the cornified envelope of the emerging epidermis was formed. By TUNEL and DNA ladder assays, we detected simultaneous cell death in the periderm and the subperiderm shortly before hatching. The absence of caspases-3, -6, and -7 activity, key enzymes of apoptosis, and the lack of typical morphological criteria of apoptosis such as cell fragmentation or membrane blebbing point to a special form of programmed cell death (PCD) leading to the desquamation of the embryonic diffusion barrier.