Search

Most tight junction (TJ) proteins build epithelial and endothelial barriers [...].

The Blood-Labyrinth Barrier (BLB) is pivotal for the maintenance of lymphatic homeostasis within the inner ear, yet the intricacies of its development and function are inadequately understood. The present investigation delves into the contribution of the Mfsd2a molecule, integral to the structural and functional integrity of the Blood-Brain Barrier (BBB), to the ontogeny and sustenance of the BLB. Our empirical findings delineate that the maturation of the BLB in murine models is not realized until approximately two weeks post-birth, with preceding stages characterized by notable permeability. Transcriptomic analysis elucidates a marked augmentation in Mfsd2a expression within the lateral wall of the cochlea in specimens exhibiting an intact BLB. Moreover, both in vitro and in vivo assays substantiate that a diminution in Mfsd2a expression detrimentally impacts BLB permeability and structural integrity, principally via the attenuation of tight junction protein expression and the enhancement of endothelial cell transcytosis. These insights underscore the indispensable role of Mfsd2a in ensuring BLB integrity and propose it as a viable target for therapeutic interventions aimed at the amelioration of hearing loss., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

Biomolecular condensates enable cell compartmentalization by acting as membraneless organelles 1 . How cells control the interactions of condensates with other cellular structures such as membranes to drive morphological transitions remains poorly understood. We discovered that formation of a tight-junction belt, which is essential for sealing epithelial tissues, is driven by a wetting phenomenon that promotes the growth of a condensed ZO-1 layer 2 around the apical membrane interface. Using temporal proximity proteomics in combination with imaging and thermodynamic theory, we found that the polarity protein PATJ mediates a transition of ZO-1 into a condensed surface layer that elongates around the apical interface. In line with the experimental observations, our theory of condensate growth shows that the speed of elongation depends on the binding affinity of ZO-1 to the apical interface and is constant. Here, using PATJ mutations, we show that ZO-1 interface binding is necessary and sufficient for tight-junction belt formation. Our results demonstrate how cells exploit the collective biophysical properties of protein condensates at membrane interfaces to shape mesoscale structures., (© 2024. The Author(s).)

Hypoxia plays an important role in the pathological process of bladder outlet obstruction. Previous research has mostly focused on the dysfunction of bladder smooth muscle cells, which are directly related to bladder contraction. This study delves into the barrier function changes of the urothelial cells under exposure to hypoxia. Results indicated that after a 5-day culture, SV-HUC-1 formed a monolayer and/or bilayer of cell sheets, with tight junction formation, but no asymmetrical unit membrane was observed. qPCR and western blotting revealed the expression of TJ-associated proteins (occludin, claudin1 and ZO-1) was significantly decreased in the hypoxia group in a time-dependent manner. No expression changes were observed in uroplakins. When compared to normoxic groups, immunofluorescent staining revealed a reduction in the expression of TJ-associated proteins in the hypoxia group. Transepithelial electrical resistance (TEER) revealed a statistically significant decrease in resistance in the hypoxia group. Fluorescein isothiocyanate-conjugated dextran assay was inversely proportional to the results of TEER. Taken together, hypoxia down-regulates the expression of TJ-associated proteins and breaks tight junctions, thus impairing the barrier function in human urothelial cells., (© 2024 The Author(s). Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Bioactive oligopeptides have gained increasing attention due to their diverse physiological functions, and these can be transported into the vasculature via transcellular and paracellular pathways. Among these, paracellular transport through the intercellular space is a passive diffusion process without energy consumption. It is currently the most frequently reported absorption route for food-derived bioactive oligopeptides. Previous work has demonstrated that paracellular pathways are mainly controlled by tight junctions, but the mechanism by which they regulate paracellular absorption of bioactive oligopeptides remains unclear. In this review, we summarized the composition of paracellular pathways across the intercellular space and elaborated on the paracellular transport mechanism of bioactive oligopeptides in terms of the interaction between oligopeptides and tight junction proteins, the protein expression level of tight junctions, the signaling pathways regulating intestinal permeability, and the properties of oligopeptides themselves. These findings contribute to a more profound understanding of the paracellular absorption of bioactive oligopeptides.

Microplastics, which are tiny plastic particles less than 5 mm in diameter, are widely present in the environment, have become a serious threat to aquatic life and human health, potentially causing ecosystem disorders and health problems. The present study aimed to investigate the effects of microplastics, specifically microplastics-polystyrene (MPs-PS), on the structural integrity, gene expression related to tight junctions, and gut microbiota in mice. A total of 24 Kunming mice aged 30 days were randomly assigned into four groups: control male (CM), control female (CF), PS-exposed male (PSM), and PS-exposed female (PSF)(n = 6). There were significant differences in villus height, width, intestinal surface area, and villus height to crypt depth ratio (V/C) between the PS group and the control group(C) (p <0.05). Gene expression analysis demonstrated the downregulation of Claudin-1, Claudin-2, Claudin-15, and Occludin, in both duodenum and jejunum of the PS group (p < 0.05). Analysis of microbial species using 16S rRNA sequencing indicated decreased diversity in the PSF group, as well as reduced diversity in the PSM group at various taxonomic levels. Beta diversity analysis showed a significant difference in gut microbiota distribution between the PS-exposed and C groups (R2 = 0.113, p<0.01), with this difference being more pronounced among females exposed to MPs-PS. KEGG analysis revealed enrichment of differential microbiota mainly involved in seven signaling pathways, such as nucleotide metabolism(p<0.05). The relative abundance ratio of transcriptional pathways was significantly increased for the PSF group (p<0.01), while excretory system pathways were for PSM group(p<0.05). Overall findings suggest that MPs-PS exhibit a notable sex-dependent impact on mouse gut microbiota, with a stronger effect observed among females; reduced expression of tight junction genes may be associated with dysbiosis, particularly elevated levels of Prevotellaceae., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Su et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

This review addresses the role of tight junction proteins at the blood-brain barrier (BBB). Their expression is described, and their role in physiological and pathological processes at the BBB is discussed. Based on this, new approaches are depicted for paracellular drug delivery and diagnostics in the treatment of cerebral diseases. Recent data provide convincing evidence that, in addition to its impairment in the course of diseases, the BBB could be involved in the aetiology of CNS disorders. Further progress will be expected based on new insights in tight junction protein structure and in their involvement in signalling pathways.

Epithelia must be able to resist mechanical force to preserve tissue integrity. While intercellular junctions are known to be important for the mechanical resistance of epithelia, the roles of tight junctions (TJs) remain to be established. We previously demonstrated that epithelial cells devoid of the TJ membrane proteins claudins and JAM-A completely lack TJs and exhibit focal breakages of their apical junctions. Here, we demonstrate that apical junctions fracture when claudin/JAM-A-deficient cells undergo spontaneous cell stretching. The junction fracture was accompanied by actin disorganization, and actin polymerization was required for apical junction integrity in the claudin/JAM-A-deficient cells. Further deletion of CAR resulted in the disruption of ZO-1 molecule ordering at cell junctions, accompanied by severe defects in apical junction integrity. These results demonstrate that TJ membrane proteins regulate the mechanical resistance of the apical junctional complex in epithelial cells., (© 2024 Nguyen et al.)

In cholinergic urticaria (CholU), small, itchy wheals are induced by exercise or passive warming and reduced sweating has been reported. Despite the described reduced muscarinic receptor expression, sweat duct obstruction, or sweat allergy, the underlying pathomechanisms are not well understood. To gain further insights, we collected skin biopsies before and after pulse-controlled ergometry and sweat after sauna provocation from CholU patients as well as healthy controls. CholU patients displayed partially severely reduced local sweating, yet total sweat volume was unaltered. However, sweat electrolyte composition was altered, with increased K + concentration in CholU patients. Formalin-fixed, paraffin-embedded biopsies were stained to explore sweat leakage and tight junction protein expression. Dermcidin staining was not found outside the sweat glands. In the secretory coils of sweat glands, the distribution of claudin-3 and -10b as well as occludin was altered, but the zonula occludens-1 location was unchanged. In all, dermcidin and tight junction protein staining suggests an intact barrier with reduced sweat production capability in CholU patients. For future studies, an ex vivo skin model for quantification of sweat secretion was established, in which sweat secretion could be pharmacologically stimulated or blocked. This ex vivo model will be used to further investigate sweat gland function in CholU patients and decipher the underlying pathomechanism(s).

Studies have revealed neurotoxicity, hepatotoxicity, and developmental and reproductive toxicity in mice exposed to aluminum. However, relatively few studies have been conducted to clarify the mechanism underlying the impact of embryonic exposure to aluminum on the development of the male reproductive system in offspring. Pregnant mice were administered aluminum chloride (AlCl 3 ) by gavage from day 12.5 of gestation until birth. Our findings demonstrated that embryonic exposure to AlCl 3 disrupted testicular development and spermatogenesis by impairing testicular architecture, reducing sperm count, and upregulating the expression of tight junction (TJ) protein between Sertoli cells (SCs). Further in vitro studies revealed that treatment with AlCl 3 stabilized TJ proteins Occludin and ZO-1 expression by inhibiting ERK signaling pathway activation, thereby upregulating Slc25a5 expression which induced ATP production leading to disruption of cytoskeletal protein homeostasis. Therefore, the study provided a new mechanistic insight into how AlCl 3 exposure interfered with testicular development and spermatogenesis while suggesting that Slc25a5 might be a target affected by AlCl 3 influencing cell metabolism., Competing Interests: Declaration of competing interest The authors declare they have no actual or potential competing financial interests., (Copyright © 2024. Published by Elsevier B.V.)

Fluorescently labelled compounds are often employed to study the paracellular properties of epithelia. For flux measurements, these compounds are added to the donor compartment and samples collected from the acceptor compartment at regular intervals. However, this method fails to detect rapid changes in permeability. For continuous transepithelial flux measurements in an Ussing chamber setting, a device was developed, consisting of a flow-through chamber with an attached LED, optical filter, and photodiode, all encased in a light-impermeable container. The photodiode output was amplified and recorded. Calibration with defined fluorescein concentration (range of 1 nM to 150 nM) resulted in a linear output. As proof of principle, flux measurements were performed on various cell lines. The results confirmed a linear dependence of the flux on the fluorescein concentration in the donor compartment. Flux depended on paracellular barrier function (expression of specific tight junction proteins, and EGTA application to induce barrier loss), whereas activation of transcellular chloride secretion had no effect on fluorescein flux. Manipulation of the lateral space by osmotic changes in the perfusion solution also affected transepithelial fluorescein flux. In summary, this device allows a continuous recording of transepithelial flux of fluorescent compounds in parallel with the electrical parameters recorded by the Ussing chamber.

This study investigates the intricate composition and spatial distribution of tight junction complex proteins during early mouse neurulation. The analyses focused on the cranial neural tube, which gives rise to all head structures. Neurulation brings about significant changes in the neuronal and non-neuronal ectoderm at a cellular and tissue level. During this process, precise coordination of both epithelial integrity and epithelial dynamics is essential for accurate tissue morphogenesis. Tight junctions are pivotal for epithelial integrity, yet their complex composition in this context remains poorly understood. Our examination of various tight junction proteins in the forebrain region of mouse embryos revealed distinct patterns in the neuronal and non-neuronal ectoderm, as well as mesoderm-derived mesenchymal cells. While claudin-4 exhibited exclusive expression in the non-neuronal ectoderm, we demonstrated a neuronal ectoderm specific localization for claudin-12 in the developing cranial neural tube. Claudin-5 was uniquely present in mesenchymal cells. Regarding the subcellular localization, canonical tight junction localization in the apical junctions was predominant for most tight junction complex proteins. ZO-1 (zona occludens protein-1), claudin-1, claudin-4, claudin-12, and occludin were detected at the apical junction. However, claudin-1 and occludin also appeared in basolateral domains. Intriguingly, claudin-3 displayed a non-canonical localization, overlapping with a nuclear lamina marker. These findings highlight the diverse tissue and subcellular distribution of tight junction proteins and emphasize the need for their precise regulation during the dynamic processes of forebrain development. The study can thereby contribute to a better understanding of the role of tight junction complex proteins in forebrain development.

Blood-brain barrier (BBB) dysfunction and hyperpermeability that occurs following traumatic and ischemic insults lead to various downstream ill effects such as cerebral edema and elevation of intracranial pressure. The inter-endothelial tight junctions that consist of tight junction proteins are critical regulators of BBB dysfunctions and hyperpermeability. The major tight junction-associated proteins of the BBB are occludin, claudins, and junctional adhesion molecules that are intracellularly linked to the adaptor protein zonula occludens-1 (ZO-1). Quantitative measurement of tight junction-associated proteins provides valuable insight into barrier integrity and mechanisms that regulate microvascular hyperpermeability. Western blot analysis is a commonly used method to separate and identify proteins in a mixture using gel electrophoresis. Understanding the changes in the expression of one or more of these proteins is critical to evaluating barrier integrity and permeability in health and disease. Furthermore, studying them will provide insight into the associated downstream signaling pathways and evaluation of therapeutic approaches for regulating BBB permeability. Herein, we have described the protocol for immunoblot analysis of ZO-1 as an indicator of tight junction integrity in brain microvascular endothelial cells., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

Dysfunction of epithelial barrier is crucial for the development of acute lung injury (ALI). This study was aimed to evaluate the role of glucose transporter 1 (GLUT1) in dysregulation of epithelial tight junction in ALI. GLUT1 was inhibited with specific antagonists WZB117 or BAY876 to see the effects on epithelial tight junction in a well-established LPS-induced mouse ALI model as well as in vitro cultured epithelial cells. Pharmacological inhibition of GLUT1 with WZB117 at either a low or high dose had no effects on lung injury and inflammation 24 h after LPS challenge, but significantly decreased the pulmonary inflammatory responses induced by LPS at 72 h with a high dose, which was verified by treatment with BAY876. WZB117 or BAY876 also recovered the expression of epithelial tight junction proteins ZO-1 and occludin. In cultured BEAS-2B and A549 cells, LPS induced increased GLUT1 expression, accompanied by decreased expression of tight junction protein ZO-1 and occludin. Blockade of GLUT1 restored LPS-induced disruption of ZO-1 and occludin in BEAS-2B rather than A549. Taken together, our results showed that GLUT1 is responsible for dysfunction of epithelial tight junctions in the late phase of LPS-induced ALI., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

L-theanine is a natural bioactive component in tea leaves and has anti-inflammatory effects. The study aimed to investigated the effects and underlying mechanisms of L-theanine on lipopolysaccharide (LPS)-induced intestinal tight junction damage in IPEC-J2 cells. Results showed that LPS induced tight junction damage by increasing reactive oxygen species production and lactate dehydrogenase (LDH) release and decreasing the mRNA expression of tight junction proteins related genes zonula occludens-1 (ZO-1, also known as Tjp1), Occludin and Claudin-1, while L-theanine reversed such an effect and attenuated the increase of p38 mitogen-activated protein kinase (p38 MAPK) mRNA expression. The p38 MAPK inhibitor (SB203580) attenuated the mRNA expression of nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (Nlrp3) inflammasome and interleukin-1β (Il-1β), and increased the mRNA expression of Tjp1, Occludin and Claudin-1, which showed a similar effect with L-theanine. In addition, NLRP3 inhibitor MCC950 attenuated the Il-1β expression and LDH release, while increased the expression of tight-junction protein-related genes. In conclusion, L-theanine could protect LPS-induced intestinal tight junction damage by inhibiting the activation of p38 MAPK-mediated NLRP3 inflammasome pathway., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Airway diseases can disrupt tight junction proteins, compromising the epithelial barrier and making it more permeable to pathogens. In people with pulmonary disease who are prone to infection with Pseudomonas aeruginosa, pro-inflammatory leukotrienes are increased and anti-inflammatory lipoxins are decreased. Upregulation of lipoxins is effective in counteracting inflammation and infection. However, whether combining a lipoxin receptor agonist with a specific leukotriene A4 hydrolase (LTA4H) inhibitor could enhance these protective effects has not to our knowledge been investigated. Therefore, we explored the effect of lipoxin receptor agonist BML-111 and JNJ26993135 a specific LTA4H inhibitor that prevents the production of pro-inflammatory LTB4 on tight junction proteins disrupted by P. aeruginosa filtrate (PAF) in human airway epithelial cell lines H441 and 16HBE-14o. Pre-treatment with BML-111 prevented an increase in epithelial permeability induced by PAF and conserved ZO-1 and claudin-1 at the cell junctions. JNJ26993135 similarly prevented the increased permeability induced by PAF, restored ZO-1 and E-cadherin and reduced IL-8 but not IL-6. Cells pre-treated with BML-111 plus JNJ26993135 restored TEER and permeability, ZO-1 and claudin-1 to the cell junctions. Taken together, these data indicate that the combination of a lipoxin receptor agonist with a LTA4H inhibitor could provide a more potent therapy., Competing Interests: The authors have declared that no competing interest exist., (Copyright: © 2023 Kalsi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Intestinal epithelium functions as a tissue barrier to prevent interaction between the internal compartment and the external milieu. Intestinal barrier function also determines epithelial polarity for the absorption of nutrients and the secretion of waste products. These vital functions require strong integrity of tight junction proteins. In fact, intestinal tight junctions that seal the paracellular space can restrict mucosal-to-serosal transport of hostile luminal contents. Tight junctions can form both an absolute barrier and a paracellular ion channel. Although defective tight junctions potentially lead to compromised intestinal barrier and the development and progression of gastrointestinal (GI) diseases, no FDA-approved therapies that recover the epithelial tight junction barrier are currently available in clinical practice. Here, we discuss the impacts and regulatory mechanisms of tight junction disruption in the gut and related diseases. We also provide an overview of potential therapeutic targets to restore the epithelial tight junction barrier in the GI tract.

Acute kidney injury (AKI) is characterized by a rapid reduction in kidney function caused by various etiologies. Tubular epithelial cell dysregulation plays a pivotal role in the pathogenesis of AKI. Tight junction (TJ) is the major molecular structure that connects adjacent epithelial cells and is critical in maintaining barrier function and determining the permeability of epithelia. TJ proteins are dysregulated in various types of AKI, and some reno-protective drugs can reverse TJ changes caused by insult. An in-depth understanding of TJ regulation and its causality with AKI will provide more insight to the disease pathogenesis and will shed light on the potential role of TJs to serve as novel therapeutic targets in AKI., (© 2023 Wiley Periodicals LLC.)

Tight junctions act as a barrier between epithelial cells to limit the transport of the paracellular substance, which is a required function in various tissues to sequestrate diverse microenvironments and maintain a normal physiological state. Tight junctions are complexes that contain various proteins, like transmembrane proteins, scaffolding proteins, signaling proteins, etc. Defects in those tight junction- related proteins can lead to hearing loss in humans which is also recapitulated in many model organisms. The disruption of the barrier between the endolymph and perilymph caused by tight junction abnormalities will affect the microenvironment of hair cells; and this could be the reason for this type of hearing loss. Besides their functions as a typical barrier and channel, tight junctions are also involved in many signaling networks to regulate gene expression, cell proliferation, and differentiation. This review will summarize the structures, localization, and related signaling pathways of hearingrelated tight junction proteins and their potential contributions to the hearing disorder., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The intestinal epithelium forms a physical barrier assembled by intercellular junctions, preventing luminal pathogens and toxins from crossing it. The integrity of tight junctions is critical for maintaining intestinal health as the breakdown of tight junction proteins leads to various disorders. Redox reactions are closely associated with energy metabolism. Understanding the regulation of tight junctions by cellular metabolism and redox status in cells may lead to the identification of potential targets for therapeutic interventions. In vitro and in vivo models have been utilized in investigating intestinal barrier dysfunction and in particular the free-living soil nematode, Caenorhabditis elegans , may be an important alternative to mammalian models because of its convenience of culture, transparent body for microscopy, short generation time, invariant cell lineage and tractable genetics.

Differentiated air-liquid interface models are the current standard to assess the mucociliary phenotype using clinically-derived samples in a controlled environment. However, obtaining basal progenitor airway epithelial cells (AEC) from the lungs is invasive and resource-intensive. Hence, we applied a tissue engineering approach to generate organotypic sinonasal AEC (nAEC) epithelia to determine whether they are predictive of bronchial AEC (bAEC) models. Basal progenitor AEC were isolated from healthy participants using a cytological brushing method and differentiated into epithelia on transwells until the mucociliary phenotype was observed. Tissue architecture was assessed using H&E and alcian blue/Verhoeff-Van Gieson staining, immunofluorescence (for cilia via acetylated α-tubulin labelling) and scanning electron microscopy. Differentiation and the formation of tight-junctions were monitored over the culture period (day 1-32) by quantifying trans-epithelial electrical resistance. End point (day 32) tight junction protein expression was assessed using Western blot analysis of ZO-1, Occludin-1 and Claudin-1. Reverse transcription qPCR-array was used to assess immunomodulatory and autophagy-specific transcript profiles. All outcome measures were assessed using R-statistical software. Mucociliary architecture was comparable for nAEC and bAEC-derived cultures, e.g. cell density P = 0.55, epithelial height P = 0.88 and cilia abundance P = 0.41. Trans-epithelial electrical resistance measures were distinct from day 1-14, converged over days 16-32, and were statistically similar over the entire culture period (global P < 0.001). This agreed with end-point (day 32) measures of tight junction protein abundance which were non-significant for each analyte (P > 0.05). Transcript analysis for inflammatory markers demonstrated significant variation between nAEC and bAEC epithelial cultures, and favoured increased abundance in the nAEC model (e.g. TGFβ and IL-1β; P < 0.05). Conversely, the abundance of autophagy-related transcripts were comparable and the range of outcome measures for either model exhibited a considerably more confined uncertainty distribution than those observed for the inflammatory markers. Organotypic air-liquid interface models of nAEC are predictive of outcomes related to barrier function, mucociliary architecture and autophagy gene activity in corresponding bAEC models. However, inflammatory markers exhibited wide variation which may be explained by the sentinel immunological surveillance role of the sinonasal epithelium., (© 2022. The Author(s).)

Occludin, tricellulin, and marvelD3 belong to the tight junction (TJ)-associated MARVEL protein family. Occludin and tricellulin jointly contribute to TJ strand branching point formation and epithelial barrier maintenance. However, whether marvelD3 has the same function remains unclear. Furthermore, the roles of the carboxy-terminal cytoplasmic tail, which is conserved in occludin and tricellulin, on the regulation of TJ strand morphology have not yet been explored in epithelial cells. We established tricellulin/occludin/marveld3 triple-gene knockout (tKO) MDCK II cells and evaluated the roles of marvelD3 in the TJ strand structure and barrier function using MDCK II cells and a mathematical model. The complexity of TJ strand networks and paracellular barrier did not change in tKO cells compared to that in tricellulin/occludin double-gene knockout (dKO) cells. Exogenous marvelD3 expression in dKO cells did not increase the complexity of TJ strand networks and epithelial barrier tightness. The expression of the carboxy-terminal truncation mutant of tricellulin restored the barrier function in the dKO cells, whereas occludin lacking the carboxy-terminal cytoplasmic tail was not expressed on the plasma membrane. These data suggest that marvelD3 does not affect the morphology of TJ strands and barrier function in MDCK II cells and that the carboxy-terminal cytoplasmic tail of tricellulin is dispensable for barrier improvement., (© 2022 New York Academy of Sciences.)

Claudin-5 is the most enriched tight junction protein at the blood-brain barrier. Perturbations in its levels of expression have been observed across numerous neurological and neuropsychiatric conditions; however, pathogenic variants in the coding sequence of the gene have never been reported previously. Here, we report the identification of a novel de novo mutation (c.178G>A) in the CLDN5 gene in two unrelated cases of alternating hemiplegia with microcephaly. This mutation (G60R) lies within the first extracellular loop of claudin-5 and based on protein modelling and sequence alignment, we predicted it would modify claudin-5 to become an anion-selective junctional component as opposed to a purely barrier-forming protein. Generation of stably transfected cell lines expressing wild-type or G60R claudin-5 showed that the tight junctions could still form in the presence of the G60R mutation but that the barrier against small molecules was clearly attenuated and displayed higher Cl- ion permeability and lower Na+ permeability. While this study strongly suggests that CLDN5 associated alternating hemiplegia is a channelopathy, it is also the first study to identify the conversion of the blood-brain barrier to an anion-selective channel mediated by a dominant acting variant in CLDN5., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Conditions that cause the loss of epithelial barrier integrity are often accompanied by dysregulation of tight junction protein expression and/or localization. Recently, we have reported that patients with mutations in SLC12A2 , the gene encoding the basolateral Na + -K + -2Cl - cotransporter (NKCC1), suffer from severe gastrointestinal deficits, including chronic gastrointestinal inflammation, gastrointestinal hemorrhage, intestinal obstruction, and constipation. Although the intestinal inflammation observed in patients with loss of NKCC1 function may or may not be due to tight junction dysfunction, we investigated whether the loss of NKCC1 function affects paracellular ion transport and epithelial barrier function. Wild-type HT29-MTX-E12 and CRISPR/Cas9-mediated NKCC1 knockout (KO) HT29 clones were tested for tight junction protein expression and localization. Tightness of epithelial cell monolayer was assessed by measurement of transepithelial electrical resistance and permeability of molecular tracers in transwell filters. Tight junction protein localization was assessed by immunofluorescence. Loss of NKCC1 expression strongly increases the expression of claudin-2 and occludin in epithelial cell monolayers. Loss of NKCC1 significantly reduces the transepithelial electrical resistance (TER) indicating an increase in paracellular ions flux, consistent with upregulation of the cation-selective and channel-forming claudin-2. In addition, NKCC1-KO monolayers showed a significant increase in the paracellular flux of small molecules like fluorescein (0.33 kDa), whereas the permeability of higher molecular weight TRITC-Dextran (4 kDa and 70 kDa) remained unchanged. Thus, NKCC1 regulates tight junction protein expression and loss of NKCC1 function affects epithelial barrier integrity.

The effectiveness of resveratrol (RES) on intestinal barrier dysfunction and colitis has been extensively studied. However, the specific effects of its microbial metabolites on gut barrier function remain unclear. Hence, we compared the protective effects of RES and its microbial metabolites dihydroresveratrol (DHR) and 3-(4-hydroxyphenyl)-propionic acid (4HPP) against intestinal barrier injury and colitis. Only 4HPP and RES significantly reduced paracellular permeability and the secretion of proinflammatory cytokines in lipopolysaccharides (LPS)-treated intestinal Caco-2 cells, which was consistent with the upregulation in tight junction (TJ) proteins. Furthermore, RES and 4HPP ameliorated intestinal barrier dysfunction and colonic inflammation in colitis mice, while DHR did not. In particular, the expressions of intestinal TJ proteins and Muc2 were restored by RES and 4HPP. The molecular mechanism involved the adenosine monophosphate-activated protein kinase (AMPK)-mediated activation of CDX2 and the regulation of the SIRT1/NF-κB pathway. These findings provide new insights into understanding the protective effects of RES against intestinal barrier damage and colitis.

Usually, duodenal barriers are investigated using intestinal cell lines like Caco-2, which in contrast to native tissue are limited in cell-type representation. Organoids can consist of all intestinal cell types and are supposed to better reflect the in vivo situation. Growing three-dimensionally, with the apical side facing the lumen, application of typical physiological techniques to analyze the barrier is difficult. Organoid-derived monolayers (ODMs) were developed to overcome this. After optimizing culturing conditions, ODMs were characterized and compared to Caco-2 and duodenal tissue. Tight junction composition and appearance were analyzed, and electrophysiological barrier properties, like paracellular and transcellular barrier function and macromolecule permeability, were evaluated. Furthermore, transcriptomic data were analyzed. ODMs had tight junction protein expression and paracellular barrier properties much more resembling the originating tissue than Caco-2. Transcellular barrier was similar between ODMs and native tissue but was increased in Caco-2. Transcriptomic data showed that Caco-2 expressed fewer solute carriers than ODMs and native tissue. In conclusion, while Caco-2 cells differ mostly in transcellular properties, ODMs reflect trans- and paracellular properties of the originating tissue. If cultured under optimized conditions, ODMs possess reproducible functionality, and the variety of different cell types makes them a suitable model for human tissue-specific investigations., (© 2022 The Authors. Annals of the New York Academy of Sciences published by Wiley Periodicals LLC on behalf of New York Academy of Sciences.)

Epithelial cells are the first line of mucosal defense. In the intestine, a single layer of epithelial cells must establish a selectively permeable barrier that supports nutrient absorption and waste secretion while preventing the leakage of potentially harmful luminal materials. Key to this is the tight junction, which seals the paracellular space and prevents unrestricted leakage. The tight junction is a protein complex established by interactions between members of the claudin, zonula occludens, and tight junction-associated MARVEL protein (TAMP) families. Claudins form the characteristic tight junction strands seen by freeze-fracture microscopy and create paracellular channels, but the functions of ZO-1 and occludin, founding members of the zonula occludens and TAMP families, respectively, are less well defined. Recent studies have revealed that these proteins have essential noncanonical (nonbarrier) functions that allow them to regulate epithelial apoptosis and proliferation, facilitate viral entry, and organize specialized epithelial structures. Surprisingly, neither is required for intestinal barrier function or overall health in the absence of exogenous stressors. Here, we provide a brief overview of ZO-1 and occludin canonical (barrier-related) functions, and a more detailed examination of their noncanonical functions., (© 2022 New York Academy of Sciences.)

Pituitary endocrine cells are supplied by Sox2-expressing stem/progenitor cells in the anterior lobe of the adult pituitary gland. These SOX2-positive cells are maintained in two types of microenvironments (niches): the marginal cell layer (MCL)-niche and the parenchymal-niche. Recently, we isolated dense SOX2-positive cell clusters from the parenchymal-niche by taking advantage of their resistance to protease treatment as parenchymal stem/progenitor cell (PS)-clusters. In the present study, by analyzing these isolated PS-clusters, we attempted to identify novel structural characteristics of pituitary stem/progenitor cell niches. Quantitative real-time PCR showed that tight junction-related genes were distinctly expressed in the isolated PS-clusters. Immunocytostaining showed that the tight junction molecules, ZO-1 and occludin, were localized in the apical membrane facing the pseudo-follicle-like structure of the isolated PS-clusters regardless of the expression of S100β, which distinguishes the sub-population of SOX2-positive cells. Furthermore, immunohistochemistry of the pituitary glands of adult rats clearly demonstrated that ZO-1 and occludin were densely present in the parenchymal-niche encircling the pseudo-follicle, while they were observed in the apical membrane in the MCL-niche facing the residual lumen. Collectively, these tight junction-related proteins might be involved in the architecture and maintenance of the plasticity of pituitary stem/progenitor cell niches.

The blood-spinal cord barrier (BSCB) is a physiological barrier between the blood and spinal cord parenchyma. This study aims to determine whether Y-27632, a Rho-associated protein kinase (ROCK) inhibitor, can protect the BSCB using in vivo models. The Evans blue fluorescence assay was used to detect leakage of the BSCB. Western blotting was used to define alterations in ROCK-related and tight junction (TJ) protein expression. Immunofluorescence triple-staining was used to evaluate histologic alterations in TJs. Locomotor function was evaluated using the open-field test, the Basso-Beattie-Bresnahan score, and footprint analysis. Two peaks of BSCB leakage after spinal cord injury (SCI) occurred at 24 h and 5 days. The ROCK inhibitor reduced the BSCB leakage at the second peak after SCI. Moreover, the ROCK inhibitor ameliorated the integrity of the BSCB and improved motor function recovery after SCI by regulating the phosphorylation of myosin phosphatase subunit-1 (MYPT1) and cofilin. ROCK inhibitors might protect the BSCB, which provides a new strategy for transitioning SCI treatment from the bench to bedside., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Ammonia is one of the major metabolites produced by intestinal microorganisms; however, its role in intestinal homeostasis is poorly understood. The present study investigated the regulation of intestinal tight junction (TJ) proteins by ammonia and the underlying mechanisms in human intestinal Caco-2 cells. Ammonia (15, 30, and 60 mM) increased the permeability of the cells in a dose-dependent manner, as indicated by reduced transepithelial electrical resistance and increased dextran flux. Immunoblot and immunofluorescence analyses revealed that the ammonia-induced increase in TJ permeability reduced the membrane localization of TJ proteins such as zonula occludens (ZO)1, ZO2, occludin, claudin-1, and claudin-3. DNA microarray analysis identified a biological pathway "response to reactive oxygen species" enriched by ammonia treatment, indicating the induction of oxidative stress in the cells. Ammonia treatment also increased the malondialdehyde content and decreased the ratio of reduced to oxidized glutathione. Meanwhile, ammonia treatment-induced mitochondrial dysfunction, as indicated by the downregulation of genes associated with the electron transport chain, reduction of the cellular ATP, NADH, and tricarboxylic acid cycle intermediate content, and suppression of the mitochondrial membrane potential. In contrast, N-acetyl cysteine reversed the ammonia-induced impairment of TJ permeability and structure without affecting the mitochondrial parameters. Collectively, ammonia impaired the TJ barrier by increasing oxidative stress in Caco-2 cells. A mitochondrial dysfunction is possibly an event preceding ammonia-induced oxidative stress. The findings of this study could potentially improve our understanding of the interplay between intestinal microorganisms and their hosts., (© 2021 Federation of American Societies for Experimental Biology.)

As a serious trauma of the neurological system, spinal cord injury (SCI) results in permanent disability, gives rise to immediate vascular damage and a wide range of matters that induce the breakage of blood spinal cord barrier (BSCB). SCI activates the expression of MMP-2/9, which are considered to accelerate the disruption of BSCB. Recent research shows that Dl-3-n-butylphthalide (NBP) exerted protective effects on blood spinal cord barrier in animals after SCI, but the underlying molecular mechanism of NBP on the BSCB undergoing SCI is unknown. Here, our research show that NBP inhibited the expression of MMP-2/9, then improved the permeability of BSCB following SCI. After the T9 level of spinal cord performed with a moderate injury, NBP was managed by intragastric administration and further performed once a day. NBP remarkably improved the permeability of BSCB and junction proteins degration, then promoted locomotion recovery. The protective effect of NBP on BSCB destruction is related to the regulation of MMP-2/9 induced by SCI. Moreover, NBP obviously inhibited the MMP-2/9 expression and junction proteins degradation in microvascular endothelial cells. In conclusion, our results indicate that MMP-2/9 are relevant to the breakdown of BSCB, NBP impairs BSCB destruction through inhibiting MMP-2/9 and promotes functional recovery subjected to SCI. NBP is likely to become a new nominee as a therapeutic to treat SCI via a transigent BSCB., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Microbes employ autoinducers of quorum sensing (QS) for population communication. Although the autoinducer of Pseudomonas aeruginosa LasI-LasR system, N-(3-oxododecanoyl)- l-homoserine lactone (3OC12), has been reported with deleterious effects on host cells, its biological effects on integrity of the intestinal epithelium and epithelial barrier are still unclear and need further investigation. In the present study, flow cytometry, transcriptome analysis and western blot technology have been adopted to investigate the potential molecular mechanisms of 3OC12 and its structurally similar analogs damage to intestinal epithelial cells. Our results indicated that 3OC12 and 3OC14 trigger apoptosis rather than necrosis and ferroptosis in intestinal epithelial cells. RNA-sequencing combined with bioinformatics analysis showed that 3OC12 and 3OC14 reduced the expression of genes from extracellular matrix (ECM)-receptor interaction pathway. Consistently, protein expressions from ECM and tight junction-associated pathway were significantly reduced after 3OC12 and 3OC14 challenge. In addition, 3OC12 and 3OC14 led to blocked cell cycle, decreased mitochondrial membrane potential, increased reactive oxygen species level and elevated Ca 2+ concentration. Reversely, the antioxidant NAC could effectively mitigate the reduced expression of ECM and tight junction proteins caused by 3OC12 and 3OC14 challenge. Collectively, this study demonstrated that QS autoinducer exposure to intestinal epithelial cells ablates the ECM and tight junctions by triggering oxidative stress and apoptosis, and finally disrupts the intestinal epithelial barrier. These findings provide a rationale for defensing QS-dependent bacterial infections and potential role of NAC for alleviating the syndrome., (© 2020 Wiley Periodicals LLC.)

Disruptions in the intestinal epithelial barrier can result in devastating consequences and a multitude of disease syndromes, particularly among preterm neonates. The association between barrier dysfunction and intestinal dysbiosis suggests that the intestinal barrier function is interactive with specific gut commensals and pathogenic microbes. In vitro and in vivo studies demonstrate that probiotic supplementation promotes significant upregulation and relocalization of interepithelial tight junction proteins, which form the microscopic scaffolds of the intestinal barrier. Probiotics facilitate some of these effects through the ligand-mediated stimulation of several toll-like receptors that are expressed by the intestinal epithelium. In particular, bacterial-mediated stimulation of toll-like receptor-2 modulates the expression and localization of specific protein constituents of intestinal tight junctions. Given that ingested prebiotics are robust modulators of the intestinal microbiota, prebiotic supplementation has been similarly investigated as a potential, indirect mechanism of barrier preservation. Emerging evidence suggests that prebiotics may additionally exert a direct effect on intestinal barrier function through mechanisms independent of the gut microbiota. In this review, we summarize current views on the effects of pro- and prebiotics on the intestinal epithelial barrier as well as on non-epithelial cell barrier constituents, such as the enteric glial cell network. Through continued investigation of these bioactive compounds, we can maximize their therapeutic potential for preventing and treating gastrointestinal diseases associated with impaired intestinal barrier function and dysbiosis.

Leaky gut is a condition of increased paracellular permeability of the intestine due to compromised tight junction barriers. In recent years, this affliction has drawn the attention of scientists from different fields, as a myriad of studies prosecuted it to be the silent culprit of various immune diseases. Due to various controversies surrounding its culpability in the clinic, approaches to leaky gut are restricted in maintaining a healthy lifestyle, avoiding irritating factors, and practicing alternative medicine, including the consumption of supplements. In the current study, we investigate the tight junction-modulating effects of processed Aloe vera gel (PAG), comprising 5-400-kD polysaccharides as the main components. Our results show that oral treatment of 143 mg/kg PAG daily for 10 days improves the age-related leaky gut condition in old mice, by reducing their individual urinal lactulose/mannitol (L/M) ratio. In concordance with in vivo experiments, PAG treatment at dose 400 μg/mL accelerated the polarization process of Caco-2 monolayers. The underlying mechanism was attributed to enhancement in the expression of intestinal tight junction-associated scaffold protein zonula occludens (ZO)-1 at the translation level. This was induced by activation of the MAPK/ERK signaling pathway, which inhibits the translation repressor 4E-BP1. In conclusion, we propose that consuming PAG as a complementary food has the potential to benefit high-risk patients.

Blood brain barrier (BBB) dysfunction developed with aging is related to brain microvascular endothelial cells (BMECs) injury and losses of tight junctions (TJs). In the present study, we found that Alisol A 24-acetate (AA), a natural compound frequently used as treatment against vascular diseases was essential for BMECs injury and TJs degradation. Our experimental results showed that AA enhanced cell viability and increased zonula occludens-1 (ZO-1), claudin-5, and occludin expression in the oxygen-glucose deprivation (OGD)-induced BMECs. The exploration of the underlying mechanism revealed that AA restrained miR-92a-3p, a noncoding RNA involved in endothelial cells senescence and TJs impairment. To test the role of the miR-92a-3p in BMECs, the cells were transfected with miR-92a-3p mimics and inhibitor. The results showed that miR-92a-3p mimics inhibited cell viability and elevated lactate dehydrogenase (LDH) levels as well as suppressed ZO-1, claudin-5 and occludin expression, while the miR-92a-3p inhibitor reversed the above results. These findings were similar to the therapeutic effects of AA in the OGD-induced BMECs. Bioinformatics analysis and dual-luciferase assay confirmed ZO-1 and occludin were the target genes of miR-92a-3p mediated AA protective roles. In summary, the data demonstrated that AA protected against BMECs damage and TJs loss through the inhibition of miR-92a-3p expression. This provided evidence for AA application in aging-associated BBB protection.

The blood-brain barrier (BBB) comprises the interface between blood, brain and cerebrospinal fluid. Its primary function, which is mainly carried out by tight junctions, is to stabilize the tightly controlled microenvironment of the brain. To study the development and maintenance of the BBB, as well as various roles their intrinsic mechanisms that play in neurological disorders, suitable measurements are required to demonstrate integrity and functional changes at the interfaces between the blood and brain tissue. Markers and plasma proteins with different molecular weight (MW) are used to measure the permeability of BBB. In addition, the expression changes of tight-junction proteins form the basic structure of BBB, and imaging modalities are available to study the disruption of BBB. In the present review, above mentioned methods are depicted in details, together with the pros and cons as well as the differences between these methods, which maybe benefit research studies focused on the detection of BBB breakdown.

Larazotide acetate (LA) is a single-chain peptide of eight amino acids that acts as a tight junction regulator to restore intestinal barrier function. LA is currently being studied in phase III clinical trials and is orally administered to adult patients with celiac disease as an adjunct therapeutic to enhance intestinal barrier function that has been disrupted by gliadin-induced immune reactivity. Mechanistically, LA is thought to act as a zonulin antagonist to reduce zonulin-induced increases in barrier permeability and has been associated with the redistribution and rearrangement of tight junction proteins and actin filaments to restore intestinal barrier function. More recently, LA has been linked to inhibition of myosin light chain kinase, which likely reduces tension on actin filaments, thereby facilitating tight junction closure. Small (rodent) and large (porcine) animal studies have been conducted that demonstrate the importance of LA as a tight junction regulatory peptide in conditions other than celiac disease, including collagen-induced arthritis in mice and intestinal ischemic injury in pigs.

Background: The mucus integrity and abnormal inflammatory response are the crucial biomarker of inflammatory bowel disease (IBD). Velvet antler (VA) has been used as traditional Chinese medicines for many years. Anti-inflammatory property was demonstrated via suppression of cyclooxygenase-2 and cytokines protein expression. And it has further proved to promote wound healing in streptozotocin-induced diabetic rats model. The aforementioned functionalities of VA extracts may be associated with the treatment of IBD. Thus, the aim of present study was to evaluate the effect of velvet antler water extracts form Formosan Sambar deer ( Rusa unicolor swinhoei , SVAE) and red deer ( Cervus elaphus , RVAE) on the barrier function and to investigate the possible mechanism using in vitro model. Methods: Human colonic epithelial cell models (Caco-2) were co-cultured with various concentrations of both SVAE and RVAE (250-500 µg mL -1 ) in dextran sulfate sodium (DSS)-induced colitis model. Trans-epithelial electrical resistance (TEER) value and the macromolecule permeability of Fluorescein isothiocyanate (FITC)-labeled dextran were measured to evaluate the integrity of monolayer of Caco-2. Western blotting was performed for analysis of protein expressions of occludin, Zonula occludens-1 (ZO-1), claudin-1, claudin-2 and myosin light chain kinase (MLCK). The cytotoxicity was conducted by MTT assay. Results: Results indicated that both SVAE and RVAE could enhance integrity of monolayer in dextran sulfate sodium (DSS)-induced colonic epithelial cell model (Caco-2) through reducing the decline of trans-epithelial electrical resistance (TEER) and macromolecule permeability at the concentration of 250 μg mL -1 . RVAE significantly increased the expression of tight junction proteins (occludin and ZO-1) while SVAE significantly reduced the activity of MLCK ( P < 0.05.). Elevated C-C chemokine ligand 20 (CCL20) production suggested that both SVAE and RVAE could enhance the repair of epithelial cell. Besides, MTT assay revealed that both extracts showed no cytotoxicity. Conclusion: Thus, SVAE and RVAE supplementation may attenuate barrier damage by enhancing the occludin and ZO-1 protein expression, decreasing MLCK expression, promoting the CCL20 production. In the future, animal study is needed for further confirmation., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Tight junctions form a selectively permeable barrier that limits paracellular flux across epithelial-lined surfaces. Small molecules (less than ∼8 Å diameter) can traverse the junction via the size- and charge-selective, high-conductance pore pathway. In contrast, the low-conductance leak pathway accommodates larger macromolecules (up to ∼100 Å diameter) and is not charge-selective. Flux across the tight junction-independent, high-conductance, non-selective, unrestricted pathway occurs at sites of epithelial damage. Cytokines can regulate each of these pathways, but commonly used measures of barrier function cannot discriminate between tight junction regulation and epithelial damage. This article describes methods for culturing intestinal epithelial cell monolayers and assessing the impact of cytokine treatment on leak and unrestricted pathway permeabilities. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation and culture of cell monolayers in Transwells Basic Protocol 2: Assessment of cytokine (IFNγ and TNF) treatment effects on barrier function Support Protocol: Immunofluorescent staining of monolayers Basic Protocol 3: Multiplex flux assay., (© 2020 Wiley Periodicals LLC.)

Loss of the blood-retinal barrier (BRB) integrity and subsequent damage to the neurovascular unit in the retina are the underlying reasons for diabetic retinopathy (DR). Damage to BRB eventually leads to severe visual impairment in the absence of prompt intervention. Diabetic macular edema and proliferative DR are the advanced stages of the disease where BRB integrity is altered. Primary mechanisms contributing to BRB dysfunction include loss of cell-cell barrier junctions, vascular endothelial growth factor, advanced glycation end products-induced damage, and oxidative stress. Although much is known about the involvement of adherens and tight-junction proteins in the regulation of vascular permeability in various diseases, there is a significant gap in our knowledge on the junctional proteins expressed in the BRB and how BRB function is modulated in the diabetic retina. In this review article, we present our current understanding of the molecular composition of BRB, the changes in the BRB junctional protein turnover in DR, and how BRB functional modulation affects vascular permeability and macular edema in the diabetic retina., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Drusen are focal deposits between the retinal pigment epithelium (RPE) and Bruch's membrane in the retina of patients with age-related macular degeneration. Amyloid-β is one of the important components of drusen, which leads to local inflammation. Furthermore, intracellular amyloid-β disrupts tight junctions of the RPE. However, the intracellular mechanisms linking intracellular amyloid-β and tight-junction disruption are not clear. In this study, intracellular amyloid-β oligomers activated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65, leading to the disorganization of tight junctions of the RPE in mice after subretinal injection of amyloid-β. Amyloid-β also triggered NF-κB activation in the RPE cells in confluent culture, which was inhibited by the suppression of the advanced glycosylation end product-specific receptor. NF-κB inhibition by an IκB kinase inhibitor prevented the suppression of expression of tight-junction proteins, zonula occuludens-1 and occludin in RPE cells. In addition, tight-junction complexes remained intact in the RPE of mice with NF-κB inhibition, although there were intracellular amyloid-β oligomers. These data suggested that NF-κB inhibition might be a therapeutic approach to prevent amyloid-β-mediated tight-junction disruption., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The blood-brain barrier (BBB) maintains the optimal microenvironment for brain function. Tight junctions (TJs) allow endothelial cells to adhere to each other, leading to the formation of a barrier that prevents the penetration of most molecules via transcellular routes. Evidence has indicated that seizure-induced vascular endothelial growth factor (VEGF) type 2 receptor (VEGFR-2) pathway activation weakens TJs, inducing vasodilatation and increasing vascular permeability and subsequent brain injury. The present study focused on investigating the expression levels of VEGF-related (VEGF-A and VEGFR-2) and TJ-related proteins (claudin-5, occludin and ZO-1) in the neocortical microvasculature of patients with drug-resistant temporal lobe epilepsy (TLE). The results obtained from hippocampal sclerosis TLE (HS-TLE) patients were compared with those obtained from patients with TLE secondary to lesions (lesion-TLE) and autopsy samples. The Western blotting and immunofluorescence results showed that VEGF-A and VEGFR-2 protein expression levels were increased in HS-TLE and lesion-TLE patients compared to autopsy group. On the other hand, claudin-5 expression was higher in HS-TLE patients and lesion-TLE patients than autopsies. The expression level of occludin and ZO-1 was decreased in HS-TLE patients. Our study described modifications to the integrity of the BBB that may contribute to the pathogenesis of TLE, in which the VEGF system may play an important role. We demonstrated that the same modifications were present in both HS-TLE and lesion-TLE patients, which suggests that seizures modify these systems and that they are not associated with the establishment of epilepsy., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Introduction: Cerebrovascular pathologies including cerebral amyloid angiopathy (CAA) and blood-brain barrier (BBB) dysregulation are prominent features in the majority of Alzheimer's disease (AD) cases., Methods: We performed neuropathologic and biochemical studies on a large, neuropathologically confirmed human AD cohort (N = 469). Amounts of endothelial tight junction proteins claudin-5 (CLDN5) and occludin (OCLN), and major AD-related molecules (amyloid beta [Aβ40], Aβ42, tau, p-tau, and apolipoprotein E) in the temporal cortex were assessed by ELISA., Results: Higher levels of soluble tau, insoluble p-tau, and apolipoprotein E (apoE) were independently correlated with lower levels of endothelial tight junction proteins CLDN5 and OCLN in AD brains. Although high Aβ40 levels, APOE ε4, and male sex were predominantly associated with exacerbated CAA severity, those factors did not influence tight junction protein levels., Discussion: Refining the molecular mechanisms connecting tau, Aβ, and apoE with cerebrovascular pathologies is critical for greater understanding of AD pathogenesis and establishing effective therapeutic interventions for the disease., (© 2020 The Authors. Alzheimer's & Dementia published by Wiley Periodicals, Inc. on behalf of Alzheimer's Association.)

Intestinal epithelial cells (IECs) exist in a metabolic state of low oxygen tension termed "physiologic hypoxia." An important factor in maintaining intestinal homeostasis is the transcription factor hypoxia-inducible factor (HIF), which is stabilized under hypoxic conditions and mediates IEC homeostatic responses to low oxygen tension. To identify HIF transcriptional targets in IEC, chromatin immunoprecipitation (ChIP) was performed in Caco-2 IECs using HIF-1α- or HIF-2α-specific antibodies. ChIP-enriched DNA was hybridized to a custom promoter microarray (termed ChIP-chip). This unbiased approach identified autophagy as a major HIF-1-targeted pathway in IEC. Binding of HIF-1 to the ATG9A promoter, the only transmembrane component within the autophagy pathway, was particularly enriched by exposure of IEC to hypoxia. Validation of this ChIP-chip revealed prominent induction of ATG9A, and luciferase promoter assays identified a functional hypoxia response element upstream of the TSS. Hypoxia-mediated induction of ATG9A was lost in cells lacking HIF-1. Strikingly, we found that lentiviral-mediated knockdown (KD) of ATG9A in IECs prevents epithelial barrier formation by >95% and results in significant mislocalization of multiple tight junction (TJ) proteins. Extensions of these findings showed that ATG9A KD cells have intrinsic abnormalities in the actin cytoskeleton, including mislocalization of the TJ binding protein vasodilator-stimulated phosphoprotein. These results implicate ATG9A as essential for multiple steps of epithelial TJ biogenesis and actin cytoskeletal regulation. Our findings have novel applicability for disorders that involve a compromised epithelial barrier and suggest that targeting ATG9A may be a rational strategy for future therapeutic intervention.

Sepsis induces both intestinal hyperpermeability and epithelial apoptosis. While each has been implicated in mediating sepsis mortality, the relationship between these two processes is unclear. We hypothesized that preventing intestinal apoptosis would prevent gut barrier dysfunction. To test this hypothesis, transgenic mice that overexpress the anti-apoptotic protein Bcl-2 in the gut epithelium (Fabpl-Bcl-2 mice) and wild-type (WT) mice were subjected to sham laparotomy or cecal ligation and puncture and orally gavaged with fluorescein isothiocyanate conjugated-dextran (FD-4) 5 h before sacrifice. Serum FD-4 concentration was assayed to measure intestinal permeability, and jejunal tight junctions were assayed for mRNA and protein expression. Baseline FD-4 concentration was similar between WT and Fabpl-Bcl-2 mice. Intestinal permeability increased 6, 12, 24, and 48 h following sepsis in WT mice; however, FD-4 concentration was significantly lower at each timepoint in Fabpl-Bcl-2 mice. In addition, there were no statistically significant changes in permeability between septic and sham transgenic mice. Intestinal mRNA expression of claudin 3, claudin 5, and occludin was lower in septic Fabpl-Bcl-2 mice, while claudin 4 mRNA levels were higher in Fabpl-Bcl-2 mice. In contrast, no differences were detected in claudins 2, 7, 15, JAM-A, or ZO-1. Protein levels followed the same trend for all tight junction mediators different between WT and Fabpl-Bcl-2 mice except occludin was significantly higher in transgenic mice. Together these results demonstrate that decreasing intestinal epithelial apoptosis prevents hyperpermeability following sepsis via tight junction alterations which may be at least partially responsible for improved survival conferred by Bcl-2 overexpression.

The tight junction (TJ) and barrier function of colonic epithelium is highly sensitive to ionizing radiation. We evaluated the effect of lysophosphatidic acid (LPA) and its analog, Radioprotein-1, on γ-radiation-induced colonic epithelial barrier dysfunction using Caco-2 and m-IC C12 cell monolayers in vitro and mice in vivo. Mice were subjected to either total body irradiation (TBI) or partial body irradiation (PBI-BM5). Intestinal barrier function was assessed by analyzing immunofluorescence localization of TJ proteins, mucosal inulin permeability, and plasma lipopolysaccharide (LPS) levels. Oxidative stress was analyzed by measuring protein thiol oxidation and antioxidant mRNA. In Caco-2 and m-IC C12 cell monolayers, LPA attenuated radiation-induced redistribution of TJ proteins, which was blocked by a Rho-kinase inhibitor. In mice, TBI and PBI-BM5 disrupted colonic epithelial tight junction and adherens junction, increased mucosal permeability, and elevated plasma LPS; TJ disruption by TBI was more severe in Lpar2 -/- mice compared to wild-type mice. RP1, administered before or after irradiation, alleviated TBI and PBI-BM5-induced TJ disruption, barrier dysfunction, and endotoxemia accompanied by protein thiol oxidation and downregulation of antioxidant gene expression, cofilin activation, and remodeling of the actin cytoskeleton. These data demonstrate that LPAR2 receptor activation prevents and mitigates γ-irradiation-induced colonic mucosal barrier dysfunction and endotoxemia., (© 2020 Federation of American Societies for Experimental Biology.)

Microbiome therapy has attracted a keen interest from both research and business sectors. Our lab has been applying this "second genome" platform to assess the functionality of herbal medicines with fulfilling results. In this study, we applied this platform to assess the potential cancer-preventive effects of three selected adaptogenic plants. The flower buds from these plants were used to constitute Preparations SL and FSP according to the receipts of two commonly consumed Chinese medicinal decoctions for gastrointestinal discomfort. Preparation SL contains Sophorae japonica and Lonicerae Japonicae, and Preparation FSP contains Sophorae japonica and Gardenia Jasminoides. SL and FSP extracts significantly (p < 0.001) lowered the polyp burden, as well as the expressions of oncogenic signaling molecules, such as MAPK/ERK, PI3K/AKT, and STAT3 in Apc Min/+ mice. The inflamed gut was alleviated by shifting M1 to M2 macrophage phenotypes and the associated immune cytokines. The other remarkable change was on the extracellular tight junction protein complex, where the occludin, ZO-1, ICAM-1, E-cadherin were significantly (p < 0.05) upregulated while the N-cadherin and β-catenin were downregulated in the treated mice. The above physiological changes in the gut epithelial barrier were companied with the changes in gut microbiome. The 16S Sequencing data revealed a marked decrease in the potential pathogens (especially Helicobacter species and hydrogen sulfide producing-bacteria) and the increase in beneficial bacteria (especially for species from the genera of Akkermansia, Barnesiella, Coprococcus, Lachnoclostridium, and Ruminococcus). The majority of which were the short-chain fatty acids (SCFAs) producers. Meanwhile SCFAs-sensing G protein-coupled receptors (GPCRs), including GPR41, GPR43, and GPR109a were also significantly upregulated. In a recent report, we proved that the bacteria-derived SCFAs plays an essential role to the anti-cancer effects of the mushroom polysaccharides and saponins in Apc Min/+ mice. In this study, we further demonstrated that butyrate treatment could enhance the extracellular tight junction protein complex as effective as the treatments with SL and FSP to the Apc Min/+ mice. Our findings provide strong evidence of the vital role of the SCFA-producers and their metabolites to the cancer-preventive properties of the SL and FSP preparations., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Tight junction (TJ) plays an important role in regulating paracellular fluid transport in salivary glands; however, little is known about the involvement of TJs in diabetes salivary glands. This study aimed to investigate the alterations of TJs and their possible contribution in diabetes-induced hyposalivation. Here, we observed that the morphologies of submandibular glands (SMGs) were impaired, characterized by enlarged acini accumulation with giant secretory granules, which were significantly reduced in atrophic ducts in SMGs of db/db mice, a spontaneous model of type-2 diabetes. However, the secretory granules were increased and scattered in the acini of diabetes parotid glands (PGs). Other ultrastructural damages including swollen mitochondria, expansive endoplasmic reticulum, and autophagosomes were observed in the diabetes group. The levels of TJ proteins including claudin-1 (Cldn1) and claudin-3 (Cldn3) were increased, whereas those of claudin-4 (Cldn4), occludin (Ocln), and zonula occludens-1 (ZO-1) were decreased in SMGs of db/db mice. Higher Cldn1 and Cldn3 and lower claudin-10 (Cldn10) and Ocln levels were observed in PGs of diabetes mice. Taken together, the structures of SMGs and PGs were impaired in diabetes mice, and the disruption of TJ integrity in both SMGs and PGs may contribute to diabetes-induced hyposalivation., (© 2020 Anatomical Society.)

Overcoming the blood-brain barrier (BBB) to enable the treatment of central nervous system (CNS) diseases is an active field of research. Modulating or opening the tight junctions (TJs) in brain endothelial cells is one method to enable a range of small-molecular-weight drugs to cross the BBB via the paracellular route. Over the past 2 decades, the molecular understanding of TJ proteins in the BBB has significantly improved, and several agonists and antagonists have been tested for modulation of the TJs. In this review, we discuss the composition of TJ proteins in the BBB and introduce indirect pharmacological TJ modulators, which target regulators of TJ proteins, and direct TJ modulators, which can selectively inhibit functions of TJ proteins., (Copyright © 2020 Elsevier Ltd. All rights reserved.)