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Allergens and Th2 cytokines affect the homeostatic environment in the airways, leading to increased mucus production by goblet cells associated with altered adherens junctional complex (AJC) and tight junction (TJ) proteins responsible for maintaining epithelial barrier function. Circadian clock-dependent regulatory mechanisms such as inflammation and epithelial barrier function are gaining more attention due to their therapeutic potential against allergic inflammatory lung diseases. Currently, there are no studies to support whether REV-ERBα activation can attenuate Th2 cytokine-induced epithelial barrier dysfunction in human bronchial epithelial cells. We hypothesized that Th2 cytokine-induced epithelial barrier dysfunction may be protected by activating REV-ERBα. Treatment with Th2 cytokines or HDM significantly reduced the cell impedance, as confirmed by transepithelial electrical resistance (TEER). However, pre-treatment with SR10067 attenuated Th2 cytokine-induced barrier dysfunction, such as decreased permeability, improved TEER, localization of AJC and TJ proteins, and mRNA and protein levels of selected epithelial barrier and circadian clock targets. Overall, we showed for the first time that REV-ERBα activation regulates altered epithelial barrier function that may have direct implications for the treatment of asthma and other allergic diseases., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Multilayered proliferation in an adherent culture as well as proliferation in a suspension culture is a characteristic feature of cancer cells. We previously showed using T47D human mammary cancer cells that nectin-4, upregulated in many cancer cells, cis-interacts with ErbB2 and its trastuzumab-resistant splice variants, p95-ErbB2 and ErbB2ΔEx16, and enhances DNA synthesis mainly through the PI3K-AKT pathway in an adherent culture. We showed here that only the combination of nectin-4 and p95-ErbB2, but not that of nectin-4 and ErbB2 or that of nectin-4 and ErbB2ΔEx16, cooperatively enhanced multilayered T47D cell proliferation through the Hippo pathway-mediated SOX2 gene expression in an adherent culture. T47D cells expressed the components of the apical junctional complex (AJC) consisting of adherens junctions (AJs) and tight junctions and cell polarity molecules, but not the AJ component afadin. The AJC and apicobasal polarity were disorganized in T47D cells in a monolayer and T47D cells stably expressing both nectin-4 and p95-ErbB2 in multilayers. These results indicate that nectin-4 and p95-ErbB2 play a stimulatory role in multilayered proliferation in an adherent culture., (© 2022 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Osmotic stress plays a crucial role in the pathogenesis of many gastrointestinal diseases. Lactobacillus casei and epidermal growth factor (EGF) effects on the osmotic stress-induced epithelial junctional disruption and barrier dysfunction were investigated. Caco-2 cell monolayers were exposed to osmotic stress in the presence or absence of L. casei or EGF, and the barrier function was evaluated by measuring inulin permeability. Tight junction (TJ) and adherens junction integrity were assessed by immunofluorescence confocal microscopy. The role of signaling molecules in the L. casei and EGF effects was determined by using selective inhibitors. Data show that pretreatment of cell monolayers with L. casei or EGF attenuates osmotic stress-induced TJ and adherens junction disruption and barrier dysfunction. EGF also blocked osmotic stress-induced actin cytoskeleton remodeling. U0126 (MEK1/2 inhibitor), the MAP kinase inhibitor, blocked EGF-mediated epithelial protection from osmotic stress. In contrast, the L. casei -mediated epithelial protection from osmotic stress was unaffected by U0126, AG1478 (EGFR tyrosine kinase inhibitor), SP600125 (JNK1/2 inhibitor), or SB202190 (P38 MAP kinase inhibitor). On the other hand, Ro-32-0432 (PKC inhibitor) blocked the L. casei -mediated prevention of osmotic stress-induced TJ disruption and barrier dysfunction. The combination of EGF and L. casei is more potent in protecting the barrier function from osmotic stress. These findings suggest that L. casei and EGF ameliorate osmotic stress-induced disruption of apical junctional complexes and barrier dysfunction in the intestinal epithelium by distinct signaling mechanisms.

Chronic alcohol alters the immune system enhancing the susceptibility to inflammation, bacterial, and viral infections in alcohol users. We have shown that alcohol causes increased permeability of mesenteric lymphatic vessels in alcohol-fed rats. The mechanisms of alcohol-induced lymphatic leakage are unknown. Endothelial cell monolayer permeability is controlled by junctional proteins complexes called tight junctions (TJ) and adherens junctions (AJ), and each can be regulated by MAPK activation. We hypothesize that ethanol induces lymphatic endothelial cell (LEC) permeability via disruption of LEC TJ through MAPK activation. An in vitro model of rat LECs was used. Ethanol-supplemented medium was added at concentrations of 0, 25, and 50 mM to confluent cells. Resistance-based barrier function, transwell permeability, cell viability, TJ, AJ, and MAPK protein activity, TJ and AJ gene expressions, and the role of p38 MAPK in barrier function regulation were measured. Ethanol increased the permeability of LECs compared to controls that was not associated with decreased cell viability. LECs treated with 50 mM ethanol showed an increase in phosphorylated levels of p38. No significant changes in TJ and AJ gene or protein expressions were observed after ethanol treatment. p38 inhibition prevented ethanol-induced increases in permeability. These findings suggest that p38 may play a role in the regulation of ethanol-induced LEC permeability, but altered permeability may not be associated with decreased TJ or AJ protein expression. Further investigation into junctional protein localization is needed to better understand the effects of ethanol on lymphatic endothelial cell-to-cell contacts and hyperpermeability.

The inclusion of a medicinal plant leaf extract (MPLE) from sage ( Salvia officinalis ) and lemon verbena ( Lippia citriodora ), rich in verbascoside and triterpenic compounds like ursolic acid, was evaluated in gilthead seabream ( Sparus aurata ) fed a low fishmeal-based diet (48% crude protein, 17% crude fat, 21.7 MJ kg -1 , 7% fishmeal, 15% fish oil) for 92 days. In particular, the study focused on the effect of these phytogenic compounds on the gut condition by analyzing the transcriptomic profiling (microarray analysis) and histological structure of the intestinal mucosa, as well as the histochemical properties of mucins stored in goblet cells. A total number of 506 differentially expressed genes (285 up- and 221 down-regulated) were found when comparing the transcriptomic profiling of the intestine from fish fed the control and MPLE diets. The gut transcripteractome revealed an expression profile that favored biological mechanisms associated to the 1) immune system, particularly involving T cell activation and differentiation, 2) gut integrity (i.e., adherens and tight junctions) and cellular proliferation, and 3) cellular proteolytic pathways. The histological analysis showed that the MPLE dietary supplementation promoted an increase in the number of intestinal goblet cells and modified the composition of mucins' glycoproteins stored in goblet cells, with an increase in the staining intensity of neutral mucins, as well as in mucins rich in carboxylated and weakly sulfated glycoconjugates, particularly those rich in sialic acid residues. The integration of transcriptomic and histological results showed that the evaluated MPLE from sage and lemon verbena is responsible for the maintenance of intestinal health, supporting gut homeostasis and increasing the integrity of the intestinal epithelium, which suggests that this phytogenic may be considered as a promising sustainable functional additive for aquafeeds., Competing Interests: JQ and JP are current NATAC BIOTECH S.L. employers. FER-L was Senior research associate by Consorcio Tecnológico de Sanidad Acuícola, Ictio Biotechnologies S.A. The remaining 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 © 2021 Salomón, Reyes-López, Tort, Firmino, Sarasquete, Ortiz-Delgado, Quintela, Pinilla-Rosas, Vallejos-Vidal and Gisbert.)

The presence of adherens junctions and the associated protein β-catenin are requirements for the development of glucose-stimulated insulin secretion (GSIS) in β-cells. Evidence indicates that modulation of β-catenin function in response to changes in glucose levels can modulate the levels of insulin secretion from β-cells but the role of β-catenin phosphorylation in this process has not been established. We find that a Ser552Ala version of β-catenin attenuates glucose-stimulated insulin secretion indicating a functional role for Ser552 phosphorylation of β-catenin in insulin secretion. This is associated with alterations F/G actin ratio but not the transcriptional activity of β-catenin. Both glucose and GLP-1 stimulated phosphorylation of the serine 552 residue on β-catenin. We investigated the possibility that an EPAC-PAK1 pathway might be involved in this phosphorylation event. We find that reduction in PAK1 levels using siRNA attenuates both glucose and GLP-1 stimulated phosphorylation of β-catenin Ser552 and the effects of these on insulin secretion in β-cell models. Furthermore, both the EPAC inhibitor ESI-09 and the PAK1 inhibitor IPA3 do the same in both β-cell models and mouse islets. Together this identifies phosphorylation of β-catenin at Ser552 as part of a cell signalling mechanism linking nutrient and hormonal regulation of β-catenin to modulation of insulin secretory capacity of β-cells and indicates this phosphorylation event is regulated downstream of EPAC and PAK1 in β-cells., (© 2021 The Author(s).)

Excessive vascular permeability occurs in inflammatory disease processes. Vascular endothelial cadherin (VE-cadherin) is an adhesion protein that controls vascular permeability. We identified monoclonal antibodies (mAbs) to human VE-cadherin that activate cell adhesion and inhibit the increased permeability of endothelial cell monolayers induced by thrombin receptor activator peptide-6 (TRAP-6). Two mAbs, 8A12c and 3A5a, reduce permeability, whereas an inhibitory mAb, 2E11d, enhances permeability. Activating mAbs also reduce permeability induced by tumor necrosis factor-α (TNF-α) and vascular endothelial cell growth factor (VEGF). The activating mAbs also stabilize the organization of the adherens junctions that are disrupted by TRAP-6, VEGF, or TNF-α. The activating mAbs act directly on the adhesive function of VE-cadherin because they did not block the accumulation of actin filaments stimulated by TRAP-6 and enhance physical cell-cell adhesion of VE-cadherin-expressing tissue culture cells. Therefore, VE-cadherin function can be regulated at the cell surface to control endothelial permeability. NEW & NOTEWORTHY Excessive vascular permeability is a serious complication of many inflammatory disease conditions. We have developed monoclonal antibodies that inhibit increases in endothelial monolayer permeability induced by several signaling factors by activating VE-cadherin mediated adhesion and stabilizing cell junctions. These antibodies and/or the mechanisms they reveal may lead to important therapeutics to treat vascular leakiness and inflammation.

Histamine-induced vascular leakage is a core process of allergic pathologies, including anaphylaxis. Here, we show that glycolysis is integral to histamine-induced endothelial barrier disruption and hyperpermeability. Histamine rapidly enhanced glycolysis in endothelial cells via a pathway that involved histamine receptor 1 and phospholipase C beta signaling. Consistently, partial inhibition of glycolysis with 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) prevented histamine-induced hyperpermeability in human microvascular endothelial cells, by abolishing the histamine-induced actomyosin contraction, focal adherens junction formation, and endothelial barrier disruption. Pharmacologic blockade of glycolysis with 3PO in mice reduced histamine-induced vascular hyperpermeability, prevented vascular leakage in passive cutaneous anaphylaxis and protected from systemic anaphylaxis. In conclusion, we elucidated the role of glycolysis in histamine-induced disruption of endothelial barrier integrity. Our data thereby point to endothelial glycolysis as a novel therapeutic target for human pathologies related to excessive vascular leakage, such as systemic anaphylaxis., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Objective: Intravenous iloprost improves Raynaud's phenomenon (RP) and promotes healing of digital ulcers in systemic sclerosis (SSc; scleroderma). Despite a short half-life, its clinical efficacy lasts weeks. Endothelial adherens junctions, which are formed by VE-cadherin clustering between endothelial cells (ECs), regulate endothelial properties including barrier function, endothelial-to-mesenchymal transition (EndoMT), and angiogenesis. We undertook this study to investigate the hypothesis that junctional disruption contributes to vascular dysfunction in SSc, and that the protective effect of iloprost is mediated by strengthening of those junctions., Methods: Dermal ECs from SSc patients and healthy controls were isolated. The effect of iloprost on ECs was examined using immunofluorescence, permeability assays, Matrigel tube formation, and quantitative polymerase chain reaction., Results: Adherens junctions in SSc were disrupted compared to normal ECs, as indicated by reduced levels of VE-cadherin and increased permeability in SSc ECs (P < 0.05). Iloprost increased VE-cadherin clustering at junctions and restored junctional levels of VE-cadherin in SSc ECs (mean ± SD 37.3 ± 4.3 fluorescence units) compared to normal ECs (mean ± SD 29.7 ± 3.4 fluorescence units; P < 0.05), after 2 hours of iloprost incubation. In addition, iloprost reduced permeability of monolayers, increased tubulogenesis, and blocked EndoMT in both normal and SSc ECs (n ≥ 3; P < 0.05). The effects in normal ECs were inhibited by a function-blocking antibody that prevents junctional clustering of VE-cadherin., Conclusion: Our data suggest that the long-lasting effects of iloprost reflect its ability to stabilize adherens junctions, resulting in increased tubulogenesis and barrier function and reduced EndoMT. These findings provide a mechanistic basis for the use of iloprost in treating SSc patients with RP and digital ulcers., (© 2020, American College of Rheumatology.)

Targeting the vascular endothelial growth factor (VEGF)/its receptor-2 (VEGFR-2) system has become a mainstay of treatment for many human diseases, including retinal diseases. We examined the therapeutic effect of recently developed N-acetylated Arg-Leu-Tyr-Glu (Ac-RLYE), a human plasminogen kringle-5 domain-derived VEGFR-2 antagonists, on the pathogenesis of diabetic retinopathy. Ac-RLYE inhibited VEGF-A-mediated VEGFR-2 activation and endothelial nitric oxide synthase (eNOS)-derived NO production in the retinas of diabetic mice. In addition, Ac-RLYE prevented the disruption of adherens and tight junctions and vascular leakage by inhibiting S-nitrosylation of β-catenin and tyrosine nitration of p190RhoGAP in the retinal vasculature of diabetic mice. Peptide treatment preserved the pericyte coverage of retinal capillaries by upregulating angiopoietin-2. These results suggest that Ac-RLYE potentially prevents blood-retinal barrier breakdown and vascular leakage by antagonizing VEGFR-2; Ac-RLYE can be used as a potential therapeutic drug for the treatment of diabetic retinopathy., (Copyright © 2020 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Inflammatory breast cancer is a highly aggressive form of breast cancer that forms clusters of tumor emboli in dermal lymphatics and readily metastasizes. These cancers express high levels of E-cadherin, the major mediator of adherens junctions, which enhances formation of tumor emboli. Previous studies suggest that E-cadherin promotes cancer when the balance between apical and basolateral cadherin complexes is disrupted. Here, we used immunohistochemistry of inflammatory breast cancer patient samples and analysis of cell lines to determine the expression of PLEKHA7, an apical adherens junction protein. We used viral transduction to re-express PLEKHA7 in inflammatory breast cancer cells and examined their aggressiveness in 2D and 3D cultures and in vivo. We determined that PLEKHA7 was deregulated in inflammatory breast cancer, demonstrating improper localization or lost expression in most patient samples and very low expression in cell lines. Re-expressing PLEKHA7 suppressed proliferation, anchorage independent growth, spheroid viability, and tumor growth in vivo. The data indicate that PLEKHA7 is frequently deregulated and acts to suppress inflammatory breast cancer. The data also promote the need for future inquiry into the imbalance between apical and basolateral cadherin complexes as driving forces in inflammatory breast cancer.

Objective: Septin 2 is localized at junctions in human microvascular endothelial monolayers. The junctional localization of septin 2 is necessary for organization of cell-cell adhesion proteins of endothelial cells. Approach and Results: Septin 2 was depleted at junctions by suppression of expression using shRNA, treatment with inflammatory cytokine, TNF (tumor necrosis factor)-α, and ectopic overexpression of septin 2 phosphatidylinositol 4,5-bisphosphate binding mutant defect in interaction with plasma membrane. Under those conditions, organizations and expression levels of various junctional proteins were analyzed. Confocal images of immunofluorescence staining showed substantial disorganization of adherens junctional proteins, nectin-2 and afadin, TJP (tight junction protein), ZO (zonula occludens)-1, and intercellular adhesion protein, PECAM-1 (platelet-endothelial cell adhesion molecule-1). Immunoblots for those proteins did not show significant changes in expression except for nectin-2 that highly increased in expression. Significant differential gene expression profiles and biological pathway analysis by septin 2 suppression and by TNF-α treatment using RNA-seq showed common overlapping pathways. The commonalities in expression may be consistent with the similar effects on the overall organization of cell-cell adhesion proteins., Conclusions: Localization of septin 2 at cell junctions are required for the arrangement of junctional proteins and the integrity of the barrier formed by endothelial monolayers.

Atherosclerosis is a chronic vascular inflammatory disease that initially starts from an arterial intima lesion and endothelial barrier dysfunction. The purpose of this study was to investigate the role of TM4SF19, a recently identified member of the transmembrane 4L six superfamily, in vascular endothelial cell adherens junctions. We found TM4SF19 expression was significantly increased in atherosclerotic plaques and sera of patients with coronary heart disease (CHD) compared with healthy people by immunohistochemistry and ELISA. In vitro, human umbilical vein endothelial cells (HUVECs) were stimulated by lipopolysaccharides (LPS). TM4SF19 and VE-cadherin expression as well as cell adherens junctions were assessed. Additionally, LPS could upregulate TM4SF19 expression and downregulate VE-cadherin expression in HUVECs in a concentration dependent manner. Overexpression of TM4SF19 substantially aggravated LPS-induced reduction of VE-cadherin expression and attenuation of vascular endothelial cell adherens junctions. However, both the decreased VE-cadherin expression and weakened cell adherens junctions induced by LPS could be dramatically reversed when the expression of TM4SF19 was depressed. This study is the first to reveal the effect of TM4SF19 on endothelial cell adherens junctions. Meanwhile, our results also provide novel therapeutic strategies for atherosclerotic diseases., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The roles of the Hippo‑Yes‑associated protein (YAP) pathway in lung injury and repair remain elusive. The present study examined the effects of systemic inhibition or stimulation of YAP activity on lung injury, repair and inflammation in a mouse model of lipopolysaccharide (LPS)‑induced lung injury. Mice were treated with or without YAP inhibitor, verteporfin, or with or without YAP stimulator, XMU‑MP‑1, and intraperitoneally injected with LPS (7.5 mg/kg). Lung injury and repair were evaluated by histological analysis and by testing for markers of lung injury. Lung inflammation was assessed by measuring tissue levels of inflammatory mediators. Lung injury was associated with a decreased, whereas lung repair was associated with an increased YAP activity evidenced by nuclear translocation. Lung injury was associated with a high level of lung inflammation and epithelial adherens junction disassembly, but not with cell proliferation or epithelial cell regeneration. The injury phase was defined as 0‑48 h post‑LPS injection, and the 48‑168 h time period was considered the repair phase. Inhibition of YAP activity at the injury phase, using verteporfin, exacerbated, whereas its stimulation, using XMU‑MP‑1, alleviated lung injury, lung inflammation and epithelial adherens junction disassembly. Inhibition or stimulation of YAP activity at the injury phase had no effects on cell proliferation or epithelial regeneration. By contrast, lung repair was associated with inflammation resolution, increased cell proliferation, epithelial regeneration and reassembly of epithelial adherens junctions. Inhibition of YAP activity at the repair phase delayed inflammation resolution, impeded lung recovery, inhibited cell proliferation and epithelial regeneration, and inhibited epithelial adherens junction reassembly. Stimulation of YAP activity at the repair phase reversed all these processes. The results of the current study demonstrated that the Hippo‑YAP activity serves a protective role against endotoxemic lung injury. The Hippo‑YAP activity alleviated lung inflammation and injury at the injury phase and promoted inflammation resolution and lung repair at the repair phase.

E-cadherin, a central component of the adherens junction (AJ), is a single-pass transmembrane protein that mediates cell-cell adhesion. The loss of E-cadherin surface expression, and therefore cell-cell adhesion, leads to increased cell migration and invasion. Treatment of colorectal cancer (CRC)-derived cells (SW-480 and HT-29) with 2.0 mM metformin promoted a redistribution of cytosolic E-cadherin to de novo formed puncta along the length of the contacting membranes of these cells. Metformin also promoted translocation from the cytosol to the plasma membrane of p120-catenin, another core component of the AJs. Furthermore, E-cadherin and p120-catenin colocalized with β-catenin at cell-cell contacts. Western blot analysis of lysates of CRC-derived cells revealed a substantial metformin-induced increase in the level of p120-catenin as well as E-cadherin phosphorylation on Ser 838/840 , a modification associated with β-catenin/E-cadherin interaction. These modifications in E-cadherin, p120-catenin and β-catenin localization suggest that metformin induces rebuilding of AJs in CRC-derived cells. Those modifications were accompanied by the inhibition of focal adhesion kinase (FAK), as revealed by a significant decrease in the phosphorylation of FAK at Tyr 397 and paxillin at Tyr 118 . These changes were associated with a reduction in the numbers, but an increase in the size, of focal adhesions and by the inhibition of cell migration. Overall, these observations indicate that metformin targets multiple pathways associated with CRC development and progression., (© 2020 Wiley Periodicals, Inc.)

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.)

Deoxynivalenol (DON) is one of the most widely distributed mycotoxins in the food chain, and the protective effect of kaempferol (KAM) pretreatment against the barrier dysfunction induced by DON in a Caco-2 cell model was investigated using a proteomic approach. The results showed that after KAM pretreatment, both the numbers of down- and up-regulated differentially expressed proteins were significantly lower than those in the DON group. Gene ontology analysis revealed that differentially expressed proteins were enriched in cell adhesion molecule binding, cell junction, and cell junction assembly. Further study demonstrated that KAM pretreatment affected the expression and assembly of tight junction and adherens junction proteins through the PKA pathway and MAPK/ERK pathway to improve the barrier integrity of the Caco-2 cell monolayer, and nutraceutical approach based on bioactive phytochemicals to improve the body's immunity might be an effective strategy to combat the adverse effects of mycotoxins on intestinal barrier function.

Allergic asthma is a common chronic lung inflammatory disease and seriously influences public health. We aim to investigate the effects of formononetin (FMN) and calycosin (CAL), 2 flavonoids in Radix Astragali, on allergic asthma and elucidate possible therapeutic targets. A house dust mite (HDM)-induced allergic asthma mouse model and TNF-α and Poly(I:C) co-stimulated human bronchial epithelial cell line (16HBE) were performed respectively in vivo and in vitro. The role of G protein-coupled estrogen receptor (GPER) was explored by its agonist, antagonist, or GPER small interfering RNA (siGPER). E-cadherin, occludin, and GPER were detected by western blotting, immunohistochemistry, or immunofluorescence. The epithelial barrier integrity was assessed by trans-epithelial electric resistance (TEER). Cytokines were examined by enzyme-linked immunosorbent assay (ELISA). The results showed that flavonoids attenuated pulmonary inflammation and hyperresponsiveness in asthmatic mice. These flavonoids significantly inhibited thymic stromal lymphopoietin (TSLP), increased occludin and restored E-cadherin in vivo and in vitro. The effects of flavonoids on occludin and TSLP were not interfered by ICI182780 (estrogen receptor antagonist), while blocked by G15 (GPER antagonist). Furthermore, compared with PPT (ERα agonist) and DPN (ERβ agonist), G1 (GPER agonist) significantly inhibited TSLP, up-regulated occludin, and restored E-cadherin. siGPER and TEER assays suggested that GPER was pivotal for the flavonoids on the epithelial barrier integrity. Finally, G1 attenuated allergic lung inflammation, which could be abolished by G15. Our data demonstrated that 2 flavonoids in Radix Astragali could alleviate allergic asthma by protecting epithelial integrity via regulating GPER, and activating GPER might be a possible therapeutic strategy against allergic inflammation., (©2020 Society for Leukocyte Biology.)

Zinc oxide nanoparticles (ZnONPs) are frequently encountered nanomaterials in our daily lives. Despite the benefits of ZnONPs in a variety of applications, many studies have shown potential health hazards of exposure to ZnONPs. We have shown that oropharyngeal aspiration of ZnONPs in mice increases lung inflammation. However, the detailed mechanisms underlying pulmonary inflammatory cell infiltration remain to be elucidated. Endothelium functions as a barrier between the blood stream and the blood vessel wall. Endothelial barrier dysfunction may increase infiltration of immune cells into the vessel wall and underlying tissues. This current study examined the effects of ZnONPs exposure on endothelial barriers. ZnONPs exposure increased leukocyte infiltration in the mouse lungs. In endothelial cells, ZnONPs reduced the continuity of tight junction proteins claudin-5 and zonula occludens-1 (ZO-1) at the cell junctions. ZnONPs induced adherens junction protein VE-cadherin internalization from membrane to cytosol and dissociation with β-catenin, leading to reduced and diffused staining of VE-cadherin and β-catenin at cell junctions. Our results demonstrated that ZnONPs disrupted both tight and adherens junctions, compromising the integrity and stability of the junction network, leading to inflammatory cell infiltration. Thus, ZnONPs exposure in many different settings should be carefully evaluated for vascular effects and subsequent health impacts., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results”.

Background: Severe neurological impairment is a problem after subarachnoid haemorrhage (SAH). Although volatile anaesthetics, such as sevoflurane, have demonstrated protective properties in many organs, their use in cerebral injury is controversial. Cerebral vasodilation may lead to increased intracranial pressure (ICP), but at the same time volatile anaesthetics are known to stabilise the SAH-injured endothelial barrier., Objective: To test the effect of sevoflurane on ICP and blood-brain barrier function., Design: Randomised study., Participants: One hundred male Wistar rats included, 96 analysed., Interventions: SAH was induced by the endoluminal filament method under ketamine/xylazine anaesthesia. Fifteen minutes after sham surgery or induction of SAH, adult male Wistar rats were randomised to 4 h sedation with either propofol or sevoflurane., Main Outcome Measures: Mean arterial pressure (MAP), ICP, extravasation of water (small), Evan's blue (intermediate) and IgG (large molecule) were measured. Zonula occludens-1 (ZO-1) and beta-catenin (β-catenin), as important representatives of tight and adherens junction proteins, were determined by western blot., Results: Propofol and sevoflurane sedation did not affect MAP or ICP in SAH animals. Extravasation of small molecules was higher in SAH-propofol compared with SAH-sevoflurane animals (79.1 ± 0.9 vs. 78.0 ± 0.7%, P = 0.04). For intermediate and large molecules, no difference was detected (P = 0.6 and P = 0.2). Both membrane and cytosolic fractions of ZO-1 as well as membrane β-catenin remained unaffected by the injury and type of sedation. Decreased cytosolic fraction of β-catenin in propofol-SAH animals (59 ± 15%) was found to reach values of sham animals (100%) in the presence of sevoflurane in SAH animals (89 ± 21%; P = 0.04)., Conclusion: This experiment demonstrates that low-dose short-term sevoflurane sedation after SAH in vivo did not affect ICP and MAP and at the same time may attenuate early brain oedema formation, potentially by preserving adherens junctions., Trial Registration: No 115/2014 Veterinäramt Zürich.

Acute Respiratory Distress Syndrome (ARDS) is a devastating disease process that involves dysregulated inflammation and decreased alveolar-capillary barrier function. Despite increased understanding of the pathophysiology, no effective targeted therapies exist to treat ARDS. Recent preclinical studies suggest that the multi-tyrosine kinase inhibitor, imatinib, which targets the Abl kinases c-Abl and Arg, has the potential to restore endothelial dysfunction caused by inflammatory agonists. Prior work demonstrates that imatinib attenuates LPS (lipopolysaccharide)-induced vascular leak and inflammation; however, the mechanisms underlying these effects remain incompletely understood. In the current study, we demonstrate that imatinib inhibits LPS-induced increase in the phosphorylation of CrkL, a specific substrate of Abl kinases, in human pulmonary endothelial cells. Specific silencing of Arg, and not c-Abl, attenuated LPS-induced pulmonary vascular permeability as measured by electrical cellular impedance sensing (ECIS) and gap formation assays. In addition, direct activation of Abl family kinases with the small molecule activator DPH resulted in endothelial barrier disruption that was attenuated by Arg siRNA. In complementary studies to characterize the mechanisms by which Arg mediates endothelial barrier function, Arg silencing was found to inhibit LPS-induced disruption of adherens junctions and phosphorylation of myosin light chains (MLC). Overall, these results characterize the mechanisms by which imatinib protects against LPS-induced endothelial barrier disruption and suggest that Arg inhibition may represent a novel strategy to enhance endothelial barrier function., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Calcitonin is expressed in the epithelium of endometrium, and modulates zonula adherens junctions which are composed of cadherin-catenins complex during the implantation window. Trophoblastic cells which have complex interaction with the epithelial cells of endometrium during implantation were demonstrated to have calcitonin receptors. Mechanism of action of calcitonin on trophoblastic cells has not yet been elucidated. Therefore, it was aimed to determine the effects of calcitonin on the expressions of β-catenin and phospho-β-catenin in a dose depended manner under the influence of progesterone and estrogen hormones (P + E) by using JAR cell line through the immunocytochemical and Western blot analyses. Moreover, adherens junctions (AJs) were ultrastructurally investigated to assess the involvement of cadherin-catenin complex in accordance with the changes in the specified parameters. Immunocytochemical analysis showed that only 10 nM calcitonin treated group had increased expression of membranous β-catenin compared to the control group, while there was decreased expression of β-catenin in the nucleus of all the experimental groups. Cytoplasmic expressions of the phospho-β-catenin decreased in all experimental groups compared to the control group while the decrease in the nuclear expression was remarkable in the groups treated with P + E, and P + E + 250 nM calcitonin. Western blot analysis showed that total β-catenin and phospho-β-catenin expressions were not significantly different. Ultrastructural analysis showed that increase in the number of AJs was noticeable in the group treated with 10 nM calcitonin. Overall, the localization and expression levels of β-catenin and phospho-β-catenin suggest that calcitonin could show its effects through the non-canonical pathway in the trophoblastic cells., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest. The authors alone are responsible for the content and writing of the paper., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Although in-vivo exposure of PM 2.5 has been suggested to initiate a disorder on vascular permeability, the effects and related mechanism has not been well defined. In this work, an obvious increase on vascular permeability has been confirmed in vivo by vein injection of PM 2.5 into Balb/c mouse. Human umbilical vein vascular endothelial cells and the consisted ex-vivo vascular endothelium were used as model to investigate the effects of PM 2.5 on the vascular permeability and the underlying molecular mechanism. Upon PM 2.5 exposure, the vascular endothelial growth factor receptor 2 on cell membrane phosphorylates and activates the downstream mitogen-activated protein kinase (MAPK)/ERK signaling. The adherens junction protein VE-cadherin sheds and the intercellular junction opens, damaging the integrity of vascular endothelium via paracellular pathway. Besides, PM 2.5 induces the intracellular reactive oxygen species (ROS) production and triggers the oxidative stress including activity decrease of superoxide dismutase, lactate dehydrogenase release and permeability increase of cell membrane. Taken together, the paracellular and transcellular permeability enhancement jointly contributes to the significant increase of endothelium permeability and thus vascular permeability upon PM 2.5 exposure. This work provides an insight into molecular mechanism of PM 2.5 associated cardiovascular disease and offered a real-time screening method for the health risk of PM 2.5 ., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The integrity of the intestinal mucosal barrier is important for the health of the host. In this study, longan pulp polysaccharides (LP) prevented the intestinal mucosal injury by increasing the expression of mucin 2, tight junction proteins zonulae occludens-1 (ZO-1), claudin-1, claudin-4, and adherens junction E-cadherin in cyclophosphamide-treated mice. To further identify the principle bioactive component of LP, four acidic polysaccharides (LPIa, LPIIa, LPIIIa, and LPIVa) were purified, and their intestinal protection activity in vitro was compared. LPIa, LPIIa, and LPIIIa displayed an ability to increase mRNA expression of ZO-1, claudin-1, occludin, and E-cadherin in differentiated Caco-2 cells, especially LPIa. LPIa has specific structure characteristics: porous surface structure, a high molecular weight (1.47 × 10 5 Da), and two specific glycosidic linkages of α-Araf-(1→ and →5)-α-Araf-(1→. These structure characteristics might primarily contribute to greater intestinal barrier protective effect of LPIa., (Copyright © 2019. Published by Elsevier Ltd.)

The pathophysiological mechanism of white matter hyperintensities of cerebral small vessel disease (CSVD) includes an impaired blood-brain barrier (BBB) with increased permeability. Neuroinflammation likely contributes to the disruption of the BBB in CSVD. Therefore, understanding the molecular mechanism of how neuroinflammation causes BBB damage is essential to preventing BBB disruption in CSVD. Matrix metalloproteinase 9 (MMP-9) contributes to BBB damage in neuroinflammatory diseases. In this study, we observed that interleukin-1β (IL-1β)-induced MMP-9 secretion in pericytes increased BBB permeability to sodium fluorescein (Na-F) by damaging the disruption of VE-cadherin, occludin, claudin-5, and zonula occludin-1 (ZO-1). Melatonin reduced BBB permeability to Na-F and inhibited the disruption of the adherens and tight junction proteins. Melatonin also downregulated MMP-9 and upregulated tissue inhibitor of metalloproteinases 1 ( TIMP-1 ) gene expression, which decreased the MMP-9/TIMP-1 ratio. In addition, nuclear translocation of NF-κB/p65 induced by IL-1β in pericytes upregulated MMP-9 expression, which was inhibited by the NF-κB inhibitor PDTC. However, the NOTCH3 inhibitor DAPT significantly inhibited NF-κB/p65 translocation to the nucleus, while melatonin in combination with DAPT significantly prevented NF-κB/p65 translocation than DAPT alone. Our results suggest that melatonin reduced MMP-9-induced permeability of the BBB. Melatonin reduced MMP-9 expression and activity, which was induced by IL-1β through the regulation of the NOTCH3/NF-κB signaling pathway in pericytes, suggesting that pericytes regulate BBB integrity and function.

Decellularized tissues offer a unique tool for developing regenerative biomaterials or in vitro platforms for the study of cell-extracellular matrix (ECM) interactions. One main challenge associated with decellularized lung tissue is that ECM components can be stripped away or altered by the detergents used to remove cellular debris. Without characterizing the composition of lung decellularized ECM (dECM) and the cellular response caused by the altered composition, it is difficult to utilize dECM for regeneration and specifically, engineering the complexities of the alveolar-capillary barrier. This study takes steps towards uncovering if dECM must be enhanced with lost ECM proteins to achieve proper epithelial barrier formation. To achieve this, the epithelial barrier function was assessed on dECM coatings with and without the systematic addition of several key basement membrane proteins. After comparing barrier function on collagen I, fibronectin, laminin, and dECM in varying combinations as an in vitro coating, the alveolar epithelium exhibited superior barrier function when dECM was supplemented with laminin as evidenced by trans-epithelial electrical resistance (TEER) and permeability assays. Increased barrier resistance with laminin addition was associated with upregulation of Claudin-18, E-cadherin, and junction adhesion molecule (JAM)-A, and stabilization of zonula occludens (ZO)-1 at junction complexes. The Epac/Rap1 pathway was observed to play a role in the ECM-mediated barrier function determined by protein expression and Epac inhibition. These findings revealed potential ECM coatings and molecular therapeutic targets for improved regeneration with decellularized scaffolds. STATEMENT OF SIGNIFICANCE: Efforts to produce a transplantable organ-scale biomaterial for lung regeneration has not been entirely successful to date, due to incomplete cell-cell junction formation, ultimately leading to severe edema in vivo. To fully understand the process of alveolar junction formation on ECM-derived biomaterials, this research has characterized and tailored decellularized ECM (dECM) to mitigate reductions in barrier strength or cell attachment caused by abnormal ECM compositions or detergent damage to dECM. These results indicate that laminin-driven Epac signaling plays a vital role in the stabilization of the alveolar barrier. Addition of laminin or Epac agonists during alveolar regeneration can reduce epithelial permeability within bioengineered lungs., (Copyright © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Morphological changes in the allograft of rat anterior cerebral vesicle at the early stages after transplantation into the peripheral nerve of an adult rat were studied by immunohistochemical methods. Immunohistochemical reaction to bromodeoxyuridine showed that the delay of mitotic division in neural stem/progenitor cells in the grafts occurred during S/G2 stage. In transplants of rat embryonic neocortex (E13), changes in the cell cycle of neural stem/progenitor cells in 3 h after transplantation into the nerve correlated with abnormal distribution of adherens junctions and interkinetic nuclear migration.

Endothelial colony-forming cells (ECFCs), a proliferative subpopulation of endothelial progenitor cells, are involved in angiogenesis and endothelial repair. In this study, we investigated endothelial barrier characteristics of ECFCs, whether vitamin D supports cell-cell adhesion and barrier integrity, and how it affects ECFC mobilization and actin dynamics. Although ECFC barrier was disrupted under inflammatory conditions, this effect was rescued by vitamin D treatment, leading to higher stability of an ECFC monolayer. Furthermore, vitamin D enhanced ECFC mobilization toward directional migration. In addition, immunocytochemistry, quantitative real-time PCR, and immunoblotting analysis showed that vitamin D increased endothelial interconnections through vascular endothelial cadherin (VE-cadherin) junctions and by impacting cell dynamics through cofilin and VE-cadherin phosphorylation. Our results suggest that vitamin D treatment efficiently counteracts inflammation in an ECFC monolayer, resulting in higher ECFC barrier integrity. This study provides evidence of a new beneficial effect of vitamin D for ECFC homeostasis.-Schröder-Heurich, B., von Hardenberg, S., Brodowski, L., Kipke, B., Meyer, N., Borns, K., von Kaisenberg, C. S., Brinkmann, H., Claus, P., von Versen-Höynck, F. Vitamin D improves endothelial barrier integrity and counteracts inflammatory effects on endothelial progenitor cells.

Lipocalin-2 (LCN2) is a stress protein, and can be hyper-produced by many kinds of cells after exposure to injury or disease conditions. In this study, we asked whether LCN2 may play a protective role in cerebral endothelium. After focal cerebral ischemia in rats, plasma levels of LCN2 were significantly elevated at 6, 12, and 24 hrs, and persisted until 3 days post-stroke. To assess the vascular mechanisms of LCN2, we used brain endothelial cell cultures to investigate its effects on neutrophil adhesion and endothelial barrier integrity. LCN2 did not affect neutrophil adhesion to endothelial cells either under normal conditions or after TNFα stimulation. TNFα significantly increased endothelial permeability, and LCN2 rescued endothelial permeability. Concomitantly, LCN2 restored the membrane distribution of the tight junction protein ZO-1 and the adherens junction protein VE-cadherin. Our findings suggest that elevated LCN2 in the blood after ischemic stroke might affect endothelial function, in part by reducing damage to endothelial junctional proteins and maintain blood-brain barrier integrity., Competing Interests: The authors have declared that no competing interests exist.

Endothelial hyperpermeability represents an initiating step in early atherosclerosis and it often occurs as a result of endothelial barrier dysfunction. Asiatic acid, a major triterpene isolated from Centella asiatica (L.) Urban, has previously been demonstrated to protect against tumor necrosis factor (TNF)-α-induced endothelial barrier dysfunction. The present study aimed to investigate the mechanisms underlying the barrier protective effect of asiatic acid in human aortic endothelial cells (HAECs). The localization of F-actin, diphosphorylated myosin light chain (diphospho-MLC), adherens junctions (AJs) and tight junctions (TJs) was studied using immunocytochemistry techniques and confocal microscopy. Their total protein expressions were examined using western blot analysis. The endothelial permeability was assessed using In Vitro Vascular Permeability Assay kits. In addition, intracellular redistribution of the junctional proteins was evaluated using subcellular fractionation kits. We show that asiatic acid stabilized F-actin and diphospho-MLC at the cell periphery and prevented their rearrangement stimulated by TNF-α. However, asiatic acid failed to attenuate cytochalasin D-induced increased permeability. Besides, asiatic acid abrogated TNF-α-induced structural reorganization of vascular endothelial (VE)-cadherin and β-catenin by preserving their reticulum structures at cell-cell contact areas. In addition, asiatic acid also inhibited TNF-α-induced redistribution of occludin and zona occludens (ZO)-1 in different subcellular fractions. In conclusion, the barrier-stabilizing effect of asiatic acid might be associated with preservation of AJs and prevention of TJ redistribution caused by TNF-α. This study provides evidence to support the potential use of asiatic acid in the prevention of early atherosclerosis, which is initiated by endothelial barrier dysfunction., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Vascular endothelial protein tyrosine phosphatase (VE-PTP) is essential for endothelial cells (ECs) adherens junction and vascular homeostasis; however, the regulatory mechanism of VE-PTP transcription is unknown, and a drug able to promote VE-PTP expression in ECs has not yet been reported in the literature. In this study, we used human ECs as a model to explore small molecule compounds able to promote VE-PTP expression, and found that atorvastatin, a HMG-CoA reductase inhibitor widely used in the clinic to treat hypercholesterolemia-related cardiovascular diseases, strongly promoted VE-PTP transcription in ECs through activating the VE-PTP promoter and upregulating the expression of the transcription factor, specificity protein 1 (SP1). Additionally, atorvastatin markedly reduced VE-cadherin-Y731 phosphorylation induced by cigarette smoke extract and significantly enhanced stability of endothelial adherens junctions. Together, our findings reveal that atorvastatin up-regulates VE-PTP expression, increases VE-cadherin protein levels, and decreases VE-cadherin-Y731 phosphorylation to strengthen EC adherens junctions and maintain vascular cell monolayer integrity, offering a new mechanism of atorvastatin against CSE-induced disruption of vascular integrity and relevant cardio-cerebrovascular disease., (Copyright © 2018. Published by Elsevier Inc.)

The Notch ligand delta-like ligand 4 (Dll4), upregulated by VEGF, is a key regulator of vessel morphogenesis and function, controlling tip and stalk cell selection during sprouting angiogenesis. Inhibition of Dll4 results in hypersprouting, nonfunctional, poorly perfused vessels, suggesting a role for Dll4 in the formation of mature, reactive, functional vessels, with low permeability and able to restrict fluid and solute exchange. We tested the hypothesis that Dll4 controls transvascular fluid exchange. A recombinant protein expressing only the extracellular portion of Dll4 [soluble Dll4 (sDll4)] induced Notch signaling in endothelial cells (ECs), resulting in increased expression of vascular-endothelial cadherin, but not the tight junctional protein zonula occludens 1, at intercellular junctions. sDll4 decreased the permeability of FITC-labeled albumin across EC monolayers, and this effect was abrogated by coculture with the γ-secretase inhibitor N -[ N -(3,5-difluorophenacetyl)-l-alanyl]- S -phenylglycine t -butyl ester. One of the known molecular effectors responsible for strengthening EC-EC contacts is PKA, so we tested the effect of modulation of PKA on the sDll4-mediated reduction of permeability. Inhibition of PKA reversed the sDll4-mediated reduction in permeability and reduced expression of the Notch target gene Hey1. Knockdown of PKA reduced sDLL4-mediated vascular-endothelial cadherin junctional expression. sDll4 also caused a significant decrease in the hydraulic conductivity of rat mesenteric microvessels in vivo. This reduction was abolished upon coperfusion with the PKA inhibitor H89 dihydrochloride. These results indicate that Dll4 signaling through Notch activation acts through a cAMP/PKA pathway upon intercellular adherens junctions, but not tight junctions, to regulate endothelial barrier function. NEW & NOTEWORTHY Notch signaling reduces vascular permeability through stimulation of cAMP-dependent protein kinase A.

The membrane pore-forming α-toxin is an important virulence factor of Staphylococcus aureus. Target cells can remove pores from their surface, but recent work shows that α-toxin may undermine this self-defense by clinging to epithelial cell junctions. The findings could lead to the development of novel remedies against S. aureus infections., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background/aims: Ouabain, a well-known plant-derived toxin, is also a hormone found in mammals at nanomolar levels that binds to a site located in the a-subunit of Na⁺,K⁺-ATPase. Our main goal was to understand the physiological roles of ouabain. Previously, we found that ouabain increases the degree of tight junction sealing, GAP junction-mediated communication and ciliogenesis. Considering our previous results, we investigated the effect of ouabain on adherens junctions., Methods: We used immunofluorescence and immunoblot methods to measure the effect of 10 nM ouabain on the cellular and nuclear content of E-cadherin, β-catenin and γ-catenin in cultured monolayers of Marin Darby canine renal cells (MDCK). We also studied the effect of ouabain on adherens junction biogenesis through sequential Ca²⁺ removal and replenishment. Then, we investigated whether c-Src and ERK1/2 kinases are involved in these responses., Results: Ouabain enhanced the cellular content of the adherens junction proteins E-cadherin, β-catenin and γ-catenin and displaced β-catenin and γ-catenin from the plasma membrane into the nucleus. Ouabain also increased the expression levels of E-cadherin and β-catenin in the plasma membrane after Ca²⁺ replenishment. These effects on adherens junctions were sensitive to PP2 and PD98059, suggesting that they depend on c-Src and ERK1/2 signaling. The translocation of β-catenin and γ-catenin into the nucleus was specific because ouabain did not change the localization of the tight junction proteins ZO-1 and ZO-2. Moreover, in ouabain-resistant MDCK cells, which express a Na⁺,K⁺-ATPase α1-subunit with low affinity for ouabain, this hormone was unable to regulate adherens junctions, indicating that the ouabain receptor that regulates adherens junctions is Na⁺,K⁺-ATPase., Conclusion: Ouabain (10 nM) upregulated adherens junctions. This novel result supports the proposition that one of the physiological roles of this hormone is the modulation of cell contacts., Competing Interests: The authors declare they have no conflicts of interest., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Hippo signaling is a critical pathway that integrates extrinsic and intrinsic mechanical cues to regulate organ size. Despite its essential role in organogenesis, little is known about its role in cell fate specification and differentiation. Here, we unravel a novel and unexpected role of the Hippo pathway effector Taz ( wwtr1 ) in controlling the size, shape and fate of a unique cell in the zebrafish ovary. We show that wwtr1 mutant females are infertile. In teleosts, fertilization occurs through the micropyle, a funnel-like opening in the chorion, formed by a unique enlarged follicle cell, the micropylar cell (MC). We describe here, for the first time, the mechanism that underlies the differentiation of the MC. Our genetic analyses show that Taz is essential for MC fate acquisition and subsequent micropyle formation in zebrafish. We identify Taz as the first bona fide MC marker and show that Taz is specifically and strongly enriched in the MC precursor. Altogether, we performed the first genetic and molecular characterization of the MC and propose that Taz is a key regulator of MC fate.This article has an associated 'The people behind the papers' interview., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Objective: Immune complex (IC) deposition activates polymorphonuclear neutrophils (PMNs), increases vascular permeability, and leads to organ damage in systemic lupus erythematosus and rheumatoid arthritis. The bioactive lipid sphingosine 1-phosphate (S1P), acting via S1P receptor 1 (S1P 1 ), is a key regulator of endothelial cell (EC) barrier function. This study was undertaken to investigate whether augmenting EC integrity via S1P 1 signaling attenuates inflammatory injury mediated by ICs., Methods: In vitro barrier function was assessed in human umbilical vein endothelial cells (HUVECs) by electrical cell-substrate impedance sensing. Phosphorylation of myosin light chain 2 (p-MLC-2) and VE-cadherin staining in HUVECs were assessed by immunofluorescence. A reverse Arthus reaction (RAR) was induced in the skin and lungs of mice with S1P 1 deleted from ECs (S1P 1 EC-knockout [ECKO] mice) and mice treated with S1P 1 agonists and antagonists., Results: S1P 1 agonists prevented loss of barrier function in HUVECs treated with IC-activated PMNs. S1P 1 ECKO and wild-type (WT) mice treated with S1P 1 antagonists had amplified RAR, whereas specific S1P 1 agonists attenuated skin and lung RAR in WT mice. ApoM-Fc, a novel S1P chaperone, mitigated EC cell barrier dysfunction induced by activated PMNs in vitro and attenuated lung RAR. Expression levels of p-MLC-2 and disruption of VE-cadherin, each representing manifestations of cell contraction and destabilization of adherens junctions, respectively, that were induced by activated PMNs, were markedly reduced by treatment with S1P 1 agonists and ApoM-Fc., Conclusion: Our findings indicate that S1P 1 signaling in ECs modulates vascular responses to IC deposition. S1P 1 agonists and ApoM-Fc enhance the EC barrier, limit leukocyte escape from capillaries, and provide protection against inflammatory injury. The S1P/S1P 1 axis is a newly identified target to attenuate tissue responses to IC deposition and mitigate end-organ damage., (© 2018, American College of Rheumatology.)

Despite the established oncogenic and profibrotic functions of enhancer of zeste homolog 2 (EZH2), a methyltransferase that induces histone H3 lysine 27 trimethylation (H3K27me3), its role in acute kidney injury (AKI) remains unclear. In this study, we demonstrated that EZH2 and H3K27me3 were upregulated in the murine kidney with AKI induced by either ischemia-reperfusion (I/R) or folic acid (FA). Pharmacologic inhibition of EZH2 with 3-deazaneplanocin A (3-DZNeP) prevented tubular injury in both models as demonstrated by reduced renal dysfunction, diminished neutrophil gelatinase-associated lipocalin expression and decreased renal tubular cell death. Injury to the kidney resulted in reduced expression of E-cadherin and ZO-1, whereas EZH2 inhibition largely preserved their expression. Moreover, 3-DZNep was effective in counteracting the increased expression of matrix metalloproteinase (MMP)-2 and MMP-9, as well as the phosphorylation of Raf-1 and ERK1/2 in the injured kidney. Conversely, blocking EZH2 reversed the decrease of tissue inhibitor of metalloproteinase (TIMP)-2 and metalloproteinase (TIMP)-3, and Raf kinase inhibitor protein (RKIP) in the kidney after acute injury. Similarly, oxidant injury to cultured kidney proximal tubular epithelial cells caused a decrease in the expression of E-cadherin, ZO-1, TIMP-2/-3, and RKIP, as well as an increase in the expression of MMP-2/9 and phosphorylation of Raf-1 ERK1/2. Blocking EZH2 with 3-DZNep or SiRNA hindered these responses. Thus, these results suggest that targeting EZH2 protects against AKI through a mechanism associated with the preservation of adhesion/junctions, reduction of matrix metalloproteinases and attenuation of the Raf-1/ERK1/2 pathway.

Xerostomia and hyposalivation are debilitating side effects for patients treated with ionizing radiation for head and neck cancer. Despite technological advances, collateral damage to the salivary glands remains a significant problem for patients and severely diminishes their quality of life. During the wound healing process, restoration of junctional contacts is necessary to maintain polarity, structural integrity, and orientation cues for secretion. However, little is known about whether these structural molecules are impacted following radiation damage and more importantly, during tissue restoration. We evaluated changes in adherens junctions and cytoskeletal regulators in an injury model where mice were irradiated with 5 Gy and a restoration model where mice injected postradiation with insulin-like growth factor 1 (IGF1) are capable of restoring salivary function. Using coimmunoprecipitation, there is a decrease in epithelial (E)-cadherin bound to β-catenin following damage that is restored to untreated levels with IGF1. Via its adaptor proteins, β-catenin links the cadherins to the cytoskeleton and part of this regulation is mediated through Rho-associated coiled-coil containing kinase (ROCK) signaling. In our radiation model, filamentous (F)-actin organization is fragmented, and there is an induction of ROCK activity. However, a ROCK inhibitor, Y-27632, prevents E-cadherin/β-catenin dissociation following radiation treatment. These findings illustrate that radiation induces a ROCK-dependent disruption of the cadherin-catenin complex and alters F-actin organization at stages of damage when hyposalivation is observed. Understanding the regulation of these components will be critical in the discovery of therapeutics that have the potential to restore function in polarized epithelium.

Aflatoxin M1 (AFM1) is a common mycotoxin in dairy milk, and it is typically concurrently present with other mycotoxins that may represent a threat to food safety. However, knowledge of how AFM1, alone or in combination with other mycotoxins, may affect human intestinal epithelial integrity remain to be established. We employed transcriptome and proteome analysis integrated with biological validation to reveal the molecular basis underlining the effect of exposure to AFM1, ochratoxin A (OTA), or both on the intestinal epithelial integrity of differentiated Caco-2 cells. Exposure to 4 μg/mL of OTA was found to disrupt human gut epithelial integrity, whereas 4 μg/mL of AFM1 did not. The integrated transcriptome and proteome analysis of AFM1 and OTA, alone or in combination, indicate the synergistic effect of the two mycotoxins in disrupting intestinal integrity. This effect was mechanistically linked to a broad range of pathways related to intestinal integrity enriched by down-regulated genes and proteins, associated with focal adhesion, adheren junction, and gap junction pathways. Furthermore, the cross-omics analysis of mixed AFM1 and OTA compared to OTA alone suggest that kinase family members, including myosin light-chain kinase, mitogen-activated protein kinases, and protein kinase C, are the potential key regulators in modulating intestinal epithelial integrity. These findings provide novel insight into the synergistic detrimental role of multiple mycotoxins in disrupting intestinal integrity and, therefore, identify potential targets to improve milk safety related to human health.

Blood-brain barrier (BBB) disruption and dysfunction result in brain edema, which is responsible for more than half of all deaths after severe traumatic brain injury (TBI). Fibroblast growth factor 21 (FGF21) has a potential neuroprotective function in the brain. However, the effects and underlying possible mechanism of action on BBB integrity following TBI remain unknown. The purpose of the current study was to determine the effects of FGF21 on BBB protection and TBI treatment. The effects of recombinant human FGF21 (rhFGF21) on BBB integrity and on tight junction (TJ) and adhesion junction (AJ) proteins were investigated both in a TBI mouse model and an in vitro BBB disruption model established with tumor necrosis factor alpha (TNF-α)-induced human brain microvascular endothelial cells (HBMECs). The ability of rhFGF21 to form an FGF21/FGFR1/β-klotho complex was confirmed by in vitro β-klotho small interfering RNA (siRNA) transfection and FGFR1 co-immunoprecipitation. In addition, the specific FGFR1 and peroxisome proliferator-activated receptor gamma (PPARγ) inhibitors PD173074 and GW9662, respectively, were applied to further explore the possible mechanism of rhFGF21 in BBB maintenance after TBI. rhFGF21 markedly reduced neurofunctional behavior deficits and cerebral edema degree, preserved BBB integrity, and recued brain tissue loss and neuron apoptosis in the mouse model after TBI. Both in vivo and in vitro, rhFGF21 upregulated TJ and AJ proteins, thereby preserving the BBB. Moreover, rhFGF21 activated PPARγ in TNF-α-induced HBMECs through formation of an FGF21/FGFR1/β-klotho complex. rhFGF21 protected the BBB through FGF21/FGFR1/β-klotho complex formation and PPARγ activation, which upregulated TJ and AJ proteins.

To understand the adverse effects of cholesterol crystals on vascular homeostasis, we have studied their effects on endothelial barrier function. Cholesterol crystals increased endothelial barrier permeability in a dose and time dependent manner. In addition, cholesterol crystals induced tyrosine phosphorylation of VE-cadherin and α-catenin, disrupting endothelial AJ and its barrier function and these effects required xanthine oxidase-mediated H 2 O 2 production, SHP2 inactivation and Frk activation. Similarly, feeding C57BL/6 mice with cholesterol-rich diet increased xanthine oxidase expression, H 2 O 2 production, SHP2 inactivation and Frk activation leading to enhanced tyrosine phosphorylation of VE-cadherin and α-catenin, thereby disrupting endothelial AJ and increasing vascular permeability. Resolvin D1, a specialized proresolving mediator, prevented all these adverse effects of cholesterol crystals and cholesterol-rich diet in endothelial cells and mice, respectively. Based on these observations, it is likely that cholesterol crystals via disrupting AJ increase vascular permeability, a critical event of endothelial dysfunction and specialized proresolving mediators such as Resolvin D1 exert protection against these effects., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Oral mucositis is a common side effect of cancer chemotherapy, with significant adverse impact on the delivery of anti-neoplastic treatment. There is a lack of consensus regarding the role of oral commensal microorganisms in the initiation or progression of mucositis because relevant experimental models are non-existent. The goal of this study was to develop an in vitro mucosal injury model that mimics chemotherapy-induced mucositis, where the effect of oral commensals can be studied. A novel organotypic model of chemotherapy-induced mucositis was developed based on a human oral epithelial cell line and a fibroblast-embedded collagen matrix. Treatment of organotypic constructs with 5-fluorouracil (5-FU) reproduced major histopathologic characteristics of oral mucositis, such as DNA synthesis inhibition, apoptosis and cytoplasmic vacuolation, without compromising the three-dimensional structure of the multilayer organotypic mucosa. Although structural integrity of the model was preserved, 5-FU treatment resulted in a widening of epithelial intercellular spaces, characterized by E-cadherin dissolution from adherens junctions. In a neutrophil transmigration assay we discovered that this treatment facilitated transport of neutrophils through epithelial layers. Moreover, 5-FU treatment stimulated key proinflammatory cytokines that are associated with the pathogenesis of oral mucositis. 5-FU treatment of mucosal constructs did not significantly affect fungal or bacterial biofilm growth under the conditions tested in this study; however, it exacerbated the inflammatory response to certain bacterial and fungal commensals. These findings suggest that commensals may play a role in the pathogenesis of oral mucositis by amplifying the proinflammatory signals to mucosa that is injured by cytotoxic chemotherapy., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Endothelium-dependent, nitric oxide-mediated dilatation is impaired in aging arteries. The dysfunction reflects increased production of reactive oxygen species (ROS), is reversed by inhibiting superoxide with superoxide dismutase (SOD) mimics, and is assumed to reflect superoxide-mediated inactivation of nitric oxide. However, the dysfunction also reflects Src-dependent degradation and loss of vascular-endothelial (VE)-cadherin from adherens junctions, resulting in a selective impairment in the ability of the junctions to amplify endothelial dilatation. Experiments therefore tested the hypothesis that SOD mimics might restore endothelial dilation in aging arteries by inhibiting Src and protecting endothelial adherens junctions. Tail arteries from young and aging Fisher 344 rats were processed for functional (pressure myograph), biochemical (immunoblot), and morphological (immunofluorescence) analyses. Cell-permeable SOD mimics [manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) or tempol] did not affect acetylcholine-induced dilatation in young arteries but increased responses and restored normal dilator function in aging arteries. In aging arteries, MnTMPyP decreased Src activity (immunoblots of Tyr 416 phosphorylated compared with total Src), increased the intensity and width of VE-cadherin staining at endothelial junctions, and increased VE-cadherin levels in Triton X-100-insoluble lysates, which represents the junctional protein. Because of aging-induced junctional disruption, inhibiting VE-cadherin clustering at adherens junctions with a function-blocking antibody does not affect acetylcholine-induced dilatation in aging arteries. However, the antibody prevented SOD mimics from restoring acetylcholine-induced dilatation in aging arteries. Therefore, SOD mimics improve impaired adherens junctions in aging endothelium, which is essential for SOD mimics to restore endothelium-dependent dilatation in aging arteries. The results suggest an important new pathological role for ROS in aging endothelium, namely, disruption of adherens junctions. NEW & NOTEWORTHY Aging-induced endothelial dysfunction is reversed by SOD mimics. This study demonstrates that they improve impaired adherens junctions in aging endothelium and that their restoration of endothelial dilatation is dependent on increased junctional activity. The results suggest a novel role for oxygen radicals in vascular aging, namely, disruption of adherens junctions.

Treatment of epithelial cells with interferon-γ and TNF-α (IFN/TNF) results in increased paracellular permeability. To identify relevant proteins mediating barrier disruption, we performed proximity-dependent biotinylation (BioID) of occludin and found that tagging of MARCKS-related protein (MRP; also known as MARCKSL1) increased ∼20-fold following IFN/TNF administration. GFP-MRP was focused at the lateral cell membrane and its overexpression potentiated the physiological response of the tight junction barrier to cytokines. However, deletion of MRP did not abrogate the cytokine responses, suggesting that MRP is not required in the occludin-dependent IFN/TNF response. Instead, our results reveal a key role for MRP in epithelial cells in control of multiple actin-based structures, likely by regulation of integrin signaling. Changes in focal adhesion organization and basal actin stress fibers in MRP-knockout (KO) cells were reminiscent of those seen in FAK-KO cells. In addition, we found alterations in cell-cell interactions in MRP-KO cells associated with increased junctional tension, suggesting that MRP may play a role in focal adhesion-adherens junction cross talk. Together, our results are consistent with a key role for MRP in cytoskeletal organization of cell contacts in epithelial cells., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

The development of three-dimensional models of reconstituted mouse epidermis (RME) has been hampered by the difficulty to maintain murine primary keratinocyte cultures and to achieve a complete epidermal stratification. In this study, a new protocol is proposed for the rapid and convenient generation of RME, which reproduces accurately the architecture of a normal mouse epidermis. During RME morphogenesis, the expression of differentiation markers such as keratins, loricrin, filaggrin, E-cadherin and connexins was followed, showing that RME structure at day 5 was similar to those of a normal mouse epidermis, with the acquisition of the natural barrier function. It was also demonstrated that RME responded to skin-relevant proinflammatory cytokines by increasing the expression of antimicrobial peptides and chemokines, and inhibiting epidermal differentiation markers, as in the human system. This new model of RME is therefore suitable to investigate mouse epidermis physiology further and opens new perspectives to generate reconstituted epidermis from transgenic mice., (Copyright © 2017 John Wiley & Sons, Ltd.)

Correction of barrier dysfunction and inflammation in acute lung injury (ALI) represents an important problem. Previous studies demonstrate barrier-protective and anti-inflammatory effects of bioactive lipid prostacyclin and its stable analog iloprost (ILO). We generated a phospholipase resistant synthetic phospholipid with iloprost attached at the sn-2 position (ILO-PC) and investigated its biological effects. In comparison to free ILO, ILO-PC caused sustained endothelial cell (EC) barrier enhancement, linked to more prolonged activation of Rap1 and Rac1 GTPases and their cytoskeletal and cell junction effectors: cortactin, PAK1, p120-catenin and VE-cadherin. ILO and ILO-PC equally efficiently suppressed acute, Rho GTPase-dependent EC hyper-permeability caused by thrombin. However, ILO-PC exhibited more sustained barrier-protective and anti-inflammatory effects in the model of chronic EC dysfunction caused by bacterial wall lipopolysacharide (LPS). ILO-PC was also more potent inhibitor of NFκB signaling and lung vascular leak in the murine model of LPS-induced ALI. Treatment with ILO-PC showed more efficient ALI recovery over 3 days after LPS challenge than free ILO. In conclusion, this study describes a novel synthetic phospholipid with barrier-enhancing and anti-inflammatory properties superior to existing prostacyclin analogs, which may be used as a prototype for future development of more efficient treatment for ALI and other vascular leak syndromes.

Accumulating evidences indicate that alcohol might play a causative in oral cancer. Unfortunately, in vitro cell systems, uncovering the molecular background of the underlying cell transformation process, are rare. Therefore, this study was conducted, to identify molecular changes and characterize their putative cell behavioral consequences in epitheloid (EPI) and fibroblastoid (FIB) oral keratinocyte phenotypes, arising from chronical alcohol treatment. Concerning adherens junctions (AJs), both EPI and FIB showed membrane-bound β-catenin, but exhibited differences for E-cadherin and zyxin. While EPI revealed E-cadherin/β-catenin membrane co-localization, which in parts also applied for zyxin, FIB membranes were devoid of E-cadherin and exhibited marginal zyxin expression. Fetal calf serum (FCS) administration in starved cells promoted proliferation in both keratinocyte phenotypes, whereat EPI and FIB yielded a strikingly modified FCS sensitivity on the temporal scale. Impedance measurement-based cell index detection yielded proliferation stimulation occurring much earlier in FIB (<20h) compared to EPI (>45h). Nuclear preference of the proliferation-associated YAP co-transcription factor in FIB was FCS independent, while it required FCS in EPI. Taken together, the lack of membrane-inherent E-cadherin/β-catenin co-localization together with low zyxin - reveals perturbation of AJ integrity in FIB. Regarding cell behavior, perturbed AJs in FIB correlate with temporal proliferation sensitivity towards FCS. CYF of 5.6 strongly suggests involvement of chromatin-bound YAP in FIB's proliferation temperosensitivity. These molecular differences detected for EPI and FIB are part of the underlying cell transformation process of alcohol-induced oral carcinogenesis, and indicate FIB being in a more advanced transformation stage., (Copyright © 2017 Elsevier B.V. All rights reserved.)

After spinal cord injury (SCI), the destruction of blood-spinal cord barrier (BSCB) is shown to accelerate gathering of noxious blood-derived components in the nervous system, leading to secondary neurodegenerative damages. SCI activates endoplasmic reticulum stress (ER stress), which is considered to evoke secondary damages of neurons and glia. Recent evidence indicates that Dl-3-n-butylphthalide (NBP) has the neuroprotective effect in ischaemic brain injury, but whether it has protective effects on SCI or not is largely unclear. Here, we show that NBP prevented BSCB disruption after SCI via inhibition of ER stress. Following a moderate contusion injury of the T9 level of spinal cord, NBP was administered by oral gavage and further treated once a day. NBP significantly attenuated BSCB permeability and breakdown of adherens junction (AJ) and tight junction (TJ) proteins, then improved locomotion recovery following SCI. The protective role of NBP on BSCB disruption is associated with the restrain of ER stress caused by SCI. Furthermore, NBP considerably constrained the expression of ER stress-associated proteins and degradation of TJ and AJ in human brain microvascular endothelial cells (HBMECs) treated with TG. In conclusion, our results indicate that ER stress is associated with the disruption of BSCB integrity after injury, NBP attenuates BSCB disruption via inhibiting ER stress and improve functional recovery following SCI., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Key Points: Ageing-induced endothelial dysfunction contributes to organ dysfunction and progression of cardiovascular disease. VE-cadherin clustering at adherens junctions promotes protective endothelial functions, including endothelium-dependent dilatation. Ageing increased internalization and degradation of VE-cadherin, resulting in impaired activity of adherens junctions. Inhibition of VE-cadherin clustering at adherens junctions (function-blocking antibody; FBA) reduced endothelial dilatation in young arteries but did not affect the already impaired dilatation in old arteries. After junctional disruption with the FBA, dilatation was similar in young and old arteries. Src tyrosine kinase activity and tyrosine phosphorylation of VE-cadherin were increased in old arteries. Src inhibition increased VE-cadherin at adherens junctions and increased endothelial dilatation in old, but not young, arteries. Src inhibition did not increase dilatation in old arteries treated with the VE-cadherin FBA. Ageing impairs the activity of adherens junctions, which contributes to endothelial dilator dysfunction. Restoring the activity of adherens junctions could be of therapeutic benefit in vascular ageing., Abstract: Endothelial dilator dysfunction contributes to pathological vascular ageing. Experiments assessed whether altered activity of endothelial adherens junctions (AJs) might contribute to this dysfunction. Aortas and tail arteries were isolated from young (3-4 months) and old (22-24 months) F344 rats. VE-cadherin immunofluorescent staining at endothelial AJs and AJ width were reduced in old compared to young arteries. A 140 kDa VE-cadherin species was present on the cell surface and in TTX-insoluble fractions, consistent with junctional localization. Levels of the 140 kDa VE-cadherin were decreased, whereas levels of a TTX-soluble 115 kDa VE-cadherin species were increased in old compared to young arteries. Acetylcholine caused endothelium-dependent dilatation that was decreased in old compared to young arteries. Disruption of VE-cadherin clustering at AJs (function-blocking antibody, FBA) inhibited dilatation to acetylcholine in young, but not old, arteries. After the FBA, there was no longer any difference in dilatation between old and young arteries. Src activity and tyrosine phosphorylation of VE-cadherin were increased in old compared to young arteries. In old arteries, Src inhibition (saracatinib) increased: (i) 140 kDa VE-cadherin in the TTX-insoluble fraction, (ii) VE-cadherin intensity at AJs, (iii) AJ width, and (iv) acetylcholine dilatation. In old arteries treated with the FBA, saracatinib no longer increased acetylcholine dilatation. Saracatinib did not affect dilatation in young arteries. Therefore, ageing impairs AJ activity, which appears to reflect Src-induced phosphorylation, internalization and degradation of VE-cadherin. Moreover, impaired AJ activity can account for the endothelial dilator dysfunction in old arteries. Restoring endothelial AJ activity may be a novel therapeutic approach to vascular ageing., (© 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.)

Resolvins are a novel class of lipid mediators that play an important role in the resolution of inflammation, although the underlying mechanisms are not very clear. To explore the anti-inflammatory mechanisms of resolvins, we have studied the effects of resolvin D1 (RvD1) on lipopolysaccharide (LPS)-induced endothelial barrier disruption as it is linked to propagation of inflammation. We found that LPS induces endothelial cell (EC) barrier disruption via xanthine oxidase (XO)-mediated reactive oxygen species (ROS) production, protein tyrosine phosphatase SHP2 inactivation and Fyn-related kinase (Frk) activation leading to tyrosine phosphorylation of α-catenin and VE-cadherin and their dissociation from each other affecting adherens junction (AJ) integrity and thereby increasing endothelial barrier permeability. RvD1 attenuated LPS-induced AJ disassembly and endothelial barrier permeability by arresting tyrosine phosphorylation of α-catenin and VE-cadherin and their dislocation from AJ via blockade of XO-mediated ROS production and thereby suppression of SHP2 inhibition and Frk activation. We have also found that the protective effects of RvD1 on EC barrier function involve ALX/FPR2 and GPR32 as inhibition or neutralization of these receptors negates its protective effects. LPS also increased XO activity, SHP2 cysteine oxidation and its inactivation, Frk activation, α-catenin and VE-cadherin tyrosine phosphorylation and their dissociation from each other leading to AJ disruption with increased vascular permeability in mice arteries and RvD1 blocked all these effects. Thus, RvD1 protects endothelial AJ and its barrier function from disruption by inflammatory mediators such as LPS via a mechanism involving the suppression of XO-mediated ROS production and blocking SHP2 inactivation., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)