Stimulatory role of nectin-4 and p95-ErbB2 in multilayered T47D cell proliferation.

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.

Lactobacillus casei and Epidermal Growth Factor Prevent Osmotic Stress-Induced Tight Junction Disruption in Caco-2 Cell Monolayers.

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.

Medicinal Plant Leaf Extract From Sage and Lemon Verbena Promotes Intestinal Immunity and Barrier Function in Gilthead Seabream (Sparus aurata).

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.

Ethanol-induced lymphatic endothelial cell permeability via MAP-kinase regulation.

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.

PLEKHA7, an Apical Adherens Junction Protein, Suppresses Inflammatory Breast Cancer in the Context of High E-Cadherin and p120-Catenin Expression.

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.

A role for PAK1 mediated phosphorylation of ß-catenin Ser552 in the regulation of insulin secretion.

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

Enhanced endothelial barrier function by monoclonal antibody activation of vascular endothelial cadherin.

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.

Glycolysis is integral to histamine-induced endothelial hyperpermeability.

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.

Dissecting the Cellular Mechanism of Prostacyclin Analog Iloprost in Reversing Vascular Dysfunction in Scleroderma.

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.

Human plasminogen-derived N-acetyl-Arg-Leu-Tyr-Glu antagonizes VEGFR-2 to prevent blood-retinal barrier breakdown in diabetic mice.

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 ß-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.

Junctional Localization of Septin 2 Is Required for Organization of Junctional Proteins in Static Endothelial Monolayers.

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.

YAP activity protects against endotoxemic acute lung injury by activating multiple mechanisms.

The roles of the Hippo­Yes­associated 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, XMU­MP­1, 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 0­48 h post­LPS injection, and the 48­168 h time period was considered the repair phase. Inhibition of YAP activity at the injury phase, using verteporfin, exacerbated, whereas its stimulation, using XMU­MP­1, 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 Hippo­YAP activity serves a protective role against endotoxemic lung injury. The Hippo­YAP activity alleviated lung inflammation and injury at the injury phase and promoted inflammation resolution and lung repair at the repair phase.

TM4SF19 aggravates LPS-induced attenuation of vascular endothelial cell adherens junctions by suppressing VE-cadherin expression.

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.

A proteomic study on the protective effect of kaempferol pretreatment against deoxynivalenol-induced intestinal barrier dysfunction in a Caco-2 cell model.

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.

LPAR2 receptor activation attenuates radiation-induced disruption of apical junctional complexes and mucosal barrier dysfunction in mouse colon.

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-ICC12 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-ICC12 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.

Exposure to Zinc Oxide Nanoparticles Disrupts Endothelial Tight and Adherens Junctions and Induces Pulmonary Inflammatory Cell Infiltration.

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 ß-catenin, leading to reduced and diffused staining of VE-cadherin and ß-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.

Frontline Science: Two flavonoid compounds attenuate allergic asthma by regulating epithelial barrier via G protein-coupled estrogen receptor: Probing a possible target for allergic inflammation.

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 (ERß 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.

Metformin inhibition of colorectal cancer cell migration is associated with rebuilt adherens junctions and FAK downregulation.

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 ß-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 Ser838/840 , a modification associated with ß-catenin/E-cadherin interaction. These modifications in E-cadherin, p120-catenin and ß-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 Tyr397 and paxillin at Tyr118 . 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.

Arg mediates LPS-induced disruption of the pulmonary endothelial barrier.

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.

Sevoflurane sedation attenuates early cerebral oedema formation through stabilisation of the adherens junction protein beta catenin in a model of subarachnoid haemorrhage: A randomised animal study.

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 (ß-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 ±â€Š0.9 vs. 78.0 ±â€Š0.7%, 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 ß-catenin remained unaffected by the injury and type of sedation. Decreased cytosolic fraction of ß-catenin in propofol-SAH animals (59 ±â€Š15%) was found to reach values of sham animals (100%) in the presence of sevoflurane in SAH animals (89 ±â€Š21%; 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.