Redistribution Of ZO-1 Research Articles

Effect of Metformin on Sertoli Cell Fatty Acid Metabolism and Blood-Testis Barrier Formation.

Sertoli cells (SCs) are essential to maintaining germ cell development. Metformin, the main pharmacologic treatment for pediatric type 2 diabetes, is administered to children during SC maturation. The present study aimed to analyze whether metformin affects SC energy metabolism and blood-testis barrier (BTB) integrity. Primary SC cultures were used for the in vitro studies. In vivo effects were studied in Sprague-Dawley rats treated with 200 mg/kg metformin from Pnd14 to Pnd30. Metformin decreased fatty acid oxidation and increased 3-hydroxybutyrate production in vitro. Moreover, it decreased the transepithelial electrical resistance across the monolayer and induced ZO-1 redistribution, suggesting an alteration of cell junctions. In vivo, a mild but significant increase in BTB permeability and ZO-1 expression was observed in the metformin group, without changes in testicular histology and meiosis progression. Additionally, adult rats that received metformin treatment during the juvenile period showed no alteration in BTB permeability or daily sperm production. In conclusion, metformin exposure may affect BTB permeability in juvenile rats, but this seems not to influence spermatogenesis progression. Considering the results obtained in adult animals, it is possible to speculate that metformin treatment during the juvenile period does not affect testicular function in adulthood.

Abstract 12294: Poldip2 Mediates Brain Vascular Permeability by Regulating ZO-1 Phosphorylation and Localization at the Interendothelial Border

Introduction: Poldip2 is a novel regulator of cerebral vascular permeability involved in aggravating blood-brain barrier disruption following ischemic stroke; however, the underlying mechanisms are incompletely understood. Tight junctions are crucial for the formation of endothelial barriers and are composed of transmembrane proteins and adaptor proteins such as ZO-1. Cerebral ischemia and inflammation lead to ZO-1 redistribution resulting in vascular permeability. Hypothesis: We hypothesized that Poldip2 may mediate ZO-1 phosphorylation and promote brain endothelial monolayer permeability following cerebral ischemia. Methods: Cerebral ischemia was induced in endothelial-specific Poldip2 knockout and control mice. Vascular permeability was assessed by Evans blue dye extravasation. For in vitro analysis, primary rat brain microvascular endothelial cells were used to investigate the effect of Poldip2 depletion by siRNA on TNF-α-induced permeability and trans-endothelial electrical resistance (TEER). Immunocytochemistry was carried out to investigate the effects of Poldip2 depletion or overexpression on ZO-1 localization. Results: Endothelial-specific Poldip2 deletion abolished Evans blue dye extravasation after cerebral ischemia induction (p<0.05). Poldip2 knockdown in vitro suppressed TNF-α-induced endothelial cell (EC) monolayer permeability (FITC-dextran and biotin-avidin assays, p<0.05 and p<0.001) and prevented decreases in TEER (p<0.01). Poldip2 depletion prevented TNF-α-induced ZO-1 disruption at interendothelial junctions (p<0.05). Poldip2 overexpression increased endothelial permeability (p<0.05), decreased TEER (p<0.05), reduced ZO-1 localization at cell-cell junctions (p<0.0001) and enhanced reactive oxygen species (ROS) production (p<0.05). Treatment with the antioxidant N-acetyl cysteine (NAC) abrogated Poldip2-induced ZO-1 disruption (p<0.0001). Immunoprecipitation studies demonstrated that Poldip2 overexpression induced tyrosine phosphorylation of ZO-1 (p<0.0001), an effect prevented by NAC treatment (p<0.01). Conclusions: Poldip2 induces ROS-mediated tyrosine phosphorylation of the TJ protein ZO-1 and promotes EC permeability following cerebral ischemia

Lactiplantibacillus plantarum YZX28 alleviated intestinal barrier dysfunction induced by enterotoxigenic Escherichia coli via inhibiting its virulence factor production

Probiotics are emerging as a promising approach to treating foodborne infections or gastrointestinal diseases. This study aimed to explore the protective effect of Lactiplantibacillus plantarum YZX28 (YZX28) on intestinal barrier dysfunction induced by enterotoxigenic Escherichia coli K80 (ETEC K80). Caco-2 cells were used for in vitro co-culture experiments of enterotoxin-producing ETEC K80 and YZX28. The barrier permeability (higher phenol red flux and decreased TEER) and levels of pro-inflammatory cytokines TNF-α and IL-8 increased by ETEC K 80 were attenuated by YZX28, and the mRNA levels of tight junctions, including ZO-1, occludin, claudin-1, and cingulin were significantly increased by YZX28 intervention. The redistribution of ZO-1, claudin-1, and actin fibers in Caco-2 cells induced by ETEC K 80 was restored. Furthermore, YZX28 could effectively reduce the adhesion level of ETEC to Caco-2 cells, alleviate the decrease of cell viability induced by ETEC, and inhibit the expression of ETEC virulence factors, including enterotoxin STa (est-A), STb (est-B), LT (elt) as well as fimbria adhesin FaeG and reduced the corresponding cAMP, cGMP, and CEACAM6 levels. This strain may be a potential therapeutic agent for treating intestinal-related problems caused by the intestinal pathogen ETEC.

Epithelial monolayer development and tight junction assembly on nanopillar arrays

Nanostructured materials provide an outstanding opportunity to both stimulate and measure cellular processes. In the context of tight junctions, it was previously reported that transient application of a nanotopographic surface over the apical brush border membrane of epithelial monolayers triggers redistribution of ZO‐1, claudins, and F‐actin that increases paracellular macromolecular flux. In excitable tissues, nanomaterials have been used to apply and measure electrical signals, such action potentials. As a first step towards translating these technologies for use in analysis of epithelial function, we sought to culture monolayers composed of transporting epithelia over nanopillar arrays without perturbing cellular structure or function. Madin‐Darby Canine kidney I (MDCKI) cells were cultured on collagen‐coated silicon chips with ~1.5 µm diameter nanopillar arrays. Fluorescence and scanning electron microscopy were used to assess the impact of height on nanopillar‐epithelial interactions. Monolayers formed over and were largely unaffected by short nanopillars. These nanopillars were located beneath basal epithelial surfaces and were not preferentially located within lateral intercellular spaces or beneath ZO‐1‐containing junctions. In contrast, tall nanopillars that exceeded cell height disrupted MDCK monolayer growth. Cells interacted with, encircled, and extended cytoplasm over the top of tall nanopillars, and dense ZO‐1 and F‐actin accumulations occasionally surrounded apical membranes adjacent to nanopillars. Finally, when grown over arrays composed of nanopillars 1 – 2 µm less than cell height, MDCKI grew more often between nanopillars. As a result, nanopillars were more frequently present within lateral intercellular spaces beneath junctions. Apical complex structure was intact, as assessed by fluorescence microscopy of ZO‐1, occludin, claudin‐2, F‐actin, and E‐cadherin. Apical microvilli were also unaffected. These data establish the conditions required for growth of mature, correctly assembled epithelial monolayers in which nanopillars are present within lateral intercellular spaces. This sets the stage for synthesis of functionalized nanopillar arrays for perturbation and analysis of epithelial biology.

Blood\u2013brain barrier dysfunction in l-ornithine induced acute pancreatitis in rats and the direct effect of l-ornithine on cultured brain endothelial cells

BackgroundIn severe acute pancreatitis (AP) the CNS is affected manifesting in neurological symptoms. Earlier research from our laboratory showed blood–brain barrier (BBB) permeability elevation in a taurocholate-induced AP model. Here we aimed to further explore BBB changes in AP using a different, non-invasive in vivo model induced by l-ornithine. Our goal was also to identify whether l-ornithine, a cationic amino acid, has a direct effect on brain endothelial cells in vitro contributing to the observed BBB changes.MethodsAP was induced in rats by the intraperitoneal administration of l-ornithine-HCl. Vessel permeability and the gene expression of the primary transporter of l-ornithine, cationic amino acid transporter-1 (Cat-1) in the brain cortex, pancreas, liver and lung were determined. Ultrastructural changes were followed by transmission electron microscopy. The direct effect of l-ornithine was tested on primary rat brain endothelial cells and a triple co-culture model of the BBB. Viability and barrier integrity, including permeability and TEER, nitrogen monoxide (NO) and reactive oxygen species (ROS) production and NF-κB translocation were measured. Fluorescent staining for claudin-5, occludin, ZO-1, β-catenin, cell adhesion molecules Icam-1 and Vcam-1 and mitochondria was performed. Cell surface charge was measured by laser Doppler velocimetry.ResultsIn the l-ornithine-induced AP model vessel permeability for fluorescein and Cat-1 expression levels were elevated in the brain cortex and pancreas. On the ultrastructural level surface glycocalyx and mitochondrial damage, tight junction and basal membrane alterations, and glial edema were observed. l-ornithine decreased cell impedance and elevated the BBB model permeability in vitro. Discontinuity in the surface glycocalyx labeling and immunostaining of junctional proteins, cytoplasmic redistribution of ZO-1 and β-catenin, and elevation of Vcam-1 expression were measured. ROS production was increased and mitochondrial network was damaged without NF-κB, NO production or mitochondrial membrane potential alterations. Similar ultrastructural changes were seen in l-ornithine treated brain endothelial cells as in vivo. The basal negative zeta potential of brain endothelial cells became more positive after l-ornithine treatment.ConclusionWe demonstrated BBB damage in the l-ornithine-induced rat AP model suggesting a general, AP model independent effect. l-ornithine induced oxidative stress, decreased barrier integrity and altered BBB morphology in a culture BBB model. These data suggest a direct effect of the cationic l-ornithine on brain endothelium. Endothelial surface glycocalyx injury was revealed both in vivo and in vitro, as an additional novel component of the BBB-related pathological changes in AP.

CRH/CRHR1 modulates cerebrovascular endothelial cell permeability in association with S1PR2 and S1PR3 under oxidative stress

Corticotrophin-releasing hormone (CRH) has been demonstrated to participate in vascular inflammation and permeability. Our previous studies have shown that blockade of S1PR2 or CRHR1 inhibited H2O2-induced brain endothelial hyperpermeability via inhibiting cPLA2 phosphorylation. However, little is known about the linkage between S1PRs and CRHR1 in oxidative stress-induced cerebrovascular endothelial hyperpermeability. Here we observed the opposite effects of S1PR2 to those of S1PR3 on the monolayer permeability of bEnd3 cells in response to H2O2. Interestingly, activation of CRHR1 was found to reverse the effects resulting from blockade/silencing of both S1PR2 and S1PR3. In bEnd3 monolayer, blockade/knockdown of S1PR2 reduced the endothelial hyperpermeability and suppressed the tight junction protein ZO-1 redistribution caused by H2O2, along with the inhibition of p38, ERK and cPLA2 phosphorylation. On the contrary, suppression/silencing of S1PR3 further promoted H2O2-induced endothelial hyperpermeability and ZO-1 redistribution, accompanied by the increased phosphorylation of p38, ERK and cPLA2. In the presence of CRH, the effects resulting from the suppression of both S1PR2 and S1PR3 were abolished. Our results elucidate a possible linkage between CRHR1 and S1PR2/S1PR3 involving in the regulation of endothelial monolayer permeability under oxidative stress condition.

Notoginsenoside R1 intervenes degradation and redistribution of tight junctions to ameliorate blood-brain barrier permeability by Caveolin-1/MMP2/9 pathway after acute ischemic stroke

BackgroundThe leakage of blood-brain barrier (BBB) is main pathophysiological change in acute stage of ischemic stroke, which not only deteriorates neurological function, but also increases the risk of hemorrhagic transformation after thrombolysis. Purpose/Study DesignThis article investigates the efficacy of Notoginsenoside R1, an active ingredient of Panax notoginseng, on BBB permeability and explores related mechanisms after acute ischemic stroke. MethodsIn vivo, male Sprague-Dawley rats (260–280 g) were selected and randomly divided into 6 groups: sham group, model group, low, middle and high doses of Notoginsenoside R1 groups and positive drug Dl-3-n-Butylphthalide group. Except for sham group, rats were performed with permanent middle cerebral artery occlusion model in each group. Twelve hours later, rats were evaluated for Bederson neurological function, and BBB integrity by Evans blue leak imaging; Triphenyltetrazolium chloride staining was used to detect the volume of cerebral infarction. Frozen sections of rats’ brain tissue were prepared for detection of MMPs activity in situ zymography. Peripheral tissue of cerebral infarction was collected and tested the expression of MMP2, 9 and tight junction proteins (zo1, claudin5, occludin) by western blot. In vitro, transwell endothelial barrier model was established by bEnd.3 cells. Oxygen glucose deprivation (OGD) was chosen to simulate the hypoxic environment. Suitable OGD stimulation time as well as Notoginsenoside R1 and Dl-3-n-Butylphthalide optimal dose concentrations were determined through transwell leakage and CCK8 assay. Furthermore, endothelial subcellular component proteins were extracted. The change of zo1, claudin5, occludin and caveolin1 was detected by western blot. ResultsNotoginsenoside R1 treatment significantly reduced BBB leakage and cerebral infarction volume, weakened neurological deficits in post-stroke rats. Moreover, it inhibited the activity of MMPs in infarcted cortex and striatum, down-regulated MMP2, 9 and up-regulated zo1 and claudin5 expressions in penumbra. In vitro, Notoginsenoside R1 treatment decreased OGD-induced endothelial barrier permeability, restored expressions of zo1, claudin5 on cellular membrane and cytoplasm, as well as mediated membrane redistribution of occludin and caveolin1 from actin cytoskeletal fraction. ConclusionsNotoginsenoside R1 treatment attenuates BBB permeability, cerebral infarction volume and neurological impairments in rats with acute cerebral ischemia. The mechanisms might be related to intervening degradation and redistribution of zo1, caludin5 and occludin by caveolin1/ MMP2/9 pathway. More effects and mechanisms of Notoginsenoside R1 on rehabilitation of stroke are worthy to be explored in the future.

Microtubule Stabilization Protects Hypothermia-Induced Damage to the Cytoskeleton and Barrier Integrity of the Corneal Endothelial Cells.

Purpose: To determine the impact of hypothermia on the barrier function of donor corneal endothelium, thereby enhancing the success of corneal transplantation. Methods: Primary cultures of porcine endothelial cells were subjected to hypothermia (15 h; 4°C). The impact on microtubule assembly, peri-junctional actomyosin ring (PAMR), and ZO-1 was assessed by immunocytochemistry with and without pretreatment with a microtubule-stabilizing agent (Epothilone B; EpoB; 100 nM) and a p38 MAP kinase inhibitor (SB-203580; 20 μM). In addition, EpoB-loaded PLGA nanoparticles (ENPs) prepared by nanoprecipitation technique and coated with poly-L-lysine (PLL-ENPs) were administered one-time for sustained intracellular delivery of EpoB. Results: Exposure to hypothermia led to microtubule disassembly concomitant with the destruction of PAMR and the displacement of ZO-1 at the cellular periphery, suggesting a loss in barrier integrity. These adverse effects were attenuated by pretreatment with EpoB or SB-203580. PLL-ENPs possessed a zeta potential of ∼26 mV and a size of ∼110 nm. Drug loading and entrapment efficiency were 5% (w/w) and ∼87%, respectively, and PLL-ENPs showed a biphasic release in vitro: burst phase (1 day), followed by a sustained phase (∼4 weeks). Pretreatment with PLL-ENPs (0.4 mg/mL) for 24 h stabilized the microtubules and opposed the hypothermia-induced damage to PAMR and the redistribution of ZO-1. Conclusions: Hypothermia induces microtubule disassembly via activation of p38 MAP kinase and subsequently breaks down the barrier function of the endothelium. Sustained intracellular delivery of EpoB using nanoparticles has the potential to overcome endothelial barrier failure during prolonged cold storage of donor cornea.

The loss of endogenous Neuregulin‐4 impairs intestinal epithelial recovery from LPS‐induced injury.

Growth factor receptors such as EGFR/erbB family members can preserve intestinal integrity by the activation of anti‐apoptotic pathways and stabilization of epithelial tight junction components. Previously we have shown that the activation of ErbB4 by its specific ligand Neuregulin‐4 (NRG4) promotes epithelial cell survival through Src and PI3 kinase/AKT pathways and reduces intestinal inflammation. However, it is unclear whether ErbB4 is an essential regulator of the intestinal barrier and if endogenous NRG4 is required for the response to intestinal inflammation. In this study we investigated the role of endogenous NRG4 as it pertains to intestinal barrier function and cell survival in vivo.METHODSNRG4 knockout mice and wild type littermates were challenged with lipopolysaccharide (LPS) injection. Intestinal permeability was determined by gavaging with 4‐kDa FITC‐dextran and collecting blood for fluorescence analysis after 4 or 24 h. The relative mRNA level of inflammatory cytokines was determined by taqman gene assays using full thickness ileal homogenates. Fixed ileal tissue for analysis of apoptosis was assessed by TUNEL and cleaved caspase‐3 staining, proliferation was evaluated by KI67 staining, and tight junction localization was determined by ZO‐1 staining.RESULTSThe loss of endogenous NRG4 in unchallenged mice did not alter baseline intestinal permeability, apoptosis, or proliferation. Interestingly, baseline inflammatory cytokine levels were reduced in the NRG4‐null mice compared to WT littermates. NRG4 KO mice showed significantly increased permeability to 4‐kDa FITC‐dextran (P<0.05) and enterocyte apoptosis (P<0.05) 24 hours after LPS‐induced injury, which was not seen 4 hours after LPS challenge. Furthermore, ZO‐1 redistribution was apparent within the base of the crypt 24 hours after LPS challenge in the NRG4 KO mice, while redistribution was only visible after 4 hours in WT mice.CONCLUSIONThe loss of endogenous NRG4 impairs the recovery of intestinal epithelial barrier after LPS‐induced injury. Further investigation of this recovery mechanism may reveal more effective strategies for targeting epithelial barrier dysfunction seen in intestinal diseases such as inflammatory bowel disease.Support or Funding Information2 R01 DK095004 06A1

Activation of CRHR1 contributes to cerebral endothelial barrier impairment via cPLA2 phosphorylation in experimental ischemic stroke

The activation of corticotrophin-releasing hormone receptor (CRHR) 1 is implicated in neuronal injury in experimental stroke. However, little is known about the relationship between CRHR1 activation and brain endothelial barrier impairment after ischemia and reperfusion (I/R). Recently we have demonstrated that the activation of extracellular signal-regulated kinase (Erk) 1/2 as well as p38 is required for hydrogen peroxide (H2O2)-increased cytosolic phospholipase A2 (cPLA2) phosphorylation in bEnd3 cells. Using this in vitro ischemic-like model, we found that both blockade and interference of CRHR1 inhibited H2O2-enhancd p38, Erk1/2 and cPLA2 phosphorylation and in turn suppressed monolayer hyperpermeability and ZO-1 redistribution. Then using the transient middle cerebral artery occlusion (tMCAO) mouse model, we revealed that CRHR1 antagonist NBI27914 pretreatment attenuated cPLA2 phosphorylation, Evans blue dye (EBD) extravasation, tight junction disruption and mitochondrial cytochrome c release. CRHR1 interference also inhibited cortical vascular hyperpermeability. Furthermore, NBI27914 administration attenuated neurovascular injury. After 30 min MCAO with 7 days reperfusion CRHR1 interference alleviated hippocampal blood-brain barrier (BBB) leakage and improved spatial cognitive dysfunction. Thus, our study demonstrates that during ischemic stroke the activation of endothelial CRHR1 contributes to BBB impairment via cPLA2 phosphorylation.

Extracellular vesicles and soluble factors secreted by Escherichia coli Nissle 1917 and ECOR63 protect against enteropathogenic E. coli-induced intestinal epithelial barrier dysfunction

BackgroundEnteric pathogens have developed mechanisms to disrupt tight junctions and increase gut permeability. Many studies have analysed the ability of live probiotics to protect intestinal epithelial cells against tight junction damage caused by bacterial pathogens. Escherichia coli Nissle 1917 (EcN) is among the probiotics that positively modulates the intestinal epithelial barrier by regulating expression and distribution of tight junction proteins. We previously reported that regulation of ZO-1, claudin-14 and claudin-2 is mediated by EcN secreted factors, either free-released or associated with outer membrane vesicles (OMVs). Factors secreted by commensal ECOR63 elicited comparable effects in intact epithelial T-84 and Caco-2 cell monolayers.ResultsHere we analyse the ability of OMVs and soluble secreted factors to protect epithelial barrier function in polarized T-84 and Caco-2 cells infected with enteropathogenic Escherichia coli (EPEC). Transepithelial electrical resistance, paracellular permeability, mRNA levels and subcellular distribution of tight junction proteins were monitored in the absence or presence of EcN and ECOR63 extracellular fractions. EPEC downregulated expression of ZO-1 ZO-2, occludin and claudin-14 and altered the subcellular localization of ZO-1, occludin and F-actin cytoskeleton. OMVs and soluble factors secreted by EcN and ECOR63 counteracted EPEC-altered transepithelial resistance and paracellular permeability, preserved occludin and claudin-14 mRNA levels, retained ZO-1 and occludin at tight junctions in the cell boundaries and ameliorated F-actin disorganization. Redistribution of ZO-1 was not accompanied by changes at mRNA level.ConclusionThis study provides new insights on the role of microbiota secreted factors on the modulation of intestinal tight junctions, expanding their barrier-protective effects against pathogen-induced disruption.

Gold nanoparticles regulate tight junctions and improve cetuximab effect in colon cancer cells.

Aim: Colon cancer (CC) is the second cause of cancer death worldwide. The use of nanoparticles for drug delivery has been increasing in cancer clinical trials over recent years. Materials & methods: We evaluated cytotoxicity of citrate-capped gold nanoparticles (GNPs) and the role they play on cell-cell adhesion. We also used GNP for delivery of cetuximab into different CC cell lines. Results: CC cells with well-formed tight junctions impair GNP uptake. Noncytotoxic concentration of GNP increases paracellular permeability in Caco-2 cells in a reversible way, concomitantly to tight junctions proteins CLDN1 and ZO-1 redistribution. GNP functionalized with cetuximab increases death of invasive HCT-116 CC cells. Conclusion: GNP can be used for drug delivery and can improve efficiency of CC therapy.

S1PR2 antagonist alleviates oxidative stress-enhanced brain endothelial permeability by attenuating p38 and Erk1/2-dependent cPLA2 phosphorylation

Both sphingosine-1-phosphate receptor-2 (S1PR2) and cytosolic phospholipase A2 (cPLA2) are implicated in the disruption of cerebrovascular integrity in experimental stroke. However, the role of S1PR2 in induction of cPLA2 phosphorylation during cerebral ischemia-induced endothelial dysfunction remains unknown. This study investigated the effect of S1PR2 blockade on oxidative stress-induced cerebrovascular endothelial barrier impairment and explored the possible mechanisms. In bEnd3 cells, cPLA2 inhibitor CAY10502 as well as S1PR2 antagonist JTE013 profoundly suppressed hydrogen peroxide (H2O2)-induced changes of paracellular permeability and ZO-1 localization. Besides p38, extracellular signal-regulated kinase (Erk) 1/2 is required for H2O2-increased cPLA2 phosphorylation and endothelial permeability. Pharmacological and genetic inhibition of S1PR2 significantly suppressed their phosphorylation in response to H2O2. Especially lentivirus-mediated knockdown of S1PR2 inhibited H2O2-induced ZO-1 redistribution and paracellular hyperpermeability. Using the permanent middle cerebral artery occlusion (pMCAO) mouse model, we found JTE013 pretreatment markedly reduced Evans blue dye (EBD) extravasation and reversed the decrease in VE-cadherin, occludin, claudin-5 and CD31 expression in infarcted hemisphere. Lentivirus-mediated S1PR2 knockdown also attenuated EBD extravasation. Furthermore, JTE013 pretreatment attenuated neurological deficit, brain edema and infarction volume. Therefore, our findings suggest the protective effect of JTE013 on brain endothelial barrier integrity is likely mediated by suppressing p38 and Erk1/2-dependent cPLA2 phosphorylation under oxidative stress.

Autophagy- and MMP-2/9-mediated Reduction and Redistribution of ZO-1 Contribute to Hyperglycemia-increased Blood–Brain Barrier Permeability During Early Reperfusion in Stroke

Post-stroke hyperglycemia during early reperfusion increases blood–brain barrier (BBB) permeability and subsequently aggravates brain injury and clinical prognosis. The decreased level of tight junction proteins (TJPs) has been reported but the underlying mechanism remains largely elusive. Herein we designed to investigate the detailed molecular events in brain microvascular endothelial cells (BMECs) ex and in vivo. After oxygen–glucose deprivation (OGD) for 90 min and reperfusion with 8 or 16 mM glucose for 30 min, glucose at 16 mM caused significant decrease in the TJP expression and particularly ZO-1 redistribution from membrane to cytoplasm of BMECs. High glucose also markedly promoted the secretion of MMP-2/9 and oxidative/nitrosative stress, enhanced autophagy and increased the Caveolin-1 and LAMP-2 expression. Moreover, in vivo experiments demonstrated that rapamycin-enhanced autophagy further caused ZO-1 reduction and the increased BBB permeability. Therefore, high-glucose exposure in the early reperfusion causes the BBB disruption, with MMP-2/9-mediated extracellular degradation, caveolin-1-mediated intracellular translocation and autophagy-lysosome-mediated degradation of ZO-1 protein all together involved in the process. The role of MMP-2/-9 and autophagy in the modulation of paracellular permeability was confirmed by pharmacological inhibition. Therefore, our findings may provide new insights into targeting ZO-1 regulation for the purpose of significantly improving the clinical prognosis of ischemic stroke.

Characterization of cell-cell junction changes associated with the formation of a strong endothelial barrier

ABSTRACTA principal function of endothelial cells is the formation of a barrier between the blood and tissues. This barrier arises from the physical connections at cell-cell junctions, which includes cytoskeletal tight junction and adherens junction proteins. Methods that alter barrier function must therefore affect these cell-cell connections. The blood brain barrier (BBB) represents perhaps the most selective endothelial barrier, which arises from endothelial cell interactions with astrocytes and pericytes. Even in non-central nervous system (CNS) endothelial cells, barrier properties can be enhanced, mimicking the BBB, through induction of intercellular junctions, by either direct co-culture with astrocytes, supplementation with astrocyte conditioned medium (ACM) and/or pharmacologic enhancement of cAMP. To understand how cell-cell junctions change during endothelial barrier enhancement, we examined the effects of ACM and/or cAMP donors added to standard media on human umbilical vein endothelial cells (HUVEC). HUVEC cultured with cAMP-elevating agents had the most enhanced barrier function as measured by Electric Cell-substrate Impedance Sensing (ECIS®), a real-time, label-free, impedance based method of studying cell barrier properties. However, subtle differences in actin and cell-cell junction proteins were seen across all four culture conditions. cAMP-elevating agents also triggered the redistribution of ZO-1 and VE-cadherin to cell-cell junctions, and intensified the actin microfilament network at the cell cortex. Using a VE-cadherin FRET-force sensor, we observed a decrease in VE-cadherin force in HUVEC cultured with ACM with cAMP donors. Our data indicate cAMP elevation induces both junctional strengthening and reduced VE-cadherin forces. Additionally, treatment with an inhibitor of formin, which reduced actin stress fibers, enhanced barrier function. These data suggest that barrier function is modulated both through the trafficking of proteins to cell-cell junctions, and through the modulation and a relaxation of mechanical force through adherens junctions as intercellular junctional complexes become established.

Cathelicidin-BF ameliorates lipopolysaccharide-induced intestinal epithelial barrier disruption in rat

AimsThe present study examined the effect of the antimicrobial peptide cathelicidin-BF (CBF) on LPS-induced mucosal injury and intestinal epithelial barrier dysfunction in a rat model and in the porcine intestinal epithelial cell line. Main methodsChanges in barrier integrity were assessed in intestinal epithelium and IPEC-J2 monolayers by measuring nutrient absorption and transepithelial electrical resistance (TER), and the permeability of intestinal epithelium was examined by measuring plasma d-lactate and diamine oxidase levels. The expression levels of tight junction (TJ) proteins were quantified by real-time PCR, and immunofluorescence was used to analyse the location and distribution of TJs in cells. Key findingsIn vivo, CBF improved epithelial barrier function through attenuating the alterations of the mucosal structure, nutrient absorption and TER in the jejunum, and preventing the down-regulation of TJ proteins in LPS-induced rat intestinal epithelium. In vitro, CBF prevented the disruption and the re-distribution of ZO-1 and occludin, and suppressed the increase in inflammatory cytokine levels in LPS-induced IPEC-J2. The CBF-induced upregulation of zonula occludens-1 and occludin was prevented by U0126 or SB203580, suggesting the involvement of the MEK and p38 MAPK pathways in the CBF-induced changes in tight junctions. SignificanceOur results showed that CBF prevents LPS-induced intestinal epithelial barrier dysfunction, suggesting its potential as a therapeutic agent for the prevention of LPS-mediated intestinal diseases. We found that exogenous CBF had protective effects on LPS-induced intestinal epithelial barrier disruption in rats and on epithelial damage in IPEC-J2 cells.

Identification of an Occludin Regulatory Motif that Confers Dynamics to Epithelial Tight Junctions (TJs) and Adherens Junctions (AJs)

Occludin is a transmembrane protein of epithelial TJs, the function of which remains unclear. Previous studies in Caco‐2 cells showed that the C‐terminal domain of occludin harbors a conserved phosphorylation hotspot, which we designated as 'Occludin Regulatory Motif' (ORM). To determine the functional significance of ORM, we cloned a stable, occludin‐deficient MDCK cell line (MDCKOKD) by shRNA transfection, and introduced GFP‐tagged human occludin deletion mutant (OclDM: Δ388‐415; lacking ORM) or wild type occludin (OclWT) and cloned stable cell lines. Confocal imaging of live cells, immunofluorescence staining and co‐immunoprecipitation analyses revealed a co‐localization of OclDM with ZO‐1 at the intercellular junctions similar to OclWT. Post seeding barrier development was faster in MDCKOKD‐OclDM and MDCKOKD‐Vec cells compared to those in MDCKOKD‐OclWT and native MDCK cells. FRAP analysis demonstrated that deletion of ORM significantly reduced the mobile fraction of occludin and elevated the proportion of detergent‐insoluble fraction of occludin. Incubation of MDCKOKD‐OclWT cell monolayers in low calcium medium (LCM) resulted in redistribution of OclWT from the junctions, but LCM had no effect on the distribution of OclDM. LCM reduced barrier function and induced ZO‐1 redistribution in MDCKOKD‐OclWT and native MDCK cells, but not in MDCKOKD‐VecorMDCKOKD‐OclDM cells, indicating the role of ORM in TJ dynamics. Depletion of occludin or lack of ORM also attenuated LCM‐mediated redistribution of E‐cadherin and β‐catenin and reorganization of actin cytoskeleton, indicating the role of ORM in AJs. In summary, results indicate a potential role for ORM in conferring dynamics to epithelial apical junctional complexes.

Ethanol impairs intestinal barrier function in humans through mitogen activated protein kinase signaling: a combined in vivo and in vitro approach.

BackgroundEthanol-induced gut barrier disruption is associated with several gastrointestinal and liver disorders.AimSince human data on effects of moderate ethanol consumption on intestinal barrier integrity and involved mechanisms are limited, the objectives of this study were to investigate effects of a single moderate ethanol dose on small and large intestinal permeability and to explore the role of mitogen activated protein kinase (MAPK) pathway as a primary signaling mechanism.MethodsIntestinal permeability was assessed in 12 healthy volunteers after intraduodenal administration of either placebo or 20 g ethanol in a randomised cross-over trial. Localization of the tight junction (TJ) and gene expression, phosphorylation of the MAPK isoforms p38, ERK and JNK as indicative of activation were analyzed in duodenal biopsies. The role of MAPK was further examined in vitro using Caco-2 monolayers.ResultsEthanol increased small and large intestinal permeability, paralleled by redistribution of ZO-1 and occludin, down-regulation of ZO-1 and up-regulation of myosin light chain kinase (MLCK) mRNA expression, and increased MAPK isoforms phosphorylation. In Caco-2 monolayers, ethanol increased permeability, induced redistribution of the junctional proteins and F-actin, and MAPK and MLCK activation, as indicated by phosphorylation of MAPK isoforms and myosin light chain (MLC), respectively, which could be reversed by pretreatment with either MAPK inhibitors or the anti-oxidant L-cysteine.ConclusionsAdministration of moderate ethanol dosage can increase both small and colon permeability. Furthermore, the data indicate a pivotal role for MAPK and its crosstalk with MLCK in ethanol-induced intestinal barrier disruption.Trial RegistrationClinicalTrials.gov NCT00928733

HGF and the regulation of tight junctions in human prostate cancer cells.

Hepatocyte growth factor (HGF) may impact the metastasis of prostate cancer via its action on prostate stem cells or their progeny. Tight junctions (TJs) are crucial to the process of metastasis and have been previously shown to be regulated by HGF. The present study aimed to evaluate the effect of HGF on the function of TJs in human prostate epithelial, prostate stem cell-like and prostate cancer cell lines. Four human prostate cancer cell lines (PC-3, DU-145, PZHPV-7, CaHPV-10), normal adult prostate parental epithelial cells (RWPE-1) and a stem cell-like derivative of RWPE-1 (WPE-STEM) were used to assess HGF-induced changes in TJs. A significant difference was noted in the behaviour between the WPE-STEM, RWPE-1 and the cancer cell lines which was HGF concentration-dependent. However, in the WPE-STEM cells, the effect was biphasic, with the cells seemingly resistant to HGF-modulated TJ disruption. Closer examination revealed that HGF affected the redistribution of ZO-1, ZO-2 and ZO-3 away from the TJs of confluent cells with concurrent loss of claudin-1 and claudin-5, and western blot analysis revealed a loss in TJ protein expression of ZO-1 and ZO-2. We demonstrated for the first time that HGF regulates TJ function in human prostate cells. Moreover, this regulation was dependent on the tumourigenicity of the cells, with the most aggressive cells most susceptible and the stem cell-like cells least susceptible. These data offer an intriguing glimpse of how TJs affect the behaviour of prostate cancer cells and how HGF modulates the expression and function of the molecules maintaining TJ structure and function.

VEGF Increases Paracellular Permeability in Brain Endothelial Cells via Upregulation of EphA2

Neurological disorders are associated with an increase in the permeability of human brain microvascular endothelial cells (HBMEC). Our previous findings have indicated that EphA2 could increase the permeability of HBMEC. Recent evidence has linked EphA2 and vascular endothelial growth factor (VEGF) to abnormalities in the vascular response. However, it is unclear whether EphA2 is involved in the VEGF-induced changes in the permeability of HBMEC. Here, changes in permeability were determined by measuring transendothelial electrical resistance (TEER) and the flux of FITC-dextran. We found that knockdown of EphA2 in HBMEC abolished the VEGF-induced reduction in TEER and increase in flux of fluorescent dextran. Moreover, VEGF-induced redistribution of ZO-1 and the recruitment of detergent-soluble occludin and claudin-5 were also prevented. Further results showed that VEGF increased EphA2 expression in a time- and dose-dependent manner, which was inhibited by a neutralizing antibody against VEGFR2 or SU1498. VEGF-induced EphA2 expression was suppressed in the brain endothelium following treatments with the PI3K inhibitor LY294002, Akt inhibitor or transfection with the dominant-negative PI3K mutants (Δp110). Similar results were obtained when ERK1/2 activation was inhibited by PD98059 or ERK1/2 siRNA transfection. Our data suggest that VEGF upregulates the expression of EphA2 in HBMEC through binding to VEGFR2 and subsequently activating the intracellular PI3K/Akt and ERK1/2 signaling pathways, which contribute to an increase in paracellular permeability. These data reveal a novel role for VEGF as a regulator of EphA2 expression in the brain endothelial cells and provide insights into the molecular mechanisms of VEGF-mediated changes in paracellular permeability.