Distribution Of Zonula Occludens-1 Research Articles

Aspergillus fumigatus Supernatants Disrupt Bronchial Epithelial Monolayers: Potential Role for Enhanced Invasion in Cystic Fibrosis.

Aspergillus fumigatus is the most commonly isolated fungus in chronic lung diseases, with a prevalence of up to 60% in cystic fibrosis patients. Despite this, the impact of A. fumigatus colonisation on lung epithelia has not been thoroughly explored. We investigated the influence of A. fumigatus supernatants and the secondary metabolite, gliotoxin, on human bronchial epithelial cells (HBE) and CF bronchial epithelial (CFBE) cells. CFBE (F508del CFBE41o-) and HBE (16HBE14o-) trans-epithelial electrical resistance (TEER) was measured following exposure to A. fumigatus reference and clinical isolates, a gliotoxin-deficient mutant (ΔgliG) and pure gliotoxin. The impact on tight junction (TJ) proteins, zonula occludens-1 (ZO-1) and junctional adhesion molecule-A (JAM-A) were determined by western blot analysis and confocal microscopy. A. fumigatus conidia and supernatants caused significant disruption to CFBE and HBE TJs within 24 h. Supernatants from later cultures (72 h) caused the greatest disruption while ΔgliG mutant supernatants caused no disruption to TJ integrity. The ZO-1 and JAM-A distribution in epithelial monolayers were altered by A. fumigatus supernatants but not by ΔgliG supernatants, suggesting that gliotoxin is involved in this process. The fact that ΔgliG conidia were still capable of disrupting epithelial monolayers indicates that direct cell-cell contact also plays a role, independently of gliotoxin production. Gliotoxin is capable of disrupting TJ integrity which has the potential to contribute to airway damage, and enhance microbial invasion and sensitisation in CF.

Zonula occludens-1 distribution and barrier functions are affected by epithelial proliferation and turnover rates.

Zonula occludens-1 (ZO-1) is a scaffolding protein of tight junctions, which seal adjacent epithelial cells, that is also expressed in adherens junctions. The distribution pattern of ZO-1 differs among stratified squamous epithelia, including that between skin and oral buccal mucosa. However, the causes for this difference, and the mechanisms underlying ZO-1 spatial regulation, have yet to be elucidated. In this study, we showed that epithelial turnover and proliferation are associated with ZO-1 distribution in squamous epithelia. We tried to verify the regulation of ZO-1 by comparing normal skin and psoriasis, known as inflammatory skin disease with rapid turnover. We as well compared buccal mucosa and oral lichen planus, known as an inflammatory oral disease with a longer turnover interval. The imiquimod (IMQ) mouse model, often used as a psoriasis model, can promote cell proliferation. On the contrary, we peritoneally injected mice mitomycin C, which reduces cell proliferation. We examined whether IMQ and mitomycin C cause changes in the distribution and appearance of ZO-1. Human samples and mouse pharmacological models revealed that slower epithelial turnover/proliferation led to the confinement of ZO-1 to the uppermost part of squamous epithelia. In contrast, ZO-1 was widely distributed under conditions of faster cell turnover/proliferation. Cell culture experiments and mathematical modelling corroborated these ZO-1 distribution patterns. These findings demonstrate that ZO-1 distribution is affected by epithelial cell dynamics.

197 Tissue proliferation and turnover spatially regulates tight junctions in squamous epithelia

Zonula occludens-1 (ZO-1) is an epithelial junctional protein that participates in adherens junctions (AJs) and tight junctions (TJs). In simple epithelia, ZO-1 localization shifts from AJ to TJ during the maturation of cell adhesion. We recently showed that ZO-1 is more multilayered in buccal mucosa than in skin. However, what causes this difference and how ZO-1 is spatially regulated have not been elucidated. Here we show that epithelial turnover and proliferation dictate ZO-1 distribution in the squamous epithelia.

Intestinal mucosal barrier in functional constipation: Dose it change?

BACKGROUNDThe intestinal mucosal barrier is the first line of defense against numerous harmful substances, and it contributes to the maintenance of intestinal homeostasis. Recent studies reported that structural and functional changes in the intestinal mucosal barrier were involved in the pathogenesis of several intestinal diseases. However, no study thoroughly evaluated this barrier in patients with functional constipation (FC).AIMTo investigate the intestinal mucosal barrier in FC, including the mucus barrier, intercellular junctions, mucosal immunity and gut permeability.METHODSForty FC patients who fulfilled the Rome IV criteria and 24 healthy controls were recruited in the Department of Gastroenterology of China-Japan Friendship Hospital. The colonic mucus barrier, intercellular junctions in the colonic epithelium, mucosal immune state and gut permeability in FC patients were comprehensively examined. Goblet cells were stained with Alcian Blue/Periodic acid Schiff (AB/PAS) and counted. The ultrastructure of intercellular junctional complexes was observed under an electron microscope. Occludin and zonula occludens-1 (ZO-1) in the colonic mucosa were located and quantified using immunohistochemistry and quantitative real-time polymerase chain reaction. Colonic CD3+ intraepithelial lymphocytes (IELs) and CD3+ lymphocytes in the lamina propria were identified and counted using immunofluorescence. The serum levels of D-lactic acid and zonulin were detected using enzyme-linked immunosorbent assay.RESULTSCompared to healthy controls, the staining of mucus secreted by goblet cells was darker in FC patients, and the number of goblet cells per upper crypt in the colonic mucosa was significantly increased in FC patients (control, 18.67 ± 2.99; FC, 22.42 ± 4.09; P = 0.001). The intercellular junctional complexes in the colonic epithelium were integral in FC patients. The distribution of mucosal occludin and ZO-1 was not altered in FC patients. No significant differences were found in occludin (control, 5.76E-2 ± 1.62E-2; FC, 5.17E-2 ± 1.80E-2; P = 0.240) and ZO-1 (control, 2.29E-2 ± 0.93E-2; FC, 2.68E-2 ± 1.60E-2; P = 0.333) protein expression between the two groups. The mRNA levels in occludin and ZO-1 were not modified in FC patients compared to healthy controls (P = 0.145, P = 0.451, respectively). No significant differences were observed in the number of CD3+ IELs per 100 epithelial cells (control, 5.62 ± 2.06; FC, 4.50 ± 2.16; P = 0.070) and CD3+ lamina propria lymphocytes (control, 19.69 ± 6.04/mm2; FC, 22.70 ± 11.38/mm2; P = 0.273). There were no significant differences in serum D-lactic acid [control, 5.21 (4.46, 5.49) mmol/L; FC, 4.63 (4.31, 5.42) mmol/L; P = 0.112] or zonulin [control, 1.36 (0.53, 2.15) ng/mL; FC, 0.94 (0.47, 1.56) ng/mL; P = 0.185] levels between FC patients and healthy controls.CONCLUSIONThe intestinal mucosal barrier in FC patients exhibits a compensatory increase in goblet cells and integral intercellular junctions without activation of mucosal immunity or increased gut permeability.

A Dual Zinc plus Arginine formulation protects against tumor necrosis factor-alpha-induced barrier dysfunction and enhances cell proliferation and migration in an in vitro gingival keratinocyte model

ObjectiveTo investigate the effects of Dual Zinc plus Arginine formulations (aqueous solution and dentifrice) on tumor necrosis factor-alpha (TNF-α)-induced barrier dysfunction as well as on cell proliferation and migration in an in vitro gingival keratinocyte model. DesignGingival keratinocytes were seeded onto the membrane of a double-chamber system in the absence and presence of recombinant TNF-α and the formulations under investigation. The barrier function was assessed by determination of transepithelial electrical resistance (TER) and paracellular transport of fluorescein isothiocyanate (FITC)-dextran. The distribution of zonula occludens-1 (ZO-1) and occludin was visualized by immunofluorescence microscopy. The effects of the formulations on keratinocyte cell proliferation were determined using a fluorescent cell tracker dye, while a migration assay kit was used to investigate their effects on cell migration. ResultsUnder conditions where TNF-α induces loss of keratinocyte barrier integrity, the Dual Zinc plus Arginine formulations (aqueous solution and dentifrice) protected the keratinocyte tight junction against the damages since they prevented the TNF-α-induced drop in TER and increase in FITC-dextran paracellular flux in the in vitro model. The treatment of keratinocytes with the formulations markedly mitigated the altered distribution of ZO-1 and occludin. Both formulations increased the proliferation of keratinocytes and alleviated the negative impact caused by TNF-α. Lastly, the formulations increased the migration capacity of keratinocytes. ConclusionsThe ability of the Dual Zinc plus Arginine formulations to protect the barrier integrity of gingival keratinocytes from TNF-α-induced damage and to promote their proliferation and migration suggests that they may offer benefits for oral health.

Eugenol attenuates inflammatory response and enhances barrier function during lipopolysaccharide-induced inflammation in the porcine intestinal epithelial cells.

Eugenol (4-allyl-2-methoxyphenol) is an essential oil component, possessing antimicrobial, anti-inflammatory, and antioxidative properties; however, the effect of eugenol on porcine gut inflammation has not yet been investigated. In this study, an in vitro lipopolysaccharide (LPS)-induced inflammation model in porcine intestinal epithelial cells (IPEC-J2) has been set up. Cells were pretreated with 100 μM (16.42 mg/L) eugenol for 2 h followed by 10 μg/mL LPS stimulation for 6 h. Proinflammatory cytokine secretion; reactive oxygen species; gene expression of proinflammatory cytokines, tight junction proteins, and nutrient transporters; the expression and distribution of zonula occludens-1 (ZO-1); transepithelial electrical resistance (TEER); and cell permeability were measured to investigate the effect of eugenol on inflammatory responses and gut barrier function. The results showed that eugenol pretreatment significantly suppressed the LPS-stimulated interleukin-8 level and the mRNA abundance of tumor necrosis factor-α and restored the LPS-stimulated decrease of the mRNA abundance of tight junction proteins, such as ZO-1 and occludin, and the mRNA abundance of nutrient transporters, such as B0 1 system ASC sodium-dependent neutral amino acid exchanger 2, sodium-dependent glucose transporter 1, excitatory amino acid transporter 1, and peptide transporter 1. In addition, eugenol improved the expression and even redistribution of ZO-1 and tended to increase TEER value and maintained the barrier integrity. In conclusion, a low dose of eugenol attenuated inflammatory responses and enhanced selectively permeable barrier function during LPS-induced inflammation in the IPEC-J2 cell line.

Expression of R345W-Fibulin-3 Induces Epithelial-Mesenchymal Transition in Retinal Pigment Epithelial Cells.

PurposeTo investigate the role of protein misfolding in retinal pigment epithelial (RPE) cell dysfunction, the effects of R345W-Fibulin-3 expression on RPE cell phenotype were studied.MethodsPrimary RPE cells were cultured to confluence on Transwells and infected with lentivirus constructs to express wild-type (WT)- or R345W-Fibulin-3. Barrier function was assessed by evaluating zonula occludens-1 (ZO-1) distribution and trans-epithelial electrical resistance (TER). Polarized secretion of vascular endothelial growth factor (VEGF), was measured by Enzyme-linked immunosorbent assay (ELISA). Differentiation status was assessed by qPCR of genes known to be preferentially expressed in terminally differentiated RPE cells, and conversion to an epithelial–mesenchymal transition (EMT) phenotype was assessed by a migration assay.ResultsCompared to RPE cells expressing WT-Fibulin-3, ZO-1 distribution was disrupted and TER values were significantly lower in RPE cells expressing R345W-Fibulin-3. In cells expressing mutant Fibulin-3, VEGF secretion was attenuated basally but not in the apical direction, whereas Fibulin-3 secretion was reduced in both the apical and basal directions. Retinal pigment epithelial signature genes were downregulated and multiple genes associated with EMT were upregulated in the mutant group. Migration assays revealed a faster recovery rate in ARPE-19 cells overexpressing R345W-Fibulin-3 compared to WT.ConclusionsThe results suggest that expression of R345W-Fibulin-3 promotes EMT in RPE cells.

Effects of short chain fatty acid on barrier disruption of human intestinal epithelial cell induced by endotoxin/lipopolysaccharide and the related mechanism

Objective: To investigate the effects of short chain fatty acid (SCFA) on barrier disruption of human intestinal epithelial cell induced by endotoxin/lipopolysaccharide (LPS) and the related mechanism. Methods: The human intestinal epithelial cell line Caco-2 was used to reproduce monolayer-cells. Cells were divided into control group, LPS group, and SCFA+ LPS group according to the random number table. Cells in control group were only routinely cultured with DMEM medium. Cells in LPS group were cultured with DMEM medium and LPS with final mass concentration of 10 μg/mL. Cells in SCFA+ LPS group were cultured with DMEM medium, LPS and SCFA (consisting of 0.5 mmol/L acetate, 0.01 mmol/L propionate, and 0.01 mmol/L butyrate) with final mass concentration of 10 μg/mL. At post culture hour (PCH) 0, 1, 2, 6, 12, and 24, transepithelial electrical resistance (TER) of cells was determined with an ohmmeter, with sample number of 72. Another portion of cells were divided and treated as above, and then Western blotting was employed to detect the protein expressions of zonula occludens 1 (ZO-1), occludin, and claudin-1 at PCH 24, with sample number of 6. Another portion of cells were divided and treated as above and then immunofluorescence was used to observe cellular morphology and distribution of ZO-1. Data were processed with analysis of variance of factorial design, one-way analysis of variance, least-significant difference test, and Bonferroni correction. Results: (1) Compared with that in control group, TER of cells in LPS group was significantly reduced from PCH 1 to 24 (P<0.01), while TER of cells in SCFA+ LPS group showed no obvious change (P>0.05). TER of cells in SCFA+ LPS group was significantly higher than that in LPS group from PCH 1 to 24 (P<0.01). (2) Compared with the protein expressions of ZO-1, occludin, and claudin-1 of cells in control group (1.25±0.10, 1.17±0.04, and 1.24±0.20), those of cells in LPS group (0.74±0.23, 0.76±0.11, and 0.77±0.11) at PCH 24 were significantly decreased (P<0.05), while those of cells in SCFA+ LPS group (1.23±0.46, 1.05±0.09, and 1.01±0.13) showed no significant differences (P>0.05). Protein expressions of occludin and claudin-1 of cells in SCFA+ LPS group were significantly higher than those in LPS group at PCH 24 (P<0.05). Protein expression of ZO-1 of cells in SCFA+ LPS group was higher than that in LPS group at PCH 24 with no significant difference (P>0.05). (3) At PCH 24, cells in control group were compact in arrangement with pebble-like appearance, and ZO-1 was distributed smoothly and continuously along the cell membrane. In LPS group, cells were sparse in arrangement with change in appearance, and ZO-1 was distributed uncontinuously along the cell membrane with curls and breaks. In SCFA+ LPS group, the appearance of cells and distribution of ZO-1 were remarkably ameliorated compared with those in LPS group. Conclusions: SCFA can alleviate the barrier disruption of human intestinal epithelial cell induced by LPS through interdicting the abnormal distribution of ZO-1 and decrease of TER and tight junction proteins' expressions.

Green tea polyphenols enhance gingival keratinocyte integrity and protect against invasion by Porphyromonas gingivalis.

The gingival epithelium, a stratified squamous tissue that acts as an interface between the external environment and the underlying connective tissue, plays an active role in maintaining periodontal health. The aim of the present study was to investigate the ability of green tea catechins to enhance gingival epithelial barrier function and protect against the disruption of epithelial integrity induced by Porphyromonas gingivalis. Both the green tea extract and epigallocatechin-3-gallate (EGCG) dose- and time-dependently increased the transepithelial electrical resistance (TER) of a gingival keratinocyte model and decreased the permeability of the cell monolayer to fluorescein isothyocyanate-conjugated 4.4-kDa dextran. This was associated with the increased expression of zonula occludens-1 (ZO-1) and occludin, two tight junction proteins. Treating the gingival keratinocyte monolayer with P. gingivalis caused a reduction in TER and affected the distribution of ZO-1 and occludin, allowing P. gingivalis to translocate through the cell monolayer. These deleterious effects mediated by P. gingivalis were abolished by the green tea extract and EGCG. This protection may be in part related to the ability of tea catechins to inhibit the protease activities of P. gingivalis. Given the above properties, green tea catechins may represent promising preventive and therapeutic molecules against periodontal disease.

Changes to the bladder epithelial barrier are associated with ketamine-induced cystitis.

The aim of the present study was to investigate the changes of the bladder epithelial barrier in the pathogenesis of ketamine-induced cystitis (KIC). A total of 60 female mice were randomly allocated into control and ketamine groups, which received daily intraperitoneal injections of saline and ketamine, respectively. Micturition behavior was recorded in 2-h intervals at the end of 4, 8 and 12 weeks, and bladders were harvested for subsequent analyses. Routine hematoxylin and eosin staining was performed on the bladders and histopathological changes were analyzed using light microscopy. The distribution of zonula occludens-1 (ZO-1) protein was determined by immunohistochemical analysis. The ultrastructure of umbrella cells was observed using a transmission electron microscope (TEM). Ketamine-addicted mice exhibited a significantly increased frequency of micturitions following 8 and 12 weeks of ketamine treatment (P<0.05 and P<0.01, respectively). Suburothelial congestion and infiltration of mononuclear cells was observed in ketamine-addicted mice following 8 and 12 weeks of treatment. Immunohistochemical examination demonstrated that there was an increased abnormal distribution of ZO-1 in the bladders of ketamine-treated mice compared with control mice. TEM analysis demonstrated that the surface of bladder urothelium became flattened, the tight junctions between umbrella cells became thinner and the endothelial cells exhibited cell body shrinkage, chromatin condensation and layer denudation in mice treated with ketamine. The present study indicated that the structural and functional changes to the bladder epithelial barrier caused by long-term use of ketamine may be key mechanisms in the development of KIC.

Effect of hydrogen on endotoxin-induced expression of zonula occludens-1 in human colon epithelial cells

Objective To investigate the effect of hydrogen on endotoxin-induced expression of zonula occludens-1 (ZO-1) in human colon epithelial cells (Caco-2 cells). Methods Caco-2 cells were cultured routinely, seeded in Transwell chambers or wells, and randomly divided into 4 groups (n=45 each) using a random number table: control group (group C); hydrogen-rich culture medium group (group H); endotoxin group (group E); hydrogen-rich culture medium + endotoxin group (group HE). The cells were cultured in high-glucose DMEM culture medium in group C. The cells were incubated in hydrogen-rich culture medium containing hydrogen 0.6 mmol/L in group H. The cells were incubated in high-glucose DMEM culture medium containing 50 μg/ml lipopolysaccharide in group E. The cells were incubated in hydrogen-rich culture medium containing 50 μg/ml lipopolysaccharide and 0.6 mmol/L hydrogen in group HE.Transepithelial electrical resistance (TEER) was measured before incubation or culture, and at 6, 12 and 24 h of incubation or culture.The viability of Caco-2 cells was measured by methyl thiazolyl tetrazolium assay at 24 h of incubation or culture.The expression of ZO-1 mRNA in Caco-2 cells was determined using real-time reverse transcriptase polymerase chain reaction at 6, 12 and 24 h of incubation or culture.The distribution of ZO-1 in Caco-2 cells was observed by immunofluorescence at 24 of incubation or culture. Results Compared with group C, TEER was significantly decreased at 6, 12 and 24 h of incubation or culture, and the expression of ZO-1 mRNA was significantly down-regulated in E and HE groups (P 0.05). Compared with group E, TEER was significantly increased at 6, 12 and 24 h of incubation or culture, and the expression of ZO-1 mRNA was significantly up-regulated in group HE (P<0.05). The distribution of ZO-1 protein in cell membrane became discontinuous, and the distribution of ZO-1 protein in cytoplasm was significantly increased in group E. Compared with group E, the distribution of ZO-1 protein in cell membrane was significantly increased and gradually became continuous, and the distribution of ZO-1 protein in cytoplasm was significantly decreased in group HE. Conclusion The mechanism by which hydrogen reduces the damage to human colon epithelial cell barrier is related to up-regulation of ZO-1 expression and improvement in the redistribution of ZO-1 protein. Key words: Hydrogen; Endotoxemia; Tight junctions; Intestines

Beneficial effects of fenofibric acid on overexpression of extracellular matrix components, COX-2, and impairment of endothelial permeability associated with diabetic retinopathy

In the Fenofibric Acid (FA) Intervention and Event Lowering in Diabetes (FIELD) study, FA, a lipid-lowering drug, has been shown to significantly reduce macular edema in diabetic patients. In the present study, we investigated whether FA reduces vascular permeability by inhibiting cyclooxygenase-2 (COX-2), a critical mediator of inflammation, and reducing overexpression of fibronectin (FN) and collagen IV (Coll IV), two basement membrane (BM) components upregulated in diabetic retinopathy. Rat retinal endothelial cells (RRECs) were grown in normal (N:5 mM glucose) or high glucose (HG:30 mM glucose) medium with or without FA for 7 days. Total protein isolated from these cells was assessed for FN, Coll IV, COX-2, and zonula occludens-1 (ZO-1), a tight junction protein, using Western blot analysis. In addition, the distribution and localization of ZO-1 was determined by immunofluorescence microscopy, and cell monolayer permeability was studied by in vitro permeability (IVP) assay. RRECs grown in HG medium showed significant increase in FN, Coll IV, and COX-2 expression (179%, 144%, 139% of N respectively), and a decrease in ZO-1 expression (48% of N) compared to those of N cells. Cells grown in HG medium supplemented with FA significantly reduced FN, Coll IV, and COX-2 expression by 47%, 32%, and 34% respectively, with concomitant increase in ZO-1 expression by 42%. In parallel studies, IVP assays showed a significant increase (139% of N) in cell monolayer permeability in RRECs grown in HG medium, which was significantly reduced with FA treatment. Additionally, immunostaining results indicated FA prevents HG-induced downregulation of ZO-1. The findings indicate that the beneficial effect of FA in reducing excess permeability is mediated, at least in part, by downregulating abnormal overexpression of BM components and inflammatory factors and preventing compromised tight junctions associated with diabetic retinopathy.

Effects of interleukin-13 and montelukast on the expression of zonula occludens-1 in human podocytes.

PurposeThe aim of this study was to investigate whether pathologic changes in zonula occludens-1 (ZO-1) are induced by interleukin-13 (IL-13) in the experimental minimal-change nephrotic syndrome (MCNS) model and to determine whether montelukast, a leukotriene receptor antagonist, has an effect on ZO-1 restoration in cultured human podocytes.Materials and MethodsHuman podocytes cultured on bovine serum albumin-coated plates were treated with different doses of IL-13 and montelukast and then examined for distribution using confocal microscopy and for ZO-1 protein levels using Western blotting.ResultsZO-1 was internalized and shown to accumulate in the cytoplasm of human podocytes in an IL-13 dose-dependent manner. High doses (50 and 100 ng/mL) of IL-13 decreased the levels of ZO-1 protein at 12 and 24 h (both p<0.01; n=3), which were significantly reversed by a high dose (0.5 µM) montelukast treatment (p<0.01; n=3).ConclusionOur results suggest that IL-13 alters the expression of ZO-1, and such alterations in the content and distribution of ZO-1 may be relevant in the pathogenesis of proteinuria in the MCNS model.

Benzalkonium chloride suppresses rabbit corneal endothelium intercellular gap junction communication.

PurposeGap junction intercellular communication (GJIC) plays a critical role in the maintenance of corneal endothelium homeostasis. We determined if benzalkonium chloride (BAK) alters GJIC activity in the rabbit corneal endothelium since it is commonly used as a drug preservative in ocular eyedrop preparations even though it can have cytotoxic effects.MethodsThirty-six adult New Zealand albino rabbits were randomly divided into three groups. BAK at 0.01%, 0.05%, and 0.1% was applied twice daily to one eye of each of the rabbits in one of the three groups for seven days. The contralateral untreated eyes were used as controls. Corneal endothelial morphological features were observed by in vivo confocal microscopy (IVCM). Immunofluorescent staining resolved changes in gap junction integrity and localization. Western blot analysis and RT-PCR evaluated changes in levels of connexin43 (Cx43) and tight junction zonula occludens-1 (ZO-1) gene and protein expression, respectively. Cx43 and ZO-1 physical interaction was detected by immunoprecipitation (IP). Primary rabbit corneal endothelial cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing BAK for 24 hours. The scrape-loading dye transfer technique (SLDT) was used to assess GJIC activity.ResultsTopical administration of BAK (0.05%, 0.1%) dose dependently disrupted corneal endothelial cell morphology, altered Cx43 and ZO-1 distribution and reduced Cx43 expression. BAK also markedly induced increases in Cx43 phosphorylation status concomitant with decreases in the Cx43-ZO-1 protein-protein interaction. These changes were associated with marked declines in GJIC activity.ConclusionsThe dose dependent declines in rabbit corneal endothelial GJIC activity induced by BAK are associated with less Cx43-ZO-1 interaction possibly arising from increases in Cx43 phosphorylation and declines in its protein expression. These novel changes provide additional evidence that BAK containing eyedrop preparations should be used with caution to avoid declines in corneal transparency resulting from losses in GJIC activity and endothelial function.

Evaluation of the protective potential of brain microvascular endothelial cell autophagy on blood-brain barrier integrity during experimental cerebral ischemia-reperfusion injury.

Brain microvascular endothelial cell (BMVEC) injury induced by ischemia-reperfusion (I/R) is the initial phase of blood-brain barrier (BBB) disruption, which results in a poor prognosis for ischemic stroke patients. Autophagy occurs in ischemic brain and has been shown to exhibit protective effects on endothelial cell against stress. However, the potential effects of BMVEC autophagy on BBB permeability during I/R and the mechanisms underlying these effects have yet to be elucidated. In the current study, we answered these questions by using chemical modulators of autophagy, including rapamycin and lithium carbonate acting, respectively, as mammalian target of rapamycin (mTOR)-dependent and mTOR-independent autophagy inducers and 3-methyladenine (3-MA) as an autophagy inhibitor. To mimic I/R injury, BMVECs were exposed to oxygen-glucose deprivation/reoxygenation (OGD/R), and a rat transient middle cerebral artery occlusion/reperfusion (MCAO/R) model was performed. All the drugs were given at 0.5h before OGD/R or MCAO/R. First, enhancement of autophagy by rapamycin and lithium carbonate attenuated, whereas suppression of autophagy by 3-MA intensified BMVEC apoptosis and the high level of ROS induced by OGD/R. In addition, rapamycin and lithium carbonate pretreatments significantly reversed the decreased level of tight junction protein zonula occludens-1 (ZO-1) induced by OGD/R and promoted the distribution of ZO-1 on cell membranes. Finally, pretreatments with rapamycin and lithium carbonate reduced evans blue extravasation and brain water content in the ischemic hemisphere of the rat. In contrast, 3-MA pretreatment exerted opposite effects both in vitro and in vivo. These results may indicate a beneficial effect of BMVEC autophagy on BBB integrity during I/R injury.

Berberine Ameliorates Ethanol-Induced Disruption of Intestinal Epithelial Tight Junction

Endotoxemia is a crucial factor contributes to alcoholic liver disease (ALD). The disruption of intestinal tight junction (TJ) caused by ethanol is one of the main underlying mechanisms. Berberine is used widely to treat gastrointestinal disorders and reportedly has beneficial effects on TJ integrity. The aims of the present study were to examine the effects of berberine on ethanol-induced intestinal epithelial TJ damage and explore the underlying mechanisms. Caco-2 cell monolayer was treated with ethanol to induce barrier damage. The effect of berberine on TJ was analyzed by measuring the transepithelial electrical resistance (TEER). Subcellular localization of TJ protein zonula occludens-1 (ZO-1) was determined by immunofluorescence microscopy. The expression levels of ZO-1, myosin light chain kinase (MLCK) and MLCK activation were analyzed by Real-time PCR and western blot. Intracellular calcium influx was measured by flow cytometry. The results showed that berberine treatment could significantly improve intestinal barrier function by decreasing intracellular calcium influx, inhibiting MLCK activation., and then reversing ZO-1 distribution. In conclusion, given that berberine also has antimicrobial and anti-inflammatory properties, the effects of berberine on endotoxins translocation are likely to be multifactorial, therefore, berberine should be a promising therapeutic option for ALD. Keywords: Alcoholic liver disease, zonula occludens-1, intracellular calcium influx, myosin light chain kinase. Read more →

Distribution of Zonula Occludens‐1 and Occludin and alterations of testicular morphology after in utero radiation and postnatal hyperthermia in rats

In utero irradiation (IR) and postnatal hyperthermia (HT) exposure cause infertility by decreasing spermatogenic colony growth and the number of sperm in rats. Four groups were used: (i) Control group, (ii) HT group (rats exposed to hyperthermia on the 10th postnatal day), (iii) IR group (rats exposed to IR on the 17th gestational day) and (iv) IR+HT group. Three and six months after the procedures testes were examined by light and electron microscopy. Some degenerated tubules in the HT group, many vacuoles in spermatogenic cells and degenerated tight junctions in the IR group, atrophic tubules and severe degeneration of tight junctions in the IR+HT group were observed. ZO-1 and occludin immunoreactivity were decreased and disorganized in the HT and IR groups and absent in the IR+HT group. The increase in the number of apoptotic cells was accompanied by a time-dependent decrease in haploid, diploid and tetraploid cells in all groups. Degenerative findings were severe after 6months in all groups. The double-hit model may represent a Sertoli cell only model of infertility due to a decrease in spermatogenic cell and alterated blood-testis barrier proteins in rat.

Altered tight junctions and fence function in NRK-52E cells induced by aristolochic acid

Aristolochic acid (AA) can accumulate in the tubulointerstitium and cause kidney-specific injuries. However, the mechanism by which AA induces nephropathy remains largely unknown. This study explored the effect of AA-I on tight junctions (TJs), and the fence function in a renal epithelial cell (REC). NRK-52E cells were exposed to different concentrations of AA-I for 4 h or 25 μM AA-I for different time. Cell viability was detected by MTT, cell apoptosis by flow cytometric analysis, the expression of zonula occludens-1 (ZO-1), E-cadherin and polarity scaffold (Par3) by western blot and immunofluorescence, cell membrane permeability by transepithelial electrical resistance (TEER). It was found that AA-I reduced the expression of ZO-1, E-cadherin, and Par3 in a concentration- and time-dependent fashion, and altered the distribution of ZO-1 and Par3 from cell membrane to cell plasma. In parallel to the reduced expression of TJ proteins, TEER exhibited a significant reduction in response to AA-I treatment in a time- and concentration-dependent manner. Meanwhile, α-SMA expression in cells was increased following AA-I treatment. In contrast, cell viability and apoptosis were unaltered with the doses of AA-I tested. Our findings show for the first time that AA-I treatment in cultured RECs induced a rapid disruption of TJ and the fence function preceding apoptosis, which indicated that aberrant expression of TJ proteins within RECs may be involved in initiating the renal tubulointerstitial disorders.

Role of Endoplasmic Reticulum Stress in Epithelial–Mesenchymal Transition of Alveolar Epithelial Cells

Endoplasmic reticulum (ER) stress has been implicated in alveolar epithelial type II (AT2) cell apoptosis in idiopathic pulmonary fibrosis. We hypothesized that ER stress (either chemically induced or due to accumulation of misfolded proteins) is also associated with epithelial-mesenchymal transition (EMT) in alveolar epithelial cells (AECs). ER stress inducers, thapsigargin (TG) or tunicamycin (TN), increased expression of ER chaperone, Grp78, and spliced X-box binding protein 1, decreased epithelial markers, E-cadherin and zonula occludens-1 (ZO-1), increased the myofibroblast marker, α-smooth muscle actin (α-SMA), and induced fibroblast-like morphology in both primary AECs and the AT2 cell line, RLE-6TN, consistent with EMT. Overexpression of the surfactant protein (SP)-C BRICHOS mutant SP-C(ΔExon4) in A549 cells increased Grp78 and α-SMA and disrupted ZO-1 distribution, and, in primary AECs, SP-C(ΔExon4) induced fibroblastic-like morphology, decreased ZO-1 and E-cadherin and increased α-SMA, mechanistically linking ER stress associated with mutant SP to fibrosis through EMT. Whereas EMT was evident at lower concentrations of TG or TN, higher concentrations caused apoptosis. The Src inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4]pyramidine) (PP2), abrogated EMT associated with TN or TG in primary AECs, whereas overexpression of SP-C(ΔExon4) increased Src phosphorylation, suggesting a common mechanism. Furthermore, increased Grp78 immunoreactivity was observed in AT2 cells of mice after bleomycin injury, supporting a role for ER stress in epithelial abnormalities in fibrosis in vivo. These results demonstrate that ER stress induces EMT in AECs, at least in part through Src-dependent pathways, suggesting a novel role for ER stress in fibroblast accumulation in pulmonary fibrosis.

Hypercholesterolaemic Serum Increases the Permeability of Endothelial Cells through Zonula Occludens‐1 with Phosphatidylinositol 3‐Kinase Signaling Pathway

Purpose. Hypercholesterolemia and tight junctions play important roles in atherosclerosis. But the relationship between these two factors is unclear. In the present study, we investigated whether hypercholesterolemic serum could change the permeability of endothelial cells through altering expression and/or distribution of tight junction protein zonula occludens-1 (ZO-1). Phosphatidylinositol 3-kinase (PI3K) signaling pathway was also examined. Materials and Methods. Cultured endothelial cells were treated with different concentration levels of hypercholesterolemic serum. The expression and distribution of ZO-1, the permeability of cultured cells and the involvement of PI3K signaling pathway were measured by various methods. Results. In the present study, we found that hypercholesterolemic serum could not change the expression of ZO-1 either in mRNA or protein level. However, hypercholesterolemic serum could change the distribution of ZO-1 in cultured endothelial cells, and increase the permeability with a dose-dependent manner. When PI3K specific inhibitor wortmannin was used, the effects induced by hypercholesterolemic serum could be partly reversed. The role of PI3K signaling pathway was further confirmed by PI3K activity assay. Conclusions. Our results suggested that although hypercholesterolemic serum could not change the expression of ZO-1, it could change the distribution and increase the permeability in endothelial cells through PI3K signaling pathway.