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tight junction [keywords]
- Escherichia coli STb Enterotoxin Dislodges Claudin-1 from Epithelial Tight Junctions. [Journal Article]
- PLoS One 2014; 9(11):e113273.
Enterotoxigenic Escherichia coli produce various heat-labile and heat-stable enterotoxins. STb is a low molecular weight heat-resistant toxin responsible for diarrhea in farm animals, mainly young pigs. A previous study demonstrated that cells having internalized STb toxin induce epithelial barrier dysfunction through changes in tight junction (TJ) proteins. These modifications contribute probably to the diarrhea observed. To gain insight into the mechanism of increased intestinal permeability following STb exposure we treated human colon cells (T84) with purified STb toxin after which cells were harvested and proteins extracted. Using a 1% Nonidet P-40-containing solution we investigated the distribution of claudin-1, a major structural and functional TJ protein responsible for the epithelium impermeability, between membrane (NP40-insoluble) and the cytoplasmic (NP-40 soluble) location. Using immunoblot and confocal microscopy, we observed that treatment of T84 cell monolayers with STb induced redistribution of claudin-1. After 24 h, cells grown in Ca++-free medium treated with STb showed about 40% more claudin-1 in the cytoplasm compare to the control. Switching from Ca++-free to Ca++-enriched medium (1.8 mM) increased the dislodgement rate of claudin-1 as comparable quantitative delocalization was observed after only 6 h. Medium supplemented with the same concentration of Mg++ or Zn++ did not affect the dislodgement rate compared to the Ca++-free medium. Using anti-phosphoserine and anti-phosphothreonine antibodies, we observed that the loss of membrane claudin-1 was accompanied by dephosphorylation of this TJ protein. Overall, our findings showed an important redistribution of claudin-1 in cells treated with STb toxin. The loss of phosphorylated TJ membrane claudin-1 is likely to be involved in the increased permeability observed. The mechanisms by which these changes are brought about remain to be elucidated.
- Effect of γ-Irradiation on Expression of Tight and Adherens Junction Protein mRNA on In Vitro Blood-Brain Barrier Model. [JOURNAL ARTICLE]
- Bull Exp Biol Med 2014 Nov 19.
We studied the effect of γ-irradiation on HUVEC endothelial cells co-cultured with allogeneic astrocytes. This 2D in vitro model of the blood-brain barrier has the same parameters as cerebral microvascular endothelial cells forming the blood-brain barrier and allows reproducing its functions in vivo. Dose-dependent changes in cell morphology and violation of monolayer integrity were observed. Real-time PCR and immunocytochemical analysis revealed changes in the expression of tight (ZO-1, claudin-5) and adherens junction protein (vascular endothelial cadherin, β-catenin) mRNA. Expression of tight and adherens junction proteins mRNA decreased in 2, 24, and 48 h after irradiation in doses of 2, 4, and 6 Gy. Significant dosedependent changes were found only for β-catenin mRNA expression in 2 h after exposition. This model of blood-brain barrier in vitro can be used for studying the molecular mechanisms regulating permeability of cerebral endothelium under normal conditions and after pathological exposures, e.g. γ-irradiation.
- Modulation of VEGF-induced retinal vascular permeability by peroxisome proliferator-activated receptor-β/δ [JOURNAL ARTICLE]
- Invest Ophthalmol Vis Sci 2014 Nov 18.
Vascular endothelial growth factor-induced retinal vascular permeability contributes to diabetic macular edema (DME), a serious vision threatening condition. Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) antagonist/reverse agonist, GSK0660, inhibits VEGF-induced human retinal microvascular endothelial cell (HRMEC) proliferation, tubulogenesis, and oxygen-induced retinal vasculopathy in newborn rats. These VEGF-induced HRMEC behaviors and VEGF-induced disruption of endothelial cell junctional complexes may well share molecular signaling events. Thus, we sought to examine the role of PPARβ/δ in VEGF-induced retinal hyper permeability. Trans-endothelial electrical resistance measurements were performed on HRMEC monolayers to assess permeability. Claudin-1/-5 localization in HRMEC monolayers was determined by immunocytochemistry. Erk 1/2 phosphorylation, VEGFR1 and R2 were assayed by western blot analysis. VEGFR1/R2 expression was measured by qRT-PCR. Lastly, retinal vascular permeability was assayed in vivo by Evans blue extravasation. HRMEC monolayers treated with VEGF for 24 hours showed decreased TEER values that were completely reversed by the highest concentration of GSK0660 (10µM) and PPARβ/δ-directed siRNA (20μM). In HRMEC treated with VEGF, GSK0660 stabilized tight junctions as evidenced by Claudin-1 staining, reduced phosphorylation of Erk1/2, and reduced VEGFR1/2 expression. PPARβ/δ siRNA had a similar effect on VEGFR expression and Claudin-1 supporting the specificity of GSK0660 in our experiments. Lastly, GSK0660 significantly inhibited VEGF-induced retinal vascular permeability and reduced retinal VEGFR1/R2 levels in C57BL/6 mice. These data suggest a protective effect for PPARβ/δ antagonism against VEGF-induced vascular permeability, possibly through reduced VEGFR expression. Therefore, antagonism/reverse agonism of PPARβ/δ siRNA may represent a novel therapeutic methodology against retinal hyperpermeability and is worthy of future investigation.
- The impact of microglial activation on blood-brain barrier in brain diseases. [Journal Article, Review]
- Front Cell Neurosci 2014.:362.
The blood-brain barrier (BBB), constituted by an extensive network of endothelial cells (ECs) together with neurons and glial cells, including microglia, forms the neurovascular unit (NVU). The crosstalk between these cells guarantees a proper environment for brain function. In this context, changes in the endothelium-microglia interactions are associated with a variety of inflammation-related diseases in brain, where BBB permeability is compromised. Increasing evidences indicate that activated microglia modulate expression of tight junctions, which are essential for BBB integrity and function. On the other hand, the endothelium can regulate the state of microglial activation. Here, we review recent advances that provide insights into interactions between the microglia and the vascular system in brain diseases such as infectious/inflammatory diseases, epilepsy, ischemic stroke and neurodegenerative disorders.
- Zinc and gastrointestinal disease. [Journal Article, Review]
- World J Gastrointest Pathophysiol 2014 Nov 15; 5(4):496-513.
This review is a current summary of the role that both zinc deficiency and zinc supplementation can play in the etiology and therapy of a wide range of gastrointestinal diseases. The recent literature describing zinc action on gastrointestinal epithelial tight junctions and epithelial barrier function is described. Zinc enhancement of gastrointestinal epithelial barrier function may figure prominently in its potential therapeutic action in several gastrointestinal diseases.
- Involvement of RhoA/ROCK1 signaling pathway in hyperglycemia-induced microvascular endothelial dysfunction in diabetic retinopathy. [Journal Article]
- Int J Clin Exp Pathol 2014; 7(10):7268-77.
Diabetic retinopathy (DR) is a well-known serious complication of diabetes mellitus (DM), and can eventually advance to end-stage blindness. In the early stage of DR, endothelial cell barrier disorganized primarily and tight junction (TJ) protein composition transformed subsequently. The small GTPase RhoA and its downstream effector Rho-associated coiled-coil containing protein kinase 1 (ROCK1) regulate a mass of cellular processes, including cell adherence, proliferation, permeability and apoptosis. Although RhoA inhibitors have provided substantial clinical benefit as hypertonicity therapeutics, their use is limited by complex microenvironment as DR. While ample evidence indicates that TJ can be influenced by the RhoA/ROCK1 signaling, the underlying mechanisms remain incompletely understood. Here, we have uncovered a significant signaling network involved in diabetic retinal microvascular endothelial dysfunction (RMVED). Our results indicated that the activation of RhoA/ROCK1 pathway due to high glucose played a key role in microvascular endothelial cell dysfunction (MVED) by way of directly inducing TJ proteins over-expression during DR. We demonstrated that inhibition of RhoA/ROCK1 may attenuate the hypertonicity of endothelial cell caused by high glucose microenvironment meanwhile. Besides, chemical and pharmacological inhibitors of RhoA/ROCK1 pathway may partly block inflammation due to DR. Simultaneously, the apoptosis aroused by high glucose was also prevented considerably by fasudil, a kind of pharmacological inhibitor of RhoA/ROCK1 pathway. These findings indicate that RhoA/ROCK1 signaling directly modulates MVED, suggesting a novel therapeutic target for DR.
- Dysregulation of JAM-A plays an important role in human tumor progression. [REVIEW]
- Int J Clin Exp Pathol 2014; 7(10):7242-7248.
Junctional adhesion molecule A (JAM-A) is a transmembrane protein that belongs to the immunoglobulin (Ig) superfamily. Evidence determines that JAM-A plays a role in numerous cellular processes, including tight junction assembly, leukocyte migration, platelet activation, angiogenesis and virus binding. Recent research suggests that JAM-A is dysregulated in various cancers and is vital for tumor progression. JAM-A is implicated in carcinogenesis via different signal pathways such as TGF-β1 signaling. Furthermore, JAM-A expression in cancers is usually associated with certain outcome of patients and might be a prognostic indicator. In this review, the correlation between JAM-A expression and human cancers will be described.
- Role of Nrf2 Dysfunction in Uremia-Associated Intestinal Inflammation and Epithelial Barrier Disruption. [JOURNAL ARTICLE]
- Dig Dis Sci 2014 Nov 16.
Gut inflammation is prevalent in chronic kidney disease (CKD) and likely contributes to systemic inflammation via disruption of the epithelial tight junction with subsequent endotoxin and bacterial translocation.To study the expression profile of inflammatory and tight junction proteins in the colon from CKD rats compared to healthy controls, and demonstrate the role of Nrf2 (transcription factor nuclear factor erythroid 2-related factor 2) using a potent Nrf2 activator.CKD was induced via 5/6 nephrectomy in Sprague-Dawley rats, and dh404 (2 mg/kg/day) was used to study the effects of systemic Nrf2 activation. The experimental groups included sham, CKD and CKD+ dh404 rats. Blood and colon tissues were analyzed after a 10-week study period.Colon from CKD rats showed histological evidence of colitis, depletion of epithelial tight junction proteins, significant reduction of Nrf2 and its measured target gene products (NQO1, catalase, and CuZn SOD), activation of NFkB, and upregulation of pro-inflammatory molecules (COX-2, MCP-1, iNOS, and gp91(phox)). Treatment with dh404 attenuated colonic inflammation, restored Nrf2 activity and levels of NQO1, catalase and CuZn SOD, decreased NFkB and lowered expression of COX-2, MCP-1, iNOS, and gp91(phox). This was associated with restoration of colonic epithelial tight junction proteins (occludin and claudin-1).CKD rats exhibited colitis, disruption of colonic epithelial tight junction, activation of inflammatory mediators, and impairment of Nrf2 pathway. Treatment with an Nrf2 activator restored Nrf2 activity, attenuated colonic inflammation, and restored epithelial tight junction proteins.
- Charge Transport Across Insulating Self-Assembled Monolayers: Non-equilibrium Approaches and Modeling To Relate Current and Molecular Structure. [JOURNAL ARTICLE]
- ACS Nano 2014 Nov 25.
This paper examines charge transport by tunneling across a series of electrically insulating molecules with the structure HS(CH2)4CONH(CH2)2R) in the form of self-assembled monolayers (SAMs), supported on silver. The molecules examined were studied experimentally by Yoon et al. (Angew. Chem. Int. Ed. 2012, 51, 4658-4661), using junctions of the structure AgS(CH2)4CONH(CH2)2R//Ga2O3/EGaIn. The tail group R had approximately the same length for all molecules, but a range of different structures. Changing the R entity over the range of different structures (aliphatic to aromatic) does not influence the conductance significantly. To rationalize this surprising result, we investigate transport through these SAMs theoretically, using both full quantum methods and a generic, independent-electron tight-binding toy model. We find that the highest occupied molecular orbital, which is largely responsible for the transport in these molecules, is always strongly localized on the thiol group. The relative insensitivity of the current density to the structure of the R group is due to a combination of the couplings between the carbon chains and the transmission inside the tail. Changing from saturated to conjugated tail groups increases the latter but decreases the former. This work indicates that significant control over SAMs largely composed of nominally insulating groups may be possible when tail groups are used that are significantly larger than those used in the experiments of Yoon et al.1.
- Loss of EP2 Receptor Subtype in Colonic Cells Compromise Epithelial Barrier Integrity by Altering Claudin-4. [Journal Article]
- PLoS One 2014; 9(11):e113270.
Prostaglandin E2 (PGE2) is a bioactive lipid mediator that exerts its biological function through interaction with four different subtypes of E-Prostanoid receptor namely EP1, EP2, EP3 and EP4. It has been known that EP2 receptor is differentially over-expressed in the epithelia of inflamed human colonic mucosa. However, the significance of the differential expression in altering epithelial barrier function is not known. In this study, we used Caco-2 cells expressing EP2 receptor, either high (EP2S) or low (EP2A), as a model epithelia and determined the barrier function of these cell monolayers by measuring the trans epithelial resistance (TER). Basal TER of EP2A (but not EP2S) monolayer was significantly lower suggesting a loss of colonic epithelial barrier integrity. In comparison, the TER of wild type Caco-2 was decreased in response to an EP2 receptor specific antagonist (AH-6809) indicating an important role for EP2 receptor in the maintenance of epithelial barrier function. The decrease TER in EP2A monolayer corresponded with a significant loss of the tight junction (TJ) protein claudin-4 without affecting other major TJ proteins. Similarly, EP2 receptor antagonism/siRNA based silencing significantly decreased claudin-4 expression in EP2S cells. Surprisingly, alteration in claudin-4 was not transcriptionally regulated in EP2A cells but rather undergoes increased proteosomal degradation. Moreover, among the TER compromising cytokines examined (IL-8, IL-1β, TNF-α, IFN-γ) only IFN-γ was significantly up regulated in EP2A cells. However, IFN-γ did not significantly decreased claudin-4 expression in Caco-2 cells indicating no role for IFN-γ in degrading claudin-4. We conclude that differential down-regulation of EP2 receptor play a major role in compromising colonic epithelial barrier function by selectively increasing proteosomal degradation of claudin-4.