Tight Junction Protein Zona Occludens-1 Research Articles

Short communication: Differential loss of bovine mammary epithelial barrier integrity in response to lipopolysaccharide and lipoteichoic acid

In the mammary gland, the blood–milk barrier prevents an uncontrolled intermixture of blood and milk constituents and hence maintains the osmotic gradient to draw water into the mammary secretion. During mastitis, the permeability of the blood–milk barrier is increased, which is reflected by the transfer of blood constituents into milk and vice versa. In this study, we aimed to investigate changes in the barrier function of mammary epithelial cells in vitro as induced by cell wall components of different pathogens. Primary bovine mammary epithelial cells from 3 different cows were grown separately on Transwell (Corning Inc., Corning, NY) inserts. The formation of tight junctions between adjacent epithelial cells was shown by transmission electron microscopy and by immunofluorescence staining of the tight junction protein zona occludens-1. The integrity of the epithelial barrier was assayed by means of transepithelial electrical resistance, as well as by diffusion of the fluorophore Lucifer yellow across the cell layer. The release of lactate dehydrogenase (LDH) was used as an indicator for cytotoxic effects. In response to a 24-h challenge with bacterial endotoxin, barrier integrity was reduced after 3 or 7h, respectively, in response to 0.5mg/mL lipopolysaccharide (LPS) from Escherichia coli or 20mg/mL lipoteichoic acid (LTA) from Staphylococcus aureus. No paracellular leakage was observed in response to 0.2mg/mL LPS or 2mg/mL LTA. Although LPS and LTA affected barrier permeability, most likely by opening the tight junctions, only LPS caused cell damage, reflected by increased LDH concentrations in cell culture medium. These results prove a pathogen-specific loss of blood–milk barrier integrity during mastitis, which is characterized by tight junction opening by both LPS and LTA and by additional epithelial cell destruction through LPS.

ZO-2 silencing induces renal hypertrophy through a cell cycle mechanism and the activation of YAP and the mTOR pathway.

Renal compensatory hypertrophy (RCH) restores normal kidney function after disease or loss of kidney tissue and is characterized by an increase in organ size due to cell enlargement and not to cell proliferation. In MDCK renal epithelial cells, silencing of the tight junction protein zona occludens 2 (ZO-2 KD) induces cell hypertrophy by two mechanisms: prolonging the time that cells spend at the G1 phase of the cell cycle due to an increase in cyclin D1 level, and augmenting the rate of protein synthesis. The latter is triggered by the nuclear accumulation and increased transcriptional activity of Yes-associated protein (YAP), the main target of the Hippo pathway, which results in decreased expression of phosphatase and tensin homologue. This in turn increased the level of phosphatidylinositol (3,4,5)-triphosphate, which transactivates the Akt/mammalian target of rapamycin pathway, leading to activation of the kinase S6K1 and increased synthesis of proteins and cell size. In agreement, in a rat model of uninephrectomy, RCH is accompanied by decreased expression of ZO-2 and nuclear expression of YAP. Our results reveal a novel role of ZO-2 as a modulator of cell size.

The extracellular matrix protein laminin-10 promotes blood-brain barrier repair after hypoxia and inflammation in vitro.

BackgroundThe blood–brain barrier (BBB) of the central nervous system (CNS) is essential for normal brain function. However, the loss of BBB integrity that occurs after ischaemic injury is associated with extracellular matrix (ECM) remodelling and inflammation, and contributes to poor outcome. ECM remodelling also contributes to BBB repair after injury, but the precise mechanisms and contribution of specific ECM molecules involved are unknown. Here, we investigated the mechanisms by which hypoxia and inflammation trigger loss of BBB integrity and tested the hypothesis ECM changes could contribute to BBB repair in vitro.MethodsWe used an in vitro model of the BBB, composed of primary rat brain endothelial cells grown on collagen (Col) I-, Col IV-, fibronectin (FN)-, laminin (LM) 8-, or LM10-coated tissue culture plates, either as a single monolayer culture or on Transwell® inserts above mixed glial cell cultures. Cultures were exposed to oxygen-glucose deprivation (OGD) and/or reoxygenation, in the absence or the presence of recombinant interleukin-1β (IL-1β). Cell adhesion to ECM molecules was assessed by cell attachment and cell spreading assays. BBB dysfunction was assessed by immunocytochemistry for tight junction proteins occludin and zona occludens-1 (ZO-1) and measurement of trans-endothelial electrical resistance (TEER). Change in endothelial expression of ECM molecules was assessed by semi-quantitative RT-PCR.ResultsOGD and/or IL-1 induce dramatic changes associated with loss of BBB integrity, including cytoplasmic relocalisation of membrane-associated tight junction proteins occludin and ZO-1, cell swelling, and decreased TEER. OGD and IL-1 also induced gene expression of key ECM molecules associated with the BBB, including FN, Col IV, LM 8, and LM10. Importantly, we found that LM10, but not FN, Col IV, nor LM8, plays a key role in maintenance of BBB integrity and reversed most of the key hallmarks of BBB dysfunction induced by IL-1.ConclusionsOur data unravel new mechanisms of BBB dysfunction induced by hypoxia and inflammation and identify LM10 as a key ECM molecule involved in BBB repair after hypoxic injury and inflammation.

Th17 Cytokines Disrupt the Airway Mucosal Barrier in Chronic Rhinosinusitis.

Cytokine mediated changes in paracellular permeability contribute to a multitude of pathological conditions including chronic rhinosinusitis (CRS). The purpose of this study was to investigate the effect of interferons and of Th1, Th2, and Th17 cytokines on respiratory epithelium barrier function. Cytokines and interferons were applied to the basolateral side of air-liquid interface (ALI) cultures of primary human nasal epithelial cells (HNECs) from CRS with nasal polyp patients. Transepithelial electrical resistance (TEER) and permeability of FITC-conjugated dextrans were measured over time. Additionally, the expression of the tight junction protein Zona Occludens-1 (ZO-1) was examined via immunofluorescence. Data was analysed using ANOVA, followed by Tukey HSD post hoc test. Our results showed that application of interferons and of Th1 or Th2 cytokines did not affect the mucosal barrier function. In contrast, the Th17 cytokines IL-17, IL-22, and IL-26 showed a significant disruption of the epithelial barrier, evidenced by a loss of TEER, increased paracellular permeability of FITC-dextrans, and discontinuous ZO-1 immunolocalisation. These results indicate that Th17 cytokines may contribute to the development of CRSwNP by promoting a leaky mucosal barrier.

Prolactin/Jak2 directs apical/basal polarization and luminal linage maturation of mammary epithelial cells through regulation of the Erk1/2 pathway

Tissue development/remodeling requires modulations in both cellular architecture and phenotype. Aberration in these processes leads to tumorigenesis. During the pregnancy/lactation cycle the mammary epithelial cells undergo complex morphological and phenotypic programs resulting in the acquisition of apical/basal (A/B) polarization and cellular maturation necessary for proper lactation. Still the hormonal regulations and cellular mechanisms controlling these events are not entirely elucidated. Here we show that prolactin (PRL)/Jak2 pathway in mammary epithelial cells uniquely signals to establish A/B polarity as determined by the apical localization of the tight junction protein zona occludens 1 (ZO-1) and the basal/lateral localization of E-cadherin, and the apical trafficking of lipid droplets. As well, our results indicate that this pathway regulates mammary stem cell hierarchy by inducing the differentiation of luminal progenitor (EpCAMhi/CD49fhi) cells to mature luminal (EpCAMhi/CD49flow) cells. Moreover, our data indicate that PRL/Jak2 coordinates both of these cellular events through limiting the mitogen activated protein kinase (Erk1/2) pathway. Together our findings define a novel unifying mechanism coupling mammary epithelial cell A/B polarization and terminal differentiation.

Chrysin inhibits diabetic renal tubulointerstitial fibrosis through blocking epithelial to mesenchymal transition.

Renal fibrosis is a crucial event in the pathogenesis of diabetic nephropathy (DN). The process known as epithelial to mesenchymal transition (EMT) contributes to the accumulation of matrix proteins in kidneys, in which renal tubular epithelial cells play an important role in progressive renal fibrosis. The current study investigated that chrysin (5,7-dihydroxyflavone) present in bee propolis and herbs, inhibited renal tubular EMT and tubulointerstitial fibrosis due to chronic hyperglycemia. Human renal proximal tubular epithelial cells (RPTEC) were incubated in media containing 5.5 mM glucose, 27.5 mM mannitol (as an osmotic control), or 33 mM glucose (HG) in the absence and presence of 1-20 μM chrysin for 72 h. Chrysin significantly inhibited high glucose-induced renal EMT through blocking expression of the mesenchymal markers vimentin, α-smooth muscle actin, and fibroblast-specific protein-1 in RPTEC and db/db mice. Chrysin reversed the HG-induced down-regulation of the epithelial marker E-cadherin and the HG-enhanced N-cadherin induction in RPTEC. In addition, chrysin inhibited the production of collagen IV in tubular cells and the deposition of collagen fibers in mouse kidneys. Furthermore, chrysin blocked tubular cell migration concurrent with decreasing matrix metalloproteinase-2 activity, indicating epithelial cell derangement and tubular basement membrane disruption. Chrysin restored the induction of the tight junction proteins Zona occludens protein-1 (ZO-1) and occludin downregulated in diabetic mice. Chrysin inhibited renal tubular EMT-mediated tubulointerstitial fibrosis caused by chronic hyperglycemia. Therefore, chrysin may be a potent renoprotective agent for the treatment of renal fibrosis-associated DN. • Glucose increases renal tubular epithelial induction of vimentin, α-SMA and FSP-1. • Glucose enhances renal EMT by blocking tubular epithelial E-cadherin expression. • Chrysin inhibits tubular EMT-mediated tubulointerstitial fibrosis in mouse kidneys. • Chrysin restores renal tubular induction of ZO-1 and occludin downregulated in diabetic mice. • Chrysin blocks glucose-induced renal tubular cell migration with reducing MMP-2 activity.

Micro- and nano-topography to enhance proliferation and sustain functional markers of donor-derived primary human corneal endothelial cells

One of the most common indications for corneal transplantation is corneal endothelium dysfunction, which can lead to corneal blindness. Due to a worldwide donor cornea shortage, alternative treatments are needed, but the development of new treatment strategies relies on the successful in vitro culture of primary human corneal endothelial cells (HCECs) because transformed cell lines and animal-derived corneal endothelial cells are not desirable for therapeutic applications. Primary HCECs are non-proliferative in vivo and challenging to expand in vitro while maintaining their characteristic cell morphology and critical markers. Biochemical cues such as growth factors and small molecules have been investigated to enhance the expansion of HCECs with a limited increase in proliferation. In this study, patterned tissue culture polystyrene (TCPS) was shown to significantly enhance the expansion of HCECs. The proliferation of HCECs increased up to 2.9-fold, and the expression amount and localization of cell–cell tight junction protein Zona Occludens-1 (ZO-1) was significantly enhanced when grown on 1μm TCPS pillars. 250nm pillars induced an optimal hexagonal morphology of HCEC cells. Furthermore, we demonstrated that the topographical effect on tight-junction expression and cell morphology could be maintained throughout each passage, and was effectively ‘remembered’ by the cells. Higher amount of tight-junction protein expression was maintained at cell junctions when topographic cues were removed in the successive seeding. This topographic memory suggested topography-exposed/induced cells would maintain the enhanced functional markers, which would be useful in cell-therapy based approaches to enable the in situ endothelial cell monolayer formation upon delivery. The development of patterned TCPS culture platforms could significantly benefit those researching human corneal endothelial cell cultivation for cell therapy, and tissue engineering applications.

Aquaporins and blood–brain barrier permeability in early edema development after traumatic brain injury

Traumatic brain injury (TBI) is a major contributor to mortality and morbidity. The pathophysiology involves development of brain edema. Therapeutic options are limited as the mechanisms are not fully understood. Changes in the function of the blood–brain barrier (BBB), as well as variations in aquaporin expression, have been proposed to be involved in the development of the edema but the contribution of each factor has not been fully elucidated. In order to evaluate these mechanisms, in a potential window of opportunity, the early dynamic response was studied using an animal model causing a moderate TBI. Sprague-Dawley rats were subjected to blunt controlled head trauma and followed for up to four days by magnetic-resonance-imaging, immunohistofluorescence, immunohistochemistry, and quantitative protein analysis. Non-traumatized animals served as controls. TBI resulted in a midline shift and a decrease in Apparent Diffusion Coefficient, indicating a hemispheric enlargement due to cytotoxic edema. The tight junction protein Zona Occludens-1 was decreased (−25%) and associated with an increased IgG permeability (+20%) in the perilesional brain tissue in accordance with a BBB breakdown. The total amount of AQP4 protein decreased (−20%). The disruption of the BBB lasted for 4 days while the impact on AQP4 levels disappeared between day 1 and 4. Our findings shows that blunt focal brain injury results in an early development of brain edema involving both cytotoxic and vasogenic components, a persistent BBB breakdown and a temporary decrease in AQP4, and indicates that both types of edemas and mechanisms should be targeted in TBI treatment.

Electroacupuncture activates enteric glial cells and protects the gut barrier in hemorrhaged rats

To investigate whether electroacupuncture ST36 activates enteric glial cells, and alleviates gut inflammation and barrier dysfunction following hemorrhagic shock. Sprague-Dawley rats were subjected to approximately 45% total blood loss and randomly divided into seven groups: (1) sham: cannulation, but no hemorrhage; (2) subjected to hemorrhagic shock (HS); (3) electroacupuncture (EA) ST36 after hemorrhage; (4) vagotomy (VGX)/EA: VGX before hemorrhage, then EA ST36; (5) VGX: VGX before hemorrhage; (6) α-bungarotoxin (BGT)/EA: intraperitoneal injection of α-BGT before hemorrhage, then EA ST36; and (7) α-BGT group: α-BGT injection before hemorrhage. Morphological changes in enteric glial cells (EGCs) were observed by immunofluorescence, and glial fibrillary acidic protein (GFAP; a protein marker of enteric glial activation) was evaluated using reverse transcriptase polymerase chain reaction and western blot analysis. Intestinal cytokine levels, gut permeability to 4-kDa fluorescein isothiocyanate (FITC)-dextran, and the expression and distribution of tight junction protein zona occludens (ZO)-1 were also determined. EGCs were distorted following hemorrhage and showed morphological abnormalities. EA ST36 attenuated the morphological changes in EGCs at 6 h, as compared with the VGX, α-BGT and HS groups. EA ST36 increased GFAP expression to a greater degree than in the other groups. EA ST36 decreased intestinal permeability to FITC-dextran (760.5 ± 96.43 ng/mL vs 2466.7 ± 131.60 ng/mL, P < 0.05) and preserved ZO-1 protein expression and localization at 6 h after hemorrhage compared with the HS group. However, abdominal VGX and α-BGT treatment weakened or eliminated the effects of EA ST36. EA ST36 reduced tumor necrosis factor-α levels in intestinal homogenates after blood loss, while vagotomy or intraperitoneal injection of α-BGT before EA ST36 abolished its anti-inflammatory effects. EA ST36 attenuates hemorrhage-induced intestinal inflammatory insult, and protects the intestinal barrier integrity, partly via activation of EGCs.

ET-65 * THE KETOGENIC DIET REDUCES CARBONIC ANHYDRASE 9 AND ALTERS THE EXPRESSION OF PROTEINS INVOLVED WITH GLIOMA GROWTH AND PROGRESSION IN HYPOXIC CONDITIONS

Patients with malignant gliomas have a median survival of approximately 15 months following diagnosis, regardless of currently available treatments which include surgery, radiation and chemotherapy. Improvement in the survival of these patients requires the design of new therapeutic modalities that take advantage of common phenotypes. One such phenotype is metabolic dysregulation - a hallmark of cancer cells. It has been postulated that one approach to treating gliomas may be by metabolic alteration such as that which occurs through the use of the ketogenic diet (KD). The KD is high-fat, low-carbohydrate diet that induces ketosis and has been utilized for the non-pharmacologic treatment of refractory epilepsy. We and others have shown that this diet enhances survival and potentiates standard therapy in mouse models of glioma, yet the anti-tumor mechanisms are not fully understood. We have previously shown that the KD reduces peritumoral edema and tumor vasculature in a mouse model of glioma. We now show that tumors from animals fed the KD show reduced expression of the hypoxia marker carbonic anhydrase 9 (CA9) when compared to animals fed a standard rodent diet (SD). Protein analyses showed that the KD alters the expression of the tight junction protein zona occludens 1 (ZO-1), and the membrane water channel aquaporin-4 (AQPN4), which are regulated by tumor hypoxia and have been implicated in peritumoral edema. We also see a reduction in phosphorylated NF-κB which may result in reduced transcription of hypoxia-related genes. Further analysis showed that the KD reduced expression of vascular endothelial growth factor receptor 2 (VEGFR2), which plays a key role in hypoxia-induced angiogenesis. Taken together our data offers insight into the anti-edema and anti-angiogenic mechanisms underlying the KD. A greater understanding of the effects of the KD as an adjuvant therapy will allow for a more rational approach to its clinical use.

Blood–brain barrier dysfunction in mice induced by lipopolysaccharide is attenuated by dapsone

Blood–brain barrier (BBB) dysfunction is a key event in the development of many central nervous system (CNS) diseases, such as septic encephalopathy and stroke. 4,4′-Diaminodiphenylsulfone (DDS, Dapsone) has displayed neuroprotective effect, but whether DDS has protective role on BBB integrity is not clear. This study was designed to examine the effect of DDS on lipopolysaccharide (LPS)-induced BBB disruption and oxidative stress in brain vessels. Using in vivo multiphoton imaging, we found that DDS administration significantly restored BBB integrity compromised by LPS. DDS also increased the expression of tight junction proteins occludin, zona occludens-1 (ZO-1) and claudin-5 in brain vessels. Level of reactive oxygen species (ROS) was reduced by DDS treatment, which may due to decreased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and NOX2 expression. Our results showed that LPS-induced BBB dysfunction could be attenuated by DDS, indicated that DDS has a therapeutic potential for treating CNS infection and other BBB related diseases.

Phospholipid transfer protein (PLTP) deficiency impaired blood–brain barrier integrity by increasing cerebrovascular oxidative stress

Phospholipid transfer protein (PLTP) regulates lipid metabolism and plays an important role in oxidative stress. PLTP is highly expressed in blood–brain barrier (BBB), but the role of PLTP in BBB integrity is not clear. In this study, BBB permeability was detected with in vivo multiphoton imaging and Evans blue assay. We found that PLTP deficient mice exhibited increased BBB permeability, as well as decreased expression of tight junction proteins occludin, zona occludens-1 (ZO-1) and claudin-5 in brain vessels. Cerebrovascular oxidative stress increased in PLTP deficient mice, including increased levels of reactive oxygen species (ROS) and lipid peroxidation marker 4-hydroxy-2-nonenal (HNE) and reduced superoxide dismutase (SOD) activity. Dietary supplementation of antioxidant vitamin E increased BBB integrity and tight junction proteins expression via reducing cerebrovascular oxidative stress. These findings indicated an essential role of PLTP in maintaining BBB integrity, possibly through its ability to transfer vitamin E, and modulate cerebrovascular oxidative stress.

Neurovascular protection by post‐ischemic intravenous injections of the lipoxin A4 receptor agonist, BML‐111, in a rat model of ischemic stroke

Resolution of inflammation is an emerging new strategy to reduce damage following ischemic stroke. Lipoxin A4 (LXA4 ) is an anti-inflammatory, pro-resolution lipid mediator with high affinity binding to ALX, the lipoxin A4 receptor. Since LXA4 is rapidly inactivated, potent analogs have been created, including the ALX agonist BML-111. We hypothesized that post-ischemic intravenous administration of BML-111 would provide protection to the neurovascular unit and reduce neuroinflammation in a rat stroke model. Animals were subjected to 90 min of middle cerebral artery occlusion (MCAO) and BML-111 was injected 100 min and 24 h after stroke onset and animals euthanized at 48 h. Post-ischemic treatment with BML-111 significantly reduced infarct size, decreased vasogenic edema, protected against blood-brain barrier disruption, and reduced hemorrhagic transformation. Matrix metalloproteinase-9 and matrix metalloproteinase-3 were significantly reduced following BML-111 treatment. Administration of BML-111 dramatically decreased microglial activation, as seen with CD68, and neutrophil infiltration and recruitment, as assessed by levels of myeloperoxidase and intracellular adhesion molecule-1. The tight junction protein zona occludens-1 was protected from degradation following treatment with BML-111. These results indicate that post-ischemic activation of ALX has pro-resolution effects that limit the inflammatory damage in the cerebral cortex and helps maintain blood-brain barrier integrity after ischemic stroke.

Short-Chain Fatty Acids Activate AMP-Activated Protein Kinase and Ameliorate Ethanol-Induced Intestinal Barrier Dysfunction in Caco-2 Cell Monolayers

Short-chain fatty acids (SCFAs) have been shown to promote intestinal barrier function, but their protective effects against ethanol-induced intestinal injury and underlying mechanisms remain essentially unknown. The aim of the study was to analyze the influence of SCFAs on ethanol-induced barrier dysfunction and to examine the role of AMP-activated protein kinase (AMPK) as a possible mechanism using Caco-2 monolayers. The monolayers were treated apically with butyrate (2, 10, or 20 mmol/L), propionate (4, 20, or 40 mmol/L), or acetate (8, 40, or 80 mmol/L) for 1 h before ethanol (40 mmol/L) for 3 h. Barrier function was analyzed by measurement of transepithelial resistance and permeation of fluorescein isothiocyanate-labeled dextran. Distribution of the tight junction (TJ) proteins zona occludens-1, occludin, and filamentous-actin (F-actin) was examined by immunofluorescence. Metabolic stress was determined by measuring oxidative stress, mitochondrial function, and ATP using dichlorofluorescein diacetate, dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide, and bioluminescence assay, respectively. AMPK was knocked down by small interfering RNA (siRNA), and its activity was assessed by a cell-based ELISA. Exposure to ethanol significantly impaired barrier function compared with controls (P < 0.0001), disrupted TJ and F-actin cytoskeleton integrity, and induced metabolic stress. However, pretreatment with 2 mmol/L butyrate, 4 mmol/L propionate, and 8 mmol/L acetate significantly alleviated the ethanol-induced barrier dysfunction, TJ and F-actin disruption, and metabolic stress compared with ethanol-exposed monolayers (P < 0.0001). The promoting effects on barrier function were abolished by inhibiting AMPK using either compound C or siRNA. These observations indicate that SCFAs exhibit protective effects against ethanol-induced barrier disruption via AMPK activation, suggesting a potential for SCFAs as prophylactic and/or therapeutic factors against ethanol-induced gut leakiness.

Intraluminal acid activates esophageal nodose C fibers after mast cell activation

Acid reflux in the esophagus can induce esophageal painful sensations such as heartburn and noncardiac chest pain. The mechanisms underlying acid-induced esophageal nociception are not clearly understood. In our previous studies, we characterized esophageal vagal nociceptive afferents and defined their responses to noxious mechanical and chemical stimulation. In the present study, we aim to determine their responses to intraluminal acid infusion. Extracellular single-unit recordings were performed in nodose ganglion neurons with intact nerve endings in the esophagus using ex vivo esophageal-vagal preparations. Action potentials evoked by esophageal intraluminal acid perfusion were compared in naive and ovalbumin (OVA)-challenged animals, followed by measurements of transepithelial electrical resistance (TEER) and the expression of tight junction proteins (zona occludens-1 and occludin). In naive guinea pigs, intraluminal infusion with either acid (pH = 2-3) or capsaicin did not evoke an action potential discharge in esophageal nodose C fibers. In OVA-sensitized animals, following esophageal mast cell activation by in vivo OVA inhalation, intraluminal acid infusion for about 20 min started to evoke action potential discharges. This effect is further confirmed by selective mast cell activation using in vitro tissue OVA challenge in esophageal-vagal preparations. OVA inhalation leads to decreased TEER and zona occludens-1 expression, suggesting an impaired esophageal epithelial barrier function after mast cell activation. These data for the first time provide direct evidence of intraluminal acid-induced activation of esophageal nociceptive C fibers and suggest that mast cell activation may make esophageal epithelium more permeable to acid, which subsequently may increase esophageal vagal nociceptive C fiber activation.

Effects of rhein on intestinal epithelial tight junction in IgA nephropathy

To investigate the effects of rhein on intestinal epithelial tight junction proteins in rats with IgA nephropathy (IgAN). Twenty-eight female Sprague-Dawley rats were randomly divided into four groups (7 per group): Control, IgAN, Rhein-treated, and Rhein-prevented. Bovine serum albumin, lipopolysaccharide and CCl4 were used to establish the rat model of IgA nephropathy. The Rhein-treated group was given rhein from week 7 until the rats were sacrificed. The Rhein-prevented group was given rhein from week 1. Animals were sacrificed at the end of week 10. We observed the changes in the intestinal epithelial tight junctions using transmission electron microscopy, and expression of intestinal epithelial tight junction proteins zona occludens protein (ZO)-1 and occludin by immunofluorescence using laser confocal microscopy. Changes in mRNA and protein expression of ZO-1 and occludin were measured by reverse transcriptase polymerase chain reaction and Western blotting. The ratio of urinary lactulose/mannitol was measured by high performance liquid chromatography (HPLC) for assessing the intestinal permeability. In the control group, the tight junctions lied between epithelial cells on the top of the outer side of the cell membrane, and appeared in dense dotted crystal structures, the neighboring cells were binded tightly with no significant gap, and the tight junction protein ZO-1 and occludin were evenly distributed in the intestinal epithelial cells at the top of the junction. Compared with the control group, in the IgAN group, the structure of the tight junction became obscured and the dotted crystal structures had disappeared; the fluorescence of ZO-1 and occludin was uneven and weaker (5.37 ± 1.27 vs 10.03 ± 1.96, P < 0.01; 4.23 ± 0.85 vs 12.35 ± 4.17, P < 0.01); the mRNA expression of ZO-1 and occludin decreased (0.42 ± 0.19 vs 0.92 ± 0.24, P < 0.01; 0.40 ± 0.15 vs 0.97 ± 0.25, P < 0.01); protein expression of ZO-1 and occludin was decreased (0.85 ± 0.12 vs 1.98 ± 0.43, P < 0.01; 0.72 ± 0.15 vs 1.38 ± 0.31, P < 0.01); and the ratio of urinary lactulose/mannitol increased (3.55 ± 0.68 vs 2.72 ± 0.21, P < 0.01). In the Rhein-prevented and Rhein-treated groups, compared with the IgAN group, the intestinal epithelial tight junctions were repaired; fluorescence of ZO-1 and occludin was stronger (11.16 ± 3.52 and 8.81 ± 2.30 vs 5.37 ± 1.27, P < 0.01; 10.97 ± 3.40 and 9.46 ± 2.40 vs 4.23 ± 0.85, P < 0.01); mRNA of ZO-1 and occludin increased (0.81 ± 0.17 and 0.64 ± 0.16 vs 0.42 ± 0.19, P < 0.01; 0.82 ± 0.22 and 0.76 ± 0.31 vs 0.40 ± 0.15, P < 0.01); protein expression of ZO-1 and occludin was increased (2.07 ± 0.41 and 1.57 ± 0.23 vs 0.85 ± 0.12, P < 0.01; 1.34 ± 0.21 and 1.15 ± 0.17 vs 0.72 ± 0.15, P < 0.01); and the ratio of urinary lactulose/mannitol decreased (2.83 ± 0.43 and 2.87 ± 0.18 vs 3.55 ± 0.68, P < 0.01). Rhein can enhance the expression of ZO-1 and occludin, repair damaged tight junctions, and protect the intestinal barrier.

Histamine suppresses epidermal keratinocyte differentiation and impairs skin barrier function in a human skin model

BackgroundDefects in keratinocyte differentiation and skin barrier are important features of inflammatory skin diseases like atopic dermatitis. Mast cells and their main mediator histamine are abundant in inflamed skin and thus may contribute to disease pathogenesis.MethodsHuman primary keratinocytes were cultured under differentiation-promoting conditions in the presence and absence of histamine, histamine receptor agonists and antagonists. The expression of differentiation-associated genes and epidermal junction proteins was quantified by real-time PCR, Western blot, and immunofluorescence labeling. The barrier function of human skin models was tested by the application of biotin as tracer molecule.ResultsThe addition of histamine to human keratinocyte cultures and organotypic skin models reduced the expression of the differentiation-associated proteins keratin 1/10, filaggrin, and loricrin by 80–95%. Moreover, the addition of histamine to skin models resulted in the loss of the granular layer and thinning of the epidermis and stratum corneum by 50%. The histamine receptor H1R agonist, 2-pyridylethylamine, suppressed keratinocyte differentiation to the same extent as did histamine. Correspondingly, cetirizine, an antagonist of H1R, virtually abrogated the effect of histamine. The expression of tight junction proteins zona occludens-1, occludin, claudin-1, and claudin-4, as well as that of desmosomal junction proteins corneodesmosin and desmoglein-1, was down-regulated by histamine. The tracer molecule biotin readily penetrated the tight junction barrier of skin cultures grown in the presence of histamine, while their diffusion was completely blocked in nontreated controls.ConclusionsOur findings suggest a new mechanism by which mast cell activation and histamine release contribute to skin barrier defects in inflammatory skin diseases.

Particulate matter air pollution disrupts endothelial cell barrier via calpain-mediated tight junction protein degradation.

BackgroundExposure to particulate matter (PM) is a significant risk factor for increased cardiopulmonary morbidity and mortality. The mechanism of PM-mediated pathophysiology remains unknown. However, PM is proinflammatory to the endothelium and increases vascular permeability in vitro and in vivo via ROS generation.ObjectivesWe explored the role of tight junction proteins as targets for PM-induced loss of lung endothelial cell (EC) barrier integrity and enhanced cardiopulmonary dysfunction.MethodsChanges in human lung EC monolayer permeability were assessed by Transendothelial Electrical Resistance (TER) in response to PM challenge (collected from Ft. McHenry Tunnel, Baltimore, MD, particle size >0.1 μm). Biochemical assessment of ROS generation and Ca2+ mobilization were also measured.ResultsPM exposure induced tight junction protein Zona occludens-1 (ZO-1) relocation from the cell periphery, which was accompanied by significant reductions in ZO-1 protein levels but not in adherens junction proteins (VE-cadherin and β-catenin). N-acetyl-cysteine (NAC, 5 mM) reduced PM-induced ROS generation in ECs, which further prevented TER decreases and atteneuated ZO-1 degradation. PM also mediated intracellular calcium mobilization via the transient receptor potential cation channel M2 (TRPM2), in a ROS-dependent manner with subsequent activation of the Ca2+-dependent protease calpain. PM-activated calpain is responsible for ZO-1 degradation and EC barrier disruption. Overexpression of ZO-1 attenuated PM-induced endothelial barrier disruption and vascular hyperpermeability in vivo and in vitro.ConclusionsThese results demonstrate that PM induces marked increases in vascular permeability via ROS-mediated calcium leakage via activated TRPM2, and via ZO-1 degradation by activated calpain. These findings support a novel mechanism for PM-induced lung damage and adverse cardiovascular outcomes.

Cell-Free Supernatants from Probiotic Lactobacillus casei and Lactobacillus rhamnosus GG Decrease Colon Cancer Cell Invasion In Vitro

Probiotics have been shown to have a preventative role in colorectal carcinogenesis but research concerning their prophylactic potential in the later stages of colorectal cancer, specifically metastasis is limited. This study explored the potential of cell-free supernatants (CFS) from 2 probiotic Lactobacillus sp., Lactobacillus casei and Lactobacillus rhamnosus GG, to inhibit colon cancer cell invasion by influencing matrix metalloproteinase-9 (MMP-9) activity and levels of the tight junction protein zona occludens-1 (ZO-1) in cultured metastatic human colorectal carcinoma cells. HCT-116 cells were treated with CFS from L. casei, L. rhamnosus, or Bacteroides thetaiotaomicron (a gut commensal); or with uninoculated bacterial growth media. Treatment with CFS from both Lactobacillus sp. decreased colorectal cell invasion but treatment with CFS from B. thetaiotaomicron did not. CFS from both Lactobacillus sp. decreased MMP-9 and increased ZO-1 protein levels. L. rhamnosus CFS also lowered MMP-9 activity. To begin elucidating the secreted bacterial factor conveying these responses, Lactobacillus sp. CFS were fractionated into defined molecular weight ranges and cell invasion assessed. Fractionation revealed that the inhibitory activity was contained primarily in the >100 kDa and 50–100 kDa fractions, suggesting the inhibitory compound may be a macromolecule such as a protein, nucleic acid, or a polysaccharide.

GLP-2 enhances barrier formation and attenuates TNFα-induced changes in a Caco-2 cell model of the intestinal barrier

IntroductionTight junctions are intercellular permeability seals that regulate paracellular transport across epithelia. Tight junction function, expression and localisation of constituent proteins are significantly altered by cytokines such as TNFα. Glucagon-like peptide-2 (GLP-2) is an intestinotrophic enteroendocrine peptide. It is not known whether GLP-2 regulates the barrier or tight junctions. The aim of this study was to investigate whether GLP-2 has an effect on tight junction function or protein expression, alone or in response to TNFα exposure. MethodsCaco-2 cells were grown to confluence on filters in the presence or absence of GLP-2. The time course of transepithelial electrical resistance developing across the monolayer was measured; tight junction protein expression was quantified by immunoblotting. At day 20, TNFα in the presence or absence of GLP-2 was added. Changes in TEER and tight junction proteins expression were quantified. Both TNFα and GLP-2 were added on the basolateral side. ResultsGLP-2 exposed Caco-2 cell monolayers showed a significant increase in transepithelial electrical resistance compared to that in untreated control cells. At the same time, expression of the tight junction proteins occludin and zona occludens-1 (ZO-1) was increased at day 17 post-seeding (1.6-fold; p=0.037 and 4.7 fold; p=0.039 respectively). Subsequent TNFα exposure induced a significant 9.3-fold (p<0.001) decrease in transepithelial electrical resistance and a corresponding reduction in the expression of ZO-1 (5.3 fold; p<0.01). However, the TNFα-induced reduction in transepithelial electrical resistance in GLP-2-exposed cells was highly attenuated to 1.8-fold (p<0.01). No change in tight junction protein expression was noted in GLP-2 exposed cells after cytokine exposure. ConclusionGLP-2 enhances formation of the epithelial barrier and its constituent proteins in Caco-2 cells, and diminishes the effects of TNFα. If these effects are replicated in vivo the GLP-2 receptor may present a therapeutic target in intestinal inflammation.