Zorka Djukic

Role of e-cadherin in the pathogenesis of gastroesophageal reflux disease

OBJECTIVES:An early event in the pathogenesis of gastroesophageal reflux disease (GERD) is an acid-induced increase in junctional (paracellular) permeability in esophageal epithelium (EE). The molecular events that account for this change are unknown. E-cadherin is a junctional protein important in barrier function in EE. Therefore, defects in barrier function in EE were sought in GERD as well as whether their presence correlated with abnormalities in e-cadherin.METHODS:Endoscopic biopsies of EE from GERD (n=20; male 10; female 10; mean age 50±10 years) and subjects with a healthy esophagus (controls; n=23; male 11; female 12; mean age 51±11 years) were evaluated in mini-Ussing chambers and by western blot and immunochemistry; and serum analyzed by enzyme-linked immunosorbent assay (ELISA). A role for e-cadherin was also assessed using a unique conditional knockout of e-cadherin in adult mouse esophagus.RESULTS:EE from GERD patients had lower electrical resistance and higher fluorescein flux than EE from controls; and the findings in GERD associated with cleavage of e-cadherin. Cleavage of e-cadherin in GERD was documented in EE by the presence of a 35-kDa, C-terminal fragment of the molecule on western blot and by an increase in soluble N-terminal fragments of the molecule in serum. Activation of the membrane metalloproteinase, A Disintegrin And Metalloproteinase (ADAM-10), was identified as a likely cause for cleavage of e-cadherin by western blot and immunostaining and a role for e-cadherin in the increased junctional permeability in EE from GERD supported by showing increased permeability after deletion of e-cadherin in mouse EE.CONCLUSIONS:The EE in GERD has increased junctional permeability and this is in association with proteolytic cleavage of e-cadherin. As loss of e-cadherin can, alone, account for the increase in junctional permeability, cleavage of e-cadherin likely represents a critical molecular event in the pathogenesis of GERD, and identification of cleaved fragments may, if confirmed in larger studies, be valuable as a biomarker of GERD.

Nrf2 deficiency impairs the barrier function of mouse oesophageal epithelium.

Objective As a major cellular defence mechanism, the Nrf2/Keap1 pathway regulates expression of genes involved in detoxification and stress response. Here we hypothesise that Nrf2 is involved in oesophageal barrier function and plays a protective role against gastro-oesophageal reflux disease (GERD). Design Human oesophageal biopsy samples, mouse surgical models and Nrf2−/− mice were used to assess the role of the Nrf2/Keap1 pathway in oesophageal barrier function. Trans-epithelial electrical resistance (TEER) was measured with mini-Ussing chambers. HE staining and transmission electron microscopy were used to examine tissue morphology, while gene microarray, immunohistochemistry, western blotting and chromatin immunoprecipitation (ChIP) analysis were used to assess gene expression. Results Nrf2 was expressed in normal oesophageal epithelium and activated in GERD of both humans and mice. Nrf2 deficiency and gastro-oesophageal reflux in mice, alone or in combination, reduced TEER and increased intercellular space in oesophageal epithelium. Nrf2 target genes and gene sets associated with oxidoreductase activity, mitochondrial biogenesis and energy production were downregulated in the oesophageal epithelium of Nrf2−/− mice. Consistent with the antioxidative function of Nrf2, a DNA oxidative damage marker (8OHdG) dramatically increased in oesophageal epithelial cells of Nrf2−/− mice compared with those of wild-type mice. Interestingly, ATP biogenesis, Cox IV (a mitochondrial protein) and Claudin 4 (Cldn4) expression were downregulated in the oesophageal epithelium of Nrf2−/− mice, suggesting that energy-dependent tight junction integrity was subject to Nrf2 regulation. ChIP analysis confirmed the binding of Nrf2 to Cldn4 promoter. Conclusions Nrf2 deficiency impairs oesophageal barrier function through disrupting energy-dependent tight junction.

Defective barrier function in neosquamous epithelium.

OBJECTIVES:Radiofrequency ablation (RFA) of Barretts esophagus (BE) is a common strategy for the prevention of esophageal adenocarcinoma (EAC). After RFA, the ablated esophagus heals on acid suppressive therapy, and is re-populated with a stratified squamous epithelium, referred to as “neosquamous epithelium (NSE).” Because the ability of the NSE to protect the underlying tissue from recurrent insult by reflux is unclear, we assessed the barrier function of NSE by comparing it to that of the native upper squamous epithelium (USE) in subjects having undergone RFA.METHODS:At varying intervals following RFA, the barrier function of NSE and USE were assessed in endoscopic biopsies by light and electron microscopy, and by measurement of electrical resistance (RT) and fluorescein flux in mini-Ussing chambers. Chamber results were further compared with results from control biopsies (healthy distal esophagus). A claudin expression profile in the tight junctions (TJs) of NSE and USE was determined using Quantitative reverse transcriptase PCR. Differential expression of claudin-4 between NSE and USE was assayed by immunoblots.RESULTS:USE was histologically normal whereas NSE showed dilated intercellular spaces and marked eosinophilia. NSE was also more permeable than USE and healthy controls, having lower mean RT and higher fluorescein fluxes. Abnormally low RT values for NSE were unrelated to the time period following RFA (or number of prior RFA sessions), being abnormal even 26 months after RFA. Abnormal permeability in NSE was associated with significantly lower values for claudin-4 and claudin-10 than in USE.CONCLUSIONS:NSE commonly exhibits defective barrier function. As this defect will make it vulnerable to injury, inflammation, and destruction by acidic and weakly acidic refluxates, it may in part explain incidences of recurrence of BE following ablation.

Enterococcus faecalis Gelatinase Mediates Intestinal Permeability via Protease-Activated Receptor 2

ABSTRACT Microbial protease-mediated disruption of the intestinal epithelium is a potential mechanism whereby a dysbiotic enteric microbiota can lead to disease. This mechanism was investigated using the colitogenic, protease-secreting enteric microbe Enterococcus faecalis. Caco-2 and T-84 epithelial cell monolayers and the mouse colonic epithelium were exposed to concentrated conditioned media (CCM) from E. faecalis V583 and E. faecalis lacking the gelatinase gene (gelE). The flux of fluorescein isothiocyanate (FITC)-labeled dextran across monolayers or the mouse epithelium following exposure to CCM from parental or mutant E. faecalis strains indicated paracellular permeability. A protease-activated receptor 2 (PAR2) antagonist and PAR2-deficient (PAR2−/−) mice were used to investigate the role of this receptor in E. faecalis-induced permeability. Gelatinase (GelE) purified from E. faecalis V583 was used to confirm the ability of this protease to induce epithelial cell permeability and activate PAR2. The protease-mediated permeability of colonic epithelia from wild-type (WT) and PAR2−/− mice by fecal supernatants from ulcerative colitis patients was assessed. Secreted E. faecalis proteins induced permeability in epithelial cell monolayers, which was reduced in the absence of gelE or by blocking PAR2 activity. Secreted E. faecalis proteins induced permeability in the colonic epithelia of WT mice that was absent in tissues from PAR2−/− mice. Purified GelE confirmed the ability of this protease to induce epithelial cell permeability via PAR2 activation. Fecal supernatants from ulcerative colitis patients induced permeability in the colonic epithelia of WT mice that was reduced in tissues from PAR2−/− mice. Our investigations demonstrate that GelE from E. faecalis can regulate enteric epithelial permeability via PAR2.

Gastroesophageal reflux activates the NF-κB pathway and impairs esophageal barrier function in mice.

The barrier function of the esophageal epithelium is a major defense against gastroesophageal reflux disease. Previous studies have shown that reflux damage is reflected in a decrease in transepithelial electrical resistance associated with tight junction alterations in the esophageal epithelium. To develop novel therapies, it is critical to understand the molecular mechanisms whereby contact with a refluxate impairs esophageal barrier function. In this study, surgical models of duodenal and mixed reflux were developed in mice. Mouse esophageal epithelium was analyzed by gene microarray. Gene set enrichment analysis showed upregulation of inflammation-related gene sets and the NF-κB pathway due to reflux. Significance analysis of microarrays revealed upregulation of NF-κB target genes. Overexpression of NF-κB subunits (p50 and p65) and NF-κB target genes (matrix metalloproteinases-3 and -9, IL-1β, IL-6, and IL-8) confirmed activation of the NF-κB pathway in the esophageal epithelium. In addition, real-time PCR, Western blotting, and immunohistochemical staining also showed downregulation and mislocalization of claudins-1 and -4. In a second animal experiment, treatment with an NF-κB inhibitor, BAY 11-7085 (20 mg·kg⁻¹·day⁻¹ ip for 10 days), counteracted the effects of duodenal and mixed reflux on epithelial resistance and NF-κB-regulated cytokines. We conclude that gastroesophageal reflux activates the NF-κB pathway and impairs esophageal barrier function in mice and that targeting the NF-κB pathway may strengthen esophageal barrier function against reflux.

Lateral Cell Membranes and Shunt Resistance in Rabbit Esophageal Epithelium

Background and AimsThe structures that contribute to shunt resistance (Rs) in esophageal epithelium are incompletely understood, with 35–40% of Rs known to be calcium-dependent, reflecting the role of e-cadherin. Two calcium-independent candidates for the remaining ~60% of Rs have been identified: the glycoprotein matrix (GPM) within stratum corneum of esophageal epithelium, and the lateral cell membranes (LCMs) from neighboring cells.MethodsTo determine the contribution of GPM and LCMs to Rs, rabbit esophageal epithelium was mounted in Ussing chambers so that transepithelial resistance (RT), a marker of Rs, could be monitored during luminal exposure to either glycosidases for disruption of the GPM or to hypertonic urea for separation of the LCMs.ResultsGlycosidases had no effect on RT. In contrast, hypertonic urea reduced RT, increased fluorescein flux and widened the intercellular spaces. That urea reduced RT, and so Rs, by widening the intercellular spaces, and not by altering the e-cadherin-dependent apical junctional complex, was supported by the ability of: (a) calcium-free solution to reduce RT beyond that produced by urea, (b) hypertonic urea to reduce RT beyond that produced by calcium free solution, (c) hypertonic sucrose to collapse the intercellular spaces and raise RT, and (d) empigen, a zwitterionic detergent, to non-osmotically widen the intercellular spaces and reduce RT.ConclusionThese data indicate that the LCMs from neighboring cells are a major contributor to shunt resistance in esophageal epithelium. As resistor, they are distinguishable from the apical junctional complex by their sensitivity to (luminal) hypertonicity and insensitivity to removal of calcium.

1060 Cleavage of E-Cadherin is More Specific Than Dilated Intercellular Spaces (DIS) Within Esophageal Epithelium (EE) for the Diagnosis of Gastroesophageal Reflux Disease (GERD)

INTRODUCTION: Maintenance of closure of the lower esophageal sphincter (LES) in resting state is central to the prevention of gastroesophageal reflux disease (GERD). There are many factors which contribute to the maintenance/failure of the “reflux barrier” of the LES. Here we present a detailed analysis of the anatomy and corresponding biomechanics of the gastroesophageal junction (GEJ) with the AIM of identifying structural differences, and consequent functional differences, in GEJ between normal subjects and GERD patients. METHODS: Three dimensional (3D) reconstructions of the GEJ from transverse magnetic resonance images (MRI) of 12 healthy volunteers and 12 reflux patients were studied for different breathing and meal phases. The orientation of the GEJ and the proximal stomach (angle φ in fig. 1), and the angle of His (θ in fig. 1) were evaluated with respect to an anatomical reference of the midpoint of the vertebrae. Biomechanical analysis of the GEJ was performed for both groups of subjects using the finite element (FE) methods to assess the mechanical consequences of the structural parameters of the GEJ. RESULTS: The angle of His increased with meal and inspiration in both normals and patients, however it always remained more acute (p=0.002) in volunteers (47.23±1.65°) than patients (54.62±1.66°). Interestingly, the orientation of GEJ and proximal stomach in patients was similar to volunteers during fasting, but became more postero-anterior during subsequent fed states. In addition the span of gastric contact with GEJ increased significantly with meal in volunteers (p=0.002) whereas patients showed no such change. FE simulations based on real geometry of the GEJ from 3D reconstruction showed higher intraluminal pressure in the GEJ with pressurization of the stomach when the angle of His was more acute. CONCLUSIONS: The difference in orientation of the GEJ and proximal stomach is possibly the reason for changes in angle of His between normals and patients. Structural and biomechanical differences in the GEJ between normals and patients indicate existence of a virtual “flap valve” in normals which might be dysfunctional in GERD patients.