Human Esophageal Mucosa Research Articles

Exploring the clinical relevance of "Angico Gum": an effective biopolymer for topical protection of oesophageal mucosa in gastroesophageal reflux disease patients.

Angico gum (AG) (Anadenanthera colubrina var. Cebil [Griseb.] Altschul) is utilized by some Brazilian communities to alleviate symptoms from gastroesophageal reflux disease. Here, we aimed to investigate the "in vitro" topical protective capacity of AG on human esophageal mucosa. Biopsies of the distal esophageal mucosa were collected from 35 patients with heartburn (24 non-erosive and 11 with erosive oesophagitis (EE)) and mounted in Üssing chambers. AG was applied topically, followed by exposure with acid solution (pH 2.0 or pH 1.0), where transepithelial electrical resistance (TER) and The transepithelial permeability for fluorescein was assessed. The incubation of the AG labeled with FITC in the esophageal mucosa was localized by fluorescence microscopy. Pretreatment with AG prevented the drop in TER induced by acid solution, as well as significantly decreases the fluorescein permeability in non-erosive patients. The protective effect of AG was sustained for up to 120 min both in biopsies of non-erosive and erosive esophagitis. Confocal microscope images showed mucosal luminal adherence of FITC-labeled AG. AG had a prolonged topical protective effect against acid solution in mucosal biopsies of patients with non-erosive and erosive esophagitis.

Proteomic analysis of the esophageal epithelium reveals key features of eosinophilic esophagitis pathophysiology.

Eosinophilic esophagitis (EoE) is a chronic non-IgE-mediated allergic disease of the esophagus. An unbiased proteomics approach was performed to investigate pathophysiological changes in esophageal epithelium. Additionally, an RNAseq-based transcriptomic analysis in paired samples was also carried out. Total proteins were purified from esophageal endoscopic biopsies in a cohort of adult EoE patients (n = 25) and healthy esophagus controls (n = 10). Differentially accumulated (DA) proteins in EoE patients compared to control tissues were characterized to identify altered biological processes and signaling pathways. Results were also compared with a quantitative proteome dataset of the human esophageal mucosa. Next, results were contrasted with those obtained after RNAseq analysis in paired samples. Finally, we matched up protein expression with two EoE-specific mRNA panels (EDP and Eso-EoE panel). A total of 1667 proteins were identified, of which 363 were DA in EoE. RNA sequencing in paired samples identified 1993 differentially expressed (DE) genes. Total RNA and protein levels positively correlated, especially in DE mRNA-proteins pairs. Pathway analysis of these proteins in EoE showed alterations in immune and inflammatory responses for the upregulated proteins, and in epithelial differentiation, cornification and keratinization in those downregulated. Interestingly, a set of DA proteins, including eosinophil-related and secreted proteins, were not detected at the mRNA level. Protein expression positively correlated with EDP and Eso-EoE, and corresponded with the most abundant proteins of the human esophageal proteome. We unraveled for the first time key proteomic features involved in EoE pathogenesis. An integrative analysis of transcriptomic and proteomic datasets provides a deeper insight than transcriptomic alone into understanding complex disease mechanisms.

Real-time delineation of basement membrane in human oral and esophageal mucosa with micro-optical coherence tomography

Basement membrane is a thin, extracellular matrix-like structure, separating the epithelium from the stroma and having a vital role in determining cancer progression. Identification of this diagnostically important structure with histology is invasive and labor-intensive. Micro-optical coherence tomography (µOCT) which has the capability to recognize subcellular-level microstructures underneath the surface offers the possibility to resolve basement membrane noninvasively in situ and in vivo. In this study, we conducted numerical analysis of the back-scattering intensity of basement membrane and experimental µOCT imaging of human labial mucosa in vivo and esophageal mucosa ex vivo, in an attempt to characterize the basement membrane underlying stratified squamous epithelial tissues. The results disclose that the layered structure of mucosa could be clearly delineated and the basement membrane presented as a steep, low-scattering linear structure in between the relatively high-scattering epithelium and stroma. Besides, according to the knowledges on its ultrastructure, we assume the low-scattering signal is collectively determined by basal lamina and reticular lamina since the former alone is extremely thin to be recognized by µOCT. This study clarifies the scattering features of basement membrane and the capability of real-time delineation of basement membrane which will help to determine the progression of epithelial cancers without the need of resection and histology.

Glutathione S-transferase omega 2 regulates cell growth and the expression of E-cadherin via post-transcriptional down-regulation of β-catenin in human esophageal squamous cells.

Glutathione S-transferase omega 2 (GSTO2), which belongs to the superfamily of GST omega class, lacks any appreciable GST activity. Although GSTO2 exhibits thioltransferase and glutathione dehydrogenase activities, its precise expression and physiological functions are still unclear. In the present study, we found that GSTO2 is exclusively expressed in the basal cell layer in Ki67-negative non-proliferative cells in the human esophageal mucosa. GSTO2 overexpression in esophageal squamous cell carcinoma (ESCC) cell lines inhibited cell growth and colony formation, and GSTO2-transfected cells formed smaller tumors in nude mice compared with mock-transfected cells. Interestingly, GSTO2 induction suppressed the expressions of E-cadherin and β-catenin at the cell-cell contact site. We quantified the phosphorylation levels of key proteins of MAPK signaling pathway and identified phosphorylation of p38. Additionally, HSP27, a downstream molecule of p38, was accelerated in GSTO2-transfected cells, unlike in mock-transfected cells. When GSTO2-transfected cells were treated with a p38 inhibitor, the expression of β-catenin and the membrane localization of E-cadherin was recovered. We next examined GSTO2 expression in 61 ESCC tissues using quantitative reverse transcription polymerase chain reaction and immunostaining. The results showed that GSTO2 mRNA and protein were significantly reduced in ESCC compared with normal tissues. When human ESCC cell lines were treated with 5-aza-2'-deoxycytidine, a DNA-methyltransferase inhibitor, GSTO2 transcription was induced, suggesting that aberrant hypermethylation is the cause of the down-regulated expression. Our results indicate that GSTO2 expression inhibits the membrane localization of E-cadherin, probably by modulation of the p38 signaling pathway. Down-regulation of GSTO2 by DNA hypermethylation contributes to the growth and progression of ESCC.

Cashew gum, a biopolymer, topically protects oesophageal mucosa in non erosive reflux disease: A promising translational study

This study aimed to investigate a standardized biopolymer, cashew gum (CG), in human oesophageal mucosa and mice with experimentally-induced non-erosive reflux disease (NERD). Human oesophageal biopsies from NERD patients were collected to evaluate the mucosal protection of CG through transepithelial electrical resistance (TER), mucosal permeability, and mucoadhesiveness tests. A surgical model of NERD in mice was induced, and barrier functions followed by suggestive oesophageal inflammatory hallmarks were evaluated. Pre-coating of CG was effective in human oesophageal mucosa by attenuating drop of TER and mucosal permeability. Labelled-CG adheres to human oesophageal mucosa for up to 1 h. In animal studies, CG improved parameters of barrier function (TER and mucosal permeability) in distal oesophagus mucosa. CG also promoted sequential support by reducing inflammatory hallmarks of oesophageal damage. CG confers topical oesophageal mucosal protection due to its mucoadhesiveness and anti-inflammatory profile. Long-duration mucoprotective products can be further explored as first-line/adjuvant NERD therapy.

Development and Application of a Functional Human Esophageal Mucosa Explant Platform to Eosinophilic Esophagitis

There is an increasing prevalence of esophageal diseases but intact human tissue platforms to study esophageal function, disease mechanisms, and the interactions between cell types in situ are lacking. To address this, we utilized full thickness human donor esophagi to create and validate the ex vivo function of mucosa and smooth muscle (n = 25). Explanted tissue was tested for contractile responses to carbachol and histamine. We then treated ex vivo human esophageal mucosa with a cytokine cocktail to closely mimic the Th2 and inflammatory milieu of eosinophilic esophagitis (EoE) and assessed alterations in smooth muscle and extracellular matrix function and stiffening. We found that full thickness human esophagus as well as the individual layers of circular and longitudinal muscularis propria developed tension in response to carbachol ex vivo and that mucosa demonstrated squamous cell differentiation. Treatment of mucosa with Th2 and fibrotic cytokines recapitulated the majority of the clinical Eosinophilic Esophagitis Diagnostic Profile (EDP) on fluidic transcriptional microarray. Transforming growth factor-beta-1 (TGFβ1) increased gene expression of fibronectin, smooth muscle actin, and phospholamban (p < 0.001). The EoE cocktail also increased stiffness and decreased mucosal compliance, akin to the functional alterations in EoE (p = 0.001). This work establishes a new, transcriptionally intact and physiologically functional human platform to model esophageal tissue responses in EoE.

Observation of the cytoarchitecture of the human esophageal mucosa with special attention to the lamina muscularis mucosae and the distribution of lymphatic vessels.

The cytoarchitecture of the esophageal mucosa was examined by using light microscopy, transmission electron microscopy, and scanning electron microscopy. The cytoarchitecture of the muscularis mucosae varied greatly among the cervical, thoracic, and abdominal esophagus, especially in the cervical esophagus, the muscularis mucosae suffered a loss and the distribution of lymphatic vessels also varied according to the site. It was suggested that these morphological differences would have a strong influence on the infiltration of esophageal cancer and the mode of lymph node metastasis.

Mapping Local Cytosolic Enzymatic Activity in Human Esophageal Mucosa with Porous Silicon Nanoneedles.

Porous silicon nanoneedles can map Cathepsin B activity across normal and tumor human esophageal mucosa. Assembling a peptide-based Cathepsin B cleavable sensor over a large array of nano-needles allows the discrimination of cancer cells from healthy ones in mixed culture. The same sensor applied to tissue can map Cathepsin B activity with high resolution across the tumor margin area of esophageal adenocarcinoma.

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model.

The incidence of both esophageal adenocarcinoma and its precursor, Barrett's Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa. A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa. This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett's Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

Abstract 1926: Identification of stem cell by immunohistochemistry in benign and malignant human esophageal mucosa

Abstract Introduction: Esophageal squamous cell carcinoma (ESCC) response to treatment if still unsatisfactory with high mortality rate. Objective: to identify the role of cancer stem cell in ESCC by studying the tumor by immunohistochemistry, with the concept of identifying the possible cell for the progression of the tumor due to its uncontrolled proliferation. Materials and methods: endoscopic biopsy and resected specimens of confirmed ESCC tissue were used for the study - fresh tumor tissue with no treatment, post treated residual tumor tissue, recurrent tumor tissue and adjoining esophageal mucosa. Primary immunomarkers used in the study were : i) alpha 6 integrin, ii) CD71, iii) p63, iv) stem cell antigen1, v) cytokeratin 6, vi) Bim1, vii) oct4, viii) nucleostemin, ix) Ki67, x). CD133, x) CD44, xi) CD24 and xii) Aldehyde dehydrogenase. Study was done routine immunohistochemistry, double immunostaining and tissue micro array. Apropriate detection system was used. Results: There was 250 tumor tissue altogether majority with well differentiated morphology. All tumors showed variable range of positivity for the all the markers, lesser number of positive cells for CD24, CD44 and CD133. Even in double immunostaining, majority of the tumor cells showed co-expressions for the various immunomarkers. Different tumor samples from different patient population groups do not any significant differences. Mucosal dysplastic samples also showed similar pattern of positive staining as by the tumor cells. Ki67 posivity was variable, higher percentage of positivity in poorly differentiated tumor areas and related with the nucleostemin positive staining pattern. Conclusion: These observations indicate that these markers are non-specific and positivity does not specifically meant the presence of cancer stem cell. Possibly expression of these markers may indicate cell proliferative pathway markers. Note: This abstract was not presented at the meeting. Citation Format: Kim Vaiphei, Saroj Kant Sinha, Rakesh Kochhar. Identification of stem cell by immunohistochemistry in benign and malignant human esophageal mucosa. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1926. doi:10.1158/1538-7445.AM2014-1926

Pulsatile exposure to simulated reflux leads to changes in gene expression in a 3D model of oesophageal mucosa

Oesophageal exposure to duodenogastroesophageal refluxate is implicated in the development of Barrett's metaplasia (BM), with increased risk of progression to oesophageal adenocarcinoma. The literature proposes that reflux exposure activates NF-κB, driving the aberrant expression of intestine-specific caudal-related homeobox (CDX) genes. However, early events in the pathogenesis of BM from normal epithelium are poorly understood. To investigate this, our study subjected a 3D model of the normal human oesophageal mucosa to repeated, pulsatile exposure to specific bile components and examined changes in gene expression. Initial 2D experiments with a range of bile salts observed that taurochenodeoxycholate (TCDC) impacted upon NF-κB activation without causing cell death. Informed by this, the 3D oesophageal model was repeatedly exposed to TCDC in the presence and absence of acid, and the epithelial cells underwent gene expression profiling. We identified ~300 differentially expressed genes following each treatment, with a large and significant overlap between treatments. Enrichment analysis (Broad GSEA, DAVID and Metacore™; GeneGo Inc) identified multiple gene sets related to cell signalling, inflammation, proliferation, differentiation and cell adhesion. Specifically NF-κB activation, Wnt signalling, cell adhesion and targets for the transcription factors PTF1A and HNF4α were highlighted. Our data suggest that HNF4α isoform switching may be an early event in Barrett's pathogenesis. CDX1/2 targets were, however, not enriched, suggesting that although CDX1/2 activation reportedly plays a role in BM development, it may not be an initial event. Our findings highlight new areas for investigation in the earliest stages of BM pathogenesis of oesophageal diseases and new potential therapeutic targets.

Detection of Esophageal Squamous Cell Carcinoma by Cathepsin B Activity in Nude Mice

Background and ObjectiveDespite great progress in treatment, the prognosis for patients with esophageal squamous cell carcinoma (ESCC) remains poor, highlighting the importance of early detection. Although upper endoscopy can be used for the screening of esophagus, it has limited sensitivity for early stage disease. Thus, development of new diagnosis approach to improve diagnostic capabilities for early detection of ESCC is an important need. The aim of this study was to assess the feasibility of using cathepsin B (CB) as a novel imaging target for the detection of human ESCC by near-infrared optical imaging in nude mice.MethodsInitially, we examined specimens from normal human esophageal tissue, intraepithelial neoplasia lesions, tumor in situ, ESCC and two cell lines including one human ESCC cell line (Eca-109) and one normal human esophageal epithelial cell line (HET-1A) for CB expression by immunohistochemistry and western blot, respectively. Next, the ability of a novel CB activatable near-infrared fluorescence (NIRF) probe detecting CB activity presented in Eca-109 cells was confirmed by immunocytochemistry. We also performed in vivo imaging of tumor bearing mice injected with the CB probe and ex vivo imaging of resected tumor xenografts and visceral organs using a living imaging system. Finally, the sources of fluorescence signals in tumor tissue and CB expression in visceral organs were identified by histology.ResultsCB was absent in normal human esophageal mucosa, but it was overexpressed in ESCC and its precursor lesions. The novel probe for CB activity specifically detected ESCC xenografts in vivo and in vitro.ConclusionsCB was highly upregulated in human ESCC and its precursor lesions. The elevated CB expression in ESCC allowed in vivo and in vitro detection of ESCC xenografts in nude mice. Our results support the usefulness of CB activity as a potential imaging target for the detection of human ESCC.

Angiotensin IV and the human esophageal mucosa: An exploratory study in healthy subjects and gastroesophageal reflux disease patients

The human esophageal mucosa expresses various components of the renin-angiotensin system (RAS), e.g. the main effector peptide angiotensin II (AngII). The aim of this study was to investigate the esophageal presence of angiotensin III (AngIII) and angiotensin IV (AngIV) forming enzymes and the AngIV receptor (AT4R). The aim was also to study the actions of AngIV and to look for aberrations in patients with gastroesophageal reflux disease (GERD). Esophageal biopsies were collected from healthy volunteers (n: 19) and individuals with erosive reflux disease (n: 14). Gene transcripts and protein expression of aminopeptidase A, -B and -M, and the AT4R were investigated by reverse transcriptase polymerase chain reaction (rt-PCR), western blot (WB) and immunohistochemistry (IHC). The functional impact of AngIV was examined in an Ussing chamber. Aminopeptidase A, -B and -M and the AT4R were expressed in the esophageal epithelium. The AT4R was less prominent in certain areas in the mucosa of reflux patients. AngIV influenced the esophageal epithelial ion transport. The impact was lower in patients with GERD. The AT4R and formation enzymes of AngIII and AngIV are present in the human esophageal epithelium. Moreover, the present results suggest that AngIV exert regulatory impact on the epithelium and that RAS is involved in mucosal aberrations associated with GERD.

1H-NMR based metabonomic profiling of human esophageal cancer tissue

BackgroundThe biomarker identification of human esophageal cancer is critical for its early diagnosis and therapeutic approaches that will significantly improve patient survival. Specially, those that involves in progression of disease would be helpful to mechanism research.MethodsIn the present study, we investigated the distinguishing metabolites in human esophageal cancer tissues (n = 89) and normal esophageal mucosae (n = 26) using a 1H nuclear magnetic resonance (1H-NMR) based assay, which is a highly sensitive and non-destructive method for biomarker identification in biological systems. Principal component analysis (PCA), partial least squares-discriminant analysis (PLS-DA) and orthogonal partial least-squares-discriminant anlaysis (OPLS-DA) were applied to analyse 1H-NMR profiling data to identify potential biomarkers.ResultsThe constructed OPLS-DA model achieved an excellent separation of the esophageal cancer tissues and normal mucosae. Excellent separation was obtained between the different stages of esophageal cancer tissues (stage II = 28; stage III = 45 and stage IV = 16) and normal mucosae. A total of 45 metabolites were identified, and 12 of them were closely correlated with the stage of esophageal cancer. The downregulation of glucose, AMP and NAD, upregulation of formate indicated the large energy requirement due to accelerated cell proliferation in esophageal cancer. The increases in acetate, short-chain fatty acid and GABA in esophageal cancer tissue revealed the activation of fatty acids metabolism, which could satisfy the need for cellular membrane formation. Other modified metabolites were involved in choline metabolic pathway, including creatinine, creatine, DMG, DMA and TMA. These 12 metabolites, which are involved in energy, fatty acids and choline metabolism, may be associated with the progression of human esophageal cancer.ConclusionOur findings firstly identify the distinguishing metabolites in different stages of esophageal cancer tissues, indicating the attribution of metabolites disturbance to the progression of esophageal cancer. The potential biomarkers provide a promising molecular diagnostic approach for clinical diagnosis of human esophageal cancer and a new direction for the mechanism study.

Prostaglandin E2 mediates acid-induced heartburn in healthy volunteers

Prostaglandin E(2) (PGE(2)) plays a major role in pain processing and hypersensitivity. This study investigated whether PGE(2) levels are increased in the esophageal mucosa after acid infusion and whether increases in PGE(2) are associated with heartburn. Furthermore, expression of the PGE(2) receptor EP1 was investigated in human esophageal mucosa. Fourteen healthy male volunteers were randomized to 30-min lower esophageal acid (1% HCl) or saline perfusion. Before and after acid perfusion, endoscopic biopsies were taken from the distal esophagus. PGE(2) concentration (pg/mg protein) and EP1 mRNA and protein in biopsy samples were measured by ELISA, RT-PCR, and Western blotting. Symptom status of heartburn was evaluated with a validated categorical rating scale with a higher values corresponding to increasing intensity. PGE(2) levels in the esophageal mucosa significantly increased after acid infusion (before vs. after acid infusion: 23.2 ± 8.6 vs. 68.6 ± 18.3, P < 0.05), but not after saline infusion (before vs. after saline infusion: 9.3 ± 2.5 vs. 9.0 ± 3.2, NS). Time to first sensation (min) after acid infusion was less than after saline (saline vs. acid infusion: 22.1 ± 4.1 vs. 5.4 ± 1.5, P < 0.05). Intensity of heartburn in the acid-infusion group was also significantly greater compared with saline (saline vs. acid infusion: 54.3 ± 13.1 vs. 178.5 ± 22.8, P < 0.01). Changes in PGE(2) levels in the esophagus correlated with symptom intensity score (r = 0.80, P = 0.029). EP1 mRNA and protein expression were observed in the normal human esophageal mucosa. Esophageal PGE(2) expression is associated with mucosal acid exposure and heartburn.

The renin–angiotensin system in the esophageal mucosa of healthy subjects and patients with reflux disease

Objective. Components of the renin–angiotensin system (RAS) were recently discovered in the esophagus, which could be of interest in relation to gastroesophageal reflux disease (GERD). The present study was undertaken to confirm and further investigate the expression of RAS in healthy and refluxed exposed human esophageal mucosae. Methods.Esophageal biopsies were obtained from healthy subjects (n = 34) and individuals with erosive reflux disease (ERD, n = 28). Evaluation of general morphology and histological signs of reflux as well as investigation of gene transcript, protein expression and localization of various RAS components using RT-PCR, ELISA, western blot and immunohistochemistry were performed. Physiological effects of the AT2R were investigated in Ussing chamber experiments. Results. The study confirmed histological signs of reflux in ERD and expression of ACE, AT1R, AT2R and CatD in all examined specimens. In addition, the main effector peptide AngII, the pro-hormone AGT, the Mas receptor and the angiotensin-forming enzymes renin, CMA, CatG and NEP were present. Individuals with reflux disease had higher transcription activity of ACE and AT1R, increased protein levels of AT2R and lower levels of MasR. AT2R stimulation increased the ion currents in healthy epithelium, whereas epithelium from individuals with reflux disease exhibited no significant response. Conclusions. The study demonstrated that a local RAS is present in the human esophageal epithelium. Some RAS components were significantly altered in individuals diagnosed with ERD suggesting involvement in the pathophysiology of GERD.

In vitro assessment of epithelial electrical resistance in human esophageal and jejunal mucosae and in Caco-2 cell layers

Objective. There is a need for a technique allowing studies of human mucosal specimens collected during different clinical conditions. This study elucidates if square wave pulse analysis discriminates between epithelial and transmural electrical resistance and if there is an association with transepithelial permeability of molecular probes. Methods.Mucosae from esophagus (surgical resections: n = 14; endoscopic biopsies: n = 15) and jejunum (n = 12) and Caco-2 cell monolayers were investigated in Ussing chambers. Transmural and epithelial electrical resistance were recorded by the use of standardized current pulses. Permeability was assessed using two fluorescein-labeled probes (weight 376 and 4000 Da). Results. Baseline epithelial electrical resistance was higher in esophageal mucosa (∼280 Ω*cm2), than in jejunal (∼10 Ω*cm2) and Caco-2 cells (∼140 Ω*cm2). The subepithelial contribution to the transmural resistance was higher in jejunal preparations (+88%) and Caco-2 cells (+75%), than in esophageal (+30%). During hypoxia the subepithelial resistance was unchanged, whereas the epithelial resistance decreased significantly in jejunal mucosa and Caco-2 cells. These findings coincided with increased transepithelial probe permeability and signs of disturbed morphology. Esophageal epithelia were resistant to hypoxia. However, exposure to deoxycholic acid and trypsin abolished the esophageal epithelial resistance and increased probe permeability. Endoscopic esophageal biopsies from patients with erosive reflux disease exhibited significantly lower epithelial resistance and higher current than healthy subjects. Conclusion.Square wave pulse analysis in Ussing chambers is suitable for assessment of epithelial electrical resistance that can reflect transepithelial permeability of molecular probes with known size. Moreover, the technique discriminated between healthy and reflux-diseased esophageal mucosal biopsies.

Trypsin impaired epithelial barrier function and induced IL-8 secretion through basolateral PAR-2: a lesson from a stratified squamous epithelial model

Immune-mediated injury by the protease-activated receptor-2-interleukin-8 (PAR-2-IL8) pathway may underlie the development of gastroesophageal reflux disease (GERD). However, the localization of PAR-2 and the mechanism of PAR-2 activation remain unclear. This study aimed to address these questions on an esophageal stratified squamous epithelial model and in the human esophageal mucosa of GERD patients. Normal human esophageal epithelial cells were cultured with the air-liquid interface system to establish the model. SLIGKV-NH2 (PAR-2 synthetic agonist), trypsin (PAR-2 natural activator), and weak acid (pH 4, 5, and 6) were added to either the apical or basolateral compartment to evaluate their effects on transepithelial electrical resistance (TEER) and IL-8 production. PAR-2 localization was examined both in the cell model and biopsies from GERD patients by immunohistochemistry. Apical trypsin stimulation induced IL-8 accompanied by decreased TEER in vitro, whereas the effective concentration from the basolateral side was 10 times lower. SLIGKV-NH2 from basolateral but not apical stimulation induced IL-8 production. Apical weak acid stimulation did not influence TEER or IL-8 production. Immunohistochemistry showed intense reactivity of PAR-2 in the basal and suprabasal layers after stimulation with trypsin. A similar PAR-2 reactivity that was mainly located at the basal and suprabasal layers was detected in GERD patients. In conclusion, the activation of the PAR-2-IL-8 pathway probably occurred at the basal and suprabasal layers, while the esophageal epithelial barrier may influence the activation of PAR-2. Under proton pump inhibitor therapy, refluxed trypsin may remain active and be a potential agent in the pathogenesis of refractory GERD.

HCl-induced and ATP-dependent upregulation of TRPV1 receptor expression and cytokine production by human esophageal epithelial cells

The pathogenesis of gastroesophageal reflux disease (GERD) remains elusive, but recent evidence suggests that early secretion of inflammatory cytokines and chemokines by the mucosa leads to influx of immune cells followed by tissue damage. We previously showed that exposure of esophageal mucosa to HCl causes ATP release, resulting in activation of acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine acetyltransferase (lyso-PAF AT), the enzyme responsible for the production of platelet-activating factor (PAF). In addition, HCl causes release of IL-8 from the esophageal mucosa. We demonstrate that esophageal epithelial cells secrete proinflammatory mediators in response to HCl and that this response is mediated by ATP. Monolayers of the human esophageal epithelial cell line HET-1A were exposed to acidified cell culture medium (pH 5) for 12 min, a total of seven times over 48 h, to simulate the recurrent acid exposure clinically occurring in GERD. HCl upregulated mRNA and protein expression for the acid-sensing transient receptor potential cation channel, subfamily vanilloid member 1 (TRPV1), lyso-PAF AT, IL-8, eotaxin-1, -2, and -3, macrophage inflammatory protein-1α, and monocyte chemoattractant protein-1. The chemokine profile secreted by HET-1A cells in response to repeated HCl exposure parallels similar findings in erosive esophagitis patients. In HET-1A cells, the TRPV1 agonist capsaicin reproduced these findings for mRNA of the inflammatory mediators lyso-PAF AT, IL-8, and eotaxin-1. These effects were blocked by the TRPV1 antagonists iodoresiniferatoxin and JNJ-17203212. These effects were imitated by direct application of ATP and blocked by the nonselective ATP antagonist suramin. We conclude that HCl/TRPV-induced ATP release upregulated secretion of various chemoattractants by esophageal epithelial cells. These chemoattractants are selective for leukocyte subsets involved in acute inflammatory responses and allergic inflammation. The data support the validity of HET-1A cells as a model of the response of the human esophageal mucosa in GERD.

Low dose aspirin prevents duodenoesophageal reflux induced mucosal changes in wistar rat esophagus by MAP kinase mediated pathways

BackgroundInvestigations of molecular mechanisms behind the progression of neoplastic changes in the esophagus have uncovered the role of the COX & 5-Lox pathways. Human squamous esophageal mucosa produces relatively large amounts of eicosanoids in the presence of inflammation. Laboratory and epidemiological data suggest that aspirin and non-steroidal anti-inflammatory drugs may be chemo preventive through their inhibitory effect on COX25, 10. Cell culture studies have shown that the members of the mitogen activated protein (MAP) kinase family plays an important role in the development of bile acid-induced carcinogenesis. Differences in MAPK pathways activated by bile exposure were also noted in esophageal squamous cell lines and biopsies from patients with GERD. The protective role of aspirin and its molecular mechanism is not well understood. Aims1.The effect of duodenal reflux on esophageal mucosa.2.The role of aspirin in preventing duodenal reflux induced esophageal mucosa changes.3.If it is proven to be preventive, the mechanism of its action. A duodenal reflux rat animal model was used by an end- to-side esophago duodenostomy. MethodsTotal of 56 rats was included. 3 were "Naive control" animals which did not undergo the surgical procedure. The remaining animals were divided into two groups: Surgery alone (experimental) and Surgery + aspirin [therapy group], esophagoduodenostomy. At 40 weeks, the rats were euthanized and appropriate esophageal samples were analysed for histopathology and p38 & ERK MAP kinases, VEGF, protease activity and caspase 3 activities. ResultsThe presence of gross mucosal nodularity was seen in 21 and 10 rats of the experimental and therapy group respectively (p = 0.03; Table 1). Reflux-associated changes such as basal cell hyperplasia were more common in the experimental group, however this association did not reach statistical significance (p = 0.15; Table 1). Epithelial hyperplasia was seen more in the experimental group, which was prevented by aspirin [p < 0.01]. Papillomatosis, as shown in Fig. 4 was more common in the experimental group (p = 0.02). Activation of p38 & ERK MAP kinases was prevented in aspirin group (p < 0.05, CI −1.796–−0.014). Examination of protease activity by zymographic analysis of the esophageal samples revealed a number of gelatinolytic bands in 50% rats of the experimental group, not observed in the therapy group. No significant changes were seen in Caspase 3 [Normal areas −99.74 & nodular areas – 100.34 percent of controls] or VEGF [mean 0.64, sd ± 0.76 Vs 0.69 ± 0.96] activity. ConclusionsOur data demonstrated that low dose aspirin reduced the incidence of duodenoesophageal reflux induced histological changes in the esophagus by preventing activation of proliferative & anti-apoptotic MAP kinases such as p38 & ER as well as protease activity. Though Barretts’ changes and adenocarcinoma have not developed, it could explain the role of duodenoesophageal reflux in the development of different histological but potential premalignant lesions and molecular level changes which are prevented by low dose aspirin.