Upregulation of miRNA-143, -145, -192, and -194 in esophageal epithelial cells upon acidic bile salt stimulation.

Barrett's esophagus (BE) is a metaplastic condition of the distal esophagus that occurs because of chronic gastroesophageal reflux. Previous studies have identified BE-specific microRNAs (miRNAs) in comparison with normal squamous epithelium (SQ). We hypothesized that BE-specific miRNAs could be induced in esophageal SQ cells by exposure to acid and/or bile salts. We aimed to determine whether BE-specific miRNAs are upregulated in an esophageal SQ cell line (Het-1A) in an environment with acid and/or bile salts and whether this is nuclear factor-κB (NF-κB) dependent. Acid and/or bile salt incubations were performed in Het-1A cells. Experiments were performed with or without inhibiting the NF-κB pathway. Quantitative reverse transcriptase polymerase chain reaction was performed to determine expression of miRNA-143, -145, -192, -194, cyclo-oxygenase-2 (COX2), mucin 2 (MUC2), and sex determining region Y-box 9. For validation, we determined levels of these miRNAs in biopsies from patients with reflux esophagitis and normal SQ. Significantly increased expression levels of miRNA-143 (2.7-fold), -145 (2.6-fold), -192 (2.0-fold), -194 (2.2-fold), COX2, MUC2, and sex determining region Y-box 9 were found upon acidic bile salt incubation, but not upon acid or bile salt alone. NF-κB pathway inhibition significantly decreased miRNA-143, -192, -194, COX2, and MUC2 expression. Additionally, miRNA-143, -145 and -194 expression was increased in reflux esophagitis biopsies compared with normal SQ, but no changes were found in miRNA-192 expression. Our findings suggest that upregulation of BE-specific miRNAs by acidic bile may be an early event in the transition of SQ to BE and that their expression is partly regulated by the NF-κB pathway.

High throughput techniques for characterizing the expression profile of Barrett's esophagus.

Barrett's esophagus (BE) is the metaplastic change of the normal lined squamous epithelium of the distal esophagus to a columnar type of epithelium as a result of chronic long-standing gastroesophageal reflux disease. Patients with BE have a significantly increased risk of developing an esophageal adenocarcinoma, with an estimated annual incidence varying from 0.4 to 1.8%. Over the last 3 decades, the incidence of BE and its associated adenocarcinoma has increased in Western countries at a rate that exceeds that of any other malignancy. Despite all the research performed on BE, there is still an inadequate understanding of the biological basis of this mucosal transformation. With the upcoming modern high throughput technologies, important progression has been made in unraveling the expression profiles and gaining more insight in the biology of BE and esophageal adenocarcinoma. Several studies reported genome, transcriptome, proteome, and kinome investigations using high throughput techniques. These studies were conducted to find biomarkers that can be used to detect BE patients with increased risk for malignant progression or to obtain more insight in the mechanism underlying BE development. In the following review, we first discuss the different techniques that are currently available and summarize findings in this field, including several recent publications of our group.

The effect of oral administration of ursodeoxycholic acid and high-dose proton pump inhibitors on the histology of Barrett's esophagus.

Bile acids may play a role in the pathogenesis of Barrett's esophagus (BE). Bile composition can be influenced by oral administration of ursodeoxycholic acid (UDCA). We prospectively investigated the effect of proton pump inhibitors (PPI) supplemented with UDCA in vivo in patients with BE. Patients with no or low-grade dysplasia who were clinically asymptomatic on PPI were eligible for the study. In order to exclude the effects of acid reflux, all patients were initially treated with 40 mg esomeprazole (ESO) twice daily for 6 months and continued on this dose till the end of the study (t = 12 months). During a period of 6 months (t = 6 month - t = 12 month) patients were treated with oral UDCA (600 mg twice daily). Patients underwent endoscopy at t = 0 months, t = 6 months and t = 12 months with multiple biopsies of the distal and proximal BE segment, normal squamous and gastric cardia. In addition, pH was measured at t = 0 months and t = 6 months using a BRAVO wireless pH capsule. Bile was sampled at the beginning of the UDCA treatment and 6 months later (t = 6 month and t = 12 month). All biopsies were reviewed for the extent of metaplasia, dysplasia, and acute and chronic inflammation. In addition, proliferation (Ki67), differentiation (villin, cytokeratins 7 and 20) and inflammation (COX-2) were investigated by immunohistochemistry (IHC). Nine patients (mean age 60 years, median BE length 7 cm) were included, of whom six had no dysplasia and three had low-grade dysplasia. pH measurements revealed a normal acid exposure in most patients at t = 0 and t = 6 months. In addition, bile composition analysis demonstrated the efficacy of UDCA. Combining the results of both phases of the study, no significant changes were seen in any of the histological or IHC parameters. Differentiation and proliferation parameters showed no significant changes. In this study, in BE patients who were clinically asymptomatic on PPI, increasing the PPI dose to the maximum for 6 months followed by the addition of UDCA for 6 months did not result in significant histological or IHC changes in their BE.

Gene expression profile comparison of Barrett's esophagus epithelial cell cultures and biopsies.

Barrett's esophagus (BE) is a metaplastic process in which the normal squamous epithelium of the distal esophagus is replaced by columnar lined epithelium. The aim was to gain more insight in the process of metaplasia and to identify which genes are specifically expressed by the epithelial cells and the surrounding tissues in BE. Hereto, the gene expression profile of a BE epithelial primary cell culture was compared to that of a BE biopsy. To specifically obtain the epithelial cell layer, epithelial cells from biopsies of BE were cultured using a Barrett specific culturing medium. Serial analysis of gene expression was applied to obtain a transcription library of the primary epithelial cell culture. The transcriptome was analyzed and compared to a previously described transcriptome of a BE biopsy. Validation of results by reverse transcriptase-polymerase chain reaction was performed using tissues of 16 BE patients and 16 primary cell cultures. Over 43,000 tags were sequenced. Genes specifically expressed by the Barrett epithelial cells were for instance Lipocalin 2 and Cyclin D1, whereas annexin A10, trefoil factor (TFF)1 and TFF2 were specifically expressed in the BE biopsies. The comparison of the gene expression profiles of BE primary cultured epithelial cells with BE biopsy defines a subset of genes that are specifically expressed by the epithelial cells and another subset that most likely is expressed by the underlying stromal tissues in the BE biopsy specimens.

Cell culture models for studying the development of Barrett's esophagus: a systematic review.

BACKGROUND: Barrett's esophagus (BE) is a premalignant condition caused by chronic gastroesophageal reflux. BE patients have an increased risk of developing esophageal adenocarcinoma (EAC). As many aspects of this condition are still unknown, there is a need for in vitro models to study BE development. AIM: To review the literature on cell lines and incubation conditions for studying BE development. METHODS: A literature search was performed using PubMed, EMBASE and the Cochrane library, combining the words esophagus, cell line, culture, Barrett's, bile, acid, exposure, reflux and adenocarcinoma. RESULTS: A wide range of cell lines and incubation conditions to study BE development have been reported. The most commonly used cell lines are derived from epithelium from patients with BE or EAC. A 25-minute incubation with 200 µM bile salts induced cell proliferation and Akt phosphorylation. However, increased CDX2 and MUC2 expression was only observed with longer incubations or higher bile salt concentrations. Two-hundred µM bile at pH 6 showed a higher toxicity to EAC cells than the same concentration at pH 7. Multiple 5-minute exposures with 200 µM bile at pH 4 or pH 7 increased CK8/18 and COX2 in BE epithelial cells. CONCLUSIONS: Two-hundred µM conjugated primary or secondary bile salts at pH 4 for multiple short exposures is able to induce BE specific factors in BE cell lines. In SQ and EAC cell lines; however, higher concentrations of secondary bile salts for 8 h are needed to induce BE specific molecules. Due to the high variability in reported methods, it is difficult to determine the most effective in vitro setup for studying the development of BE.