Yuji Tamagawa

Notch signaling pathway and Cdx2 expression in the development of Barrett's esophagus

Cdx2 expression in esophageal stem cells induced by reflux bile acids may be an important factor for development of Barretts esophagus, whereas Notch signaling is a molecular signaling pathway that plays an important role in the determination of cell differentiation. ATOH1 (a factor associated with Notch signaling) plays an important role in differentiation of stem cells into goblet cells. However, the relationship between the Notch signaling pathway and Cdx2 expression in the development of Barretts esophagus has not been explored. The aim of this study was to investigate the interrelationship between Notch signaling and Cdx2 in esophageal epithelial cells. The expressions of Cdx2, MUC2, and intracellular signaling molecules related to Notch signaling (Notch1, Hes1, and ATOH1) were examined using real-time polymerase chain reaction (PCR) and immunohistochemical staining with biopsy specimens obtained from esophageal intestinal metaplasia (IM) with goblet cells (IM(+)) and columnar epithelium not accompanied by goblet cells (IM(−)). For in vitro experiments, we employed human esophageal epithelial cell lines (OE33, OE19, and Het-1A). After forced Cdx2 expression by applying a Cdx2 expression vector to the cells, changes in the expressions of Notch1, Hes1, ATOH1, Cdx2, and MUC2 were analyzed by real-time PCR and western blot analysis. Changes in expressions of Notch1, Hes1, ATOH1, Cdx2, and MUC2 in cells were analyzed following stimulation with bile acids in the presence or absence of Cdx2 blocking with Cdx2-siRNA. Suppressed Hes1 and enhanced ATOH1 and MUC2 expressions were identified in IM(+) specimens. Forced expression of Cdx2 in cells suppressed Hes1, and enhanced ATOH1 and MUC2 expressions, whereas bile acids suppressed Hes1, and enhanced ATOH1, Cdx2, and MUC2 expressions. On the other hand, these effects were blocked by siRNA-based Cdx2 downregulation. Enhanced expression of Cdx2 by stimulation with bile acids may induce intestinal differentiation of esophageal columnar cells by interaction with the Notch signaling pathway.

Prevalence of gastroesophageal reflux disease in children, adults, and elderly in the same community.

The prevalence of gastroesophageal reflux disease (GERD) in adults is increasing in Japan as well as worldwide likely due to increasing obesity and the decreasing rate of Helicobacter pylori infection. However, data regarding the prevalence of GERD in children and adolescents in Japan are lacking. We investigated the prevalence of GERD in children, adults, and elderly living in the same community.

Fatty acid synthase expression in Barrett's esophagus: implications for carcinogenesis.

Goals To investigate the relationship between fatty acid synthase (FASN) expression and the clinicopathological characteristics of Barretts esophagus and its carcinogenesis. Background FASN, a key enzyme of the fatty acid biosynthetic pathway, is overexpressed not only in various types of cancer, but also in premalignant conditions. Therefore, FASN overexpression is considered to be indicative of a possible premalignant stage. Study Patients (N=354) with endoscopically and histologically proven Barretts esophagus were enrolled. Mucin phenotyping of Barretts esophagus, expression of FASN and COX-2, cellular proliferation, and apoptosis were evaluated immunohistochemically in biopsy samples, and factors influencing FASN expression were determined by multivariate logistic regression analysis. To evaluate if gastric reflux induces FASN expression, esophageal adenocarcinoma cells were treated with bile acid and low pH, and the effect of a FASN inhibitor on cell proliferation was assessed. Results Expression of FASN protein was observed in 52.2% of patients with Barretts esophagus by immunohistochemistry; this expression pattern was retained in esophageal adenocarcinoma. Intestinal mucin phenotype, COX-2, increased stromal angiogenesis, and elevated proliferating cell nuclear antigen index were confirmed to be positive independent factors for FASN expression. In the esophageal adenocarcinoma cell line SEG-1, FASN mRNA was induced by bile acid with low pH. Cell proliferation was strongly suppressed by the FASN inhibitor C75. Conclusions FASN is strongly expressed in the intestinal mucin phenotype of Barretts esophagus, in which Barretts glandular cells display elevated cellular proliferation, angiogenesis, and COX-2 expression. Exposure of the lower esophagus to bile acid with low pH may induce FASN in Barretts esophagus.

Bile acids induce Delta-like 1 expression via Cdx2-dependent pathway in the development of Barrett’s esophagus

Crosstalk between the Notch signaling pathway and Caudal-related homeobox 2 (Cdx2) has important roles in the development of Barrett’s esophagus (BE). We investigated the expression and function of the Notch signaling ligand Delta-like 1 (Dll1) during the development of BE. We determined the expression levels of Dll1 and intracellular signaling molecules related to Notch signaling ((Notch1, Hairy/enhancer of split 1 (Hes1), and Atonal homolog 1 (ATOH1)) in human esophageal squamous and Barrett’s epithelium samples. Next, those expression levels in esophageal squamous cells (Het-1A) and Barrett’s esophageal cells (CP-A and BAR-T) following stimulation with either bile acids or gamma-secretase inhibitor were investigated. Finally, changes in those expression levels following transfection of a Cdx2 or Dll1 expression vector into Het-1A cells were examined. In addition, changes in those expression levels following knockdown of Cdx2 or Dll1 in CP-A cells were also examined. Dll1 was found to be upregulated and localized in the cell membrane and cytoplasm in BE. Bile acids enhanced cytoplasmic expression of Dll1 in CP-A cells, while cleaved Notch1 expression did not change, suggesting lack of a Dll1 agonistic effect on Notch signaling. Cells transfected with Cdx2 revealed significantly enhanced Dll1, while forced expression of Dll1 enhanced ATOH1, Cdx2, and MUC2 expression levels. Nevertheless, enhanced Dll1 did not induce Hes1 expression, suggesting that Dll1 may primarily function as an intracellular signaling molecule and not a Notch agonistic ligand in the canonical pathway. In addition, knockdown of Cdx2 completely abrogated any increase in Dll1 expression upon treatment with bile acids. Our results revealed a novel function of Dll1: facilitation of intestinal metaplasia in conjunction with Cdx2 expression. Furthermore, they suggest that intracellular induction of Dll1 expression in esophageal epithelial cells due to Cdx2 induction in response to bile acids has important roles in BE development.

Crosstalk between TLR5 and Notch1 signaling in epithelial cells during intestinal inflammation.

During intestinal inflammation, a variety of signaling events are activated to perform several cell functions. Although the distinct roles of these pathways have been elucidated, the effects of their crosstalk activities remain to be clarified. We evaluated the crosstalk between two evolutionary conserved cell signaling systems, toll-like-receptor (TLR) 5 and Notch1, in intestinal epithelial cells during inflammation. Significant induction of the expression of Notch1 and Jagged1 was observed in the distal part of the colon, together with abundant localization of Notch1 intracellular domain (N1ICD) in the surface epithelium of inflamed colonic mucosa. By targeting intestinal epithelial cells, it was shown that recombination-signal-binding-protein-Jκ (RBP-Jκ)-mediated Notch functions are dependent on a flagellin-TLR5-mediated pathway. Conversely, using a γ-secretase inhibitor, we demonstrated that Notch synergistically increases TLR5‑mediated NF-κB activation. In addition, the effects of Notch on the NF-κB target gene interleukin-6 (IL-6) expression were revealed by evaluating the RBP-Jκ responsive element in the IL-6 promoter in vitro. Modulation of TLR5 and Notch crosstalk by transient blocking of Notch during the acute phase of colitis was beneficial for ameliorating colonic inflammation as well as disease status. In conclusion, the results suggest the effectiveness of Notch-targeted drug strategy for the treatment of intestinal inflammation.

Esophageal triamcinolone acetonide–filling method: a novel procedure to prevent stenosis after extensive esophageal endoscopic submucosal dissection (with videos)

BACKGROUND AND AIMS Endoscopic submucosal dissection (ESD) for extensive esophageal carcinomas may cause severe stenosis requiring endoscopic balloon dilations (EBDs). A standard prevention method has not been established. We propose the esophageal triamcinolone acetonide (TA)-filling method as a novel local steroid administration procedure. METHODS We enrolled 22 consecutive patients with early esophageal cancer who were treated using either subcircumferential or circumferential ESD (15 and 7 procedures, respectively) in this case series. Esophageal TA filling was performed on the day after ESD and 1 week later and was performed again if mild stenosis was found on follow-up. EBD with TA filling was performed only for severe stenosis that prevented endoscope passage. The primary endpoint was the incidence of severe stenosis. Secondary endpoints were the total number of EBDs and additional TA filling, dysphagia score, time to stenosis and to complete re-epithelialization, and any adverse events. RESULTS The incidence of severe stenosis was 4.5% (1/22; confidence interval, .1%-22.8%), and EBD was performed 2 times in 1 patient. Mild stenosis was found in 9 patients. Additional TA filling was performed in 45.5% of patients (10/22; median, 5 times; range, 1-13). The dysphagia score deteriorated to 1 to 2 in 31.8% (7/22) but showed a final score of 0 after complete re-epithelialization in 90.9% (20/22). The median time to stenosis was 3 weeks (range, 3-4) and that to complete re-epithelialization was 7 weeks (range, 4-36). No severe adverse events occurred. CONCLUSIONS The esophageal TA-filling method is highly effective for preventing severe stenosis after extensive esophageal ESD.

Impact of the composition of gastric reflux bile acids on Barrett's oesophagus.

BACKGROUND The effect of the composition of reflux bile acids, especially the ratio of hydrophobic to hydrophilic ones, on the development of Barretts oesophagus has not been fully investigated in human studies. AIMS To evaluate the influence of the bile acid composition of gastric juice on Barretts oesophagus, a prospective study was designed. METHODS Fifty patients with and 100 patients without Barretts oesophagus were enrolled. For all enrolled patients, gastric juice was collected by the endoscopic procedure for bile acid analysis. The ratio of hydrophobic to hydrophilic bile acids (bile hydrophobicity ratio, BHR) was calculated from 6 kinds of bile acids analysed in gastric juice. The relationship between the ratio and clinico-pathological factors of Barretts oesophagus was investigated. RESULTS The mean of BHR of patients with Barretts oesophagus was significantly higher than that of patients without Barretts oesophagus (0.26 ± 0.05 vs. 0.08 ± 0.02, p<0.05). In multivariate analysis, a high BHR value was a predictor for the presence of Barretts oesophagus (OR 5.74, p<0.001). In patients with Barretts oesophagus, the BHR correlated with COX-2 protein expression and with accelerated cellular proliferation. CONCLUSIONS Patients with Barretts oesophagus had a higher BHR in the gastric juice than those without.

202 Omeprazole and Fluticasone Inhibit IL-13-Stimulated Eotaxin-3 Expression by Esophageal Epithelial Cells Through Different Mechanisms and With Additive Effects: Rationale for Combining PPIs With Topical Steroids for EoE Patients

Introduction: Eosinophilic esophagitis (EoE) often presents with symptoms that are considered to relate to esophageal dysfunctions. Our previous study demonstrated that TRPA1 plays important role in tissue mast cell activation-induced increase in the excitability of esophageal vagal nodose C fibers. The present study aims to determine whether prolonged antigen exposure in vivo sensitizes TRPA1 in esophageal vagal afferents in a guinea pig model of EoE. Method: Antigen challenge-induced responses in esophageal mucosa were first studied by histological assessments and Ussing chamber methods. TRPA1 functions in vagal sensory neurons were then studied by calcium imaging and by whole-cell patch clamp recordings in DiI-labeled esophageal nodose and jugular neurons. Extracellular single-fiber recordings were performed in nodose and jugular C-fiber neurons using ex vivo esophagealvagal preparations with intact nerve endings in the esophagus. Results: (1) Prolonged antigen challenge (0.1% Ovalbumin, 30-second/day for 2-week) in antigen-sensitized guinea pigs increased inflammation scores from 2.0±0.47 (naive) to 4.33±0.27 (OVA-2w) in the esophagus (p<0.05). OVA challenge also increased the infiltrations of both mast cells (from 8.2±2.1 to 63.5±4.5 in mucosa, and from 14.0±2.4 to 43.1±4.5 in muscle layers, both p<0.05) and eosinophils (from 2.6±0.9 to 63.5±20.0 in mucosa, and from 0 to 5.2±2.55/ in muscle layers, both p<0.05) in the cross-sections of the esophagus. (2) Antigen challenge decreased the transepithelial resistance in esophageal epithelium from 564.7±63.4 V·cm2 to 356.0±45.5V·cm2 (p<0.05). (3) Antigen challenge increased TRPA1 agonist AITC-induced calcium influx in both nodose (from 43.5% to 66.7%) and jugular neurons (from 38.9% to 51.9%)(both p<0.05). (4) Antigen challenge increased current density elicited by TRPA1 agonist AITC in DiI-labeled esophageal nodose neurons (naive vs OVA-2w: 24.3±5.4 vs 59.7 ±4.7 pA/pF, p<0.05) and jugular neurons (naive vs OVA-2w: 31.5±5.3 vs 65.8 ±6.2 pA/pF, p<0.05). (5) In naive animals, intra-esophageal infusion of TRPA1 agonist AITC did not evoke action potential discharge in either esophageal nodose or jugular C fibers. After OVA challenge, intra-esophageal infusion of AITC was able to evoke action potential discharges in both nodose C fibers (baseline vs AITC: 0.75±0.25 Hz vs 4.63±1.03 Hz, p<0.01, n=8) and Jugular C fibers (baseline vs AITC: 0.88±0.25 Hz vs 2.5±0.38Hz, p<0.01, n=8). Conclusion: These results demonstrated that prolonged antigen challenge induced allergic inflammation, decreased epithelial barrier resistance, and sensitized TRPA1 in vagal nociceptive neurons and afferent C fibers in the esophagus. These changes enabled intra-esophageal noxious chemical to activate esophageal nociceptor. This novel finding may help to better understand esophageal dysfunction in EoE.

SUPERFICIAL ESOPHAGEAL CANCER OBSERVED WITH THE PILLCAM ESO 2 IN COMBINATION WITH THE FLEXIBLE SPECTRAL IMAGING COLOR ENHANCEMENT SYSTEM

PillCam ESO2 (Given Imaging, Yoqneam, Israel), a secondgeneration esophageal capsule endoscope, is expected to provide an excellent visualization and consequently a higher rate of detection of esophageal lesions. However, the diagnostic yield of capsule endoscopy for esophageal neoplastic lesions against a high-quality charge coupled device (CCD) endoscope with the Hi-Vision system (Olympus Medical Systems, Tokyo, Japan) is still controversial. The flexible spectral imaging color enhancement (FICE) system is a new dye-less imaging technique that enhances mucosal and vascular patterns, and this computed virtual chromoendoscopy is superior to the images obtained with conventional endoscope. FICE has also been available for capsule endoscopy of the small intestine and this system possibly improves the detection of the lesion. We encountered the impressive case of a 63-year-old man with superficial cancer of the lower esophagus that was examined with the combined use of PillCam ESO2 and FICE. The cancer was histologically identified as type 0-IIc, 4.8 ¥ 3.0 cm in size and with submucosal invasion accompanied by a regional lymph node metastasis. Capsule endoscopy with PillCam ESO2 was also done after the conventional endoscopy according to the simplified ingestion procedure, but the lesion was vaguely detected as shown in Fig. 1. The FICE procedure was carried out by the following three modes: (i) light with wavelength under 500 nm was filtered out to avoid a brownish tone, such as the effect of bile; (ii) light with hemoglobin absorption wavelength (415–540 nm) was enhanced; and (iii) mode A plus mode B. The lesion was more clearly shown in FICE image by mode A compared to conventional PillCam ESO2 (Fig. 2). Thus, the combined use of the FICE system with the PillCam ESO2 demonstrated a superior outcome in the diagnostic yield of esophageal capsule endoscopy. In the future, combination use of FICE may be essential for capsule endoscopy, although large-scale, randomized and controlled studies are needed.

Ankylosaurus back sign: novel endoscopic finding in esophageal eosinophilia patients indicating proton pump inhibitor response

Background and study aims  Characteristic endoscopic findings, such as linear furrows, rings, and whitish exudates, indicate the presence of esophageal eosinophilia (EE), though no specific findings are known to distinguish eosinophilic esophagitis (EoE) from proton pump inhibitor-responsive esophageal eosinophilia (PPI-REE). Here, we present a novel endoscopic finding in some EE patients possessing a linear longitudinal arrangement of whitish nodules with the appearance of the back of an Ankylosaurus dinosaur, termed Ankylosaurus back sign (ABS), and evaluations of its significance in affected patients. Patients and methods  Fifty-five patients diagnosed with EE (≥ 15 eosinophils/high power field) who were treated at our hospital and shown to evaluate a PPI response were enrolled. Endoscopic findings at baseline and clinical parameters were retrospectively reviewed. Furthermore, the clinicopathological features of patients with ABS, as well as the relationship between its presence and PPI response were evaluated. Results  Fifty-five patients (47 males, 8 females) with EE (17 with EoE, 38 with PPI-REE) were evaluated, of whom 50 (90.9 %) had linear furrows, the most frequently found feature, while ABS was found in 9 (16.4 %). Inter-observer agreement was substantial for ABS (κ 0.77). Interestingly, all patients with ABS had PPI-REE. Our findings revealed that the presence of ABS was closely associated with reflux esophagitis (RE) in patients with PPI-REE. Conclusions  Although ABS was less frequent than typical endoscopic findings such as linear furrows in EE, this novel finding was closely associated with PPI-REE accompanied with RE. The clinical implications of ABS in patients with EE should be investigated further.