Nitric oxide could promote development of Barrett's esophagus by S-nitrosylation-induced inhibition of Rho-ROCK signaling in esophageal fibroblasts.

Barrett's esophagus arises in the process of wound healing in distal esophageal epithelium damaged by gastroesophageal reflux disease. Differentiation of fibroblast into myofibroblasts, a smooth muscle cell-like phenotype and tissue contraction are crucial processes in wound healing. No study has evaluated mechanism by which luminal esophageal nitric oxide (NO) affect Rho-associated coiled coil-forming protein kinase (Rho-ROCK) signaling pathway, a key factor of tissue contraction, in stromal fibroblasts to develop Barrett's esophagus. Using esophageal fibroblasts, we performed collagen-based cell contraction assays and evaluated influence of Rho-ROCK signaling in the exposure to acidic bile salts and NOC-9, which is an NO donor. We found that enhanced cell contraction induced by acidic bile salts was inhibited by NO, accompanied by decrease in phosphorylated myosin light chain expression and stress fiber formation. NO directly S-nitrosylated GTP-RhoA and consequently blocked Rho-ROCK signaling. Moreover, exposure to NO and Y27632, a Rho-ROCK signaling inhibitor, decreased α-SMA expression and increased bone morphogenetic protein-4 (BMP4) expression and secretion. These findings could account for the increased expression of BMP4 in the columnar epithelial cells and stromal fibroblasts in human Barrett's esophagus. NO could impair wound contraction by blocking the Rho-ROCK signaling pathway and promote the development of Barrett's esophagus.NEW & NOTEWORTHY Barrett's esophagus is the condition where esophageal epithelium damaged by gastroesophageal reflux disease (GERD) is abnormally healed via replacing of metaplastic columnar epithelium, but very few studies have conducted focusing wound healing in the development of Barrett's esophagus. Esophageal luminal nitric oxide inhibits Rho-ROCK signaling pathway in esophageal fibroblasts, which leads to delay tissue contraction, a pivotal step in proper wound healing. Moreover, this inhibition increases tissue BMP4 expression. Impaired wound healing could be related to Barrett's esophagus.

Decreased Expression of NRF2 Target Genes after Alcohol Exposure in the Background Esophageal Mucosa of Patients with Esophageal Squamous Cell Carcinoma.

Patients with esophageal squamous cell carcinoma (ESCC) might have a specific mechanism for the carcinogenesis by alcohol consumption in the background esophageal mucosa, and nuclear factor erythroid 2-related factor 2 (NRF2), which plays a protective role against esophageal carcinogenesis, and barrier dysfunction might be associated with this phenomenon. This study aimed to confirm this hypothesis. Twenty patients with superficial ESCCs (ESCC patients) and 20 age- and sex-matched patients without ESCC (non-ESCC patients) were enrolled. Biopsy samples were obtained from non-neoplastic esophageal mucosa: one for histological evaluation, one for quantitative real-time polymerase chain reaction (PCR), and two for the mini-Ussing chamber system to measure transepithelial electrical resistance (TEER) and, thereafter, for PCR. The TEER after acetaldehyde or both acetaldehyde and ethanol exposure did not differ significantly between ESCC and non-ESCC patients. Unlike non-ESCC patients, mRNA levels of NRF2 target genes and claudin4 in ESCC patients tended to decrease after the exposure, with a significant difference between no exposure and both acetaldehyde and ethanol exposure in NRF2 target genes (p < 0.05). Furthermore, in ESCC patients, the decreased tendency of mRNA levels of NRF2 target genes after the exposure was more pronounced in high-risk states, such as aldehyde dehydrogenase 2 (ALDH2) Lys alleles (Glu/Lys + Lys/Lys), Lugol-voiding lesion grade C, and drinking history. In conclusion, the protective role of NRF2 against carcinogenesis from alcohol exposure might be disrupted in the background esophageal mucosa of ESCC patients, which might lead to a high incidence of metachronous ESCC.

The Impact of Duodenal Mucosal Vulnerability in the Development of Epigastric Pain Syndrome in Functional Dyspepsia.

An unidentified cause of functional dyspepsia (FD) is closely associated with medication resistance. Acid suppression is a traditional and preferential method for the treatment of FD, but the efficacy of this treatment varies between epigastric pain syndrome (EPS) and postprandial syndrome (PDS): it is efficient in the former but not much in the latter. Transepithelial electrical resistance (TEER), a surrogate of mucosal barrier function, was measured under pH 3 and pH 5 acidic conditions using duodenal biopsy specimens obtained from the patients with EPS and PDS and asymptomatic healthy controls. The infiltration of inflammatory cells to the duodenal mucosa was accessed by immunohistochemical analysis. The duodenal mucosal TEER in EPS patients was decreased by exposure to the acidic solution compared to that of the controls and the PDS patients. The decrease in TEER of the EPS patients was observed even under pH 5 weak acidic condition and was correlated to degree of the epigastric pain. Moreover, the duodenal mucosa of EPS patients presented an increase in mast cells and plasma cells that expressed Ig-E. Duodenal mucosal vulnerability to acid is likely to develop EPS.

The Ameliorating Effect of Switching to Vonoprazan: A Novel Potassium-Competitive Acid Blocker in Patients with Proton Pump Inhibitor Refractory Non-Erosive Reflux Disease.

INTRODUCTION: Weakly acidic reflux has been reported to be the major cause of symptoms in patients with proton pump inhibitor (PPI)-refractory nonerosive reflux disease (NERD) undergoing PPI treatment. We previously reported that reflux at pH 4-5 was the main factor that induced symptoms in such patients. The present study aimed to elucidate the symptom-ameliorating effect of vonoprazan (VPZ) by evaluating the change in the pH value of the refluxate using multichannel intraluminal impedance and pH (MII-pH) monitoring. METHODS: We retrospectively evaluated the records of MII-pH monitoring in 29 symptom index (SI) and/or symptom association probability (SAP)-positive patients with PPI-refractory NERD. After switching to VPZ 20 mg/day, we performed MII-pH monitoring again. We assessed the change in the score of the frequency scale for the symptoms of gastroesophageal reflux disease (FSSG), the pH value of the refluxate, and the percent times of intragastric pH <4 or <5 before and after switching. We divided the patients into the following 2 groups according to the FSSG score after switching: effective and noneffective groups. RESULTS: Among of the 29 SI/SAP-positive patients, 16 underwent switching to VPZ. Furthermore, of these 16 patients, 10 underwent MII-pH monitoring again after switching. The FSSG score decreased, the pH value of the refluxate increased, and the percent times of intragastric pH <4 or <5 reduced after switching when compared with the findings before switching. In the effective group, both the proportion of reflux at pH <4 and that of reflux at pH 4-5 decreased while taking VPZ when compared with the findings while taking double-dose PPI. In the noneffective group, the proportion of reflux at pH <4 decreased but that of reflux at pH 4-5 increased and that of reflux at pH <5 did not change overall while taking VPZ. In addition, the percent times of intragastric pH <5 values were low in the effective group. CONCLUSION: Symptom suppression appears to be inadequate in patients with persistent reflux at pH 4-5 even with VPZ 20 mg/day. Strong acid suppressive therapy with VPZ to increase the pH value of the refluxate to ≥5 is useful for symptom improvement.

Impaired Mucosal Integrity in Proximal Esophagus Is Involved in Development of Proton Pump Inhibitor-Refractory Nonerosive Reflux Disease.

BACKGROUND AND OBJECTIVE: Weakly acidic reflux reaching to the proximal esophagus is closely related to the perception of gastroesophageal reflux in patients with nonerosive reflux disease despite treatment with a proton pump inhibitor (PPI). However, little is known about the involvement of the patients' mucosal integrity of the proximal esophagus. METHODS: We recruited 15 symptomatic nonerosive gastroesophageal reflux disease (GERD) patients with a positive symptom index despite PPI treatment and 11 healthy asymptomatic volunteers as controls. The biopsy specimens obtained from the proximal and distal esophagus were applied to a mini-Ussing chamber system to measure transepithelial electrical resistance (TEER) against a pH 4 weak acid. The esophageal biopsy samples were subjected to quantitative real-time PCR and immunohistochemical analysis. RESULTS: In the proximal esophagus, the weak acid exposure reduced the TEER in the PPI-refractory patients compared to that in the controls. The frequency of the reflux extending to the proximal esophagus had a significant correlation with the reduction in the proximal esophageal TEER in the patients. The reduced TEER in the proximal esophagus was accompanied by an increase in IL-8 and IL-1ß mRNA and a decrease in occludin mRNA levels. The proximal esophageal mucosa in the patients presented infiltration of CD3-positive lymphocytes and an increased expression of solute carrier organic anion transporter family member 2A1 (SLCO2A1), a passage gate of reflux symptom-evoking molecules. CONCLUSIONS: The reflux perception is related to an impairment of the proximal esophageal mucosal integrity in patients with nonerosive reflux disease despite PPI.

Sox2 induces tumorigenesis and angiogenesis of early-stage esophageal squamous cell carcinoma through secretion of Suprabasin.

Early stage of esophageal squamous cell carcinoma (ESCC) is known to be accompanied by angiogenesis and morphological changes of microvessels. Transcription factor Sox2 is amplified in various cancers including ESCC, but the role of Sox2 in the carcinogenesis and angiogenesis has not been determined. Hence, we aimed to investigate the role of Sox2 in the early stage of ESCC. We found that the expression of Sox2 was significantly higher in early-stage ESCC tissues than that in their adjacent normal tissues. We then established Sox2-inducible normal human esophageal squamous cell line (HetSox2) to investigate the role of Sox2 in esophageal carcinogenesis and angiogenesis in vitro. Sox2 overexpression led to increased cell proliferation and spheroid formation. The culture supernatant of Sox2-overexpressing HetSox2 induced migration and sprouting of endothelial cell line HUVEC (human umbilical vein endothelial cell). As for the mechanism, we found that the expression of secreted protein Suprabasin was directly induced by Sox2. Suprabasin enhanced proliferation of normal human esophageal squamous cells when added to the culture. Moreover, Suprabasin enhanced migration and sprouting of HUVEC cells, which were observed with the culture supernatant of Sox2-overexpressing HetSox2. This angiogenic effect of Suprabasin was abolished by inhibiting AKT phosphorylation, which suggested its dependence on AKT signaling. Finally, we showed that Suprabasin expression and the density of microvessels were significantly higher in ESCC tissues with high Sox2 expression. Our study suggested that increased Sox2 expression in esophageal squamous cells induced Suprabasin expression, and as a result initiated the carcinogenesis via increased cell proliferation and angiogenesis.

Influence of the pH Value of Refluxate and Proximal Extent on Heartburn Perception in Patients with Proton Pump Inhibitor-Refractory Non-Erosive Reflux Disease.

BACKGROUND AND OBJECTIVES: Weakly acidic reflux has been reported as the major cause of symptom occurrence in patients with proton pump inhibitor (PPI)-refractory non-erosive reflux disease (NERD). This study is aimed at clarifying whether the pH value of weakly acidic reflux affects the induction of symptoms. METHODS: We retrospectively evaluated the records of combined multichannel intraluminal impedance and pH monitoring in 57 patients with PPI-refractory NERD. Weakly acidic refluxes were divided into 3 categories based on the pH value of the refluxate: pH 4-5, 5-6, and 6-7. RESULTS: A total of 29 patients were positive in the symptom index. The symptom provocation rate in reflux of pH 4-5 (19%) was much higher than in that of pH 5-6 (11%) and pH 6-7 (12%). In the reflux at pH 4-5, the symptom provocation rate in the proximal reflux was higher than that in the distal reflux (p < 0.05), whereas the reflux at pH 5-6 and pH 6-7 was not significantly different in the symptom provocation rate between the proximal and distal refluxes. CONCLUSION: Reflux at pH <5 reaching the proximal esophagus was the main factor in the induced symptoms of patients with PPI-refractory NERD.

Strong Intra-Esophageal Reflux May Contribute to the Development of Barrett's Adenocarcinoma and Affect the Localization.

BACKGROUND: There has been no study that has directly measured the esophageal reflux factors in Barrett's adenocarcinoma (BA) using 24-h multichannel intraluminal impedance-pH monitoring (24-h MII-pH). We aimed to clarify the esophageal reflux factors in Barrett's esophagus (BE) and BA and the factors that determine the location of BA with 24-h MII-pH. METHODS: We performed 24-h MII-pH in 26 patients with superficial BA treated endoscopically (BA group) and 13 patients with BE (BE group) and examined the esophageal reflux factors (esophageal acid exposure time [AET], bolus exposure (acid, weakly acid, and alkaline), and number of reflux episodes. In the BA group, there were 16 cases in which the lesions were localized in an area in contact with the esophagogastric junction (EGJ; EGJ group), and 10 cases in which the lesions were proximal to the BE and separated from the EGJ (non-EGJ group). RESULTS: Total reflux in the bolus exposure in the BA group showed higher values compared to that in the BE group. The total of acid and weakly acid reflux of bolus exposure was significantly higher in the BA group than that in the BE group. The BA group also had greater numbers of total reflux episodes than the BE group. As for the cancer locations in BE, the cases in which the lesions were located proximally and separated from the EGJ had more AET and total reflux and acid reflux indicated by bolus exposure compared to the lesions adjacent to the EGJ. CONCLUSIONS: Stronger gastro-esophageal reflux appeared to be an important factor in the development of adenocarcinoma from BE. In addition, the cancer location in BE may be related to the intensity of esophageal reflux.

Continuous liquid-suction catheter attachment for endoscope reduces volume of liquid reflux to the mouth in esophageal endoscopic submucosal dissection.

BACKGROUND AND AIM: Pooling of liquid in the esophageal lumen can worsen the field of vision and cause liquid reflux to the mouth, which leads to aspiration pneumonia, in esophageal endoscopic submucosal dissection (ESD). We developed a continuous liquid-suction catheter attachment for the endoscope (CLCA) that has multiple tiny holes and can suction the liquid without causing mucosal injury. Thus, we aim to show the efficacy of CLCA in esophageal ESD. METHODS: This was a single-blinded, randomized controlled trial involving patients with superficial esophageal cancer. The enrolled patients were randomly assigned to the conventional ESD (C-ESD) or ESD with CLCA (CLCA-ESD) groups. Primary endpoint was volume of liquid reflux to the mouth during the ESD procedure. Secondary endpoints were incidence of aspiration pneumonia and procedure time. RESULTS: Fifty patients were enrolled in this trial. Volume of liquid reflux to the mouth was significantly lower in the CLCA-ESD group than in the C-ESD group (mean: 10 vs 73 mL, P = 0.010). Furthermore, the incidence of aspiration pneumonia on computed tomography (CT) scan between the two groups was also significantly different (4.0% vs 32.0%, P = 0.023), although no significant difference was observed through chest radiography. In addition, procedure time tended to be shorter in the CLCA-ESD group (P = 0.054). CONCLUSION: This study first showed that use of CLCA in esophageal ESD reduced the volume of liquid reflux to the mouth and contributed to decreased incidence of aspiration pneumonia on CT scan (UMIN000018167).

The Use of Higher Dose Steroids Increases the Risk of Rebleeding After Endoscopic Hemostasis for Peptic Ulcer Bleeding.

BACKGROUND: Previous studies have shown that several factors such as hemodynamic instability at admission are risk factors for rebleeding of peptic ulcer bleeding. However, whether steroid use increases the risk of rebleeding remains elusive. AIMS: This study aimed to clarify the risk factors for rebleeding after endoscopic hemostasis for peptic ulcer bleeding. METHODS: A total of 185 patients who underwent endoscopic hemostasis for peptic ulcer bleeding at our institution between 2005 and 2017 were retrospectively analyzed. We evaluated factors, including comorbid conditions, in-hospital onset, and steroid use, associated with rebleeding by logistic regression analysis. In addition, we investigated the association between the dose of steroids and rebleeding. RESULTS: The rebleeding rate after endoscopic hemostasis for peptic ulcer bleeding was 14.6%. In the multivariate analysis, the independent risk factors for rebleeding were steroid use (odds ratio 4.56, p = 0.015), multiple ulcers (4.43, p = 0.005), number of comorbidities ≥ 3 3.18, p = 0.026), hemodynamic instability (3.06, p = 0.039), and number of comorbidities ≥ 3 (2.93, p = 0.047). Furthermore, the use of higher dose steroids (≥ 20 mg per day in prednisolone; 10.55, p = 0.002), but not lower dose (< 20 mg per day in prednisolone), was an independent risk factor for rebleeding in the multivariate analysis. The relationship between steroid use and rebleeding was observed in a dose-dependent manner (p for trend = 0.002). CONCLUSIONS: This study first revealed that using higher dose steroids was an independent risk factor for rebleeding after endoscopic hemostasis for peptic ulcer bleeding, with a dose-response relation.

Estrogen-Dependent Nrf2 Expression Protects Against Reflux-Induced Esophagitis.

BACKGROUND: Gastroesophageal reflux disease is more common in males than in females. The enhanced antioxidative capacity of estrogen in females might account for the gender difference. Nuclear factor erythroid 2-related factor 2 (Nrf2) plays a pivotal role in the host defense mechanism against oxidative stress. AIMS: This study aimed to clarify the role of Nrf2 in reflux-induced esophageal inflammation, focusing on the gender difference and nitric oxide. METHODS: Gastroesophageal reflux was surgically induced in male and female rats. Nitrite and ascorbic acid were administered for 1 week to provoke nitric oxide in the esophageal lumen. Male rats with gastroesophageal reflux were supplemented with 17ß-estradiol or tert-butylhydroquinone, an Nrf2-inducing reagent. Esophageal squamous cell carcinoma KYSE30 cells were treated with 17ß-estradiol. Nrf2 expression was examined by Western blotting and quantitative real-time PCR. Antioxidant gene expression profiles were examined by a PCR array. RESULTS: In the presence of nitric oxide, reflux-induced esophageal damage was less evident, whereas esophageal expression of Nrf2 and its target genes such as Nqo1 was more evident in female or male rats supplemented with 17ß-estradiol than in male rats. 17ß-Estradiol increased nuclear Nrf2 expression in KYSE30 cells. tert-Butylhydroquinone increased tissue Nqo1 mRNA expression, leading to a reduction in reflux-induced esophageal damage. CONCLUSIONS: Estrogen-dependent Nrf2 expression might contribute to protection against the development of gastroesophageal reflux disease in females.

Leptin Aggravates Reflux Esophagitis by Increasing Tissue Levels of Macrophage Migration Inhibitory Factor in Rats.

Leptin, produced primarily by the adipose tissue, acts as a pro-inflammatory modulator, thereby contributing to the development of obesity-related disease. Although high levels of leptin in the obese are closely related to gastroesophageal reflux disease, the mechanism by which leptin influences esophageal inflammation remains unknown. Macrophage migration inhibitory factor (MIF) is produced by immune cells, such as T lymphocytes and macrophages, and MIF is known to induce the production of tumor necrosis factor α (TNF-α), interleukin 1ß (IL-1ß) and interleukin 6 (IL-6). We therefore investigated the mechanism whereby leptin aggravates reflux esophagitis, by focusing on esophageal tissue levels of MIF and CD3+ T lymphocytes, both of which are crucial for the reflux-induced epithelial damage. Esophageal inflammation was surgically induced in male Wistar rats by ligating the forestomach and narrowing the duodenum to facilitate gastroesophageal reflux, followed by administration of leptin or vehicle with an osmotic pump system for 1 week. We demonstrated that the administration of leptin exacerbated the reflux esophagitis with the apparent infiltration of CD3+ T lymphocytes and caused the significant increase in the esophageal tissue levels of MIF. Moreover, the leptin caused increases in the esophageal tissue levels of TNF-α, IL-1ß and IL-6, downstream targets of MIF. Importantly, the increases in these pro-inflammatory cytokines were accompanied by increased protein levels of phospho-STAT3 and phospho-AKT, pivotal molecules of leptin signaling pathways. In conclusion, through enhancing the MIF-induced inflammatory signaling, leptin could contribute to the development of gastroesophageal reflux disease.

Detection of Acetaldehyde in the Esophageal Tissue among Healthy Male Subjects after Ethanol Drinking and Subsequent L-Cysteine Intake.

Ethanol is oxidized by alcohol dehydrogenase to acetaldehyde, a recognized carcinogen for the esophagus. However, no previous study has measured the acetaldehyde levels in the esophageal tissue. L-cysteine has been shown to reduce the acetaldehyde levels in the saliva; however, it is unknown whether L-cysteine intake affects the acetaldehyde concentration in the esophageal tissue. The aim of this study was to measure the acetaldehyde concentration in the esophageal tissue after ethanol drinking and evaluate the effect of L-cysteine intake on the acetaldehyde levels in the esophagus. We enrolled 10 male subjects with active acetaldehyde dehydrogenase-2*1/*1 (ALDH2*1/*1) genotype and 10 male subjects with the inactive acetaldehyde dehydrogenase-2*1/*2 (ALDH2*1/*2) genotype, the mean ages of whom were 25.6 and 27.9 years, respectively. In this prospective, single-blind, placebo-controlled study using L-cysteine and placebo lozenges (first and second examination), saliva and blood were collected before and after ethanol drinking. Esophageal tissue was obtained by endoscopic biopsy at 60 minutes after drinking, and the acetaldehyde and ethanol concentrations were measured. The acetaldehyde concentration of the saliva was significantly lower in those taking L-cysteine than in those taking the placebo. Acetaldehyde in the esophageal tissue was detected only in those taking L-cysteine lozenges. There were no correlations between the acetaldehyde concentrations in the esophageal tissue and saliva or blood. In conclusion, we detected acetaldehyde in the human esophageal tissue after ethanol drinking. Unexpectedly, intake of L-cysteine lozenges appears to contribute to detection of acetaldehyde in the esophageal tissue.

Factors Associated With Metachronous Gastric Cancer Development After Endoscopic Submucosal Dissection for Early Gastric Cancer.

GOALS: To clarify the factors associated with metachronous gastric cancer development after endoscopic submucosal dissection (ESD) for early gastric cancer. BACKGROUND: Patients who undergo ESD for early gastric cancer have an appreciable risk of developing metachronous gastric cancer. However, there have been few reports on the association between life style and the development of such cancer. STUDY: Patients with early gastric cancer who underwent ESD at our institution between 2003 and 2012 were enrolled. Metachronous gastric cancer was defined as secondary gastric cancer detected >1 year after initial ESD. Factors, including age, gender, body mass index, eradication of Helicobacter pylori, cigarette smoking, drinking, and continuous use of a proton pump inhibitor, associated with metachronous gastric cancer development were evaluated by Cox proportional hazard regression analysis. RESULTS: A total of 539 patients with a mean 53.6-month follow-up period were analyzed. The 5-year cumulative incidence of secondary gastric cancer was 13.0%. Multivariate analysis exhibited that age of 60 years and above [hazard ratio (95% confidence interval)=4.05 (1.23-13.4)] and cigarette smoking [2.12 (1.19-3.78)] were independent risk factors for metachronous gastric cancer development. Furthermore, ≥20 pack-years of smoking [1.51 (1.03-2.24)] was a significant risk factor with a dose-response relationship (P for trend=0.042). There was no significant association between Helicobacter pylori eradication and metachronous gastric cancer development. CONCLUSIONS: This is the first study to demonstrate the detailed association between cigarette smoking and metachronous gastric cancer development.

The Risk Factors for Metastasis in Non-Ampullary Duodenal Neuroendocrine Tumors Measuring 20 mm or Less in Diameter.

BACKGROUND/AIMS: The treatment strategy for non-ampullary duodenal neuroendocrine tumors (NAD-NETs) ≤20 mm in diameter has not been established. In this study, we aimed to evaluate the detailed characteristics of NAD-NETs ≤20 mm in diameter to clarify the risk factors of subsequent metastasis. METHODS: The patients with NAD-NETs ≤20 mm in diameter who had been treated at 12 institutions between 1992 and 2013 were enrolled. Clinical records were retrieved, and histopathological findings of all cases were centrally reviewed by 2 pathologists. RESULTS: We studied 49 patients with a mean follow-up period of 66.5 months. Thirty-five patients were initially treated with endoscopic resection (ER), and 14 with surgery. A univariate analysis revealed the ORs and 95% CIs of the risk factors for metastasis were lymphovascular invasion (12.5 [2.01-77.9]), multiple tumors (9.75 [1.46-65.4]), a tumor size of 11-20 mm (6.67 [1.21-36.6]), and World Health Organization grade G2 (7.13 [1.16-43.9]). Five-year overall and disease-specific survival rates were 86.1 and 97.2%, respectively. CONCLUSION: This is the first study to demonstrate the risk factors of metastasis in NAD-NETs ≤20 mm in diameter. These findings may be helpful for determining the appropriate therapeutic approach and the clinical strategy of treatment following ER.

In oesophageal squamous cells, nitric oxide causes S-nitrosylation of Akt and blocks SOX2 (sex determining region Y-box 2) expression.

OBJECTIVE: Barrett's metaplasia might develop if GORD causes oesophageal squamous cells to convert into columnar cells. Acid and bile exposures upregulate columnar differentiation genes like CDX2 in oesophageal squamous cells, but it is not known if such exposures downregulate squamous differentiation genes like SOX2. In addition to acid and bile, patients with GORD also have high oesophageal concentrations of nitric oxide (NO). This study aims to determine how acid, bile salts and NO affect genes that influence oesophageal cell phenotype. DESIGN: Oesophageal squamous cells from patients with Barrett's oesophagus were exposed to acidic bile salts or NOC-9 (an NO donor). SOX2, p63 (squamous transcription factor) and CDX2 mRNAs were measured by quantitative RT-PCR. SOX2 and its regulatory Akt pathway proteins were evaluated by western blotting. S-nitrosylation by NO was blocked by dithiothreitol. Immunohistochemistry for SOX2 was performed on the oesophagus of rats with surgically induced GORD which were fed diets with and without nitrite supplementation. RESULTS: In oesophageal squamous cells, NO profoundly decreased SOX2 protein and caused a significantly greater decrease in SOX2 mRNA than did acidic bile salts. NO also decreased p63 and increased CDX2 expression. NO caused S-nitrosylation of Akt, blocking its phosphorylation. Akt pathway inhibition by LY294002 or Akt siRNA reduced SOX2 mRNA. Rats fed with nitrite-supplemented diets exhibited weaker SOX2 oesophageal staining than rats fed with normal diets. CONCLUSIONS: In oesophageal squamous cells, NO blocks SOX2 expression through Akt S-nitrosylation. NO also increases CDX2 and decreases p63 expression. By triggering molecular events preventing squamous differentiation while promoting intestinal differentiation, NO might contribute to Barrett's pathogenesis.

Estrogen Enhances Esophageal Barrier Function by Potentiating Occludin Expression.

BACKGROUND: We recently demonstrated that a female sex hormone, estrogen, suppressed esophageal epithelial injury in a reflux esophagitis model of rat, suggesting that estrogen may play an important role in controlling the progress of the gastro-esophageal reflux disease spectrum. However, the precise mechanism of the action is unclear. AIM: To investigate the potential role of estrogen in the esophageal barrier function. METHODS: Male rabbits were pretreated with either continuous release 17ß-estradiol or placebo, and the excised esophageal mucosa was subjected to Ussing chamber experiments after the 2-week pre-treatment. The mucosal side of the chamber was perfused with luminal irritants (HCl or acidified sodium nitrite), while the basal side was perfused by modified Krebs buffer. The epithelial barrier function was evaluated by the transmembrane resistance and the epithelial permeability. The intercellular space of the epithelium was investigated with transmission electron microscopy and the expression of tight junction protein, occludin, claudin-1, and claudin-4, with immunoblotting. RESULTS: Estrogen pre-treatment significantly attenuated the decrease in the transmembrane resistance and the increase in the epithelial permeability induced by luminal irritants. Furthermore, the dilation of the intercellular space induced by luminal HCl was significantly alleviated by 17ß-estradiol administration. The baseline occludin expression was significantly potentiated by 17ß-estradiol administration. CONCLUSIONS: This is the first study showing an enhancement of the esophageal barrier function by 17ß-estradiol administration. The lack of the protective effect of estrogen could be responsible for the male predominance of erosive reflux esophagitis.

Risk factors of Barrett's esophagus.

Barret's esophagus, and accompanying esophageal adenocarcinoma have been increasing over the last 3-4 decades in Western countries. Although the prevalence of the esophageal adenocarcinoma has not obviously increased in Japan, GERD, a precursor condition of the can- cer, has recently increased in that country. Recognized risk factors for Barrett's esophagus are male gender, higher age, GERD, obesity, and protective factor for the disease is H. pyloi infection. Identification of risk factors for Barrett's esophagus should be useful to estimate the future trend of esophageal adenocarcinoma in Japan.

Autocrine VEGF signaling promotes proliferation of neoplastic Barrett's epithelial cells through a PLC-dependent pathway.

BACKGROUND & AIMS: Tumor cells express vascular endothelial growth factor (VEGF), which induces angiogenesis. VEGF also activates VEGF receptors (VEGFRs) on or within tumor cells to promote their proliferation in an autocrine fashion. We studied the mechanisms of autocrine VEGF signaling in Barrett's esophagus cells. METHODS: Using Barrett's epithelial cell lines, we measured VEGF and VEGFR messenger RNA and protein, and studied the effects of VEGF signaling on cell proliferation and VEGF secretion. We studied the effects of inhibiting factors in this pathway on levels of phosphorylated phospholipase Cγ1 (PLCG1), protein kinase C, and extracellular signal-regulated kinases (ERK)1/2. We performed immunohistochemical analysis of phosphorylated VEGFR2 on esophageal adenocarcinoma tissues. We studied effects of sunitinib, a VEGFR2 inhibitor, on proliferation of neoplastic cells and growth of xenograft tumors in mice. RESULTS: Neoplastic and non-neoplastic Barrett's cells expressed VEGF and VEGFR2 messenger RNA and protein, with higher levels in neoplastic cells. Incubation with recombinant human VEGF significantly increased secretion of VEGF protein and cell number; knockdown of PLCG1 markedly reduced the recombinant human VEGF-stimulated increase in levels of phosphorylated PLCG1 and phosphorylated ERK1/2 in neoplastic cells. Esophageal adenocarcinoma tissues showed immunostaining for phosphorylated VEGFR2. Sunitinib inhibited VEGF signaling in neoplastic cells and reduced weight and volume of xenograft tumors in mice. CONCLUSIONS: Neoplastic and non-neoplastic Barrett's epithelial cells have autocrine VEGF signaling. In neoplastic Barrett's cells, VEGF activation of VEGFR2 initiates a PLCG1-protein kinase C-ERK pathway that promotes proliferation and is self-sustaining (by causing more VEGF production). Strategies to reduce autocrine VEGF signaling (eg, with sunitinib) might be used to prevent or treat cancer in patients with Barrett's esophagus.

Endoscopic findings for predicting gastric acid secretion status.

BACKGROUND AND AIM: Gastric acidic abnormalities are related to various types of diseases in Helicobacter pylori (H. pylori) infection status. However, no studies have shown correlations between many tiny endoscopic findings and the acid secretion level simultaneously. In the present study, we investigated predictive tiny endoscopic findings of hyperchlorhydria and hypochlorhydria. METHODS: A total of 223 subjects without organic diseases who underwent esophagogastroduodenoscopy and endoscopic gastrin test (EGT) for estimating gastrin-stimulated gastric acid secretory response between 1999 and 2012 at our institution were retrospectively analyzed. Two blinded expert endoscopists reviewed the images independently and recorded the endoscopic findings. RESULTS: According to the EGT values, the enrolled subjects were categorized into hyperchlorhydria, normal acid secretion, and hypochlorhydria groups. In all subjects, hematin (odds ratio [95% confidence interval] = 3.32 [1.40-7.84]) and antral erosion(2.88 [1.24-6.70]) were the predictive endoscopic findings for hyperchlorhydria, and swelling of areae gastricae (14.4 [5.74-36.1]) and open-type atrophy (15.1 [7.35-31.1]) were those for hypochlorhydria. In addition, the predictive endoscopic findings for hyperchlorhydria differed according to the H. pylori infection status, hematin in H. pylori-positive subjects and antral erosion in H. pylori-negative subjects, in contrast to those for hypochlorhydria, which were the same irrespective of the H. pylori infection status. CONCLUSIONS: We could predict the acid secretion status based on the endoscopic findings regardless of H. pylori infection status, which would be of some help for evaluating the risk for acid-related diseases.