Nobuyuki Ara

Exogenous luminal nitric oxide exacerbates esophagus tissue damage in a reflux esophagitis model of rats

Objective. Cytotoxic concentrations of nitric oxide are generated luminally at the gastroesophageal junction through the entero-salivary recirculation of dietary nitrate in humans. The site of luminal nitric oxide generation shifts to the lower esophagus when gastric acid is refluxed into the esophagus. The aim of this study was to investigate the influence of persistent administration of exogenous nitric oxide on esophageal damage. Material and methods. 0.1% sodium nitrite and/or 1% ascorbic acid was administered in an established rat acid-refluxed esophagitis model. Co-administration of both reactants in this model is thought to induce high concentrations of nitric oxide luminally in the esophagus by an acid-catalyzed chemical reaction when refluxed gastric acid is present. The tissue damage was evaluated by a macroscopic lesion index and myeloperoxidase activity. Nitrotyrosin was assessed immunohistochemically as a footprint of peroxynitrite formation. Results. Co-administration of sodium nitrite and ascorbic acid induced a 4- to 5-fold increase in the esophageal damage compared with baseline reflux esophagitis, while the damage was unchanged when either of the reagents alone was given. Nitrotyrosine was strongly stained in the tissue from the co-administration. Treatment of superoxide scavengers efficiently prevented the exacerbation of esophageal damage by exogenous nitric oxide exposure, suggesting an essential role of superoxide in esophageal damage. Conclusions. Exogenous luminal nitric oxide greatly exacerbated the tissue damage of reflux esophagitis. Diffusion of the luminal nitric oxide into the adjacent superoxide-enriched inflamed tissue of the esophagus could lead to the production of the highly toxic agent peroxynitrite, thus causing exacerbation of the esophageal damage.

A pilot study of scheduled endoscopic balloon dilation with oral agent tranilast to improve the efficacy of stricture dilation after endoscopic submucosal dissection of the esophagus.

Backgrounds and Aim: As circumferential or near-circumferential endoscopic submucosal dissection (ESD) for superficial esophageal neoplasms might evoke refractory strictures, multiple sessions of endoscopic balloon dilation (EBD) are required. We aimed to assess the effectiveness and safety of oral agent tranilast with EBD for improving the efficacy of stricture dilation after esophageal ESD. Methods: In an open-label prospective study at a single institution, 31 asymptomatic consecutive patients with superficial esophageal squamous cell carcinomas were enrolled from April 2007 to October 2010. After ESD, we performed scheduled EBD (twice weekly for 4 wk) with or without administration of oral agent tranilast for 8 weeks. Thereafter, we added additional EBD on the basis of solid criteria—for example, patient’s awareness of vomiting >1/wk and inability of passage of routine endoscope through the ESD site. We compared the rates of post-ESD strictures and the numbers of additional EBD sessions for 48 weeks after ESD and the Dysphagia score between tranilast (T)-group and none (N)-group, based on patients’ subjective symptoms, at 16, 24, and 48 weeks after ESD. Results: The percentage of post-ESD strictures in T-group was significantly lower than that in N-group (P=0.04). The median numbers of additional EBD sessions and Dysphagia score at 16 and 24 weeks after ESD in T-group were significantly smaller than those in N-group (P=0.0138, 0.002, 0.005, respectively). No adverse events and no recurrence were observed. Conclusions: We demonstrated for the first time that scheduled EBD combined with oral agent tranilast might be effective and safe for improving the efficacy of stricture dilation after esophageal ESD.

Gender differences in oesophageal mucosal injury in a reflux oesophagitis model of rats

Objective There is a strong male predominance of oesophageal adenocarcinoma, which might be related to the higher prevalence of precursor lesions such as erosive reflux oesophagitis in men compared with women. This experiment investigated the gender difference in a reflux oesophagitis model of rats and explored the potential role of oestrogen in controlling oesophageal tissue damage. Design An acid-reflux oesophagitis model was surgically produced in male and female rats, and ascorbic acid in the diet and sodium nitrite in the drinking water were administered to half of either group to provoke luminal exogenous nitric oxide (NO) as an exacerbating agent. Seven days after the surgery, the oesophagus was excised, and the injury area, myeloperoxidase activity and pro-inflammatory cytokine levels were measured. Furthermore, 17β-oestradiol was administered to ovariectomised female rats or male rats, which then underwent reflux oesophagitis surgery. Results While there was no gender difference in oesophageal damage in the baseline model, oesophageal damage was more intensively observed in males than in females in the presence of exogenous NO administration. While oesophageal damage was increased in ovariectomised rats compared with sham ovariectomised, exacerbated oesophageal damage was attenuated by the replacement of 17β-oestradiol. In addition, exacerbated oesophageal damage in male rats was suppressed by 17β-oestradiol. Conclusion This is the first study showing the prominent gender difference in the severity of oesophageal tissue damage in a gastro-oesophageal reflux disease-related animal model, highlighting the critical involvement of oestrogen in controlling gastro-oesophageal reflux disease-related oesophageal epithelial injury.

Gastric Hyposecretion in Esophageal Squamous-Cell Carcinomas

Recently, gastric fundic atrophy is reported to be an independent risk factor for esophageal squamous-cell carcinoma (ESCC). The aim of this study is to investigate the acid secretory level in ESCC in a case-control study. From April 2004 to March 2008, 100 consecutive subjects with early ESCC and 100 age- and sex-matched asymptomatic controls were prospectively enrolled. Gastrin-stimulated acid output was assessed by endoscopic gastrin test. Conditional regression analyses were used to adjust for other potential confounders. Multivariate analyses revealed a strong association between profound hypochlorhydria and ESCC with odds ratio (95% confidence interval): 6.0 (1.9–18.4). The association remained significant after adjusting for the effect of gastric atrophy as a covariate. The association became stronger as the ESCC developed more distal site of the esophagus. This study indicates that profound hypochlorhydria is a strong independent risk factor for ESCC even after adjusting for the influence of gastric atrophy.

Comparison of the Long-Term Outcomes of Endoscopic Resection for Superficial Squamous Cell Carcinoma and Adenocarcinoma of the Esophagus in Japan

OBJECTIVES:Few studies have simultaneously evaluated the long-term outcomes of endoscopic resection (ER) for squamous cell carcinoma (SCC) and adenocarcinoma (AC) of the esophagus in Japan. The objective of this study was to evaluate the long-term outcomes of ER for superficial esophageal cancer in consecutive patients.METHODS:This was a retrospective study from a single institution. From April 2001 to June 2012, 204 patients with SCC and 26 patients with AC were included from a total of 355 consecutive patients who were treated by esophageal ER at the Tohoku University Hospital. Patients with submucosal invasion deeper than 200 μm and lymphovascular involvement were excluded. The intervention followed was endoscopic therapy.RESULTS:Overall survival, disease-free survival, and recurrence rates were evaluated as long-term outcomes. In the SCC group, during the median observation time of 36.5 months (range, 6–120 months), 22 (10.8%) patients experienced metachronous recurrence, 4 (2.0%) patients experienced local recurrence, and 27 (13.2%) patients died from causes unrelated to SCC. In the AC group, during the median observation time of 45.5 months (range, 6–131 months), one patient (3.8%) experienced metachronous recurrence and two (7.7%) died from causes unrelated to AC. The cumulative 5-year overall survival rates were not significantly different between SCC (75.9%) and AC (88.9%) (P=0.120). The cumulative 5-year disease-free survival rates of SCC (57.1%) were significantly lower than those of AC (85.2%; P=0.017). The cumulative 5-year recurrence rates of SCC (32.0%) were significantly higher than those of AC (4.2%; P=0.023).CONCLUSIONS:The rate of recurrence after ER was higher in patients with SCC than that in patients with AC. These findings suggest that, by detecting AC of the esophagus earlier, a satisfactory prognosis without recurrence can be expected after ER in Japan, and more rigorous endoscopic follow-up is necessary after ER in patients with SCC than in those with AC.

484 Paradoxical Effects of H. pylori Infection on Low-Dose Aspirin Induced Gastropathy Depending on the Gastric Acid Secretion Level

Background The association of Helicobacter pylori infection with aspirin-induced gastropathy is controversial. H. pylori infection exerts diverse effects on gastric acid secretion. In this study, the interaction between H. pylori infection and aspirin was investigated with reference to the individual gastric acid secretion level in H. pylori-positive subjects.

Exogenous luminal nitric oxide exposure accelerates columnar transformation of rat esophagus

Exposure of the esophageal mucosa to refluxed gastroduodenal contents is recognized to be an important risk factor for Barretts esophagus (BE). At the human gastroesophageal junction, nitric oxide is generated luminally through the enterosalivary recirculation of dietary nitrate, and in cases with gastroesophageal reflux, the site of luminal nitric oxide generation could shift to the distal esophagus. The aim of this study is to investigate whether exogenous luminal nitric oxide could promote the development of BE in rats. Sodium nitrite plus ascorbic acid were administered to a rat surgical model of BE, in which the gastroduodenal contents were refluxed into the esophagus to generate exogenous luminal nitric oxide in the esophagus by the acid‐catalyzed chemical reaction between the 2 reagents. The emergence of BE was evaluated histologically in the early phase (several weeks) after the surgery with or without exogenous nitric oxide administration. To elucidate the histogenesis of BE, CDX2, MUC2 and MUC6 expressions were investigated immunohistochemically. Coadministration of sodium nitrite plus ascorbic acid significantly accelerated the timing of emergence and increased the area of BE compared with controls. Administration of either reagent alone did not show any promotive effects on BE formation. Immunohistochemically, the columnar epithelium thus induced was similar to the specialized intestinal metaplasia in human BE. The results of this animal model study suggest that exogenous luminal nitric oxide could be involved in the pathogenesis of the columnar transformation of the esophagus. Further studies in human are warranted.

Disruption of gastric barrier function by luminal nitrosative stress: a potential chemical insult to the human gastro-oesophageal junction

Objective: The human gastro-oesophageal junction is exposed to abundant amounts of luminal reactive nitrogen oxide species (RNOS) derived from the enterosalivary re-circulation of dietary nitrate. The aim of this study is to investigate the direct effects of luminal RNOS on the adjacent gastric barrier function using an ex vivo chamber model. Methods: A chamber model in which the rat gastric mucosal membrane was mounted between the two halves of a chamber was designed to simulate the microenvironment of the lumen and the adjacent mucosa of the gastro-oesophageal junction. On the mucosal side of the chamber, RNOS were generated by the acidification of physiological concentrations of sodium nitrite. The epithelial barrier function was evaluated by electrophysiological transmembrane resistance, and membrane permeability with [3H]mannitol flux. The expression of occludin was evaluated by immunohistochemistry and immunoblotting. Dinitrosyl dithiolato iron complex (DNIC) was also measured by means of electron paramagnetic resonance spectroscopy to confirm the diffusion of RNOS from the mucosal lumen into the mounted mucosa. Results: The administration of acidified nitrite to the mucosal lumen caused both a decrease in transmembrane resistance and an increase in epithelial permeability, suggesting a disturbance of the gastric barrier function. These changes were accompanied by a derangement of the expression of occludin. The diffusion of luminal RNOS into the mounted membrane was confirmed by showing the generation of DNIC within the tissue. Conclusions: Simulating the microenvironment of the human gastro-oesophageal junction, this study demonstrated that RNOS generated luminally at the human gastro-oesophageal junction can derange the barrier function of the adjacent tissue by disrupting the tight junction.

Cdx2 Expression and Intestinal Metaplasia Induced by H. pylori Infection of Gastric Cells Is Regulated by NOD1-Mediated Innate Immune Responses

Chronic infection with the bacterial Helicobacter pylori is a major cause of gastric and duodenal ulcer disease, gastric mucosal atrophy, and cancer. H. pylori-induced expression of the intestinal epithelial-specific transcription factor caudal-related homeobox 2 (Cdx2) contributes to intestinal metaplasia, a precursor event to gastric cancer. Given a role for the bacterial pattern recognition molecule nucleotide-binding oligomerization domain 1 (NOD1) in the innate immune response to bacterial infection, we investigated mechanisms used by NOD1 to regulate H. pylori infection and its propensity towards the development of intestinal metaplasia. We found that Cdx2 was induced by H. pylori infection in both normal and neoplastic gastric epithelial cells in a manner that was inversely related to NOD1 signaling. Mechanistic investigations revealed that Cdx2 induction relied upon activation of NF-κB but was suppressed by NOD1-mediated activation of TRAF3, a negative regulator of NF-κB. In vivo, prolonged infection of NOD1-deficient mice with H. pylori led to increased Cdx2 expression and intestinal metaplasia. Furthermore, gastric epithelial cells from these mice exhibited increased nuclear expression of the NF-κB p65 subunit and decreased expression of TRAF3. Overall, our findings illuminated a role for NOD1 signaling in attenuating H. pylori-induced Cdx2 expression in gastric epithelial cells, suggesting a rationale to augment NOD1 signaling in H. pylori-infected patients to limit their risks of accumulating precancerous gastric lesions.

Reactive nitrogen oxide species induce dilatation of the intercellular space of rat esophagus

Abstract Objective. Dilatation of the intercellular space (DIS) of the esophageal epithelium is recognized as one of the earliest histological changes in gastroesophageal reflux disease patients. At the human gastroesophageal junction, reactive nitrogen oxide species (RNOS) are generated luminally through the entero-salivary re-circulation of dietary nitrate. In cases with gastroesophageal reflux, the site of luminal RNOS generation may shift to the distal esophagus. The aim of this study was to investigate whether luminal RNOS exposure could be involved in the pathogenesis of DIS. Material and methods. Rat esophageal mucosa was studied with an Ussing chamber model. On the luminal side of the chamber, RNOS were generated by the acidification of physiologic concentrations of sodium nitrite (1.0 or 5.0 mM). Esophageal barrier function was assessed by means of electrophysiological transmembrane resistance and membrane permeability by means of 3H-mannitol flux. The dimensions of the intercellular spaces were assessed by using transmission electron microscopy. Results. Administration of acid plus sodium nitrite induced DIS of the esophageal epithelium, and this ultrastructural morphological change was accompanied by a concomitant decrease in the transmembrane resistance and an increase in the epithelial permeability. The DIS induced by luminal RNOS was also confirmed in an in vivo exposure model. Conclusions. The present animal study indicates that the RNOS generated by the acidification of salivary nitrite in the presence of refluxed gastric acid in the esophagus could be a luminal factor that is responsible for the induction of DIS. Further studies are warranted to investigate the clinical relevance of the present findings to the human situation.