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T1900 A Novel 3D Ex Vivo Model of Native Human Barrett's Oesophagus
Additional application of bile acids results in Barrett like metaplasia and constitutes a promising model for studying Barrett's Esophagus and its progression to adenocarcinoma.
endoderm to squamous or columnar epithelium. The functional relevance of these differentially expressed genes needs further investigation. The AIM of our study was to examine the functional relevance of transcriptional regulators, SOX2 and CDX2, in context of cytokeratin expression changes that are reminiscent to squamous versus columnar differentiation. METHOD: Primary squamous and Barrett's epithelial cells as well as HaCaT, a human keratinocyte cell line, were used for the experiments. Treatments included exposure to pulses of acid for 15 minutes at pH 4.5, four times daily, with bile salts for up to 6 days. Western blot, immunofluorescence and quantitative PCR were used to quantify the expression of CK7, CK10/13, CDX2 and SOX2. We used SOX2 siRNA and CDX2 siRNA to examine the effect of loss of function of these genes on the cytokeratin expression in presence or absence of acid+bile treatment. RESULTS: We found that exposure of primary squamous cells as well as HaCaT cells to acid+bile resulted in decreased SOX2 and increased CDX2 expression. As shown in the figure below, we also found that the treatment with acid+bile decreased the markers of squamous differentiation (CK10/13) and increased the columnar differentiation marker (CK7). SOX2 knockdown by siRNA in HaCaT cells resulted in decreased squamous and increased columnar differentiation markers. We next treated HaCaT cells with acid+bile to increase CDX2 expression and then treated the cells with control or CDX2 siRNA. We noted that in the presence of CDX2 siRNA, acid+bile failed to decrease the markers of squamous differentiation as well as failed to increase the columnar differentiation marker. CONCLUSION: We found that SOX2 and CDX2, transcriptional regulators that were predicted by endodermal differentiation model, have functional relevance as shown by the alteration of cytokeratin expression pattern. The mechanisms and the interplay between SOX2 and CDX2 are being investigated. Other markers predicted based on this model are also being examined.
AGA Abstracts
T1896 Stimulatory Effect of Luminal Nitric Oxide On the Development of Barrett's Esophagus in Rat Hiroyuki Endo, Kiyotaka Asanuma, Katsunori Iijima, Shuichi Ohara, Tooru Shimosegawa Aim: Many studies reported that gastric acid and bile acid could affect the development of Barrett's esophagus (BE), but the pathogenesis of BE has not been clarified. It has been demonstrated that cytotoxic concentration of nitric oxide (NO) is generated luminally at gastroesophageal junction through the entero-salivary re-circulation of dietary nitrate in human. Furthermore, the site of luminal NO generation shifts to the lower esophagus when gastric acid refluxes into the esophagus. In the present study, using a rat model, we investigated whether NO generated luminally could affect the development of BE. Method: We made a rat model of BE by performing an esophagojejunostomy and gastrojejunostomy as previously described (Tao Zhang, et.al. DDS 2007). In this model, it was proved that both gastric and duodenal contents refluxed into the esophagus with the esophagus maintained at acidic pH. After the surgery, rats were divided into two groups. One group was administrated 0.05% of sodium-nitrite (NaNO2) in tap water and 1.0% of ascorbic acid in powdered diet. (Ascorbic acid converts NaNO2 to NO in the acidic condition.) The other group was administrated a tap water and conventional diet as controls. Four and eight weeks later, rats were sacrificed and the esophagus was taken. Severity of inflammation was assessed macroscopically and incidence of BE was assessed microscopically in each group, as well. Furthermore, CDX2, MUC2, MUC5AC and MUC6 expression were investigated by immunohistochemistry in order to elucidate histogenity of Barrett's esophagus. Results: The severity of inflammation tended to be increased in NaNO2 group compared with controls. At the 4 weeks, BE was more frequently emerged significantly in NaNO2-administered group compared with controls with the incidence of 40% and 5%, respectively (P<0.05). Subsequently, at 8 weeks, BE was observed in substantial parts of both groups (77.8% in NaNO2 group v.s. 62.5% in controls). These findings suggest that administration of NaNO2 could accelerate the development of BE in the rat model. CDX2, MUC2, MUC6 immunostaining were positive and MUC5AC immunostaining was negative in the emerged columnar epithelium in the rat model, being consistent with immunohistochemical property of BE in human. Conclusion: In this study, we found that administration of nitrite accelerates the development of BE in the rat model, suggesting that NO generated luminally in the esophagus may be involved in columnar transformation of squamous epithelium of the esophagus.
T1899 Insulin Resistance As a Risk Factor for Barrett's Esophagus Katarina B. Greer, Lacie Brenner, Kayode Olowe, Beth Bednarchik, Anokh Kondru, Gary W. Falk, Dawn Dawson, William M. Grady, Joseph Willis, Gregory S. Cooper, Li Li, Amitabh Chak Background and aims: Obesity has been associated with esophageal adenocarcinoma (EAC) and its precursor Barrett's esophagus (BE), independent of gastroesophageal reflux (GERD). Hyperinsulinemia related to obesity leads to increased cellular proliferation and decreased apoptosis, which is postulated to be one mechanism for carcinogenesis. The aim of this case control study was to investigate obesity, central adiposity and hyperinsulinemia defined by insulin resistance (HOMA-IR) as a risk factor for BE. We also explored the role of total adiponectin and leptin as risk factors for Barrett's esophagus. Methods: BE patients (n=97) were recruited from consecutive patients presenting to a tertiary care institution and compared with GERD controls (n=112). Collected data included baseline demographic characteristics, anthropometric measures, fasting glucose, insulin, adiponectin and leptin concentrations. Patients were categorized as overweight or obese based on WHO criteria of calculated body mass index (BMI). Central adiposity was defined as waist hip ratio (WHR) > 0.85. Insulin resistance was estimated through homeostatic model assessment (HOMA). Proportions of obese subjects among cases and controls were compared by a chi-square test. Univariate and multivariate regression analysis was performed on all variables of interest. Results: Patients with BE had higher BMI than GERD controls (31.1 vs. 29.4 kg/m2, p=0.04). After adjustment for demographic factors, BMI emerged as a consistent risk factor for BE. Risk of BE was increased over 2 fold in obese subjects compared to normal weight controls (OR 2.76, 95% CI 1.12, 6.80). Central adiposity did not increase risk of BE in our study population (OR 1.46, 95% CI 0.26, 7.76). Patients with BE had higher baseline insulin resistance as assessed by HOMA (2.42 vs. 1.87, p=0.04). In a multivariate logistic regression analysis, insulin resistance was independently associated with the presence of BE after adjusting for age, sex, and gender (adjusted OR 1.28, 95% CI 1.05, 1.57). There was no relationship between serum adiponectin levels and BE cases status (OR 0.68 95% CI 0.28, 1.65). High levels of serum leptin did not increase BE risk in our study sample (1.68, 95% CI 0.61, 4.34). Conclusions: Obesity and insulin resistance are associated with increased risk of BE. The role of the proliferative insulin pathway in development of esophageal tissue metaplasia needs to be explored at the tissue level.
T1897 Conversion of Goblet to Non-Goblet Columnar Metaplasia of the Esophagus. a Clinical/Pathologic and Molecular Study of 10 Cases Tanya A. Rege, Carissa A. Sanchez, Xiaohong Li, David Cowan, Brian J. Reid, Patricia L. Blount, Robert D. Odze Background: In patients with Barrett's esophagus (BE), it is presumed that only esophageal columnar epithelium with goblet cells is at risk for neoplastic progression. However, we have noted, anecdotally, that a small proportion of patients with established BE convert to non-goblet columnar epithelium over the course of long term endoscopic surveillance. This phenomenon has never been investigated, and the aim of this study was to evaluate the clinical, pathologic and flow cytometric abnormalities of BE patients who have converted from goblet to non-goblet columnar epithelium, and to compare the data with a large group of “non-converters.” The results have implications with regard to the ACG definition of BE, which require goblet cells to be identified in order to establish the diagnosis. Design: During a 7 year period (2001-2008) in a prospective BE surveillance cohort in which all patients had 4 quadrant biopsies every 2 cm of esophagus, 10 BE patients out of 333 (3%) who regressed from goblet to non-goblet cell columnar epithelium of the esophagus as confirmed in their last 2 surveillance endoscopies, were identified. After exclusion of 137 patients who had cancer or EMR during surveillance, the final (N=10) cases and 186 controls were evaluated for a variety of clinical and pathologic features, including flow cytometric characteristics (increased 4N fraction, aneuploidy). Results: BE converters (mean follow-up = 59.6 months) showed a similar male/female ratio (8/2), but were significantly older (mean age: 67 years) compared to the 186 non-converters (mean follow-up = 48.7 months) (M/F:149/ 37, p=0.8, mean age: 63.7 years, p=0.07). Converters showed a significantly shorter mean length of esophageal columnar epithelium at baseline (1.6 cm, range: 1-6) and a significantly shorter length at last endoscopy (mean 1.3 cm, range 1-4) compared to non-converters (5.4 (1-17) and 5.0 (1-16), respectively, p<0.01 for both comparisons). The 10 converters showed a similar proportion of cases with tetraploidy (30%), aneuploidy (40%), or any flow abnormality (50%) compared to non-converters (23%, 16% and 31%, respectively, p> 0.05 for all comparisons). The prevalence rate of dysplasia/cancer in the two patient groups were statistically similar. Conclusions: Conversion of goblet (BE) to non-goblet columnar epithelium of the esophagus is rare (prevalence rate = 3%), but is more common in BE patients with shorter segments of columnar epithelium. Conversion of BE to non-goblet epithelium is not associated with loss of flow cytometric abnormalities, suggesting that these patients may still be at risk for cancer and should remain in endoscopic surveillance.
T1900 A Novel 3D Ex Vivo Model of Native Human Barrett's Oesophagus Natalia Scobioala-laker, Amy Reynolds, Alyson Parris, Esther M. Mitchell, Michael P. Lewis, Hugh J. Kennedy, William Stebbings, Alison Prior, Martin Phillips, Ian Beales, Mark R. Williams Barrett's oesophagus is the replacement of the normal oesophageal stratified, squamous epithelial lining of the lower oesophagus with a glandular polarised columnar epithelium. Columnar epithelial cells exhibit an intestinal metaplastic-phenotype characterised by the morphological presence of numerous glands or crypts. An understanding of the molecular and cellular basis for the development and progression of Barrett's metaplasia is required to develop effective clinical management strategies. This has been hampered by the lack of an ex vivo model of human Barrett's oesophagus. Previous work in our laboratory has developed a novel ex vivo model of the human colonic epithelium (Reynolds et al., J. Physiol 2007). AIMS: To develop a 3D ex vivo culture model of isolated Barrett's crypts that is amenable to real-time imaging. METHODS: Tissue samples of Barrett's oesophagus were obtained at endoscopy (Ethical approval ). Biopsies were incubated in a calcium-free, hepesbuffered saline solution and Barrett's crypts were liberated by vigorous shaking. Barrett's crypts were placed into a 3D culture system for up to 5 days. Viability of cultured Barrett's crypts was assessed by calcein-AM uptake and propidium iodide exclusion. The activity of a number of signalling pathways implicated in the genesis/maintenance of Barrett's crypts
T1898 SOX2 and CDx2, Transcriptional Regulators of Early Differentation, Play a Key Role in the Development of Acid and Bile-Associated Columnar Metaplasia Yuvnish Bhardwaj, Cathrine J. DeMars, Shalini Achra, Marlys Anderson, Ganapathy A. Prasad, Kenneth K. Wang, Raul A. Urrutia, Navtej Buttar BACKGROUND: The mechanism of development of Barrett's metaplasia remains to be defined. Using an endodermal differentiation model, we have previously identified a set of genes which show changes in temporal expression during embryonic differentiation of
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was investigated by immunocytochemical labelling of dephospho-beta catenin, NFkB, phospho-ERK. Proliferation status of cultured Barrett's crypts was assessed by BrDU/Ki67 labelling and digital video time-lapse microscopy. RESULTS: The isolation procedure liberated Barrett's crypts that resembled single and branched glandular structures. Both types of structure loaded with calcein-AM at any stage during a number of days in culture. Cultured Barrett's crypts excluded prodium iodide when added to the culture media. Immunolabelling of nuclear beta catenin, NFkB and phospho-ERK labelling was present throughout Barrett's crypt structures and was particular prominent at the crypt-base. Similarly, nuclear BrdU incorporation and Ki67 labelling predominated in this compartment. Real-time mitotic events were monitored by digital time-lapse video microscopy. CONCLUSIONS: We have developed a novel 3D ex vivo culture model of native human Barrett's oesophagus that is amenable to real-time imaging. Future experiments will interrogate the role of Wnt/beta catenin, MAPK and NFkB signalling pathways in the maintenance and propagation of the Barrett's phenotype.
T1903
Introduction: Barrett's esophagus (BE) is the replacement of the normal squamous esophageal epithelium with an intestinalized columnar epithelium. It is an important risk factor for esophageal adenocarcinoma in humans. The sine qua non for the diagnosis of Barrett's esophagus is the presence of intestinal mucin-producing goblet cells. Atonal homologue 1 (encoded by Atoh1, also known as Hath1 and Math1) is a basic helix-loop-helix transcription factor important for cell fate determination of the intestinal secretory cells, including goblet cells. However its expression in human Barrett's esophagus has not yet been described. Herein we report on our further modeling of BE In Vitro using Math1 expression in normal human esophageal keratinocytes. Materials and Methods: 5 paired biopsies (from BE tissue and adjacent normal squamous epithelium) were studied for Hath1 mRNA and protein expression using quantitative real-time RT-PCR and immunofluorescence. Human immortalized esophageal keratinocytes (EPC2-hTERT) were transduced with MSCV-Math1-GFP (Martine Roussel, UT Memphis) retrovirus to express Math1. Quantitative RT-PCR and western blot were to check Math1 and intestine-specific genes expression in the transfectants and control cells. WST-1 assay and BrdU incorporation were performed for cell proliferation analysis. Results: We observed significant induction of Hath1 mRNA in all 5 Barrett's samples tested. Hath1 mRNA levels were induced from 4- to 1000-fold over the normal esophagus controls. An antibody directed against Hath1/Math1 (provided by Dr. Jane Johnson, UT Southwestern Medical Center), confirmed the expression of Hath1 protein in Barrett's specimens but not in normal human esophagus. Math1 expression is maintained in the EPChTERTcells for many weeks, suggesting it does not have significant detrimental effects on keratinocyte fitness or viability. EPC2-hTERT cells that received the MSCV-Math1 retrovirus appeared to elongate and extrude cytoplasmic projections, suggesting Math1/Hath1 expression may possess important effects on cell shape and adhesion proteins. Math1 expression induced a modest increase in Muc2 (3-fold) and other intestine-specific genes mRNA levels. Conclusion: Hath1 is ectopically expressed in human Barrett's esophagus and likely contributes to the emergence of intestinal mucin-secreting goblet cells in that tissue. Ectopic Math1 expression in EPC2-hTERT keratinocytes does not inhibit proliferation or induce intestinal mucin production but does induce significant changes in cell morphology, consistent with its role promoting cell differentiation in the intestine and central nervous systems.
T1901 Retinoic Acid Causes Induction of CDx2 in a Long Term Model of Squamous Oesophagus Benjamin J. Colleypriest, John Farrant, Jonathan M. Slack, David Tosh Introduction Barrett's metaplasia is the precursor lesion for oesophageal adenocarcinoma and both are exhibiting rapidly increasing incidences. Whilst the molecular mechanisms in the progression from Barrett's metaplasia to cancer are being unravelled, the initial steps to intestinal metaplasia are less clear. There is mounting evidence that the transcription factor caudal type homeobox transcription factors 2 (cdx2) is involved in the early steps in Barrett's. Two transgenic mouse studies demonstrate that ectopic gastric expression of cdx2 results in intestinalisation. Both acid and bile, the main protagonists of Barrett's, have been shown to induce cdx2 expression in various experimental models. Recently the morphogen retinoic acid has been shown to provoke a columnar phenotype in squamous oesophageal biopsies and expression of an intestinal mucin in oesophageal cells. We demonstrate that retinoic acid invokes the induction of cdx2 in a long term model of squamous oesophagus. Methods Explants of mouse oesophageal epithelium are cultured in media supplemented with 10uM retinoic acid. The explant culture model recapitulates the repertoire of cellular phenotype found within the oesophagus. After 10 days cellular phenotype is observed using immunohistochemistry and PCR. Results Treatment of oesophageal explant culture with retinoic acid provokes the expression of cdx2, as demonstrated by PCR, which is not present in the control culture treated with vehicle alone. The outgrowth of cells surrounding the explants treated with retinoic acid demonstrate a linear shape contrasting with the circular control cultures. The number of cells expressing the squamous transcription factor p63 is decreased by retinoic acid treatment. Conclusion Retinoic acid has suggested as a candidate molecule for induction of BO and is present in the duodenal lumen. Retinoic acid suppresses epidermal keratinocyte differentiation and induces a columnar phenotype in human squamous oesophageal biopsies. Furthermore treatment of oesophageal squamous cell primary culture with RA results in expression of the intestinal mucin, muc 2. Whilst RA is accepted as a morphogen important in the expression of Hox genes during development and in pathology, this is the first time RA has been shown to induce cdx2 expression in oesophageal cells. The same pathway may be responsible for the induction of cdx2 seen with bile given that lithocholic acid, a component of refluxate, competes at the retinoid x receptor binding site.
T1904 Smoking and the Risk of Barrett's Esophagus in Women Brian C. Jacobson, Edward L. Giovannucci, Charles S. Fuchs Background: Cigarette use is associated with esophageal adenocarcinoma, and cross-sectional studies suggest an association between smoking and Barrett's esophagus (BE). We sought to examine the influence of smoking on the risk for BE using data collected prospectively as part of the ongoing Nurses' Health Study. Methods: We assessed the association between smoking and pathologically-confirmed BE (n=278) among 17,011 women who underwent upper gastrointestinal endoscopy for any reason between 1980 and 2004. Self-reported data on smoking and potential confounding variables were collected from biennial questionnaires. Unconditional logistic regression was used to determine odds ratios (OR) and 95% confidence intervals (CI) for the risk of BE using data from the questionnaire cycle just prior to the index endoscopy. Results: Among eligible women, 46% were former smokers and 10% were current smokers prior to their index endoscopy. Compared to women who never smoked, former smokers who used 1-24 cigarettes/day had a multivariate OR for BE of 1.26 (95% CI 0.96-1.66), former smokers who used >25 cigarettes/day had a multivariate OR of 1.80 (95% CI 1.19-2.72), current smokers who used 1-24 cigarettes/day had a multivariate OR of 0.98 (95% CI 0.58-1.67), and current smokers who used >25 cigarettes/day had a multivariate OR of 1.19 (95% CI 0.43-3.30). Results were similar among a cohort of women reporting regular heartburn/acid-reflux 1 or more times a week who underwent endoscopy, and among the entire Nurses' Health Study cohort (n=112,759) regardless of history of endoscopy. The risk for BE increased significantly with increasing pack-years smoked among former (P = 0.02), but not current smokers (p=0.68), especially when considering exposure >25 years prior to index endoscopy. We also saw an increased risk for BE among women who had accumulated >10 pack-years of smoking by age 30 (multivariate OR 1.71; 95% CI 1.20-2.45). There was no significant association between the number of years after smoking cessation and BE. The association between smoking and BE was not accounted for by changes in weight after smoking cessation. However, in a stratified analysis, obese (body mass index > 30 kg/m2) former smokers had twice the risk for BE than women with body mass index <20 kg/m2. Conclusions: Heavy, remote smoking is associated with an increased risk for Barrett's esophagus. This suggests a long latency period between exposure and development of the disease, even after discontinuation of smoking. Obesity may modify the association between smoking and BE.
T1902 Effect of Selective and Non-Selective Cyclooxygenase (COX)-1 and COX-2 Inhibitors and Nitric Oxide Releasing Aspirin (No-ASA) On the Chronic Esophagitis in the Rat Model of Barrett's Esophagus Jolanta Majka, Tomasz Brzozowski, Alexandra Szlachcic, Rafal Pabianczyk, Katarzyna Urbanczyk, Romana Tomaszewska, Stanislaw Konturek, Wieslaw W. Pawlik Mixed reflux of the gastroduodenal contents induces the esophageal mucosal damage and inflammation progressing to chronic esophagitis and premalignant Barrett's esophagus. Histamine H2-receptor antagonists and proton pump inhibitors (PPI) have been shown to be effective against esophageal reflux but efficacy of nonsteroidal anti-inflammatory drugs (NSAIDs), selective COX-2 inhibitors and new class NSAID nitric oxide releasing aspirin (NO-ASA) to inhibit esophageal lesions in rat model of Barrett's esophagus has not been fully clarified. Eighty rats with esophagogastroduodenal anastomosis (EGDA) resulting in chronic esophagitis were randomly divided 4 groups treated i.g. daily either with: 1) vehicle 2) aspirin (100 mg/kg-d) or NO-ASA (65 mg/kg-d), 3) celecoxib (10 mg/kg-d), a selective COX-2 inhibitor and 4) PPI pantoprazole (10 mg/kg-d) or H2-receptor antagonist, ranitidine (30 mg/kg-d). At 4 months the esophageal damage was evaluated by macroscopic and histological index score, the esophageal blood flow (EBF) was determined by H2-gas clearance method, and plasma IL-1β and TNF-α levels (ELISA) and mucosal expression of COX-2 and iNOS mRNA was evaluated by RT-PCR. Chronic esophagitis was developed in all EGDA animals followed by a decrease in the EBF and the rise in the plasma TNF-α and IL-1β levels. Histology revealed extensive esophageal ulcerations with development of columnar epithelium, formation of mucus glands in squamous epithelium, intestinal metaplasia distant to anastomosis consisting of goblet cells, infiltration of inflammatory cells including plasma cells and lymphocytes. COX-2 and iNOS mRNAs were absent in the esophageal mucosa of sham-control animals but strongly upregulated in metaplastic Barrett's epithelium. Ranitidine and ASA failed to influence the development of Barrett's esophagus but celecoxib and NOASA significantly reduced the macroscopic and histological score index of these lesions and reversed the fall of the EBF, the rise in plasma TNF-α and IL-β levels and decreased the mRNA for COX-2- and iNOS. Pantoprazole, which completely suppressed gastric acidity, significantly attenuated reflux esophagitis and expression of COX-2- and iNOS mRNA. We conclude that 1) development of chronic reflux esophagitis in rodent model of Barrett's esophagus is associated with severe morphology changes, an impairment of EBF due to overexpression of COX-2 and iNOS, and excessive expression and release of TNF-α and IL-1β, and 2) acid suppressive drugs (pantoprazole), the selective COX-2 inhibitors (celecoxib) and NO releasing ASA could be useful as the therapeutic option in the treatment of Barrett's esophagus.
T1905 Gene Groups Modulated By Bile Acid Signaling Show Altered Expression in Tissue from Barrett's Esophagus Murat Kirca, Shane P. Duggan, Fiona Behan, Dermot P. Kelleher Introduction: Barrett's esophagus (BE) is a consequence of chronic gastro-esophageal reflux disease. Bile acids are an important toxic component of the refluxate. Recent work from our laboratory using gene microarray technology has demonstrated that exposure of esophageal cells to bile acids results in regulation of over 30% of the genes previously demonstrated to be altered in genomic studies of BE and esophageal adenocarcinoma (EAC). The current study aimed to validate and examine the expression of bile acid regulated genes, as suggested by these genomic studies, in esophageal tissue derived from a cohort of BE patients. Methods: Genes significantly altered in BE and EAC tissue and additionally modulated by bile acids exposure in esophageal cells were identified by an integrative genomic approach.
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Atoh1/Math1 Is Ectopically Expressed in Human Barrett's Epithelium and Its Expression in Immortalized Human Esophageal Keratinocytes Alters Cellular Morphology Jianping Kong, Shinsuke Funakoshi, John P. Lynch
endoderm to squamous or columnar epithelium. The functional relevance of these differentially expressed genes needs further investigation. The AIM of our study was to examine the functional relevance of transcriptional regulators, SOX2 and CDX2, in context of cytokeratin expression changes that are reminiscent to squamous versus columnar differentiation. METHOD: Primary squamous and Barrett's epithelial cells as well as HaCaT, a human keratinocyte cell line, were used for the experiments. Treatments included exposure to pulses of acid for 15 minutes at pH 4.5, four times daily, with bile salts for up to 6 days. Western blot, immunofluorescence and quantitative PCR were used to quantify the expression of CK7, CK10/13, CDX2 and SOX2. We used SOX2 siRNA and CDX2 siRNA to examine the effect of loss of function of these genes on the cytokeratin expression in presence or absence of acid+bile treatment. RESULTS: We found that exposure of primary squamous cells as well as HaCaT cells to acid+bile resulted in decreased SOX2 and increased CDX2 expression. As shown in the figure below, we also found that the treatment with acid+bile decreased the markers of squamous differentiation (CK10/13) and increased the columnar differentiation marker (CK7). SOX2 knockdown by siRNA in HaCaT cells resulted in decreased squamous and increased columnar differentiation markers. We next treated HaCaT cells with acid+bile to increase CDX2 expression and then treated the cells with control or CDX2 siRNA. We noted that in the presence of CDX2 siRNA, acid+bile failed to decrease the markers of squamous differentiation as well as failed to increase the columnar differentiation marker. CONCLUSION: We found that SOX2 and CDX2, transcriptional regulators that were predicted by endodermal differentiation model, have functional relevance as shown by the alteration of cytokeratin expression pattern. The mechanisms and the interplay between SOX2 and CDX2 are being investigated. Other markers predicted based on this model are also being examined.
AGA Abstracts
T1896 Stimulatory Effect of Luminal Nitric Oxide On the Development of Barrett's Esophagus in Rat Hiroyuki Endo, Kiyotaka Asanuma, Katsunori Iijima, Shuichi Ohara, Tooru Shimosegawa Aim: Many studies reported that gastric acid and bile acid could affect the development of Barrett's esophagus (BE), but the pathogenesis of BE has not been clarified. It has been demonstrated that cytotoxic concentration of nitric oxide (NO) is generated luminally at gastroesophageal junction through the entero-salivary re-circulation of dietary nitrate in human. Furthermore, the site of luminal NO generation shifts to the lower esophagus when gastric acid refluxes into the esophagus. In the present study, using a rat model, we investigated whether NO generated luminally could affect the development of BE. Method: We made a rat model of BE by performing an esophagojejunostomy and gastrojejunostomy as previously described (Tao Zhang, et.al. DDS 2007). In this model, it was proved that both gastric and duodenal contents refluxed into the esophagus with the esophagus maintained at acidic pH. After the surgery, rats were divided into two groups. One group was administrated 0.05% of sodium-nitrite (NaNO2) in tap water and 1.0% of ascorbic acid in powdered diet. (Ascorbic acid converts NaNO2 to NO in the acidic condition.) The other group was administrated a tap water and conventional diet as controls. Four and eight weeks later, rats were sacrificed and the esophagus was taken. Severity of inflammation was assessed macroscopically and incidence of BE was assessed microscopically in each group, as well. Furthermore, CDX2, MUC2, MUC5AC and MUC6 expression were investigated by immunohistochemistry in order to elucidate histogenity of Barrett's esophagus. Results: The severity of inflammation tended to be increased in NaNO2 group compared with controls. At the 4 weeks, BE was more frequently emerged significantly in NaNO2-administered group compared with controls with the incidence of 40% and 5%, respectively (P<0.05). Subsequently, at 8 weeks, BE was observed in substantial parts of both groups (77.8% in NaNO2 group v.s. 62.5% in controls). These findings suggest that administration of NaNO2 could accelerate the development of BE in the rat model. CDX2, MUC2, MUC6 immunostaining were positive and MUC5AC immunostaining was negative in the emerged columnar epithelium in the rat model, being consistent with immunohistochemical property of BE in human. Conclusion: In this study, we found that administration of nitrite accelerates the development of BE in the rat model, suggesting that NO generated luminally in the esophagus may be involved in columnar transformation of squamous epithelium of the esophagus.
T1899 Insulin Resistance As a Risk Factor for Barrett's Esophagus Katarina B. Greer, Lacie Brenner, Kayode Olowe, Beth Bednarchik, Anokh Kondru, Gary W. Falk, Dawn Dawson, William M. Grady, Joseph Willis, Gregory S. Cooper, Li Li, Amitabh Chak Background and aims: Obesity has been associated with esophageal adenocarcinoma (EAC) and its precursor Barrett's esophagus (BE), independent of gastroesophageal reflux (GERD). Hyperinsulinemia related to obesity leads to increased cellular proliferation and decreased apoptosis, which is postulated to be one mechanism for carcinogenesis. The aim of this case control study was to investigate obesity, central adiposity and hyperinsulinemia defined by insulin resistance (HOMA-IR) as a risk factor for BE. We also explored the role of total adiponectin and leptin as risk factors for Barrett's esophagus. Methods: BE patients (n=97) were recruited from consecutive patients presenting to a tertiary care institution and compared with GERD controls (n=112). Collected data included baseline demographic characteristics, anthropometric measures, fasting glucose, insulin, adiponectin and leptin concentrations. Patients were categorized as overweight or obese based on WHO criteria of calculated body mass index (BMI). Central adiposity was defined as waist hip ratio (WHR) > 0.85. Insulin resistance was estimated through homeostatic model assessment (HOMA). Proportions of obese subjects among cases and controls were compared by a chi-square test. Univariate and multivariate regression analysis was performed on all variables of interest. Results: Patients with BE had higher BMI than GERD controls (31.1 vs. 29.4 kg/m2, p=0.04). After adjustment for demographic factors, BMI emerged as a consistent risk factor for BE. Risk of BE was increased over 2 fold in obese subjects compared to normal weight controls (OR 2.76, 95% CI 1.12, 6.80). Central adiposity did not increase risk of BE in our study population (OR 1.46, 95% CI 0.26, 7.76). Patients with BE had higher baseline insulin resistance as assessed by HOMA (2.42 vs. 1.87, p=0.04). In a multivariate logistic regression analysis, insulin resistance was independently associated with the presence of BE after adjusting for age, sex, and gender (adjusted OR 1.28, 95% CI 1.05, 1.57). There was no relationship between serum adiponectin levels and BE cases status (OR 0.68 95% CI 0.28, 1.65). High levels of serum leptin did not increase BE risk in our study sample (1.68, 95% CI 0.61, 4.34). Conclusions: Obesity and insulin resistance are associated with increased risk of BE. The role of the proliferative insulin pathway in development of esophageal tissue metaplasia needs to be explored at the tissue level.
T1897 Conversion of Goblet to Non-Goblet Columnar Metaplasia of the Esophagus. a Clinical/Pathologic and Molecular Study of 10 Cases Tanya A. Rege, Carissa A. Sanchez, Xiaohong Li, David Cowan, Brian J. Reid, Patricia L. Blount, Robert D. Odze Background: In patients with Barrett's esophagus (BE), it is presumed that only esophageal columnar epithelium with goblet cells is at risk for neoplastic progression. However, we have noted, anecdotally, that a small proportion of patients with established BE convert to non-goblet columnar epithelium over the course of long term endoscopic surveillance. This phenomenon has never been investigated, and the aim of this study was to evaluate the clinical, pathologic and flow cytometric abnormalities of BE patients who have converted from goblet to non-goblet columnar epithelium, and to compare the data with a large group of “non-converters.” The results have implications with regard to the ACG definition of BE, which require goblet cells to be identified in order to establish the diagnosis. Design: During a 7 year period (2001-2008) in a prospective BE surveillance cohort in which all patients had 4 quadrant biopsies every 2 cm of esophagus, 10 BE patients out of 333 (3%) who regressed from goblet to non-goblet cell columnar epithelium of the esophagus as confirmed in their last 2 surveillance endoscopies, were identified. After exclusion of 137 patients who had cancer or EMR during surveillance, the final (N=10) cases and 186 controls were evaluated for a variety of clinical and pathologic features, including flow cytometric characteristics (increased 4N fraction, aneuploidy). Results: BE converters (mean follow-up = 59.6 months) showed a similar male/female ratio (8/2), but were significantly older (mean age: 67 years) compared to the 186 non-converters (mean follow-up = 48.7 months) (M/F:149/ 37, p=0.8, mean age: 63.7 years, p=0.07). Converters showed a significantly shorter mean length of esophageal columnar epithelium at baseline (1.6 cm, range: 1-6) and a significantly shorter length at last endoscopy (mean 1.3 cm, range 1-4) compared to non-converters (5.4 (1-17) and 5.0 (1-16), respectively, p<0.01 for both comparisons). The 10 converters showed a similar proportion of cases with tetraploidy (30%), aneuploidy (40%), or any flow abnormality (50%) compared to non-converters (23%, 16% and 31%, respectively, p> 0.05 for all comparisons). The prevalence rate of dysplasia/cancer in the two patient groups were statistically similar. Conclusions: Conversion of goblet (BE) to non-goblet columnar epithelium of the esophagus is rare (prevalence rate = 3%), but is more common in BE patients with shorter segments of columnar epithelium. Conversion of BE to non-goblet epithelium is not associated with loss of flow cytometric abnormalities, suggesting that these patients may still be at risk for cancer and should remain in endoscopic surveillance.
T1900 A Novel 3D Ex Vivo Model of Native Human Barrett's Oesophagus Natalia Scobioala-laker, Amy Reynolds, Alyson Parris, Esther M. Mitchell, Michael P. Lewis, Hugh J. Kennedy, William Stebbings, Alison Prior, Martin Phillips, Ian Beales, Mark R. Williams Barrett's oesophagus is the replacement of the normal oesophageal stratified, squamous epithelial lining of the lower oesophagus with a glandular polarised columnar epithelium. Columnar epithelial cells exhibit an intestinal metaplastic-phenotype characterised by the morphological presence of numerous glands or crypts. An understanding of the molecular and cellular basis for the development and progression of Barrett's metaplasia is required to develop effective clinical management strategies. This has been hampered by the lack of an ex vivo model of human Barrett's oesophagus. Previous work in our laboratory has developed a novel ex vivo model of the human colonic epithelium (Reynolds et al., J. Physiol 2007). AIMS: To develop a 3D ex vivo culture model of isolated Barrett's crypts that is amenable to real-time imaging. METHODS: Tissue samples of Barrett's oesophagus were obtained at endoscopy (Ethical approval ). Biopsies were incubated in a calcium-free, hepesbuffered saline solution and Barrett's crypts were liberated by vigorous shaking. Barrett's crypts were placed into a 3D culture system for up to 5 days. Viability of cultured Barrett's crypts was assessed by calcein-AM uptake and propidium iodide exclusion. The activity of a number of signalling pathways implicated in the genesis/maintenance of Barrett's crypts
T1898 SOX2 and CDx2, Transcriptional Regulators of Early Differentation, Play a Key Role in the Development of Acid and Bile-Associated Columnar Metaplasia Yuvnish Bhardwaj, Cathrine J. DeMars, Shalini Achra, Marlys Anderson, Ganapathy A. Prasad, Kenneth K. Wang, Raul A. Urrutia, Navtej Buttar BACKGROUND: The mechanism of development of Barrett's metaplasia remains to be defined. Using an endodermal differentiation model, we have previously identified a set of genes which show changes in temporal expression during embryonic differentiation of
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was investigated by immunocytochemical labelling of dephospho-beta catenin, NFkB, phospho-ERK. Proliferation status of cultured Barrett's crypts was assessed by BrDU/Ki67 labelling and digital video time-lapse microscopy. RESULTS: The isolation procedure liberated Barrett's crypts that resembled single and branched glandular structures. Both types of structure loaded with calcein-AM at any stage during a number of days in culture. Cultured Barrett's crypts excluded prodium iodide when added to the culture media. Immunolabelling of nuclear beta catenin, NFkB and phospho-ERK labelling was present throughout Barrett's crypt structures and was particular prominent at the crypt-base. Similarly, nuclear BrdU incorporation and Ki67 labelling predominated in this compartment. Real-time mitotic events were monitored by digital time-lapse video microscopy. CONCLUSIONS: We have developed a novel 3D ex vivo culture model of native human Barrett's oesophagus that is amenable to real-time imaging. Future experiments will interrogate the role of Wnt/beta catenin, MAPK and NFkB signalling pathways in the maintenance and propagation of the Barrett's phenotype.
T1903
Introduction: Barrett's esophagus (BE) is the replacement of the normal squamous esophageal epithelium with an intestinalized columnar epithelium. It is an important risk factor for esophageal adenocarcinoma in humans. The sine qua non for the diagnosis of Barrett's esophagus is the presence of intestinal mucin-producing goblet cells. Atonal homologue 1 (encoded by Atoh1, also known as Hath1 and Math1) is a basic helix-loop-helix transcription factor important for cell fate determination of the intestinal secretory cells, including goblet cells. However its expression in human Barrett's esophagus has not yet been described. Herein we report on our further modeling of BE In Vitro using Math1 expression in normal human esophageal keratinocytes. Materials and Methods: 5 paired biopsies (from BE tissue and adjacent normal squamous epithelium) were studied for Hath1 mRNA and protein expression using quantitative real-time RT-PCR and immunofluorescence. Human immortalized esophageal keratinocytes (EPC2-hTERT) were transduced with MSCV-Math1-GFP (Martine Roussel, UT Memphis) retrovirus to express Math1. Quantitative RT-PCR and western blot were to check Math1 and intestine-specific genes expression in the transfectants and control cells. WST-1 assay and BrdU incorporation were performed for cell proliferation analysis. Results: We observed significant induction of Hath1 mRNA in all 5 Barrett's samples tested. Hath1 mRNA levels were induced from 4- to 1000-fold over the normal esophagus controls. An antibody directed against Hath1/Math1 (provided by Dr. Jane Johnson, UT Southwestern Medical Center), confirmed the expression of Hath1 protein in Barrett's specimens but not in normal human esophagus. Math1 expression is maintained in the EPChTERTcells for many weeks, suggesting it does not have significant detrimental effects on keratinocyte fitness or viability. EPC2-hTERT cells that received the MSCV-Math1 retrovirus appeared to elongate and extrude cytoplasmic projections, suggesting Math1/Hath1 expression may possess important effects on cell shape and adhesion proteins. Math1 expression induced a modest increase in Muc2 (3-fold) and other intestine-specific genes mRNA levels. Conclusion: Hath1 is ectopically expressed in human Barrett's esophagus and likely contributes to the emergence of intestinal mucin-secreting goblet cells in that tissue. Ectopic Math1 expression in EPC2-hTERT keratinocytes does not inhibit proliferation or induce intestinal mucin production but does induce significant changes in cell morphology, consistent with its role promoting cell differentiation in the intestine and central nervous systems.
T1901 Retinoic Acid Causes Induction of CDx2 in a Long Term Model of Squamous Oesophagus Benjamin J. Colleypriest, John Farrant, Jonathan M. Slack, David Tosh Introduction Barrett's metaplasia is the precursor lesion for oesophageal adenocarcinoma and both are exhibiting rapidly increasing incidences. Whilst the molecular mechanisms in the progression from Barrett's metaplasia to cancer are being unravelled, the initial steps to intestinal metaplasia are less clear. There is mounting evidence that the transcription factor caudal type homeobox transcription factors 2 (cdx2) is involved in the early steps in Barrett's. Two transgenic mouse studies demonstrate that ectopic gastric expression of cdx2 results in intestinalisation. Both acid and bile, the main protagonists of Barrett's, have been shown to induce cdx2 expression in various experimental models. Recently the morphogen retinoic acid has been shown to provoke a columnar phenotype in squamous oesophageal biopsies and expression of an intestinal mucin in oesophageal cells. We demonstrate that retinoic acid invokes the induction of cdx2 in a long term model of squamous oesophagus. Methods Explants of mouse oesophageal epithelium are cultured in media supplemented with 10uM retinoic acid. The explant culture model recapitulates the repertoire of cellular phenotype found within the oesophagus. After 10 days cellular phenotype is observed using immunohistochemistry and PCR. Results Treatment of oesophageal explant culture with retinoic acid provokes the expression of cdx2, as demonstrated by PCR, which is not present in the control culture treated with vehicle alone. The outgrowth of cells surrounding the explants treated with retinoic acid demonstrate a linear shape contrasting with the circular control cultures. The number of cells expressing the squamous transcription factor p63 is decreased by retinoic acid treatment. Conclusion Retinoic acid has suggested as a candidate molecule for induction of BO and is present in the duodenal lumen. Retinoic acid suppresses epidermal keratinocyte differentiation and induces a columnar phenotype in human squamous oesophageal biopsies. Furthermore treatment of oesophageal squamous cell primary culture with RA results in expression of the intestinal mucin, muc 2. Whilst RA is accepted as a morphogen important in the expression of Hox genes during development and in pathology, this is the first time RA has been shown to induce cdx2 expression in oesophageal cells. The same pathway may be responsible for the induction of cdx2 seen with bile given that lithocholic acid, a component of refluxate, competes at the retinoid x receptor binding site.
T1904 Smoking and the Risk of Barrett's Esophagus in Women Brian C. Jacobson, Edward L. Giovannucci, Charles S. Fuchs Background: Cigarette use is associated with esophageal adenocarcinoma, and cross-sectional studies suggest an association between smoking and Barrett's esophagus (BE). We sought to examine the influence of smoking on the risk for BE using data collected prospectively as part of the ongoing Nurses' Health Study. Methods: We assessed the association between smoking and pathologically-confirmed BE (n=278) among 17,011 women who underwent upper gastrointestinal endoscopy for any reason between 1980 and 2004. Self-reported data on smoking and potential confounding variables were collected from biennial questionnaires. Unconditional logistic regression was used to determine odds ratios (OR) and 95% confidence intervals (CI) for the risk of BE using data from the questionnaire cycle just prior to the index endoscopy. Results: Among eligible women, 46% were former smokers and 10% were current smokers prior to their index endoscopy. Compared to women who never smoked, former smokers who used 1-24 cigarettes/day had a multivariate OR for BE of 1.26 (95% CI 0.96-1.66), former smokers who used >25 cigarettes/day had a multivariate OR of 1.80 (95% CI 1.19-2.72), current smokers who used 1-24 cigarettes/day had a multivariate OR of 0.98 (95% CI 0.58-1.67), and current smokers who used >25 cigarettes/day had a multivariate OR of 1.19 (95% CI 0.43-3.30). Results were similar among a cohort of women reporting regular heartburn/acid-reflux 1 or more times a week who underwent endoscopy, and among the entire Nurses' Health Study cohort (n=112,759) regardless of history of endoscopy. The risk for BE increased significantly with increasing pack-years smoked among former (P = 0.02), but not current smokers (p=0.68), especially when considering exposure >25 years prior to index endoscopy. We also saw an increased risk for BE among women who had accumulated >10 pack-years of smoking by age 30 (multivariate OR 1.71; 95% CI 1.20-2.45). There was no significant association between the number of years after smoking cessation and BE. The association between smoking and BE was not accounted for by changes in weight after smoking cessation. However, in a stratified analysis, obese (body mass index > 30 kg/m2) former smokers had twice the risk for BE than women with body mass index <20 kg/m2. Conclusions: Heavy, remote smoking is associated with an increased risk for Barrett's esophagus. This suggests a long latency period between exposure and development of the disease, even after discontinuation of smoking. Obesity may modify the association between smoking and BE.
T1902 Effect of Selective and Non-Selective Cyclooxygenase (COX)-1 and COX-2 Inhibitors and Nitric Oxide Releasing Aspirin (No-ASA) On the Chronic Esophagitis in the Rat Model of Barrett's Esophagus Jolanta Majka, Tomasz Brzozowski, Alexandra Szlachcic, Rafal Pabianczyk, Katarzyna Urbanczyk, Romana Tomaszewska, Stanislaw Konturek, Wieslaw W. Pawlik Mixed reflux of the gastroduodenal contents induces the esophageal mucosal damage and inflammation progressing to chronic esophagitis and premalignant Barrett's esophagus. Histamine H2-receptor antagonists and proton pump inhibitors (PPI) have been shown to be effective against esophageal reflux but efficacy of nonsteroidal anti-inflammatory drugs (NSAIDs), selective COX-2 inhibitors and new class NSAID nitric oxide releasing aspirin (NO-ASA) to inhibit esophageal lesions in rat model of Barrett's esophagus has not been fully clarified. Eighty rats with esophagogastroduodenal anastomosis (EGDA) resulting in chronic esophagitis were randomly divided 4 groups treated i.g. daily either with: 1) vehicle 2) aspirin (100 mg/kg-d) or NO-ASA (65 mg/kg-d), 3) celecoxib (10 mg/kg-d), a selective COX-2 inhibitor and 4) PPI pantoprazole (10 mg/kg-d) or H2-receptor antagonist, ranitidine (30 mg/kg-d). At 4 months the esophageal damage was evaluated by macroscopic and histological index score, the esophageal blood flow (EBF) was determined by H2-gas clearance method, and plasma IL-1β and TNF-α levels (ELISA) and mucosal expression of COX-2 and iNOS mRNA was evaluated by RT-PCR. Chronic esophagitis was developed in all EGDA animals followed by a decrease in the EBF and the rise in the plasma TNF-α and IL-1β levels. Histology revealed extensive esophageal ulcerations with development of columnar epithelium, formation of mucus glands in squamous epithelium, intestinal metaplasia distant to anastomosis consisting of goblet cells, infiltration of inflammatory cells including plasma cells and lymphocytes. COX-2 and iNOS mRNAs were absent in the esophageal mucosa of sham-control animals but strongly upregulated in metaplastic Barrett's epithelium. Ranitidine and ASA failed to influence the development of Barrett's esophagus but celecoxib and NOASA significantly reduced the macroscopic and histological score index of these lesions and reversed the fall of the EBF, the rise in plasma TNF-α and IL-β levels and decreased the mRNA for COX-2- and iNOS. Pantoprazole, which completely suppressed gastric acidity, significantly attenuated reflux esophagitis and expression of COX-2- and iNOS mRNA. We conclude that 1) development of chronic reflux esophagitis in rodent model of Barrett's esophagus is associated with severe morphology changes, an impairment of EBF due to overexpression of COX-2 and iNOS, and excessive expression and release of TNF-α and IL-1β, and 2) acid suppressive drugs (pantoprazole), the selective COX-2 inhibitors (celecoxib) and NO releasing ASA could be useful as the therapeutic option in the treatment of Barrett's esophagus.
T1905 Gene Groups Modulated By Bile Acid Signaling Show Altered Expression in Tissue from Barrett's Esophagus Murat Kirca, Shane P. Duggan, Fiona Behan, Dermot P. Kelleher Introduction: Barrett's esophagus (BE) is a consequence of chronic gastro-esophageal reflux disease. Bile acids are an important toxic component of the refluxate. Recent work from our laboratory using gene microarray technology has demonstrated that exposure of esophageal cells to bile acids results in regulation of over 30% of the genes previously demonstrated to be altered in genomic studies of BE and esophageal adenocarcinoma (EAC). The current study aimed to validate and examine the expression of bile acid regulated genes, as suggested by these genomic studies, in esophageal tissue derived from a cohort of BE patients. Methods: Genes significantly altered in BE and EAC tissue and additionally modulated by bile acids exposure in esophageal cells were identified by an integrative genomic approach.
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Atoh1/Math1 Is Ectopically Expressed in Human Barrett's Epithelium and Its Expression in Immortalized Human Esophageal Keratinocytes Alters Cellular Morphology Jianping Kong, Shinsuke Funakoshi, John P. Lynch