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Dysplasia Associated Microenvironment Changes in Barrett’s Esophagus
AGA Abstracts
Sa1159 DYSPLASIA ASSOCIATED MICROENVIRONMENT CHANGES IN BARRETT'S ESOPHAGUS Yan Sun, Marlys Anderson, Kavel Visrodia, Liam Zakko, James Allen, Lori S. Lutzke, Kenneth K. Wang Background Barrett's Esophagus (BE) is the only known precancerous lesion of esophageal adenocarcinoma, however, the microenvironmental changes are not well established. We previously reported that there was increased inflammatory cytokine production of IL6 in the stromal cells in response to TNF-α and IL1-1β, particularly in the setting of high grade dysplasia (HGD). Our hypothesis was that this was due to an increase in myofibroblasts that are active secretors of IL6. Methods Fibroblasts were isolated from primary human BE biopsy samples of non-dysplasia (ND), low grade dysplasia (LGD) and high grade dysplasia. Myofibroblasts were characterized by immunocytochemistry (ICC) being positive for α-SMA and vimentin, while negative for desmin and pan-cytokeratin. Cultures were maintained as previously described. Human recombinant IL-1β protein 10 ng/ml and human TNF-α protein 50 ng/ml were used to stimulate the fibroblasts for 96 hours. The expression levels of α-SMA, vimentin, desmin and pan-cytokeratin were detected by immunofluorescence. Stained cells were imaged with an AxioVert microscope and the numbers of α-SMA, vimentin positive cells and desmin, pan-cytokeratin negative cells were counted. At least 100 DAPIpositive cells were assessed for 3 times in each group. Chi-square test and Two-way ANOVA were used for statistical analysis. Results After stimulation by IL-1β and TNF-α, all the fibroblasts appeared activated and had the properties of myofibroblasts. The positive rates of α-SMA changed from 21.11%-44.11% in ND, 16.56%-48.56% in LGD, 42.11%-64.78% in HGD, respectively; Vimentin changed from 38.33%-93.89% in ND, 11.96%-91.11% in LGD, 51.44%-88.89% in HGD, respectively. The expression levels of α-SMA and vimentin were significantly higher after stimulation in all three groups of BE cells compared with those before stimulation (P<0.05). Desmin and the epithelial marker pan-cytokeratin had almost no expressions with or without stimulation (P>0.05). The fibroblasts from HGD initially had more expressions of α-SMA and vimentin than those in ND and LGD (P<0.05). The expression levels of myofibroblasts after stimulation was gradually increased from ND, LGD to HGD, and the positive rates were 36.17%, 41.64%, 54.83%, respectively. Conclusions The stained myofibroblasts increased during the progression to esophageal cancer after stimulation by inflammatory cytokines. These findings suggest that myofibroblasts are increased in Barrett's mucosa with increased inflammatory stimulation although numbers of stromal cells are constant. This suggests that myofibroblasts are transformed from existing fibroblasts during the neoplastic process and are likely responsible for the increased IL6 production.
Figure 2 Expression levels of myofibroblasts before and after stimulation by IL-1β and TNFα in three different BE stromal cell culture. *, P<0.05, compared with the unstimulated group.
Sa1160 HOW DOES ESOPHAGEAL REFLUX INDUCE DNA DAMAGE IN ESOPHAGEAL CELLS? Olga Korolkova, Andela Horvat, Vikas Bhardwaj, Kay Washington, Wael M. El-Rifai, Alexander Zaika Background: We and others have previously demonstrated that chronic esophageal reflux causes severe DNA damage in esophageal cells leading to increased mutational rate, genomic instability and esophageal cancer. Although genotoxic effects of reflux play the primary role in esophageal tumorigenesis, it remains unclear how reflux induces DNA damage. This understanding is especially important in light of the future development of effective chemopreventive approaches for esophageal tumor. Methods and Results: To investigate the mechanisms of DNA damage induced by reflux, we established a model, in which nontransformed esophagus cells were exposed to acidic bile salts in vitro. We also analyzed GERD patients. Exposure of esophageal cells to reflux led to strong induction of reactive oxygen species (ROS) and DNA damage. ROS was found to be the critical inductor of DNA damage as inhibition of ROS by various antioxidants significantly inhibited the genotoxic effects of ROS. Among ROS species, we identified superoxide radicals (determined using electron paramagnetic resonance spectroscopy) and hydrogen peroxide (determined with Amplex-Red assay). ROS induced by reflux were found to have a dual origin. It was generated by mitochondria and by activation of NADPH oxidases (NOX) localized on the cellular membranes. We specifically identified NOX1 and NOX2 enzymes as main generators of ROS. Inhibition of these enzymes by siRNA or specific inhibitor apocynin led to significant inhibition of ROS and DNA damage induced by reflux. Our data also revealed significantly increased expression of NOX and DNA damage in esophageal biopsied from patients with GERD compared to normal controls. DNA damage was determined by staining for phosphorylated histone H2AX and Comet assay. We also dissected the mechanism of NOX activation. Our data showed that exposure of esophageal cells to acidic bile causes phosphorylation of the p47phox subunit of NOX leading to its activation. This process is mediated by protein kinase C (PKC), which is activated by acidic bile salts. Inhibition of PKC with specific inhibitors or siRNA strongly affected phosphorylation of p47phox and ROS. Conclusion: DNA damage induced by reflux is mediated by an increase in production of mitochondrial ROS and activation of NADPH oxidases. Inhibitors of ROS production such as apocynin, which not only specifically inhibits NADPH oxidases but also scavenges mitochondrial ROS, might be the most effective approach to protect esophageal epithelium against bile acidsassociated damage and tumorigenesis.
Sa1161 GLANDULAR METAPLASIA AND DYSPLASIA AT THE SQUAMOCOLUMNAR JUNCTION MIRROR BARRETT'S ESOPHAGUS IN MOUSE MODELS Sarawut Kongkarnka, Sama Sayin, Haibo Liu, Huan Deng, Timothy C. Wang, Elena Komissarova, Antonia R. Sepulveda
Figure 1 Number of α-SMA and vimentin positive cells before and after stimulation by IL1β and TNF-α in three different BE stromal cell culture. U, Unstimulated, S, Stimulated, *, P<0.05.
Introduction: Barrett's esophagus (BE) can progress to esophageal adenocarcinoma (EAC) through a metaplasia-dysplasia-carcinoma sequence driven by inflammatory responses triggered by bile/acid reflux. The transgenic mouse model of BE (L2-IL-1β) reproduces Barrettlike metaplasia and dysplasia development, and is accelerated by exposure to bile acids and/ or nitrosamines. We used this model to quantify proliferation in areas of columno-glandular metaplasia and dysplasia at the squamo-columnar junction (SCJ) induced by overexpression of IL-1β (IL), bile acid (B), testosterone (T) and N-Methyl-N-Nitrosourea (MNU). Methods: 53 mice, including 49 L2-IL-1β transgenic (IL) and 4 wild-type (WT) mice, were classified into 6 experimental groups according to the treatment received: A) WT+B, B) IL, C) IL+B, D) IL+B+T, E) IL+B+T+M and F) IL+B+M, and 5 WT mice with no treatment were a control group. Histopathologic changes at the SCJ, including presence of acute and chronic inflammation, cardia mucous gland hyperplasia (CMGH) determined by the number of mucous gland profiles greater than 4, and dysplasia, were scored. Immunohistochemistry for MUC2, CDX2, TP53 and Ki67 were done. Tissues were examined at age 12 to 20 months. Results: All 53 experimental animals had chronic and acute inflammation at the SCJ and 49 (92.4%) were accompanied by reactive or regenerative epithelial changes. CMGH was identified in 44 (83%) mice from all 6 experimental groups. L2-IL-1β (IL) mice alone had CMGH median score=13 and bile acid exposure in IL mice resulted in increased CMGH
S-239
AGA Abstracts
Sa1159 DYSPLASIA ASSOCIATED MICROENVIRONMENT CHANGES IN BARRETT'S ESOPHAGUS Yan Sun, Marlys Anderson, Kavel Visrodia, Liam Zakko, James Allen, Lori S. Lutzke, Kenneth K. Wang Background Barrett's Esophagus (BE) is the only known precancerous lesion of esophageal adenocarcinoma, however, the microenvironmental changes are not well established. We previously reported that there was increased inflammatory cytokine production of IL6 in the stromal cells in response to TNF-α and IL1-1β, particularly in the setting of high grade dysplasia (HGD). Our hypothesis was that this was due to an increase in myofibroblasts that are active secretors of IL6. Methods Fibroblasts were isolated from primary human BE biopsy samples of non-dysplasia (ND), low grade dysplasia (LGD) and high grade dysplasia. Myofibroblasts were characterized by immunocytochemistry (ICC) being positive for α-SMA and vimentin, while negative for desmin and pan-cytokeratin. Cultures were maintained as previously described. Human recombinant IL-1β protein 10 ng/ml and human TNF-α protein 50 ng/ml were used to stimulate the fibroblasts for 96 hours. The expression levels of α-SMA, vimentin, desmin and pan-cytokeratin were detected by immunofluorescence. Stained cells were imaged with an AxioVert microscope and the numbers of α-SMA, vimentin positive cells and desmin, pan-cytokeratin negative cells were counted. At least 100 DAPIpositive cells were assessed for 3 times in each group. Chi-square test and Two-way ANOVA were used for statistical analysis. Results After stimulation by IL-1β and TNF-α, all the fibroblasts appeared activated and had the properties of myofibroblasts. The positive rates of α-SMA changed from 21.11%-44.11% in ND, 16.56%-48.56% in LGD, 42.11%-64.78% in HGD, respectively; Vimentin changed from 38.33%-93.89% in ND, 11.96%-91.11% in LGD, 51.44%-88.89% in HGD, respectively. The expression levels of α-SMA and vimentin were significantly higher after stimulation in all three groups of BE cells compared with those before stimulation (P<0.05). Desmin and the epithelial marker pan-cytokeratin had almost no expressions with or without stimulation (P>0.05). The fibroblasts from HGD initially had more expressions of α-SMA and vimentin than those in ND and LGD (P<0.05). The expression levels of myofibroblasts after stimulation was gradually increased from ND, LGD to HGD, and the positive rates were 36.17%, 41.64%, 54.83%, respectively. Conclusions The stained myofibroblasts increased during the progression to esophageal cancer after stimulation by inflammatory cytokines. These findings suggest that myofibroblasts are increased in Barrett's mucosa with increased inflammatory stimulation although numbers of stromal cells are constant. This suggests that myofibroblasts are transformed from existing fibroblasts during the neoplastic process and are likely responsible for the increased IL6 production.
Figure 2 Expression levels of myofibroblasts before and after stimulation by IL-1β and TNFα in three different BE stromal cell culture. *, P<0.05, compared with the unstimulated group.
Sa1160 HOW DOES ESOPHAGEAL REFLUX INDUCE DNA DAMAGE IN ESOPHAGEAL CELLS? Olga Korolkova, Andela Horvat, Vikas Bhardwaj, Kay Washington, Wael M. El-Rifai, Alexander Zaika Background: We and others have previously demonstrated that chronic esophageal reflux causes severe DNA damage in esophageal cells leading to increased mutational rate, genomic instability and esophageal cancer. Although genotoxic effects of reflux play the primary role in esophageal tumorigenesis, it remains unclear how reflux induces DNA damage. This understanding is especially important in light of the future development of effective chemopreventive approaches for esophageal tumor. Methods and Results: To investigate the mechanisms of DNA damage induced by reflux, we established a model, in which nontransformed esophagus cells were exposed to acidic bile salts in vitro. We also analyzed GERD patients. Exposure of esophageal cells to reflux led to strong induction of reactive oxygen species (ROS) and DNA damage. ROS was found to be the critical inductor of DNA damage as inhibition of ROS by various antioxidants significantly inhibited the genotoxic effects of ROS. Among ROS species, we identified superoxide radicals (determined using electron paramagnetic resonance spectroscopy) and hydrogen peroxide (determined with Amplex-Red assay). ROS induced by reflux were found to have a dual origin. It was generated by mitochondria and by activation of NADPH oxidases (NOX) localized on the cellular membranes. We specifically identified NOX1 and NOX2 enzymes as main generators of ROS. Inhibition of these enzymes by siRNA or specific inhibitor apocynin led to significant inhibition of ROS and DNA damage induced by reflux. Our data also revealed significantly increased expression of NOX and DNA damage in esophageal biopsied from patients with GERD compared to normal controls. DNA damage was determined by staining for phosphorylated histone H2AX and Comet assay. We also dissected the mechanism of NOX activation. Our data showed that exposure of esophageal cells to acidic bile causes phosphorylation of the p47phox subunit of NOX leading to its activation. This process is mediated by protein kinase C (PKC), which is activated by acidic bile salts. Inhibition of PKC with specific inhibitors or siRNA strongly affected phosphorylation of p47phox and ROS. Conclusion: DNA damage induced by reflux is mediated by an increase in production of mitochondrial ROS and activation of NADPH oxidases. Inhibitors of ROS production such as apocynin, which not only specifically inhibits NADPH oxidases but also scavenges mitochondrial ROS, might be the most effective approach to protect esophageal epithelium against bile acidsassociated damage and tumorigenesis.
Sa1161 GLANDULAR METAPLASIA AND DYSPLASIA AT THE SQUAMOCOLUMNAR JUNCTION MIRROR BARRETT'S ESOPHAGUS IN MOUSE MODELS Sarawut Kongkarnka, Sama Sayin, Haibo Liu, Huan Deng, Timothy C. Wang, Elena Komissarova, Antonia R. Sepulveda
Figure 1 Number of α-SMA and vimentin positive cells before and after stimulation by IL1β and TNF-α in three different BE stromal cell culture. U, Unstimulated, S, Stimulated, *, P<0.05.
Introduction: Barrett's esophagus (BE) can progress to esophageal adenocarcinoma (EAC) through a metaplasia-dysplasia-carcinoma sequence driven by inflammatory responses triggered by bile/acid reflux. The transgenic mouse model of BE (L2-IL-1β) reproduces Barrettlike metaplasia and dysplasia development, and is accelerated by exposure to bile acids and/ or nitrosamines. We used this model to quantify proliferation in areas of columno-glandular metaplasia and dysplasia at the squamo-columnar junction (SCJ) induced by overexpression of IL-1β (IL), bile acid (B), testosterone (T) and N-Methyl-N-Nitrosourea (MNU). Methods: 53 mice, including 49 L2-IL-1β transgenic (IL) and 4 wild-type (WT) mice, were classified into 6 experimental groups according to the treatment received: A) WT+B, B) IL, C) IL+B, D) IL+B+T, E) IL+B+T+M and F) IL+B+M, and 5 WT mice with no treatment were a control group. Histopathologic changes at the SCJ, including presence of acute and chronic inflammation, cardia mucous gland hyperplasia (CMGH) determined by the number of mucous gland profiles greater than 4, and dysplasia, were scored. Immunohistochemistry for MUC2, CDX2, TP53 and Ki67 were done. Tissues were examined at age 12 to 20 months. Results: All 53 experimental animals had chronic and acute inflammation at the SCJ and 49 (92.4%) were accompanied by reactive or regenerative epithelial changes. CMGH was identified in 44 (83%) mice from all 6 experimental groups. L2-IL-1β (IL) mice alone had CMGH median score=13 and bile acid exposure in IL mice resulted in increased CMGH
S-239
AGA Abstracts