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Su1738 Misoprostol Protects Gastric Barrier Function After Acid Injury in an Ex Vivo Canine Model
Su1736
gastrointestinal injury; its role in the prevention of acid related injury alone is less well examined. Due to the similarities in gastric physiology between dogs and humans, we used an ex vivo canine model to study the protective effect of misoprostol on acid injury to gastric mucosa. Our previous work demonstrated the validity of an ex vivo acid injury model (pH 1.2) using the Ussing chamber system with canine tissue. For this current study, gastric mucosa was collected from 6 random unadoptable dogs from a local animal control facility that were previously selected for euthanasia by shelter veterinarians. Gastric mucosa was maintained in Ussing chambers as previously described. After a 30-minute equilibration, acid Ringer's solution (pH 1.2) was applied to the mucosal aspect of the tissue for 45 minutes. Misoprostol was also applied immediately after the equilibration period to both the mucosa and serosa. Transepithelial resistance (TER) and 3H-mannitol flux were used as indices of barrier function. Tissue samples were collected from each treatment group, stained with hematoxylin and eosin, and examined by a blinded investigator. Application of acid Ringer's solution significantly decreased TER as compared to control (p<0.001). TER of acid-injured, misoprostol-treated tissues was significantly higher than acid-injured tissue for all time periods from 60 to 195 minutes (Figure 1; p=0.008). 3H-mannitol flux was higher in acid injured tissue compared to control or acid + misoprostol treated tissue (Figure 2, mean±SE: Control 0.08±0.01, Acid injury 0.21±0.10, Acid + misoprostol 0.09±0.01, p= 0.21). There was no significant effect on morphology of control tissues after the experiment. Acid injury caused partial disruption of the superficial gastric epithelium. Misoprostol treatment partially prevented acid-mediated injury histologically. In conclusion, this model of injury provides a novel comparative model of peptic ulcer disease that can be studied ex vivo. Additionally, misoprostol provided protection from acid injury in both barrier function, as measured by mucosal TER and 3H-mannitol flux, as well as morphologically. This drug may serve clinical utility for a disease syndrome with high morbidity and mortality for prevention or treatment of ulceration due to peptic acid injury, independent of NSAID effect on ulcer injury.
AGA Abstracts
MicroRNA-222 and CUG-binding Protein 1 Repress Translation of WntReceptor Frizzled Homolog-7 and Inhibit Intestinal Epithelial Repair Yu Chen, Rao N. Jaladanki, Tongtong Zou, Lan Liu, Lan Xiao, Ran Zhuang, Myriam Gorospe, Jian-Ying Wang Wnt signaling has emerged as a key regulator of maintaining gut epithelial renewal and homeostasis. Wnt proteins locally interact with and activate the Frizzled (Fz) receptors, leading to stimulation of their target gene transcription by increasing β-catenin nuclear translocation. Our recent study shows that activation of Wnt3a/β-catenin signaling enhances intestinal epithelial repair by promoting c-Myc-regulated gene expression, but the exact mechanism that regulates Fz-receptor abundance remains unknown. microRNAs (miRNAs) and RNA-binding proteins (RBPs) regulate gene expression at the posttranscriptional level. miRNA-222 (miR-222) and RBP CUG-binding protein 1 (CUGBP1) are highly expressed in the gut mucosa and they are involved in many aspects of cellular function In Vitro and In Vivo. In this study, we test the hypothesis that miR-222 and CUGBP1 jointly regulate Fz7 expression in intestinal epithelial cells (IECs) and therefore the epithelial repair after injury. Methods: Studies were conducted in IEC-6 cells, which were derived from rat intestinal crypts. Interaction of CUGBP1 with the Fz-7 mRNA was detected by biotin pull-down and RNP-IP assays, whereas miR-222 binding to the Fz-7 transcript was examined by RNA pulldown assay using biotin-labeled miR-222 and pmir-Glo reporter system. Fz-7 translation was examined by measuring its newly synthesized protein and using chimeric luciferaseFz-7 coding region (CR) and 3'-untranslated region (UTR) reporter gene assays. CUGBP1 and miR-222 functions were investigated by siRNA silencing and ectopic gene overexpression. Epithelial repair was measured in an In Vitro wounding model. Results: miR-222 was found to bind the Fz-7 mRNA via the CR rather than its 5'- and 3'-UTRs, whereas CUGBP1 bound to both 3'-UTR and CR of the Fz-7 mRNA. Overexpression of a precursor miR-222 increased miR-222/Fz-7 mRNA complex, repressed Fz-7 translation, and reduced its protein levels (by ~60%), although it failed to alter total Fz-7 mRNA levels. miR-222 silencing decreased miR-222 association with the Fz-7 mRNA, enhanced Fz-7 translation, and increased its protein content (by ~2-fold). Ectopic CUGBP1 overexpression also repressed Fz-7 translation without effect on Fz-7 mRNA levels, while CUGBP1 silencing stimulated Fz-7 expression. Interestingly, co-transfection of the miR-222 precursor with CUGBP1 synergistically repressed Fz-7 translation; CUGBP1 silencing partially abolished the Fz-7 repression in cells overexpressing miR-222. Ectopic overexpression of CUGBP1 and miR-222 or Fz-7 silencing repressed Wnt-dependent transcriptional activity and suppressed epithelial repair after wounding Conclusions: These results indicate that CUGBP1 and miR-222 synergistically repress Fz-7 translation and inactivate Wnt signaling pathway, thus inhibiting epithelial repair after wounding. Su1737 Stimulation of Gastric Ulcer Healing by Expression of Heat Shock Protein 70 Tomoaki Ishihara, Tohru Mizushima Background & Aims: It is important in the treatment of gastric ulcers to not only prevent further ulcer formation but also to enhance ulcer healing. When cells are exposed to various gastric irritants, expression of heat shock proteins (HSPs) is induced, making the cells resistant to the irritants. A number of indirect lines of evidence suggest HSPs protect gastric mucosa from irritant-induced production of lesions. For example, geranylgeranylacetone (GGA), a clinically used anti-ulcer drug was shown to have HSP-inducing activity. We recently obtained direct evidence that HSPs, especially HSP70, are protective against irritantinduced gastric ulcers, using transgenic mice; we showed that null mice lacking Heat shock factor 1 (HSF1), a transcription factor for hsp genes or transgenic mice expressing HSP70 showed sensitive or resistant, respectively, phenotype to irritant-induced gastric ulcers. However, no data have been reported concerning the role of HSP70 in gastric ulcer healing. Gastric ulcer healing is a complex process involving inflammatory responses (such as an increase in the level of prostaglandin E2 (PGE2)) and induction of expression of growth factors, resulting in cell proliferation and migration at the gastric ulcer margin and angiogenesis in granulation tissue. In this study, we have examined the role of HSP70 in gastric ulcer healing. Methods: Gastric ulcers were produced by focal and serosal application of acetic acid to wild-type mice and transgenic mice expressing HSP70. Up-regulation of HSP70 was monitored by immunoblotting and immunohistochemical analyses. Human recombinant HSP70 was purified from E. coli cells expressing this protein. Results: Expression of HSP70 was induced in both the gastric ulcer margin and granulation tissue. Compared with wildtype mice, gastric ulcer healing was accelerated in transgenic mice expressing HSP70, and both cell proliferation at the gastric ulcer margin and angiogenesis in granulation tissue were enhanced. Furthermore, expression of growth factors and the gastric level of PGE2 increased in ulcerated tissues relative to normal tissue, and these increases were enhanced in the transgenic mice. Oral administration of GGA to wild-type mice, either prior to or after ulcer formation, not only induced expression of HSP70 in the stomach but also accelerated gastric ulcer healing. On the other hand, oral administration of purified recombinant HSP70 prior to the ulcer formation, but not after formation, stimulated gastric ulcer healing. Conclusions: This study provides the first direct evidence that HSP70 accelerates gastric ulcer healing. The results also suggest that both the HSP70 produced prior to ulcer formation and released from damaged cells, and the HSP70 produced after ulcer formation are involved in this accelerated healing process.
TER of gastric mucosa. Acid injury produces decrease in barrier function (p<0.001, acid injury versus control). Misoprostol applied with acid protects against decrease in barrier function and accelerates recovery (p=0.008, misoprostol versus acid injury). n=10
3H-mannitol flux of gastric mucosa. Acid injury increases mannitol permeability. Misoprostol attenuates this increase (p=0.21, n=5). Su1739 Dynamic Changes of Gene Expression During the Development of Mouse Esophageal Epithelium and the Role of NRF2/Keap1 Pathway Hao Chen, Jianying Li, Haiyan Li, Yuhui Hu, Whitney Tevebaugh, Jianwen Que, Xiaoxin L. Chen
Su1738 Misoprostol Protects Gastric Barrier Function After Acid Injury in an Ex Vivo Canine Model Tracy L. Hill, Anthony Blikslager, Duncan Lascelles
Objective Morphological changes during human and mouse esophageal development have been well characterized. However, changes at molecular level in the course of esophageal morphogenesis remain unclear. This study aims to globally profile critical genes and signaling pathways during the development of mouse esophagus. By using microarray analysis this study also aims to determine how Nrf2/Keap1 pathway regulates the morphogenesis of the
Peptic ulceration is a significant cause of morbidity in human medicine, resulting in 300,000 hospitalizations per year with an associated cost of 3.3 billion annually in the US; prevention of peptic acid injury is therefore a relevant and important field of study. Misoprostol, a synthetic PGE1 analog, has historically been used in the treatment of NSAID-mediated
AGA Abstracts
S-492
gastrointestinal injury; its role in the prevention of acid related injury alone is less well examined. Due to the similarities in gastric physiology between dogs and humans, we used an ex vivo canine model to study the protective effect of misoprostol on acid injury to gastric mucosa. Our previous work demonstrated the validity of an ex vivo acid injury model (pH 1.2) using the Ussing chamber system with canine tissue. For this current study, gastric mucosa was collected from 6 random unadoptable dogs from a local animal control facility that were previously selected for euthanasia by shelter veterinarians. Gastric mucosa was maintained in Ussing chambers as previously described. After a 30-minute equilibration, acid Ringer's solution (pH 1.2) was applied to the mucosal aspect of the tissue for 45 minutes. Misoprostol was also applied immediately after the equilibration period to both the mucosa and serosa. Transepithelial resistance (TER) and 3H-mannitol flux were used as indices of barrier function. Tissue samples were collected from each treatment group, stained with hematoxylin and eosin, and examined by a blinded investigator. Application of acid Ringer's solution significantly decreased TER as compared to control (p<0.001). TER of acid-injured, misoprostol-treated tissues was significantly higher than acid-injured tissue for all time periods from 60 to 195 minutes (Figure 1; p=0.008). 3H-mannitol flux was higher in acid injured tissue compared to control or acid + misoprostol treated tissue (Figure 2, mean±SE: Control 0.08±0.01, Acid injury 0.21±0.10, Acid + misoprostol 0.09±0.01, p= 0.21). There was no significant effect on morphology of control tissues after the experiment. Acid injury caused partial disruption of the superficial gastric epithelium. Misoprostol treatment partially prevented acid-mediated injury histologically. In conclusion, this model of injury provides a novel comparative model of peptic ulcer disease that can be studied ex vivo. Additionally, misoprostol provided protection from acid injury in both barrier function, as measured by mucosal TER and 3H-mannitol flux, as well as morphologically. This drug may serve clinical utility for a disease syndrome with high morbidity and mortality for prevention or treatment of ulceration due to peptic acid injury, independent of NSAID effect on ulcer injury.
AGA Abstracts
MicroRNA-222 and CUG-binding Protein 1 Repress Translation of WntReceptor Frizzled Homolog-7 and Inhibit Intestinal Epithelial Repair Yu Chen, Rao N. Jaladanki, Tongtong Zou, Lan Liu, Lan Xiao, Ran Zhuang, Myriam Gorospe, Jian-Ying Wang Wnt signaling has emerged as a key regulator of maintaining gut epithelial renewal and homeostasis. Wnt proteins locally interact with and activate the Frizzled (Fz) receptors, leading to stimulation of their target gene transcription by increasing β-catenin nuclear translocation. Our recent study shows that activation of Wnt3a/β-catenin signaling enhances intestinal epithelial repair by promoting c-Myc-regulated gene expression, but the exact mechanism that regulates Fz-receptor abundance remains unknown. microRNAs (miRNAs) and RNA-binding proteins (RBPs) regulate gene expression at the posttranscriptional level. miRNA-222 (miR-222) and RBP CUG-binding protein 1 (CUGBP1) are highly expressed in the gut mucosa and they are involved in many aspects of cellular function In Vitro and In Vivo. In this study, we test the hypothesis that miR-222 and CUGBP1 jointly regulate Fz7 expression in intestinal epithelial cells (IECs) and therefore the epithelial repair after injury. Methods: Studies were conducted in IEC-6 cells, which were derived from rat intestinal crypts. Interaction of CUGBP1 with the Fz-7 mRNA was detected by biotin pull-down and RNP-IP assays, whereas miR-222 binding to the Fz-7 transcript was examined by RNA pulldown assay using biotin-labeled miR-222 and pmir-Glo reporter system. Fz-7 translation was examined by measuring its newly synthesized protein and using chimeric luciferaseFz-7 coding region (CR) and 3'-untranslated region (UTR) reporter gene assays. CUGBP1 and miR-222 functions were investigated by siRNA silencing and ectopic gene overexpression. Epithelial repair was measured in an In Vitro wounding model. Results: miR-222 was found to bind the Fz-7 mRNA via the CR rather than its 5'- and 3'-UTRs, whereas CUGBP1 bound to both 3'-UTR and CR of the Fz-7 mRNA. Overexpression of a precursor miR-222 increased miR-222/Fz-7 mRNA complex, repressed Fz-7 translation, and reduced its protein levels (by ~60%), although it failed to alter total Fz-7 mRNA levels. miR-222 silencing decreased miR-222 association with the Fz-7 mRNA, enhanced Fz-7 translation, and increased its protein content (by ~2-fold). Ectopic CUGBP1 overexpression also repressed Fz-7 translation without effect on Fz-7 mRNA levels, while CUGBP1 silencing stimulated Fz-7 expression. Interestingly, co-transfection of the miR-222 precursor with CUGBP1 synergistically repressed Fz-7 translation; CUGBP1 silencing partially abolished the Fz-7 repression in cells overexpressing miR-222. Ectopic overexpression of CUGBP1 and miR-222 or Fz-7 silencing repressed Wnt-dependent transcriptional activity and suppressed epithelial repair after wounding Conclusions: These results indicate that CUGBP1 and miR-222 synergistically repress Fz-7 translation and inactivate Wnt signaling pathway, thus inhibiting epithelial repair after wounding. Su1737 Stimulation of Gastric Ulcer Healing by Expression of Heat Shock Protein 70 Tomoaki Ishihara, Tohru Mizushima Background & Aims: It is important in the treatment of gastric ulcers to not only prevent further ulcer formation but also to enhance ulcer healing. When cells are exposed to various gastric irritants, expression of heat shock proteins (HSPs) is induced, making the cells resistant to the irritants. A number of indirect lines of evidence suggest HSPs protect gastric mucosa from irritant-induced production of lesions. For example, geranylgeranylacetone (GGA), a clinically used anti-ulcer drug was shown to have HSP-inducing activity. We recently obtained direct evidence that HSPs, especially HSP70, are protective against irritantinduced gastric ulcers, using transgenic mice; we showed that null mice lacking Heat shock factor 1 (HSF1), a transcription factor for hsp genes or transgenic mice expressing HSP70 showed sensitive or resistant, respectively, phenotype to irritant-induced gastric ulcers. However, no data have been reported concerning the role of HSP70 in gastric ulcer healing. Gastric ulcer healing is a complex process involving inflammatory responses (such as an increase in the level of prostaglandin E2 (PGE2)) and induction of expression of growth factors, resulting in cell proliferation and migration at the gastric ulcer margin and angiogenesis in granulation tissue. In this study, we have examined the role of HSP70 in gastric ulcer healing. Methods: Gastric ulcers were produced by focal and serosal application of acetic acid to wild-type mice and transgenic mice expressing HSP70. Up-regulation of HSP70 was monitored by immunoblotting and immunohistochemical analyses. Human recombinant HSP70 was purified from E. coli cells expressing this protein. Results: Expression of HSP70 was induced in both the gastric ulcer margin and granulation tissue. Compared with wildtype mice, gastric ulcer healing was accelerated in transgenic mice expressing HSP70, and both cell proliferation at the gastric ulcer margin and angiogenesis in granulation tissue were enhanced. Furthermore, expression of growth factors and the gastric level of PGE2 increased in ulcerated tissues relative to normal tissue, and these increases were enhanced in the transgenic mice. Oral administration of GGA to wild-type mice, either prior to or after ulcer formation, not only induced expression of HSP70 in the stomach but also accelerated gastric ulcer healing. On the other hand, oral administration of purified recombinant HSP70 prior to the ulcer formation, but not after formation, stimulated gastric ulcer healing. Conclusions: This study provides the first direct evidence that HSP70 accelerates gastric ulcer healing. The results also suggest that both the HSP70 produced prior to ulcer formation and released from damaged cells, and the HSP70 produced after ulcer formation are involved in this accelerated healing process.
TER of gastric mucosa. Acid injury produces decrease in barrier function (p<0.001, acid injury versus control). Misoprostol applied with acid protects against decrease in barrier function and accelerates recovery (p=0.008, misoprostol versus acid injury). n=10
3H-mannitol flux of gastric mucosa. Acid injury increases mannitol permeability. Misoprostol attenuates this increase (p=0.21, n=5). Su1739 Dynamic Changes of Gene Expression During the Development of Mouse Esophageal Epithelium and the Role of NRF2/Keap1 Pathway Hao Chen, Jianying Li, Haiyan Li, Yuhui Hu, Whitney Tevebaugh, Jianwen Que, Xiaoxin L. Chen
Su1738 Misoprostol Protects Gastric Barrier Function After Acid Injury in an Ex Vivo Canine Model Tracy L. Hill, Anthony Blikslager, Duncan Lascelles
Objective Morphological changes during human and mouse esophageal development have been well characterized. However, changes at molecular level in the course of esophageal morphogenesis remain unclear. This study aims to globally profile critical genes and signaling pathways during the development of mouse esophagus. By using microarray analysis this study also aims to determine how Nrf2/Keap1 pathway regulates the morphogenesis of the
Peptic ulceration is a significant cause of morbidity in human medicine, resulting in 300,000 hospitalizations per year with an associated cost of 3.3 billion annually in the US; prevention of peptic acid injury is therefore a relevant and important field of study. Misoprostol, a synthetic PGE1 analog, has historically been used in the treatment of NSAID-mediated
AGA Abstracts
S-492