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Matrix Stiffness Corresponding to Strictured Small Intestine Induces a PRO-Fibrotic Response in Human Colonic Fibroblasts
(16/32) in the NEC group to 83.3% (25/30; p < 0.01) in the RAP groups. Protein levels of Beclin 1 and LC3II were markedly increased and levels of LC3I decreased in the ileum from the RAP group compared to the NEC group. Levels of p62 were not changed between groups. Protein levels of anti-apoptotic regulators Bcl-2 and Bcl-w were significantly increased in the RAP group. Conclusions: Pharmacologic induction of autophagy exacerbates the development and severity of NEC in the rat NEC model. These data provide further evidence that activation of autophagy, but not of apoptosis plays a crucial role in disease development in experimental NEC. Supported by the NIH Grant HD-39657 (to B.D.)
Intestinal CRF Receptor Subtypes Mediate Distinct Intestinal Barrier Function Responses Through Intestinal Mast Cell Activation Elizabeth L. Overman, Jean E. Rivier, Adam J. Moeser Psychological stress is a major contributing factor in the onset and exacerbation of gastrointestinal disease through poorly understood mechanisms. Our previous studies in a porcine model showed that stress-induced disturbances in mucosal barrier function are regulated by peripheral activation of CRFr subtypes CRFr1 and CRFr2. Furthermore, results from these studies suggested that CRFr1 mediates deleterious mucosal barrier responses to stress while CRFr2 may play a protective role. The objective of this study was to further elucidate the role of CRFr subtypes in stress intestinal barrier dysfunction. Porcine ileum was mounted on Ussing Chambers and exposed to CRFr1 and CRFr2 agonist treatments: 1) Control 2) CRF;(1μM;CRFr1/r2 agonist) 3) UCN2;(0.1μM CRFr2 agonist) 4) Stressin 1 (2nM;CRFr1 agonist). Intestinal barrier function was measured in terms of mucosal-to-serosal flux of 4 kDa-FITC dextran (FD4) over a 180-minute period on Ussing chambers. CRF treatment increased (p < 0.005) paracellular flux of FD4 compared with controls. Ileal mucosa treated with the CRFr1 agonist Stressin 1 exhibited the greatest FD4 flux compared with all other treatments (p< 0.001) whereas treatment with the CRFr2 agonist UCN2 had no effect on baseline ileal barrier function. However, when added in combination with Stressin 1, UCN2 inhibited (p<0.05) Stressin 1-induced increases in FD4 flux. To determine if the effects of CRFr activation on barrier function were mediated via intestinal mast cell activation, ileal tissue was pre-treated with the mast cell stabilizer cromolyn (10 -4M) and then CRFr agonists were applied to tissues. Blockade of mast cell activation prevented increases in paracellular flux induced by CRF and Stressin 1 (p <0.05). Overall, these data demonstrate CRFr1 and CRFr2 play distinct roles in regulation of stress-induce mucosal barrier dysfunction with CRFr1 mediating intestinal barrier dysfunction and CRFr2 mediating protection, potentially through negative regulation of CRFr1 pathways. In addition, these data show that CRFr1mediated intestinal barrier dysfunction is mediated through activation of intestinal MCs.
Mo1784 Oxidative Stress and Deletion of Transketolase-Like (TKTL) 1 in Murine Experimental Colitis Susanne Bentz, Theresa Pesch, Lutz Wolfram, Cheryl de Valliere, Katharina Leucht, Michael Fried, Johannes F. Coy, Martin Hausmann, Gerhard Rogler Background: TKTL1 is an enzyme involved in the pentose phosphate pathway (PPP) which provides the cells with the reducing molecule nicotinamide adenine dinucleotide phosphate (NADPH). Low levels of endogenous antioxidants have been described in the colonic mucosa of inflammatory bowel disease (IBD) patients and oxidative stress may therefore exceed the capacity of endogenous defense mechanisms, such as neutralization of reactive oxygen species (ROS) by NADPH. We investigated the role of TKTL1 during experimental dextran sodium sulfate (DSS) induced murine colitis and its potential function in ROS detoxification. Methods: Mucosal damage in TKTL1-/- and wild type (WT) mice was assessed by miniendoscopy and histology during acute DSS colitis. mRNA levels of IFN-γ, iNOS, IL-6, TNF, transketolase (TKT) and TKTL2 were determined by PCR and/or Western blotting. To assess oxidative and nitrosative stress, nitrosylation, carbonylation and anti-oxidative enzymes catalase (Cat), superoxide dismutase (SOD) 1 and 2 as well as glutathione (GSH) were determined by Western Blotting or enzymatic cycling assay. Myeloperoxidase (MPO) was determined for assessment of tissue neutrophils. Results: TKTL1 deletion or DSS treatment did not influence TKT or TKTL2 mRNA and protein expression. Mucosal damage was significantly increased in TKTL1-/- mice as compared to WT during DSS colitis as shown by miniendoscopy, a significant shorter colon and higher histological scores. Consistently higher mRNA expression of IFN-γ, iNOS, IL-6 and TNF involved in inflammation was detected. An overall rise in oxidative stress was measurable as a significantly enhanced iNOS protein expression, higher MPO activity and induction of protein modification by NO (N-Tyr) in TKTL1-/- mice. However, introduction of carbonyl groups by ROS was undetectable, neither was SOD1, SOD2, and Cat expression nor a change in GSH content. Conclusion: Experimental colitis in TKTL1-/- mice was more severe as compared to WT. This indicates a role of TKTL1 during mucosal repair and homeostasis.
Mo1782 Deficiency of BACH1 Ameliorates TNBS-Induced Colitis in Mice Thorough the Anti-Inflammatory Function of HO-1 Highly Expressed Macrophages Akihito Harusato, Yuji Naito, Tomohisa Takagi, Kazuhiko Uchiyama, Katsura Mizushima, Yasuko Hirai, Toshifumi Tsuji, Hiroyuki Yoriki, Munehiro Kugai, Ryusuke Horie, Ken Inoue, Kohei Fukumoto, Shinya Yamada, Osamu Handa, Satoshi Kokura, Hiroshi Ichikawa, Akihiko Muto, Kazuhiko Igarashi, Toshikazu Yoshikawa Introduction: BTB and CNC homolog 1 (Bach1) is known as a transcriptional repressor of heme oxygenase-1 (HO-1), which protects cells/tissues from various injuries involved in the intestinal inflammation such as inflammatory bowel disease. So, we hypothesized that Bach1 has an important effect on colonic mucosal injury by modulating HO-1 expression. The aim of this study was to investigate whether Bach1 deficiency ameliorated 2,4,6-trinitrobenzine sulfonic acid (TNBS)-induced colitis. Methods: 8-week-old female C57BL/6 (wild-type) and homozygous Bach1-/- C57BL/6 mice were subjected to this study. Colonic mucosal injuries induced by an enema administration of TNBS were evaluated macroscopically, histologically, and then biochemically using homogenates of inflamed colonic mucosa. We also evaluated the enhancement by co-treatment of an HO-1 inhibitor, tin protoporphyrin (SnPP). HO-1 expression and localization were examined using western blotting and immunofluorescent staining. As HO-1 localized mainly in macrophages in colonic mucosa, we isolated the intraperitoneal macrophages and analyzed the function of these cells. Result: TNBS-induced colonic mucosal injuries were remarkably improved in Bach1-/- mice. The administration of an HO-1 inhibitor, SnPP, cancelled the therapeutic effect against TNBS-induced colitis in Bach1-/- mice. The expression of HO-1 mRNA in colonic mucosa was significantly increased in Bach1-/- mice compared to wild-type mice. The result of immunofluorescent staining in colonic mucosa showed that HO-1 expression was mainly localized in F4/80 positive macrophages. Administration of TNBS induced expression of cytokines, such as KC, and TNF-α, which was suppressed in Bach1-/- mice. Furthermore, isolated peritoneal macrophages from Bach1-/- mice, but not from the wild type, inhibited the production of these cytokines after TNF-α stimulus. Conclusion: Disruption of the Bach1 gene caused inhibition of TNBS-induced colitis through the function of HO-1 highly expressed macrophages, indicating that regulation of Bach1 could be an important therapeutic target in the intestinal inflammation.
Mo1785 Matrix Stiffness Corresponding to Strictured Small Intestine Induces a PROFibrotic Response in Human Colonic Fibroblasts Kay L. Sauder, Laura A. Johnson, Eva Rodansky, Luz P. Blanco, Justin D. Mih, Daniel Tschumperlin, Peter D. Higgins Background: Crohn's disease is characterized by repeated cycles of inflammation and mucosal healing which ultimately progresses to intestinal fibrosis. This inexorable progression towards fibrosis suggests that fibrosis becomes inflammation-independent and auto-propagative. We hypothesized that resident colonic fibroblasts detect increased stiffening of extracellular matrix and respond with increased proliferation, a transition to myofibroblast-like morphology, and increased production of extracellular matrix components which further stiffens the matrix, creating a fibrogenesis positive feedback loop. Methods: The stiffness of fresh ex vivo small intestine from normal human small intestine and ex vivo Crohn's disease strictures were measured with a microelastometer. Collagen-coated polyacrylamide matrices corresponding to the physiological stiffness of normal (compliant) or Crohn's (fibrotic) small intestine were used as models of the intestinal myofibroblast environment. Normal human colonic fibroblasts (Ccd-18co cells) were grown on physiologically normal (0.01%, 0.02% bis-acrylamide) or pathologically stiff (0.08%, 0.16%) matrices. Cellular response was assayed for changes in cell morphology, a-smooth muscle actin (αSMA) staining, and gene expression. Results: Microelastometer measurements revealed a significant increase in colonic tissue stiffness between both normal human colon and Crohn's stricture small intestine as well as between the stricture and adjacent tissue margin. In CCD-18co cells grown on stiff matrices corresponding to Crohn's strictures, cellular proliferation increased 2 to 6-fold with increasing stiffness. Increased stiffness induced a marked change in cell morphology, to larger, stellatetype cells, with multiple dendritic processes. This morphological change was accompanied by a corresponding increase in αSMA+ bundles. Concurrent with the stiffness-induced morphological changes, growth on a stiff matrix caused increases in the expression of profibrotic (CTGF, collagen I, endothelin I, Lox) genes, and decreases in matrix proteinases (MMP1, MMP10, cathepsin O, YKL-40) and pro-inflammatory genes (IL-1β, Cox-2). Conclusions: Human intestinal fibroblasts sense changes in the matrix stiffness within a physiologically relevant range. Stiff matrices, corresponding to the stiffness of Crohn's strictures, increase cell proliferation, induce a myofibroblast-like morphology, and induce pro-fibrotic gene expression while repressing matrix remodeling genes. This may contribute to a positive feedback loop of fibrogenesis and auto-propagation of fibrosis.
Mo1783 Induction of Autophagy Increases Intestinal Injury in a Rat Model of Necrotizing Enterocolitis Christine Coursodon, Chelsea Snarrenberg, Camille Adkins-Rieck, Katerina Dvorak, Bohuslav Dvorak Background: Neonatal necrotizing enterocolitis (NEC) is a major cause of morbidity and mortality in premature infants. Autophagy is a mechanism regulating a normal turnover of cytosolic components, but is also rapidly up regulated under conditions leading to cellular stress. While a basal level of autophagy maintains normal cellular homeostasis, uncontrolled activation of autophagy leads to excessive degradation of cellular content and cell death. Previously we have shown increased autophagy in the intestinal epithelium of NEC patients and in the ileum of NEC rats. However, the role of autophagy in NEC pathogenesis remains still unclear. Aim: To evaluate the effect of a well-established inducer of autophagy Rapamycin - on development of NEC injury, protein expression of autophagic and apoptotic regulators in the rat NEC model. Methods: Premature rats were divided into the following groups: hand-fed with formula (NEC), or hand-fed with formula with 200ng/rat/day of Rapamycin (RAP). Both groups were exposed to asphyxia/cold stress to develop NEC. After 96 hours, all animals were sacrificed and ileal tissue was collected. Incidence and severity of NEC injury was evaluated. Protein levels of autophagy (Beclin 1, LC3 and p62) and apoptosis (Bcl-2 and Bcl-w) regulators were measured in the terminal ileum. Results: Administration of Rapamycin significantly increased the incidence of NEC injury from 50%
Mo1786 Spironolactone Inhibits TGFβ-Induced Fibrogenisis In Vitro but is Associated With Increased Mortality In Vivo Laura A. Johnson, Shail M. Govani, Joel Joyce, Peter D. Higgins Background: The renin-angiotensin-aldosterone system (RAAS) has been implicated in cardiac fibrosis in heart failure. Spironolactone (SPIR), an aldosterone antagonist, prevents fibrosis and mortality in patients with heart failure. Crohn's disease causes intestinal fibrosis, leading to strictures and obstruction. Studies of angiotensin-2 receptor antagonists in rodent
S-649
AGA Abstracts
AGA Abstracts
Mo1781
Intestinal CRF Receptor Subtypes Mediate Distinct Intestinal Barrier Function Responses Through Intestinal Mast Cell Activation Elizabeth L. Overman, Jean E. Rivier, Adam J. Moeser Psychological stress is a major contributing factor in the onset and exacerbation of gastrointestinal disease through poorly understood mechanisms. Our previous studies in a porcine model showed that stress-induced disturbances in mucosal barrier function are regulated by peripheral activation of CRFr subtypes CRFr1 and CRFr2. Furthermore, results from these studies suggested that CRFr1 mediates deleterious mucosal barrier responses to stress while CRFr2 may play a protective role. The objective of this study was to further elucidate the role of CRFr subtypes in stress intestinal barrier dysfunction. Porcine ileum was mounted on Ussing Chambers and exposed to CRFr1 and CRFr2 agonist treatments: 1) Control 2) CRF;(1μM;CRFr1/r2 agonist) 3) UCN2;(0.1μM CRFr2 agonist) 4) Stressin 1 (2nM;CRFr1 agonist). Intestinal barrier function was measured in terms of mucosal-to-serosal flux of 4 kDa-FITC dextran (FD4) over a 180-minute period on Ussing chambers. CRF treatment increased (p < 0.005) paracellular flux of FD4 compared with controls. Ileal mucosa treated with the CRFr1 agonist Stressin 1 exhibited the greatest FD4 flux compared with all other treatments (p< 0.001) whereas treatment with the CRFr2 agonist UCN2 had no effect on baseline ileal barrier function. However, when added in combination with Stressin 1, UCN2 inhibited (p<0.05) Stressin 1-induced increases in FD4 flux. To determine if the effects of CRFr activation on barrier function were mediated via intestinal mast cell activation, ileal tissue was pre-treated with the mast cell stabilizer cromolyn (10 -4M) and then CRFr agonists were applied to tissues. Blockade of mast cell activation prevented increases in paracellular flux induced by CRF and Stressin 1 (p <0.05). Overall, these data demonstrate CRFr1 and CRFr2 play distinct roles in regulation of stress-induce mucosal barrier dysfunction with CRFr1 mediating intestinal barrier dysfunction and CRFr2 mediating protection, potentially through negative regulation of CRFr1 pathways. In addition, these data show that CRFr1mediated intestinal barrier dysfunction is mediated through activation of intestinal MCs.
Mo1784 Oxidative Stress and Deletion of Transketolase-Like (TKTL) 1 in Murine Experimental Colitis Susanne Bentz, Theresa Pesch, Lutz Wolfram, Cheryl de Valliere, Katharina Leucht, Michael Fried, Johannes F. Coy, Martin Hausmann, Gerhard Rogler Background: TKTL1 is an enzyme involved in the pentose phosphate pathway (PPP) which provides the cells with the reducing molecule nicotinamide adenine dinucleotide phosphate (NADPH). Low levels of endogenous antioxidants have been described in the colonic mucosa of inflammatory bowel disease (IBD) patients and oxidative stress may therefore exceed the capacity of endogenous defense mechanisms, such as neutralization of reactive oxygen species (ROS) by NADPH. We investigated the role of TKTL1 during experimental dextran sodium sulfate (DSS) induced murine colitis and its potential function in ROS detoxification. Methods: Mucosal damage in TKTL1-/- and wild type (WT) mice was assessed by miniendoscopy and histology during acute DSS colitis. mRNA levels of IFN-γ, iNOS, IL-6, TNF, transketolase (TKT) and TKTL2 were determined by PCR and/or Western blotting. To assess oxidative and nitrosative stress, nitrosylation, carbonylation and anti-oxidative enzymes catalase (Cat), superoxide dismutase (SOD) 1 and 2 as well as glutathione (GSH) were determined by Western Blotting or enzymatic cycling assay. Myeloperoxidase (MPO) was determined for assessment of tissue neutrophils. Results: TKTL1 deletion or DSS treatment did not influence TKT or TKTL2 mRNA and protein expression. Mucosal damage was significantly increased in TKTL1-/- mice as compared to WT during DSS colitis as shown by miniendoscopy, a significant shorter colon and higher histological scores. Consistently higher mRNA expression of IFN-γ, iNOS, IL-6 and TNF involved in inflammation was detected. An overall rise in oxidative stress was measurable as a significantly enhanced iNOS protein expression, higher MPO activity and induction of protein modification by NO (N-Tyr) in TKTL1-/- mice. However, introduction of carbonyl groups by ROS was undetectable, neither was SOD1, SOD2, and Cat expression nor a change in GSH content. Conclusion: Experimental colitis in TKTL1-/- mice was more severe as compared to WT. This indicates a role of TKTL1 during mucosal repair and homeostasis.
Mo1782 Deficiency of BACH1 Ameliorates TNBS-Induced Colitis in Mice Thorough the Anti-Inflammatory Function of HO-1 Highly Expressed Macrophages Akihito Harusato, Yuji Naito, Tomohisa Takagi, Kazuhiko Uchiyama, Katsura Mizushima, Yasuko Hirai, Toshifumi Tsuji, Hiroyuki Yoriki, Munehiro Kugai, Ryusuke Horie, Ken Inoue, Kohei Fukumoto, Shinya Yamada, Osamu Handa, Satoshi Kokura, Hiroshi Ichikawa, Akihiko Muto, Kazuhiko Igarashi, Toshikazu Yoshikawa Introduction: BTB and CNC homolog 1 (Bach1) is known as a transcriptional repressor of heme oxygenase-1 (HO-1), which protects cells/tissues from various injuries involved in the intestinal inflammation such as inflammatory bowel disease. So, we hypothesized that Bach1 has an important effect on colonic mucosal injury by modulating HO-1 expression. The aim of this study was to investigate whether Bach1 deficiency ameliorated 2,4,6-trinitrobenzine sulfonic acid (TNBS)-induced colitis. Methods: 8-week-old female C57BL/6 (wild-type) and homozygous Bach1-/- C57BL/6 mice were subjected to this study. Colonic mucosal injuries induced by an enema administration of TNBS were evaluated macroscopically, histologically, and then biochemically using homogenates of inflamed colonic mucosa. We also evaluated the enhancement by co-treatment of an HO-1 inhibitor, tin protoporphyrin (SnPP). HO-1 expression and localization were examined using western blotting and immunofluorescent staining. As HO-1 localized mainly in macrophages in colonic mucosa, we isolated the intraperitoneal macrophages and analyzed the function of these cells. Result: TNBS-induced colonic mucosal injuries were remarkably improved in Bach1-/- mice. The administration of an HO-1 inhibitor, SnPP, cancelled the therapeutic effect against TNBS-induced colitis in Bach1-/- mice. The expression of HO-1 mRNA in colonic mucosa was significantly increased in Bach1-/- mice compared to wild-type mice. The result of immunofluorescent staining in colonic mucosa showed that HO-1 expression was mainly localized in F4/80 positive macrophages. Administration of TNBS induced expression of cytokines, such as KC, and TNF-α, which was suppressed in Bach1-/- mice. Furthermore, isolated peritoneal macrophages from Bach1-/- mice, but not from the wild type, inhibited the production of these cytokines after TNF-α stimulus. Conclusion: Disruption of the Bach1 gene caused inhibition of TNBS-induced colitis through the function of HO-1 highly expressed macrophages, indicating that regulation of Bach1 could be an important therapeutic target in the intestinal inflammation.
Mo1785 Matrix Stiffness Corresponding to Strictured Small Intestine Induces a PROFibrotic Response in Human Colonic Fibroblasts Kay L. Sauder, Laura A. Johnson, Eva Rodansky, Luz P. Blanco, Justin D. Mih, Daniel Tschumperlin, Peter D. Higgins Background: Crohn's disease is characterized by repeated cycles of inflammation and mucosal healing which ultimately progresses to intestinal fibrosis. This inexorable progression towards fibrosis suggests that fibrosis becomes inflammation-independent and auto-propagative. We hypothesized that resident colonic fibroblasts detect increased stiffening of extracellular matrix and respond with increased proliferation, a transition to myofibroblast-like morphology, and increased production of extracellular matrix components which further stiffens the matrix, creating a fibrogenesis positive feedback loop. Methods: The stiffness of fresh ex vivo small intestine from normal human small intestine and ex vivo Crohn's disease strictures were measured with a microelastometer. Collagen-coated polyacrylamide matrices corresponding to the physiological stiffness of normal (compliant) or Crohn's (fibrotic) small intestine were used as models of the intestinal myofibroblast environment. Normal human colonic fibroblasts (Ccd-18co cells) were grown on physiologically normal (0.01%, 0.02% bis-acrylamide) or pathologically stiff (0.08%, 0.16%) matrices. Cellular response was assayed for changes in cell morphology, a-smooth muscle actin (αSMA) staining, and gene expression. Results: Microelastometer measurements revealed a significant increase in colonic tissue stiffness between both normal human colon and Crohn's stricture small intestine as well as between the stricture and adjacent tissue margin. In CCD-18co cells grown on stiff matrices corresponding to Crohn's strictures, cellular proliferation increased 2 to 6-fold with increasing stiffness. Increased stiffness induced a marked change in cell morphology, to larger, stellatetype cells, with multiple dendritic processes. This morphological change was accompanied by a corresponding increase in αSMA+ bundles. Concurrent with the stiffness-induced morphological changes, growth on a stiff matrix caused increases in the expression of profibrotic (CTGF, collagen I, endothelin I, Lox) genes, and decreases in matrix proteinases (MMP1, MMP10, cathepsin O, YKL-40) and pro-inflammatory genes (IL-1β, Cox-2). Conclusions: Human intestinal fibroblasts sense changes in the matrix stiffness within a physiologically relevant range. Stiff matrices, corresponding to the stiffness of Crohn's strictures, increase cell proliferation, induce a myofibroblast-like morphology, and induce pro-fibrotic gene expression while repressing matrix remodeling genes. This may contribute to a positive feedback loop of fibrogenesis and auto-propagation of fibrosis.
Mo1783 Induction of Autophagy Increases Intestinal Injury in a Rat Model of Necrotizing Enterocolitis Christine Coursodon, Chelsea Snarrenberg, Camille Adkins-Rieck, Katerina Dvorak, Bohuslav Dvorak Background: Neonatal necrotizing enterocolitis (NEC) is a major cause of morbidity and mortality in premature infants. Autophagy is a mechanism regulating a normal turnover of cytosolic components, but is also rapidly up regulated under conditions leading to cellular stress. While a basal level of autophagy maintains normal cellular homeostasis, uncontrolled activation of autophagy leads to excessive degradation of cellular content and cell death. Previously we have shown increased autophagy in the intestinal epithelium of NEC patients and in the ileum of NEC rats. However, the role of autophagy in NEC pathogenesis remains still unclear. Aim: To evaluate the effect of a well-established inducer of autophagy Rapamycin - on development of NEC injury, protein expression of autophagic and apoptotic regulators in the rat NEC model. Methods: Premature rats were divided into the following groups: hand-fed with formula (NEC), or hand-fed with formula with 200ng/rat/day of Rapamycin (RAP). Both groups were exposed to asphyxia/cold stress to develop NEC. After 96 hours, all animals were sacrificed and ileal tissue was collected. Incidence and severity of NEC injury was evaluated. Protein levels of autophagy (Beclin 1, LC3 and p62) and apoptosis (Bcl-2 and Bcl-w) regulators were measured in the terminal ileum. Results: Administration of Rapamycin significantly increased the incidence of NEC injury from 50%
Mo1786 Spironolactone Inhibits TGFβ-Induced Fibrogenisis In Vitro but is Associated With Increased Mortality In Vivo Laura A. Johnson, Shail M. Govani, Joel Joyce, Peter D. Higgins Background: The renin-angiotensin-aldosterone system (RAAS) has been implicated in cardiac fibrosis in heart failure. Spironolactone (SPIR), an aldosterone antagonist, prevents fibrosis and mortality in patients with heart failure. Crohn's disease causes intestinal fibrosis, leading to strictures and obstruction. Studies of angiotensin-2 receptor antagonists in rodent
S-649
AGA Abstracts
AGA Abstracts
Mo1781