Protective effect of magnesium sulfate on cranial nerves ...
T1684 Protective Role for Matriptase During DSS-Induced Colitis
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Insulin-like Growth Factor (IGFBP)-3 expression increases 3.6 fold in smooth muscle cells of stricturing Crohn's disease compared to normal margins and that the increase in endogenous IGFBP-3 stimulates collagen I production. IGFBP-3 binds to the TGF-βRI/II receptor complex in these cells. AIM: To identify the TGF-βRI/II-dependent signaling pathways activated by IGFBP-3 that regulate collagen I expression and production in human intestinal smooth muscle cells. METHODS: Normal human intestinal smooth muscle cells were isolated from the muscularis propria of patients undergoing bariatric surgery and used to prepare RNA, cell lysates, or placed into primary culture. Collagen I, smad2 and smad3 mRNA expression was measured by quantitative-PCR. Collagen I production was measured by immunoblot analysis in quiescent cells incubated with rhIGFBP-3 (100ng/ml) alone or in the presence of various test agents; a TGF-βRI kinase inhibitor (500nM), the smad2/3 inhibitor SB-431542 (10μM), or an IGFBP-3 neutralizing antibody (20μg/ml). Activation of smad3 was measured by immunoblot analysis using a smad3(Ser423/425) phospho-specific antibody. RESULTS: Incubation of normal human intestinal smooth muscle cells with rhIGFBP-3 increased smad3 expression by 1.6 ± 0.1 fold and increased smad3 phosphorylation by 36 ± 8‰ above basal. Smad2 mRNA levels were unchanged. The IGFBP-3-dependent increase in collagen I mRNA expression and protein production was inhibited 25 ± 19‰ and 32‰, respectively, by the TGF-βRI kinase inhibitor, and 43 ± 30‰ and 43 ± 16‰, respectively, by the smad2/3 inhibitor SB-431542. Incubation of normal intestinal smooth muscle cells with a neutralizing antibody to IGFBP-3 decreased basal smad3 and collagen I mRNA expression by 38 ± 4‰ and 47 ± 4‰, respectively, implying that endogenous IGFBP-3 regulates smad3 and collagen I mRNA expression. CONCLUSIONS: IGFBP-3 regulates collagen I expression and production by activation of the TGF-βRI/II receptors and downstream smad3 phos- phorylation in human intestinal smooth muscle cells. The upregulation of IGFBP-3 expression in smooth muscle cells of the muscularis propria in Crohn's disease stimulates excess collagen I production and contributes to stricture formation. T1681 Inflammation-Induced Mucosal Endothelial-to-Mesenchymal Transition (EndoMT): A Novel Mechanism Contributing to the Development of Intestinal Fibrosis Florian Rieder, Sean Kessler, Franco Scaldaferri, Anja Schirbel, Gail West, Carol de La Motte, Claudio Fiocchi BACKGROUND: Intestinal fibrosis is a serious complication of inflammatory bowel disease (IBD). Recent evidence indicates that non-mesenchymal cells can contribute to fibrosis through the process of EndoMT. We investigated whether human intestinal microvascular endothelial cells (HIMEC) can undergo EndoMT under inflammatory conditions as found in IBD. METHODS: HIMEC from control (n=8) and IBD mucosa (n=8) were cultured in the presence of TGF-β1, IL-1β and TNF-α, alone or in combination, or with supernatants of resting or activated lamina propria mononuclear cells (LPMC). Cells were evaluated for morphological, phenotypic, molecular and functional changes before and after treatment. The occurrence of EndoMT was also assessed in situ by immunofluorescence in human and murine inflamed colonic tissues. RESULTS: When exposed to a combination of TGF-β1, IL-1β and TNF-α, or activated LPMC supernatants, HIMEC acquired a spindle-shape mor- phology, and increased and rearranged actin fibers like those found in intestinal myofibrob- lasts. These changes were accompanied by a progressive loss of typical endothelial cell markers, such as CD31, vWF, and VE-cadherin, and the concomitant acquisition of typical cytoskeletal components of intestinal myofibroblasts, such as FSP-1, N-cadherin and α- SMA, as demonstrated by immunocytochemistry, electron microscopy and immunoblotting. The newly acquired morphologic and phenotypic features were observed in both control and IBD HIMEC, were stable for at least 10 days, and persisted after the removal of the inducing agents. Importantly, the above changes were accompanied by functional differenti- ation, verified by a dramatic reduction in acetylated LDL-uptake, reduced migratory capacity, increased production of fibronectin, and de novo synthesis of collagen I as compared to untreated HIMEC. That EndoMT had occurred was further confirmed by comparative mic- roarray genomic analysis, which showed a transformation of HIMEC into a profibrogenic phenotype. This was validated by quantitative PCR showing upregulation of collagen I A2, tenascin C, and FSP-1, and downregulation of vWF and entactin. Finally, evidence for EndoMT was detected in situ in mucosal tissue involved by active IBD as shown by the co-localization of α-SMA and vWF in inflamed vessels, as well as in an endothelial cell-specific (Tie-2/ lacZ) reporter mouse with TNBS colitis. CONCLUSION: Under inflammatory conditions mucosal microvascular endothelial cells undergo EndoMT In Vitro and In Vivo. Therefore, mucosal EndoMT represents a novel, inflammation-driven mechanism that contributes to the emergence of intestinal fibrosis during the course of IBD. T1682 TAK1 Expression in Ileal Mucosa and Sub-Epithelial Myofibroblasts Is Correlated to a Pro-Fibrogenic Phenotype in Patients with Crohn's Disease Alessia R. Grillo, Melania Scarpa, Laura Nai, Paola Brun, Marco Scarpa, Imerio Angriman, Andrea Buda, Renata D'Incà, Giacomo C. Sturniolo, Diego Martines, Ignazio Castagliuolo Introduction: Thirty percent of Crohn's disease (CD) patients have a fibrotic phenotype causing recurrent ileal stenosis. Although intestinal sub-epithelial myofibroblasts (ISEFMs) in CD show an enhanced ability to produce and reorganize extracellular matrix proteins, the molecular mechanisms inducing this fibrogenic phenotype are not known. TAK1 (transformig growth factor activated kinase1) is a MAP3K activated by various factors able to ignite different signal transduction pathways. Since previous studies have linked TAK1 activity to hearth and kidney fibrosis, we investigated the expression of TAK1 in CD patients and assessed whether TAK1 is involved in ISEMFs fibrogenic phenotype. Methods: TAK1 and its activate posphorylated form (pTAK1) expression in ileum surgical specimens from CD and control patients and in primary ISEMFs were assessed by Western Blot and double labelling confocal immunohistochemistry (CIF). Level of TAK1 mRNA was determined in ileal biopsies from CD (n=17, without clinical and endoscopic signs of stenosis), control (n=14) and colitis (n=9) and correlated to fibrosis markers: pro-collagen1a, Timp1 and αSMA. The functional role of TAK1 in ISEMFs collagen synthesis, cellular migration (by quantitative RT-PCR and 3H-proline incorporation) and cell migration (in a wound healing A-557 AGA Abstracts assay) was determined using a specific Tak1 inhibitor and sh-RNA silencing. Results: TAK1 and p-TAK1 levels, as assessed by WB and CIF, were increased in CD patients as compared to controls. TAK1 and p-TAK mainly localized in lamina propria cells, identified as ISEMFs (αSMA positive). TAK1 mRNA increased 4,8-fold (p=0,01) as compared with controls and directly correlated with fibrotic marker expression (Tak1/pro-col1a R=0.370 p=0.022, Tak1/ αSMA R=0.420 p=0.010). Primary ISEMFs from CD patients (n=4) showed higher TAK1 and pTAK1 expression, associated to increased pro-collagen1a mRNA levels (p=0,01) versus control cells (n=5). TAK1 activation by TGF-β occurred within 30 min and was prevented by a TGF-βR inhibitor. ISEMFs treatment with a TAK1 inhibitor or TAK-1 silencing with sh-RNA significantly inhibited basal and TGF-β1 induced collagen production (by RT-PCR and 3H-proline incorporation) and migration. Summary and conclusions: We report here that: 1)TAK1 and pTAK1 are up-regulated in ISEMFs of CD patients, 2) TAK1 expression correlates with marker of fibrosis in ileal biopsies of patients without signs of stenosis, 3) TAK1 activation is required to induce a fibrogenic phenotype in ISEMFs. We speculate that TAK1 activation is a key event in the development of ileal fibrosis and may represent a marker for the development of ileal stenosis. T1683 5-ASA Impacts PI3 Kinase-Mediated β-Catenin Signaling Elizabeth Managlia, Rebecca B. Katzman, Terrence A. Barrett Background and Aims: Nuclear accumulation of βcatenin is an established indicator of activated intestinal epithelial progenitor cells. A novel antibody specific for nuclear β-catenin phosphorylated by Akt (pβ-catenin) suggests the cooperation of PI3K in activating β-catenin signaling in this cell population (He et al Nat Gen 2007). Many sporadic colorectal cancers (CRC) show evidence of dysregulation in these pathways and previous data from our lab showing induction of β-catenin target genes in colitis supports the idea that chronic intestinal inflammation activates epithelial progenitor cells. Human and mouse data suggest 5-ASA reduces colitis-induced cancer but it is unclear whether this impact is due to direct effects on epithelial signaling and/or indirect changes mediated by reduction in tissue inflammation. To examine the direct affect of 5-ASA on epithelial β-catenin signaling, HCT116 (a colon cancer cell line that exhibits high basal levels of PI3K and β-catenin signaling) cells were used. To address the clinical relevance, pβ-catenin levels were assessed in biopsies from patients with and without 5-ASA therapy. Methods and Results: Cells were plated to 80% confluency, serum starved for 16 hours and pre-treated with 30mM 5-ASA for 1 hour or left untreated. Cells were then stimulated with conditioned media for 2 hours and protein was extracted or stimulated for 24 hours to isolate RNA for low density cDNA microarrays. Western blots of p-AKT showed a 1.7-fold decrease with 5-ASA pre-treatment and pβ- catenin decreased by 2.6-fold. Microarray analyses indicated decreases in the β-catenin target genes c-myc(-2.0), cyclin D1(-1.6), and CD44v6(-1.7) as well as the PI3K related genes amphiregulin (-3.4), IGF2(-1.7) and PDGFa (-3.4). Analysis of p-AKT and pβ-catenin protein levels from human biopsies showed an average decrease of 1.4-fold and 2.4-fold respectively in patients taking 5-ASA compared to untreated colitis. Conclusion: These data suggest 5- ASA treatment reduces p-Akt and Akt phosphorylated β-catenin within activated epithelial progenitors in colitis. Data in HCT116 cells suggest that 5-ASA-mediated inhibition of PI3K signaling attenuates downstream activation of β-catenin and transcription of target genes. These data support the notion that the chemopreventative effect of 5-ASA may not be due solely to its anti-inflammatory properties, but also to an impact on epithelial progenitor cells. T1684 Protective Role for Matriptase During DSS-Induced Colitis Sarah Netzel-Arnett, Terez Shea-Donohue, Marguerite S. Buzza, Luigi Notari, Aiping Zhao, Rex Sun, Alessio Fasano, Toni Antalis Inflammatory bowel diseases (IBD) are chronic intestinal disorders whose etiology is not completely understood. Matriptase is a transmembrane serine protease highly expressed in epithelium all along the gastrointestinal (GI) tract whose function is unknown. Matriptase has been shown to activate several known regulators of GI pathophysiology including protease-activated receptor-2 (PAR2), urokinase-type plasminogen activator (uPA), and pro- hepatocyte growth factor (proHGF) suggesting it may play a functional role in intestinal physiology and diseases of the GI tract. Aim: To investigate the role of matriptase in GI pathophysiology and inflammation. Methods: IBD patient tissue specimens, including Crohn's disease (CD) and ulcerative colitis (UC) were compared with normal controls for matriptase mRNA levels by quantitative PCR. The functional role of matriptase was tested in a mouse model of intestinal injury and inflammation induced by oral administration of dextran sodium sulfate (DSS). Since the matriptase knockout mouse is perinatal lethal, a matriptase hypomorphic mouse strain expressing less than 1% matriptase mRNA levels and their littermate controls were used in these studies. Clinical symptoms including weight loss, diarrhea, and bleeding were monitored daily. Inflammation and damage to the colonic mucosa were assessed by histology. Changes in mucosal permeability were determined by transepithelial electrical resistance (TEER) of ex vivo intestinal tissues. Results: We found that in colonic specimens of CD and UC patients, disease is associated with reduced levels of matriptase expression compared to healthy controls. In the mouse model, decreased matriptase expression results in a dramatic increase in susceptibility to DSS-induced colitis as assessed by survival, clinical symptoms, and histopathology. When mice were treated with DSS for 7 days followed by water without DSS, 70% of matriptase hypomorphic mice died by day 13, while 100% of control mice survived DSS-induced injury. The clinical disease score on Day 11 was 10.4 ± 1.0 for the hypomorphic group compared with. 4.7 ± 0.3 for the control group. The matriptase hypomorphic mice also displayed an increase in intestinal permeability relative to their littermate controls. Conclusions: The reduction in matriptase expression in IBD patients and the increased susceptibility of matriptase hypo- morphic mice to DSS-induced colitis suggests disease severity may be functionally associated with low matriptase levels. Our studies demonstrate a protective role for the serine protease matriptase in the GI tract and its potential deregulation in the pathogenesis of IBD. AGA Abstracts
Transcript of T1684 Protective Role for Matriptase During DSS-Induced Colitis
Insulin-like Growth Factor (IGFBP)-3 expression increases 3.6 fold in smooth muscle cells ofstricturing Crohn's disease compared to normal margins and that the increase in endogenousIGFBP-3 stimulates collagen I production. IGFBP-3 binds to the TGF-βRI/II receptor complexin these cells. AIM: To identify the TGF-βRI/II-dependent signaling pathways activated byIGFBP-3 that regulate collagen I expression and production in human intestinal smoothmuscle cells. METHODS: Normal human intestinal smooth muscle cells were isolated fromthe muscularis propria of patients undergoing bariatric surgery and used to prepare RNA,cell lysates, or placed into primary culture. Collagen I, smad2 and smad3 mRNA expressionwas measured by quantitative-PCR. Collagen I production was measured by immunoblotanalysis in quiescent cells incubated with rhIGFBP-3 (100ng/ml) alone or in the presenceof various test agents; a TGF-βRI kinase inhibitor (500nM), the smad2/3 inhibitor SB-431542(10μM), or an IGFBP-3 neutralizing antibody (20μg/ml). Activation of smad3 was measuredby immunoblot analysis using a smad3(Ser423/425) phospho-specific antibody. RESULTS:Incubation of normal human intestinal smooth muscle cells with rhIGFBP-3 increased smad3expression by 1.6 ± 0.1 fold and increased smad3 phosphorylation by 36 ± 8‰ abovebasal. Smad2 mRNA levels were unchanged. The IGFBP-3-dependent increase in collagenI mRNA expression and protein production was inhibited 25 ± 19‰ and 32‰, respectively,by the TGF-βRI kinase inhibitor, and 43 ± 30‰ and 43 ± 16‰, respectively, by thesmad2/3 inhibitor SB-431542. Incubation of normal intestinal smooth muscle cells with aneutralizing antibody to IGFBP-3 decreased basal smad3 and collagen I mRNA expressionby 38 ± 4‰ and 47 ± 4‰, respectively, implying that endogenous IGFBP-3 regulates smad3and collagen I mRNA expression. CONCLUSIONS: IGFBP-3 regulates collagen I expressionand production by activation of the TGF-βRI/II receptors and downstream smad3 phos-phorylation in human intestinal smoothmuscle cells. The upregulation of IGFBP-3 expressionin smooth muscle cells of the muscularis propria in Crohn's disease stimulates excess collagenI production and contributes to stricture formation.
T1681
Inflammation-Induced Mucosal Endothelial-to-Mesenchymal Transition(EndoMT): A Novel Mechanism Contributing to the Development of IntestinalFibrosisFlorian Rieder, Sean Kessler, Franco Scaldaferri, Anja Schirbel, Gail West, Carol de LaMotte, Claudio Fiocchi
BACKGROUND: Intestinal fibrosis is a serious complication of inflammatory bowel disease(IBD). Recent evidence indicates that non-mesenchymal cells can contribute to fibrosisthrough the process of EndoMT. We investigated whether human intestinal microvascularendothelial cells (HIMEC) can undergo EndoMT under inflammatory conditions as foundin IBD. METHODS: HIMEC from control (n=8) and IBD mucosa (n=8) were cultured inthe presence of TGF-β1, IL-1β and TNF-α, alone or in combination, or with supernatantsof resting or activated lamina propria mononuclear cells (LPMC). Cells were evaluated formorphological, phenotypic, molecular and functional changes before and after treatment.The occurrence of EndoMT was also assessed in situ by immunofluorescence in human andmurine inflamed colonic tissues. RESULTS: When exposed to a combination of TGF-β1,IL-1β and TNF-α, or activated LPMC supernatants, HIMEC acquired a spindle-shape mor-phology, and increased and rearranged actin fibers like those found in intestinal myofibrob-lasts. These changes were accompanied by a progressive loss of typical endothelial cellmarkers, such as CD31, vWF, and VE-cadherin, and the concomitant acquisition of typicalcytoskeletal components of intestinal myofibroblasts, such as FSP-1, N-cadherin and α-SMA, as demonstrated by immunocytochemistry, electron microscopy and immunoblotting.The newly acquired morphologic and phenotypic features were observed in both controland IBD HIMEC, were stable for at least 10 days, and persisted after the removal of theinducing agents. Importantly, the above changes were accompanied by functional differenti-ation, verified by a dramatic reduction in acetylated LDL-uptake, reduced migratory capacity,increased production of fibronectin, and de novo synthesis of collagen I as compared tountreated HIMEC. That EndoMT had occurred was further confirmed by comparative mic-roarray genomic analysis, which showed a transformation of HIMEC into a profibrogenicphenotype. This was validated by quantitative PCR showing upregulation of collagen I A2,tenascin C, and FSP-1, and downregulation of vWF and entactin. Finally, evidence for EndoMTwas detected in situ in mucosal tissue involved by active IBD as shown by the co-localizationof α-SMA and vWF in inflamed vessels, as well as in an endothelial cell-specific (Tie-2/lacZ) reporter mouse with TNBS colitis. CONCLUSION: Under inflammatory conditionsmucosal microvascular endothelial cells undergo EndoMT In Vitro and In Vivo. Therefore,mucosal EndoMT represents a novel, inflammation-driven mechanism that contributes tothe emergence of intestinal fibrosis during the course of IBD.
T1682
TAK1 Expression in Ileal Mucosa and Sub-Epithelial Myofibroblasts IsCorrelated to a Pro-Fibrogenic Phenotype in Patients with Crohn's DiseaseAlessia R. Grillo, Melania Scarpa, Laura Nai, Paola Brun, Marco Scarpa, Imerio Angriman,Andrea Buda, Renata D'Incà, Giacomo C. Sturniolo, Diego Martines, Ignazio Castagliuolo
Introduction: Thirty percent of Crohn's disease (CD) patients have a fibrotic phenotypecausing recurrent ileal stenosis. Although intestinal sub-epithelial myofibroblasts (ISEFMs)in CD show an enhanced ability to produce and reorganize extracellular matrix proteins, themolecular mechanisms inducing this fibrogenic phenotype are not known. TAK1 (transformiggrowth factor activated kinase1) is a MAP3K activated by various factors able to ignitedifferent signal transduction pathways. Since previous studies have linked TAK1 activity tohearth and kidney fibrosis, we investigated the expression of TAK1 in CD patients andassessed whether TAK1 is involved in ISEMFs fibrogenic phenotype. Methods: TAK1 andits activate posphorylated form (pTAK1) expression in ileum surgical specimens from CDand control patients and in primary ISEMFs were assessed by Western Blot and doublelabelling confocal immunohistochemistry (CIF). Level of TAK1 mRNA was determined inileal biopsies from CD (n=17, without clinical and endoscopic signs of stenosis), control(n=14) and colitis (n=9) and correlated to fibrosis markers: pro-collagen1a, Timp1 andαSMA. The functional role of TAK1 in ISEMFs collagen synthesis, cellular migration (byquantitative RT-PCR and 3H-proline incorporation) and cell migration (in a wound healing
A-557 AGA Abstracts
assay) was determined using a specific Tak1 inhibitor and sh-RNA silencing. Results: TAK1and p-TAK1 levels, as assessed by WB and CIF, were increased in CD patients as comparedto controls. TAK1 and p-TAK mainly localized in lamina propria cells, identified as ISEMFs(αSMA positive). TAK1 mRNA increased 4,8-fold (p=0,01) as compared with controls anddirectly correlated with fibrotic marker expression (Tak1/pro-col1a R=0.370 p=0.022, Tak1/αSMA R=0.420 p=0.010). Primary ISEMFs from CD patients (n=4) showed higher TAK1and pTAK1 expression, associated to increased pro-collagen1a mRNA levels (p=0,01) versuscontrol cells (n=5). TAK1 activation by TGF-β occurred within 30 min and was preventedby a TGF-βR inhibitor. ISEMFs treatment with a TAK1 inhibitor or TAK-1 silencing withsh-RNA significantly inhibited basal and TGF-β1 induced collagen production (by RT-PCRand 3H-proline incorporation) and migration. Summary and conclusions: We report herethat: 1)TAK1 and pTAK1 are up-regulated in ISEMFs of CD patients, 2) TAK1 expressioncorrelates with marker of fibrosis in ileal biopsies of patients without signs of stenosis, 3)TAK1 activation is required to induce a fibrogenic phenotype in ISEMFs. We speculate thatTAK1 activation is a key event in the development of ileal fibrosis and may represent amarker for the development of ileal stenosis.
T1683
5-ASA Impacts PI3 Kinase-Mediated β-Catenin SignalingElizabeth Managlia, Rebecca B. Katzman, Terrence A. Barrett
Background and Aims: Nuclear accumulation of βcatenin is an established indicator ofactivated intestinal epithelial progenitor cells. A novel antibody specific for nuclear β-cateninphosphorylated by Akt (pβ-catenin) suggests the cooperation of PI3K in activating β-cateninsignaling in this cell population (He et al Nat Gen 2007). Many sporadic colorectal cancers(CRC) show evidence of dysregulation in these pathways and previous data from our labshowing induction of β-catenin target genes in colitis supports the idea that chronic intestinalinflammation activates epithelial progenitor cells. Human and mouse data suggest 5-ASAreduces colitis-induced cancer but it is unclear whether this impact is due to direct effectson epithelial signaling and/or indirect changes mediated by reduction in tissue inflammation.To examine the direct affect of 5-ASA on epithelial β-catenin signaling, HCT116 (a coloncancer cell line that exhibits high basal levels of PI3K and β-catenin signaling) cells wereused. To address the clinical relevance, pβ-catenin levels were assessed in biopsies frompatients with and without 5-ASA therapy. Methods and Results: Cells were plated to 80%confluency, serum starved for 16 hours and pre-treated with 30mM 5-ASA for 1 hour orleft untreated. Cells were then stimulated with conditioned media for 2 hours and proteinwas extracted or stimulated for 24 hours to isolate RNA for low density cDNA microarrays.Western blots of p-AKT showed a 1.7-fold decrease with 5-ASA pre-treatment and pβ-catenin decreased by 2.6-fold. Microarray analyses indicated decreases in the β-catenin targetgenes c-myc(-2.0), cyclin D1(-1.6), and CD44v6(-1.7) as well as the PI3K related genesamphiregulin (-3.4), IGF2(-1.7) and PDGFa (-3.4). Analysis of p-AKT and pβ-catenin proteinlevels from human biopsies showed an average decrease of 1.4-fold and 2.4-fold respectivelyin patients taking 5-ASA compared to untreated colitis. Conclusion: These data suggest 5-ASA treatment reduces p-Akt and Akt phosphorylated β-catenin within activated epithelialprogenitors in colitis. Data in HCT116 cells suggest that 5-ASA-mediated inhibition of PI3Ksignaling attenuates downstream activation of β-catenin and transcription of target genes.These data support the notion that the chemopreventative effect of 5-ASA may not be duesolely to its anti-inflammatory properties, but also to an impact on epithelial progenitor cells.
T1684
Protective Role for Matriptase During DSS-Induced ColitisSarah Netzel-Arnett, Terez Shea-Donohue, Marguerite S. Buzza, Luigi Notari, AipingZhao, Rex Sun, Alessio Fasano, Toni Antalis
Inflammatory bowel diseases (IBD) are chronic intestinal disorders whose etiology is notcompletely understood. Matriptase is a transmembrane serine protease highly expressed inepithelium all along the gastrointestinal (GI) tract whose function is unknown. Matriptasehas been shown to activate several known regulators of GI pathophysiology includingprotease-activated receptor-2 (PAR2), urokinase-type plasminogen activator (uPA), and pro-hepatocyte growth factor (proHGF) suggesting it may play a functional role in intestinalphysiology and diseases of the GI tract. Aim: To investigate the role of matriptase in GIpathophysiology and inflammation. Methods: IBD patient tissue specimens, includingCrohn's disease (CD) and ulcerative colitis (UC) were compared with normal controls formatriptase mRNA levels by quantitative PCR. The functional role of matriptase was testedin a mouse model of intestinal injury and inflammation induced by oral administration ofdextran sodium sulfate (DSS). Since the matriptase knockout mouse is perinatal lethal, amatriptase hypomorphic mouse strain expressing less than 1% matriptase mRNA levels andtheir littermate controls were used in these studies. Clinical symptoms including weightloss, diarrhea, and bleeding were monitored daily. Inflammation and damage to the colonicmucosa were assessed by histology. Changes in mucosal permeability were determined bytransepithelial electrical resistance (TEER) of ex vivo intestinal tissues. Results: We foundthat in colonic specimens of CD and UC patients, disease is associated with reduced levelsof matriptase expression compared to healthy controls. In the mouse model, decreasedmatriptase expression results in a dramatic increase in susceptibility to DSS-induced colitisas assessed by survival, clinical symptoms, and histopathology. When mice were treatedwith DSS for 7 days followed by water without DSS, 70% of matriptase hypomorphic micedied by day 13, while 100% of control mice survived DSS-induced injury. The clinicaldisease score on Day 11 was 10.4 ± 1.0 for the hypomorphic group compared with. 4.7 ±0.3 for the control group. The matriptase hypomorphic mice also displayed an increase inintestinal permeability relative to their littermate controls. Conclusions: The reduction inmatriptase expression in IBD patients and the increased susceptibility of matriptase hypo-morphic mice to DSS-induced colitis suggests disease severity may be functionally associatedwith low matriptase levels. Our studies demonstrate a protective role for the serine proteasematriptase in the GI tract and its potential deregulation in the pathogenesis of IBD.
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