Su1903 The Trans-Golgi Network Protein Aftiphilin Is Involved in Regulation of Intestinal Epithelial Permeability in Colonic Epithelial Cells

to be acid sensitive2, both receptors were targeted in RE by STW5 and O. Data further substantiate differential MAPKinase signaling in GERD. The present data might contripute to our understanding of the pathogenesis of the disease. 1Abdel-Aziz et al. DDW 2013

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Heme Oxygease-1 (HO-1) Prevents Intestinal Ischemia-Reperfusion Injury (IRI) Through Its Regulation of the Inflammasome Yusuke Horii, Kazuhiko Uchiyama, Yuma Hotta, Atsushi Majima, Toshifumi Doi, Makoto Tanaka, Yuriko Onozawa, Kentaro Suzuki, Yukiko Uehara, Hideki Horie, Osamu Dohi, Tetsuya Okayama, Naohisa Yoshida, Kazuhiro Katada, Kazuhiro Kamada, Takeshi Ishikawa, Osamu Handa, Tomohisa Takagi, Hideyuki Konishi, Yuji Naito, Akihiko Muto, Kazuhiko Igarashi, Yoshito Itoh Introduction Small intestine ischemia-reperfusion injury (IRI) is caused by such as mesenteric artery occlusion and cardiovascular surgery. If adequate treatment is carried out, it causes serious conditions such as multiple organ failure. IRI is characterized by inflammation due to oxidative stress and leukocyte recruitment. The Nlrp3 inflammasome plays a central role in the induction of inflammatory responses through the secretion of IL-1b. On the other hand, it has been reported that the antioxidant enzymes heme oxygease-1 (HO-1) and carbon monoxide (CO) which is a by-product controls the various inflammatory conditions. However, the role of the inflammasome and the mechanisms whereby HO-1 controls the inflammasome in intestinal IRI remain unclear. Methods To investigate the role of HO-1 in the control of the Nlrp3 inflammasome and disease, IRI was inducted in Bach1 KO mice by occluding the superior mesenteric artery for 45 min. Inflammatory responses in the small intestine were assessed 4h following reperfusion by measuring mucosal IL-1b, caspase1, Nlrp3 mRNA and protein expression by RT PCR and Western blotting, respectively. Results IRI was significantly attenuated in the absence of Bach1, and the protective effect was partially abolished by the co-administration of the HO-1 inhibitor (tin protoporphyrin (SnPP) (20mg/ kg)). IL-1b mRNA was increased significantly during IRI in the intestinal mucosa of WT mice but not in Bach1 KO mice (66.5% decrease p<0.05). IL-1b and caspase-1 protein were expressed by IRI in the intestinal mucosa of WT mice but not in Bach1 KO mice. The inhibitory effect of IL-1b mRNA in Bach1 KO mice was partially abolished by the administration of the HO-1 inhibitor. Conclusions These findings suggest that HO-1 modulates the small intestinal IRI through its ability to regulate the Nlrp3 inflammasome.

Modulation of GPR84, LOX-1 and mitogen-activated protein kinases in esophageal tissue Su1900 Portal Triad Clamping in Rats, BPC 157 Acutely Protect Blood Vessels in the Intestine and Caecum, and Counteracts Intestinal Lesions. A Comparison With L-NAME and L-Arginine Marijan Kolovrat, Antonija Djuzel, Marko Sever, Tamara Kralj, Katarina Kasnik, Jaksa Vukojevic, Dora Zaler, Vedrana Zivkovic, Domagoj Drmic, Danijela Kolenc, Gorana Aralica, Sven Seiwerth, Predrag Sikiric

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Background We suggested that the rescue of the blood vessels may be essential for the stable gastric pentadecapeptide BPC 157 as therapy in ulcerative colitis (Curr Med Chem. 2012;19(1):126-32.; Curr Pharm Des. 2011;17(16):1612-32.). Here, we hypothesized that BPC 157 rescues blood vessels in intestinal ischemia injury caused by the portal triad clamping. Of note, depending about the ischemia attenuation or aggravation, the number of blood vessels due to more or less branching may correspondingly decreased (aggravated ischemia) or increase (attenuated ischemia, and rescued blood vessels maintenance), and likewise, anastomosis formation should additionally contribute decreased or increased number of blood vessels presented. A comparison was carried out with the effect of the L-NAME, and L-arginine given alone and/or together. Methods In deeply anaesthetised rats, throughout 30min of the portal triad clamping, in the minute intervals, the assessment involved the most distal part of the ileum, and caecum, 1 cm long, four neighboring vessels where the all arising branches including formed anastomosis between them were all assessed as the total blood vessels number. Besides, medication (/kg) as bath at the clamped area (BPC 157 (10μg, 10ng), L-NAME (5mg), L-arginine (100mg), alone and/or together, was applied at 5min after the portal triad clamping. Results/Discussion At the 30min of the portal triad clamping, controls were presented with fewer branching and anastomosis (intestinum 14.3±1.2; caecum 122.5±5.2), BPC 157 with more branching and anastomosis (intestinum 27.5±3.2; caecum 154.5±7.2, both P<0.05, at least vs. control), markedly less intestine congestion along with few microscopical findings (Figure 1). NO-system agents presented different results, L-arginine a decrease (intestinum 9.2±1.0; caecum 63.3±6.6, both P<0.05, at least vs. control); L-NAME an effect on caecum (intestinum 13.3±2.2; caecum 102.5±5.2, P<0.05, at least vs. control); L-NAME+L-arginine (intestinum 6.3±1.0; caecum 147.5±5.2 both P<0.05, at least vs. control) suggest that these effects are at least partly NO-system related; BPC 157 overcomes the effect of L-arginine (BPC 157+L-arginine (intestinum 33.3±2.2; caecum 183.5±5.2 both P<0.05, at least vs. control); BPC 157+L-arginine+LNAME (intestinum 21.8±2.2; caecum 165.5±5.2 both P<0.05, at least vs. control); its effect was however counteracted by L-NAME: BPC 157+L-NAME (intestinum 14.5±2.2; caecum 106.5±5.2). Conclusions BPC 157 could be used along with portal triad clamping, and may rescue blood vessels in both intestine and caecum, an effect at least partly related to NO-system.

MMP-12 Modulation of Intestinal Epithelial Tight Junction Barrier Is Mediated by P38 Kinase Activation of Myosin Light Chain Kianse (MLCK) Rana Al-Sadi, Prashant K. Nighot, Thomas Y. Ma Background: Matrix metalloproteinase-12 (MMP-12) is an elastase that is engaged in the degradation and remodeling of extracellular matrix (ECM) and regulates ECM homeostasis. MMP-12 levels are markedly elevated in intestinal tissue of patients with inflammatory bowel disease (IBD) and closely correlate with the disease activity and degree of inflammation. IBD patients have a defective intestinal tight junction (TJ) barrier manifested by an increase in intestinal permeability and elevated intestinal tissue and serum levels of bacterial antigens. The role of MMP-12 in modulating intestinal epithelial TJ permeability remains unclear. Aims: the major aims of this study are to characterize the MMP-12 effect on the intestinal TJ barrier function and to elucidate the intracellular mechanisms involved. Methods: Filtergrown Caco-2 monolayers were used as an in-vitro intestinal epithelial model system. Various cell biology and molecular methodologies were utilized. Results: 1) MMP-12 (500 ng/ml) caused a time- and dose-dependent drop in Caco-2 transepithelial resistance (TER) and increase in Caco-2 inulin flux. 2) MMP-12 induced increase in Caco-2 TJ permeability was associated with an increase in myosin light chain kinase (MLCK) protein expression. 3) Inhibition of MLCK with pharmacologic inhibitor ML-7 (10 mM) prevented the MMP-12 induced drop in Caco-2 TER and increase in inulin flux. 4) Moreover, siRNA induced MLCK silencing inhibited the MMP-12 induced increase in Caco-2 TJ permeability. 5) MMP-12 caused a rapid activation of p38 kinase in Caco-2 monolayers. 6) Inhibition of p38 kinase (by pharmacologic inhibitor or by siRNA induced silencing) prevented the MMP-12 induced increase in Caco-2 TJ permeability. 7) Inhibition of p38 kinase also prevented the MMP12 induced increase in MLCK protein expression in Caco-2 monolayers. Conclusion: These data show that MMP-12 plays an important role in the modulation of Caco-2 TJ barrier function by regulating the activation of p38 kinase pathway and MLCK expression. Su1903 The Trans-Golgi Network Protein Aftiphilin Is Involved in Regulation of Intestinal Epithelial Permeability in Colonic Epithelial Cells Ivy Ka Man Law, Charalabos Pothoulakis Background and Aims: Aftiphilin (AFTPH) is localized in the trans-golgi network (TGN) and is involved in intracellular trafficking. We have recently found that AFTPH expression is downregulated in colonic epithelial cells in TNBS-induced mouse colitis and patients with ulcerative colitis (Law et al, Gut 2014). However, the role of AFTPH in epithelial cell signaling and colitis are unknown. Since epithelial cell permeability is an important feature of colitis and is related to TGN function, we examined the hypothesis that AFTPH may regulate epithelial cell polarity and permeability during colitis. Methods: AFTPH genesilencing was performed by transfection of small interfering RNA (siRNA) against AFTPH (si-AFTPH) to human NCM460 colonic epithelial cells, while AFTPH overexpression in vitro was by achieved by transduction of lentivirus-expressing AFTPH. Global gene regulation by AFTPH gene silencing in these cells was evaluated using microarray analysis (array type: U133 +2.0). Cellular localization of E-cadherin (CDH1) and tight junction protein ZO1 in NCM460 cells was examined by immunocytochemistry (ICC). Epithelial cell permeability was studied using Dextran, Alexa Fluor® 680 (10 kMW, Life Technologies) and transepithelial electric resistance (TEER) was measured with the Millicell ERS-2 (Millipore). Results: Microarray analysis showed that AFTPH gene silencing in NCM460 cells downregulated expression of genes involved in epithelial cell adhesion signaling (E-catherin and βcatenin among others). AFTPH gene-silencing also reduced the sensitivity to contact inhibition in NCM460 cells. In NCM460 cell during the growth arrest state, AFTPH gene-silencing downregulated ZO-1 protein expression (ICC and Western blot) in cell junctions, while CDH1 showed increased perinuclear localization (ICC). Knock down of AFTPH in NCM460

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cells reduced TEER by 34.3%, and increased dextran permeability (p<0.05), while lentiviral AFTPH overexpression reduced permeability (p<0.05). Conclusions: Our results indicate that gene silencing of AFTPH in human colonocytes increased epithelial permeability, possibly by regulating expression and localization of genes related to epithelial cell adhesion signaling. These are the first results suggesting that AFTPH may be a new gene regulating intestinal epithelial permeability. Acknowledgement: UCLA Vector Core and UCLA Clinical Microarray Core. Supported by NIH grant DK60729 (CP), P50 DK64539 (Project 2, CP), and the Blinder Research Foundation for Crohn's Disease (IKML).

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Background and aims: Non-invasive methods to screen for Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC) are desirable. Metabolomics involves the systematically study of small molecule metabolites that form unique chemical fingerprints associated with specific cellular or biological processes. We aimed to explore the potential role of metabolomic profiling in differentiating BE and EAC from population controls with and without reflux symptoms. Methods: Fasting plasma samples from 40 patients: 10 each with EAC, BE, Gastroesophageal reflux disease (GERD) and Non-GERD controls were obtained for comprehensive metabolomic profiling. All subjects were residents of Olmsted County, MN. Subjects with EAC and BE had both endoscopic and histologic confirmation of diagnosis. EAC subjects had early (6, 60 %) and locally advanced (4, 40 %) disease. All controls had endoscopy which was negative for BE and EAC. All subjects provided information on reflux symptoms by filling out validated reflux symptoms questionnaires. Metabolomic profiles were generated using liquid chromatography- mass spectrometry (LC-MS) analysis with standard methods by investigators who were blinded to the classification of subjects. Data from the plasma samples corresponding to each of the patient groups were analyzed using an untargeted metabolic profiling approach. Data processing and statistical analysis were performed using Masshunter DA reprocessor and Mass Profiler Professional software (Agilent Technologies Inc.) respectively. Results: Demographic details of subjects in all four groups are shown in table 1. Preliminary analyses showed a separation of the metabolomic profile of subjects with EAC when compared to BE or control subjects. Similar separation was also seen between the metabolomic profile of BE and controls. Figure 1 shows Principal component Analysis (PCA) plots of all the four patient groups and their expressed metabolites. Some of the differentially expressed metabolites were identified using accurate mass obtained by Time of flight- Mass spectrometry analysis (TOF-MS) and by comparing them against the METLIN (Metabolite and Tandem Mass spectrometry) database. These include 2-Tetrahydrothiopheneacetic acid and N-stearoyl tyrosine in patients with EAC which were upregulated when compared with BE as well as non GERD controls. Also, Dihydroshikonofuran, 2-(6'Methylthio)hexylmalic acid, Gibberellin A15 and N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) were upregulated in the BE patient group when compared with non GERD controls. Conclusion: Metabolic profiling of plasma from BE,EAC and control subjects is able to identify distinct metabolites associated with each of the disease states.This may potentially reflect differences in regulatory pathways associated with these conditions and could be utilized in early detection strategies for BE and EAC. Table 1: Baseline characteristics of subjects in the BE, EAC, GERD and Non GERD study groups

Su1904 Effect of REG Protein on Mucosal Permeability and Adhesion Molecule Expression in the Small Intestine Yoshitaka Kitayama, Hirokazu Fukui, Takahisa Yamasaki, Takashi Kondo, Tomoaki Kono, Fumihiko Toyoshima, Hisatomo Ikehara, Yoshio Ohda, Toshihiko Tomita, Tadayuki Oshima, Jiro Watari, Hiroto Miwa Backgrounds: The disturbance of mucosal barrier is a key mechanism to explain how NSAID induces the mucosal injury in the gastrointestinal tract. We have previously reported that REG protein, a tissue regeneration-associated molecule, plays a cytoprotective role in NSAIDinduced mucosal injury in the small intestine. However, it still remains unclear how REG protein confers the resistance to NSAID-induced mucosal injury. Therefore, focusing on the mucosal barrier function, we examined the effect of REG protein on mucosal permeability and adhesion molecule expression in the small intestine. Methods: The expression of adhesion molecules in the small intestine were examined by real-time RT-PCR and immunohistochemistry in Reg I-knockout (KO) and wild-type littermates. Reg I-KO and WT mice received subcutaneous indomethacin (10 mg/kg). Mice were orally administrated with FITCdextran 24 hours after the treatment of indomethacin, and blood samples were collected from the treated mice. The effect of REG protein on epithelial (Caco2 cell) permeability was examined by trans-epithelial electrical resistance (TEER) method. Moreover, Caco2 cells were treated with REG protein and examined in adhesion molecules expression by western blot. Results: The expression of E-cadherin was decreased in Reg I-KO mice compared with WT mice. Serum FITC level was significantly elevated in Reg I-KO mice than in WT one when mice were treated with indomethacin. The level of TEER was significantly increased in REG-treated group than in control. REG protein treatment (10 nM) promoted the phosphorylation of Akt in Caco2 cells and furthermore enhanced the expression of claudin 15, E-cadherin and desmoglein 2 in those cells. Conclusions: REG protein is involved in adhesion molecules expression and maintenance of mucosal permeability in the small intestine. Su1905 Intestinal Adiponectin Receptor 1 (AdipoR1) Modulates Inflammation During Colitis: a Potential Link in Adipose Tissue-Intestinal Crosstalk During Inflammatory Bowel Disease Aristea Sideri, Hon Wai Koon, David Q. Shih, Charalabos Pothoulakis, Iordanis Karagiannidis Background and aims: We presented evidence suggesting that the adiponectin-AdipoR1 axis may play a role in the communication between mesenteric preadipocytes and the intestine during IBD (Gastroenterology 2014;146:S-823). Here, we expanded these studies and examined whether mesenteric whole fat induces inflammatory responses in human colonic epithelial NCM460 cells. The potential intracellular signaling pathways regulating AdipoR1 expression and the role of AdipoR1 in mice with colitis were also investigated. Methods: Conditioned medium from mesenteric fat from Ulcerative colitis (UC), Crohn's disease (CD) and control subjects (n=8-11 individual samples per group) were added to human colonic epithelial NCM460 cell monolayers and cytokine and PPARγ mRNA levels were measured using FlexScript LDA and RT-PCR, respectively. Colonic biopsies of control, UC and CD patients (n=4) were stained for AdipoR1. AdipoR1 mRNA levels in colon tissues from TNBS and vehicle-exposed mice (n=4) were evaluated with RT-PCR. The effect of intracolonic silencing of AdipoR1 (by si- AdipoR1) in the severity (weight, colon length, histologic colitis score) of TNBS-induced colitis (48 hrs) in mice (n=4-6 per group) was also evaluated. Results: VEGFA (p=0.0698), TGFB2 (p<0.05), G-CSF (p=0.0538), and IL8 (p<0.05) were increased while CCL4 (p=0.0541), IFNγ (p=0.0981), and IL-5 (p=0.0814) mRNAs were decreased in colonocytes exposed to UC vs control fat media. IL-8 (p<0.05) and IL-12A (p<0.05) were increased, while IL-1β (p=0.0764), IL-5 (p=0.0732), IL-15 (p= 0.0785), CCL3 (p<0.0897), and PDGFA (p<0.01) mRNAs were decreased in NCM460 cells exposed to CD vs control fat media. Interestingly, VEGFA (p<0.05), TGFB2 (p<0.05), CCL4 (p<0.05) and IL-12A (p=0.0721) levels were different in colonocytes exposed to UC vs CD media. Human colonic biopsies of UC and CD patients and mice with TNBS colitis show higher AdipoR1 levels (p<0.05). PPARγ mRNA levels were decreased in colonocytes exposed to media from UC (p<0.01) and CD (p=0.0832) whole fat media, mirroring the regulation of AdipoR1. Mice with intracolonic AdipoR1 silencing lost more weight and had worse colitis scores (p<0.05). Conclusion: Media from human mesenteric fat of UC and CD patients induce disease-dependent inflammation-related responses in colonocytes, including changes in AdipoR1 levels that may be related to down regulation of PPARγ, a known regulator of AdipoR1. The exacerbation of colitis in mice after intra-colonic silencing of AdipoR1suggests a novel protective role for this receptor in the development of colitis and IBD. Supported by DK047343 (CP), P50 DK64539 (Project 2, CP), The Broad Foundation (BMRP) (IK), and the Blinder Research Foundation for Crohn's Disease (AS).

Figure 1: 3D Score plot of Principal Component Analysis (PCA) of the study groups. [ Key: Gp1 (in red) - Non-GERD controls, Gp2 (in blue) - GERD controls, Gp3 (in brown) - BE and Gp4 (in grey) - EAC group ] Su1913 Activin a Stifles Esophageal Squamous Cell Invasion in a Premalignant but Not Malignant Microenvironment Holli A. Loomans, Chase Taylor, Claudia D. Andl Background: The interactions of cancer cells with their microenvironment can promote invasion and metastasis. Within the tumor microenvironment, fibroblasts facilitate extracellular matrix remodeling, which is a critical component of not only cancer cell invasion. Activin A (Act A) signaling is intertwined with TGFβ signaling and has been implicated as a novel

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Utility of Plasma Metabolomic Profiles in Differentiating Barrett's Esophagus and Esophageal Adenocarcinoma From Control Subjects: A Pilot Study Swarup Kumar, Dhananjay Sakrikar, Michele L. Johnson, Charles Ford, Prasad G. Iyer