T1694 Sulfasalazine, But Not Sulfapyridine or 5-Aminosalicylic Acid, Induces Apoptosis in Human Intestinal Myofibroblasts

AGA Abstracts

T1694

T1696

Sulfasalazine, But Not Sulfapyridine or 5-Aminosalicylic Acid, Induces Apoptosis in Human Intestinal Myofibroblasts Kevin Hughes, Yashwant R. Mahida

Permanent Impairment of Catalase Activity Due to a Minor Expression of Catalase Protein in Peripheral White Mononuclear Cells (PWMC)of Patients with Naïve and Treated Crohns Disease (CD) Belen Beltran, Marisa Iborra, Ines Moret, Jose Luis Garcia Gimenez, Guillermo Bastida, Francisco Rausell, Federico Pallardo, Julio Ponce, Pilar Nos

INTRODUCTION: Intestinal myofibroblasts are prominent below the surface epithelium and are believed to play an important role in the pathogenesis of inflammatory bowel disease. With impaired epithelial barrier, the myofibroblasts become exposed to luminal constituents. These include the drugs sulfasalazine (SUSZ) and olsalazine (OLSZ), which may also be cleaved by luminal bacteria to sulfapyridine (SUPY) / 5-aminosalicylic acid (5ASA) and 5ASA / 5ASA, respectively. AIM: To investigate basal proliferation rate and susceptibility to cell death in myofibroblasts isolated from normal and ulcerative colitis (UC) intestinal mucosal samples. METHODS: Myofibroblasts were established in culture, following their migration out of the lamina propria of normal colonic (NC), normal ileal (NI) and UC mucosal samples that had been denuded of epithelial cells. Propidium iodide-stained cells were studied by flow cytometry to assess those undergoing DNA synthesis (events in S phase of cell cycle) and apoptosis (events in hypodiploid/sub-G1 region). To assess proliferation, myofibroblasts were cultured for 24 h in 0.1% fetal calf serum (FCS). Effects of SUSZ, SUPY, 5ASA and OLSZ were studied in myofibroblasts cultured (for 24 h) in 10% FCS. Data are expressed as mean(SEM). RESULTS: Compared to those isolated from NC (n=11), greater proportion of myofibroblasts isolated from UC samples (n=9) were in S phase [6.2(0.8)% vs 10.0(1.2)%; p=0.01]. There was no significant difference in the basal rate of apoptosis in these cells [0.7(0.1)% vs 1.1(0.2)%]. Exposure to SUSZ resulted in a dose dependant increase in the number of events in the sub-G1 region in myofibroblasts isolated from NC (n=5), NI (n=5) and UC (n=3) samples (Table). Apoptosis was confirmed by Hoechst staining. By contrast, there were no significant differences in the sub-G1 regions of myofibroblasts exposed to similar concentrations of SUPY [10 mM: 1.16(0.21)%], 5ASA [10 mM: 1.10(0.20)%] or OLSZ [10 mM: 0.69(0.18)%], compared to control medium [0.71(0.22)%]. CONCLUSIONS: 1. Compared to NC, myofibroblasts isolated from UC mucosa proliferate more rapidly. In Vivo, this may lead to disruption of mucosal function in UC. 2. In contrast to SUPY, 5 ASA or OLSZ, SUSZ induces apoptosis in intestinal myofibroblasts. Thus, compared to others, SUSZ may possess additional biological activities In Vivo. Percent myofibroblast events in sub-G1 region

Background: CD patients have an increased oxidative stress damage in their peripheral leukocyte and decreased plasma antioxidants. We have shown that PWMC of active CD patients have an increased oxidative stress, which depends on H2O2 production (1). Aims: 1. To analyze catalase (CAT) antioxidant activity in active and inactive CD patients, in order to clarify the origin of the H2O2 in CD. 2. To study the correlation between CAT activity and enzyme concentration. Methods: Blood samples from healthy subjects (n=20) and from patients (n=20)at onset of CD and yet to begin any medication were obtained. Experiments were repeated when clinical remission was achieved . Disease sactivity was scored with Harvey-Bradshaw index. PWMC were isolated by Ficoll-Histopaque sedimentation. CAT activity was measured spectrophotometrically by enzymatic assays. Statistical analysis was performed with the Mann-Whitney test. CAT expression was studied in active CD patients, inactive CD patient and controls subjects by western blotting (WB) using anti-CAT monoclonal antibody. Results: Harvey-Bradshaw index values were: Active CD (8.72 ± 2.22), inactive CD (1± 1.2). Inactive patients had the experiment performed when they were a mean time of 8.7 ± 3 months in remission. CAT activity in PWMC of active and inactive CD is lower than healthy subjects (17,22 ± 9,44 (p=0.0016 vs control) in active-CD and 10,38 ± 4,47 (p=0.0003 vs control) in inactive CD and 33,51 ± 15,9 U/mg in controls. WB results have shown diminished levels of CAT in active and inactive CD patients than in control subjects (figure 1). Conclusions: The inhibition of CAT activity in PWMC of active CD will help to the generation of H2O2. CAT inhibition is permanent in CD as inactiveCD patients do not recover CAT activity. The results suggest a correlation between activity and CAT protein levels. This event could contribute to PWMC immunological functioning and thus could have an impact on the physiopathology of CD. (1) Beltrán, B. et al. Gastroenterology 2006; 130-4 (suppl 2): A 360.

T1697 Resveratrol-Induced Cell Cycle Arrest and Apoptosis in Rat Intestinal Smooth Muscle Cells Phyllissa Schmiedlin-Ren, Ellen M. Zimmermann

p<0.0001 (ANOVA)

Smooth muscle cell hyperplasia and increased collagen synthesis cause fibrotic strictures that often complicate Crohn's disease. We have shown that resveratrol results in decreased cell numbers and reduced collagen synthesis in intestinal smooth muscle cells (ISMC) isolated from Lewis strain rats. This rat strain is susceptible to experimental Crohn's disease, with prominent fibrosis not seen in other models. Resveratrol (Res; 1 uM) has been reported to inhibit proliferation of human aortic vascular smooth muscle cells and to stimulate apoptosis (25 uM). Its reported effect on apoptosis in other cell lines has been variable. Aim: to determine the mechanism of the Res effect on rat ISMC numbers. Methods: Rat ISMC, isolated from the muscularis externa of Lewis rat colons by collagenase digestion, maintain expression of smooth muscle alpha-actin through >20 passages. Cells of passages 11-12 were exposed to varying concentrations of Res. Cell cycle was assessed by flow cytometry of propidium iodide stained cells. Apoptosis was assessed by measuring caspase-3 activation (luminescence in Promega Caspase-Glo 3/7 Assay) and by TUNEL-flow cytometry (Phoenix Flow Systems). Results: Res (25, 50, and 100 uM; 24 h) resulted in cell cycle arrest in Sphase (% of cells in S-phase expressed as fold control was 1.46, 3.90, and 5.29, respectively; p = 0.033 for Res 100 uM); no effect was seen with Res ≤10 uM. Increased caspase activation was seen at 2 days with 10, 25, and 50 uM Res (1.20, 1.37, and 1.57 fold control, respectively; p<0.02). Following 1 day treatments, significant changes in caspase activation were not seen with 10 uM Res, while caspase activation was markedly diminished with doses ≥50 uM (p<0.00004). This latter finding may be explained at least in part by differing cell numbers present in the wells at the end of the treatment period due to cell cycle arrest. TUNEL-flow cytometry showed an increase of slightly greater that 2-fold in the numbers of apoptotic cells with doses in the range of 50-100 uM; this approached significance with p<0.06. Conclusion: Resveratrol causes cell cycle arrest in rat intestinal smooth muscle cells. An increase in apoptosis appears to also be induced, but at a lower level. This data is consistent with potential utility of Res in the treatment of intestinal fibrosis.

T1695 Aquaporine 8: A New Histological Marker for Colonic Inflammation in Crohn's Disease? Simone C. Wolfkamp, Pieter Stokkers, Esther W. Vogels, Noor L. Bekkali, Fiebo J. Ten Kate, Anje A. Te Velde Introduction: Gene expression of Aquaporine 8 (Aqp8), a waterchannel permeable to water and glycerol and present in different parts of the gastrointestinal tract, has been shown to be downregulated in DSS, TNBS and CD4+CD45RBhigh transfer colitis models in mice. Lightcycler analysis of Aqp8 gene expression levels in human inflammatory bowel disease (IBD) patients confirmed this downregulation in macroscopically affected colon compared with nonaffected colon. No expression was observed in the ileum. We investigated the presence of AQP8 in colonic and ileac specimens of human IBD patients through immunohistochemical staining. Objective: To confirm our previous data on protein level by studying the presence of AQP8 in the colon and the ileum in both Crohn's Disease (CD) and Ulcerative Colitis (UC) patients, as well as healthy controls. Materials and Methods: Either surgical resection specimens or endoscopic biopsy specimens were obtained from CD and UC patients and controls. Endoscopic biopsy specimens were obtained from affected and non-affected areas in the same patient for comparison. Immunohistochemical stainings with AQP8 antibody were used to examine the presence of AQP8 in the colon as well as the ileum. Hence, presence of AQP8 was verified using the same technique in our DSS mice specimens. Results: Although AQP8 was present in all colonic human specimens, there was a distinct difference in expression pattern. In healthy controls AQP8 was diffusely expressed throughout the epithelial layer of the crypts both apical and basal. UC patients showed a similar pattern. However, this expression pattern was different in CD patients, where expression of AQP8 appeared (rather)as small vesicle-like structures. This remarkable pattern was present in all CD patients tested. No expression of AQP8 was seen in the colonic samples of the CD4+CD45RB high transfer mice. In concordance with our Lightcycler analysis, no expression of AQP8 was found in the ileum of the CD and UC patients. Conclusion: AQP8 has a distinct histological recognisable pattern in colonic specimens of CD patients. This could suggest a possible role for AQP8 as histological marker for colonic inflammation in IBD. Furthermore, as the Aqp8 gene is located on IBD locus 8, our findings could suggest a possible role for Aqp8 in the pathogenesis of CD. The vesicle-like pattern visible in all CD patients could possibly be subscribed to a protein trafficking problem. This is currently under investigation

AGA Abstracts

T1698 Tissue Nonspecific Alkaline Phosphatase Is Induced in Enterocytes in Response to Inflammation/Oxidative Stress Rocio Lopez-Posadas, Raquel Gonzalez, Isabel M. Ballester, Isabel Romero-Calvo, Maria Dolores Suarez, Antonio Zarzuelo, Olga Martinez-Augustin, Fermin Sanchez de Medina Alkaline phosphatase (AP) activity is substantially increased in intestinal inflammation (Biochem Pharmacol 2004;68:2317). This is partly accounted for by augmented epithelial AP activity. Here we explore this phenomenon at the cellular level. Intestinal epithelial cells (IEC18, Caco2, HT29, HCA7) have increased (2-8 fold) AP activity when treated with tert-butyl hydroperoxide (tButOOH), hydrogen peroxide or monochloramine, all of which produce oxidative stress. This was accompanied by a higher sensitivity to the AP inhibitors, levamisole and homoarginine, and to heat. These treatments resulted in glutathione depletion and substantial cell toxicity. We selected the nontumoral IEC18 cell line for subsequent experiments. AP upregulation could be detected 3-9 h after tButOOH exposure and peaked at 12-24 h. In addition to oxidants, we tested the effect of IL-1β, TNF and IFN-γ, which

A-560