Skin andintestinal woundhealing: Effect of inhibiting CD18 dependent neutrophil recruitment

A554 AGA ABSTRACTS

GASTROENTEROLOGY Vol. 118, No.4

2910 VILLUS ATROPHY IN A MOUSE MODEL OF TOTAL PAREN· TERAL NUTRITION: APOPTOSIS AS A CAUSE AND POTENTIAL MECHANISMS. Hua Yang, Yongyi Fan, Paul A. Antony, Daniel H. Teitelbaum, Univ of Michigan, Ann Arbor, MI. A major consequence of total parenteral nutrition (TPN) is the development of villus atrophy. We have previously demonstrated that TPN results in a significant increase in intraepitheliallymphocyte (JEL) derived interferon gamma (INF-'Y) expression. Because INF-'Y can promote an apoptotic process via regulation of FAS and FAS ligand (FAS-L), we hypothesized that the cause of TPN-induced villus atrophy would be an apoptotic process, and that this would be mediated via altered cytokine expression. Methods: C57BLl6 mice were randomized to intravenous TPN or oral feeding (Control) and were sacrificed after 7 days. Small bowel epithelial cells and JEL were isolated and studied for atrophy by histology; apoptosis using cell surface expression of phosphatidylserine; and mRNA was isolated for reverse transcriptase polymerase chain reaction (PCR) to detect INF-'Y (competitive PCR, expressed as attomoles/l00 M-g mRNA) and Fas and Fas-L (PCR using beta actin for relative expression). Results (mean::'::SD) were statistically compared with unpaired t-tests, * p<0.05. Results: TPN resulted in a significant loss of villus height compared to controls (310::'::42M-m vs. 210::'::45M-m, respectively). Apoptosis significantly increased in small bowel epithelial cells (see Table), whereas there was no change in epithelial necrosis (TPN 15.5::'::7.8% vs. Control 17.3::'::6.4%). Along with these epithelial apoptotic changes, IEL INF-'Y expression significantly rose in the TPN group. FAS expression by epithelial cells showed little change, however, there was a significant increase in IEL FAS-L expression with TPN. Conclusions: TPN-associated villus atrophy is associated with a significant increase in epithelial apoptosis. Concomitant with this apoptosis was a significant rise in JEL INF-'Y mRNA expression and an increase in FAS-L mRNA expression. It is possible that the increase in INF-'Y with TPN results in an increase in FAS-L expression and may be one of the mediators of the observed apoptosis.

differentiate, presumably reflecting formation of mature adherens junctions. 2. EGF receptor and SHC but not PLC-'Yl associate with .the cytoskeleton as CaC02 cells differentiate. 3. EGF results in SHC phosphorylation and shift to the CSK in differentiating CaC02 cells. 4. Phosphotyrosyl proteins are primarily associated with the cytoskeleton in differentiated CaC02 cells. 5. EGF results in a much greater EGFR tyrosine phosphorylation in differentiating CaC02 cells. These data suggest that EGFR receptor signaling is regulated by intestinal cell differentiation, possibly via its association with the cytoskeleton.

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INTESTINAL GROWTH ADAPTATION AND GLUCAGON-LIKE PEPTIDE 2 (GLP-2) IN PLASMA AND INTESTINAL TISSUE FOLLOWING SMALL BOWEL RESECTION OR ILEAL-JEJUNAL TRANSPOSITION IN RATS. Jesper Thulesen, Bolette Hartmann, Hannelouise Kissow, Palle Bekker Jeppesen, Cathrine Orskov, Jens Juul Holst, Steen Seier Poulsen, Univ of Copenhagen, Copenhagen, Denmark. Introduction: Glucagon-like peptide 2 (GLP-2) is produced by endocrine cells in the mucosa of the small intestine and colon. It has recently been demonstrated as a regulator of intestinal growth and may be of importance in the mediation of intestinal growth adaptation. This study investigates circulating and intestinal levels of GLP-2 in conditions with compensatory intestinal growth following either 50% proximal or 50% distal small bowel resection, or ileal-jejunal transposition. Methods: Female Wistar rats were allocated to the following groups: resection of (I) proximal or (2) distal half ofthe small intestine; sham-operated (3) proximal or (4) distal half; (5) ileal-jejunal transposition, and (6) sham-operated for ileal-jejunal transposition. Intestinal weight, length and morphometric data were obtained. Levels of GLP-2 in plasma and intestinal tissue were measured. Results: Adaptive small bowel growth after two weeks was more pronounced following proximal resection than following distal small bowel resection. Compensatory growth of the colon was observed both in the distally and proximally resected rats. Two weeks after surgery, circulating GLP-2 levels were increased three-fold in the proximally resected group, and two-fold in the distally resected group. Tissue GLP-2 levels were not changed in resected rats. Ileal-jejunal transposition led to pronounced growth of the transposed segment and a less pronounced growth of the remaining intestinal segments, including the colon. Plasma GLP-2 levels were increased two-fold, whereas GLP-2 levels were unchanged in the intestinal segments, including the transposed segment. Conclusions: The present data indicate that post-resectional intestinal growth correlates with circulating levels of GLP-2, whereas intestinal tissue levels of GLP-2 remain rather unchanged, suggesting that GLP-2 primarily act in an endocrine manner to stimulate intestinal growth.

EGF RECEPTOR SIGNALING PATHWAYS SHIFT TO THE CY· TOSKELETON IN DIFFERENTIATING CAC02 CELLS. John F. Thompson, Tiong The', Jose A. Ortega, Univ of Miami Sch of Medicine, Miami, FL. Epidermal growth factor signaling pathways regulate a variety of functions in both proliferating and differentiated intestinal epithelial cells. This laboratory has previously demonstrated that EGF receptor signaling is associated with the cytoskeleton and regulated by cell density in IEC-6 cells. To determine if EGFR signaling is regulated in a similar fashion in differentiating intestinal epithelial cells, both soluble and cytoskeletal fractions of CaC02 cells were studied at different stages of differentiation. METHODS: CaC02 cells were seeded on Transwell plates in DME-FI21 5%FBS to reach confluence at 7 days post seeding. At 4, 7, and 14 days post seeding, cells were solubilized in 0.2% Triton X-loo for soluble fraction (SOL) and then RlPA for cytoskeletal fraction (CSK). SOL and CSK distribution of J3-catenin, E-cadherin, EGFR, phospholipase C'Y, SHC and phosphotyrosyl proteins was determined by immunoblot of equal aliquots of SOL and CSK fractions from each time point post seeding. RESULTS: The adherens junctions proteins, J3-catenin and E-cadherin, as well as EGFR, demonstrated a marked redistribution to the CSK postconfluence. SHC, but not PLC'Y-1, also shifted to the CSK fraction as CaC02 cells differentiated. Basolaterally administed EGF resulted in a SHC doublet,indicating SHC phosphorylation, as well as an enhanced shift of SHC to the CSK. Phosphotyrosyl proteins were distributed equally between both SOL and CSK in undifferentiated CaC02 cells but demonstrated a marked shift to the CSK fraction in differentiated cells. Also, EGF stimulation resulted in EGFR phosphorylation primarily in the CSK fraction at each time point. However, the magnitude of EGFR phosphorylation was much greater in 7 day post seeding differentiating cells. CONCLUSIONS l . {3-catenin and E-cadherin associate with the CSK as CaC02 cells

SKIN AND INTESTINAL WOUND HEALING: EFFECT OF INHIBITING CD1S DEPENDENT NEUTROPHIL RECRUITMENT. Leif Torkvist, Peter Mansson, Johan Raud, Jorgen Larsson, Henrik Thorlacius, Dept of Surg, Karolinska Inst, Stockholm, Sweden. Anastomotic leakage and flap necrosis are major complications in gastrointestinal surgery and reconstructive. In these circumstances, postoperative tissue injury may to some extent be caused by recruitment of leukocytes which are capable of releasing toxic products, including oxygen free radicals, proteases and vasoactive substances. CD II/CD 18 is an important adhesion molecule mediating firm adhesion and transendothelial migration of leukocytes. The objective of the present study was to investigate the effects of inhibiting the function of CDl8 on dermal and intestinal infiltration of neutrophils and on the healing of surgical skin flaps and colonic anastomosis. For this purpose, we created a dorsal skin flap or an end-toend anastomosis in the large bowel of Sprauge-Dawley rats. Anastomotic breaking strength and skin necrosis were analysed 3 and 6 days after surgery, respectively. Tissue myeloperoxidase (MPO) was used as marker of neutrophil recruitment. Pre-treatment with a monoclonal antibody directed against rat CDl8 (WT.3) at 2.0 mglkg, but not 0.75 mg/kg, significantly improved flap survival, i.e. the survival was 80% in the anti-CDl8 group as compared to 38% in the control group treated with an iso- and subtype matched control antibody. Moreover, it was observed that skin MPO increased markedly 24 h after surgery. Notably, administration of WT.3 (2 mglkg) decreased this dermal MPO accumulation by more than 80%. Similarly, it was found that injection of WT.3 (2 mglkg) reduced anastomotic MPO by more than 90%. However, this reduction in neutrophil infiltration had no effect on the reconstitution of the integrity of the anastomosis 3 days after surgery, i.e. the anastomotic breaking strength

Group Control

TPN

Apoptosis ('!o)

INF-y (N=10)

FAS

FAS·L

N=8

(N=4)

(N=4)

15.6±7.7 64.4±22.1 '

O.28±O.14 ' O.78±O.20'

103.9±17.4 116.5±206

118.7±21.2'

88.1±19.4

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April 2000

was I.S :t 0.4 Nand 1.3 :t 0.6 N in control and WT.3 treated rats, respectively. Taken together, these findings suggest that specific inhibition of CD 18 function and reduced neutrophil recruitment may improve the survival of experimental skin flaps and, thus, may represent a potential target for therapeutic intervention. In contrast, we also found that blocking CD18-dependent neutrophil infiltration in the intestine did not change the breaking strength of colonic anastomosis. Thus, neutrophils may influence the wound healing process differently in specific organs. This needs to be considered when applying an anti-inflammatory treatment regime in order to improve tissue healing.

2914 HUMAN GASTRIC EPITHELIAL PROLIFERATION IS COORDI· NATED BY INSULIN·LIKE GROWTH FACTORS AND THEIR BINDING PROTEINS. Eric Tremblay, Pierre Chailler, Daniel Menard, Univ de Sherbrooke, Sherbrooke, PQ, Canada. IGF peptides (IGFl,IGF2) are known to be produced in fetal and adult gastric tissues and IGF2 is an autocrine factor for gut-derived cell lines. However their possible contribution to the regulation of cell proliferation during development or regeneration of human gastric mucosa is unknown, as the modulatory effect of IGF binding proteins (IGFBPs). AIMS: To evaluate the ability of human gastric mucosa to produce IGFBPs and to test the effects of native IGFs on epithelial cell proliferation as well as that of truncated IGFs which do not interact with IGFBPs. METHODS: Western! FarWestern blots and immunofluorescence were performed to characterize the expression of IGFl-receptor and IGFBP-l to -6. The effects of growth factors on DNA synthesis were determined in fetal gastric explants maintained in serum-free organ culture. RESULTS: All gastric epithelial cells expressed the IGFI-R. IGFBP-l protein detected in gastric tissue likely derived from an extragastric source: immunoreactivity was found in all mucosal cell types (endocrine pathway of delivery) and its total amount did not increase in culture. IGFBP-2 to -6 proteins were rather associated with epithelial cells and differentially localized along the pit-gland axis. They were produced endogenously and modulated in culture. Addition of IGFI to gastric explants stimulated 3H-thymidine incorporation into DNA after 24-48 h and increased the epithelial labeling index. Illustrating the counter regulatory effect of endogenous IGFBPs on IGF action, truncated R3IGFl was more potent than IGF1. DeI(l-6)IGF2 was the most potent inducer whereas IGF2 remained without significant effect (even at 200ng/mL). The last finding probably reflects the presence of IGFBP-2 and -6, two IGF2 carriers intensely synthesized in the pit/neck region comprising the proliferative compartment and abundantly secreted during culture. CONCLUSION: This study provides new evidence for the involvement of an intragastric IGF/lGFBP system in the fine regulation of epithelial cell division whereby the specific influence of IGF2 appears to be tightly regulated by IGFBP isoforms preferentially associated with this growth factor and proliferative cells. (Supported by the Medical Research Council of Canada; D.M. is a member of the MRC Research Group on Functional Development and Physiopathology of the Digestive Tract.)

2915 THE SRC-RELATED INTESTINAL KINASE SIK IS PRESENT IN THE NUCLEUS AND PHOSPHORYLATES THE RNA·BINDING PROTEIN SAM68. Angela L. Tyner, Jason Derry, Stephane Richard, Taiping Chen, Xin Ye, Univ of Illinois Coil of Medicine, Chicago, IL; McGill Univ, Montreal, Quebec, Canada. Sik is an epithelial-specific intracellular tyrosine kinase that is expressed in differentiating cells of the gastrointestinal tract. Using a variety of techniques including transfection, immunoprecipitation. immunoblotting and confocal microscopy, we have identified the first substrate of Sik, the RNA binding protein Sam68. The cellular function of Sam68 (Src-associated in mitosis, 68 kDa) is not well understood, but its ability to functionally substitute for the HIV-l Rev protein suggests a role in RNA export and posttranscriptional gene regulation. Introduction of Sik and Sam68 expression constructs into the HT-29 human colon adenocarcinoma cell line results in Sam68 tyrosine phosphorylation. As a consequence of phosphorylation by Sik, the ability of Sam68 to bind RNA is inhibited. Sik and Sam68 colocalize in distinct nuclear structures termed SNBs (Sam68 Nuclear Bodies) in HT·29 cells. We found that Sik interacts with Sam68 through its SH3 and SH2 domains, and that the proline rich P3 region of Sam68 is required for Sik SH3 binding. While Sam68 may be phosphorylated by Src-family members during mitosis when the nuclear membrane breaks down, Sik is the first identified tyrosine kinase that is capable of phosphorylating Sam68 within the nucleus where it resides during most of the cell cycle. Sik phosphorylation of Sam68 within the nucleus may regulate gene expression associated with intestinal epithelial cell differentiation at the posttranscriptional level.

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2916 MEVASTATIN POTENTIATES BUTYRATE·INDUCED ANTI· PROLIFERATIVE EFFECT AND INDUCES APOPTOSIS IN CO· LON CANCER CELLS. Astrid Waechtershaeuser, Bora Akoglu, Wolfgang F. Caspary, Juergen Stein, J W Goethe Univ, Frankfurt/Main, Germany. 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA-) inhibitors (HRI) prevent formation of mevalonate from HMG-CoA and thereby inhibit cholesterol synthesis. Intermediates in the cholesterol synthetic pathway, like farnesyl- and geranylgeranylpyrophosphate, are needed for isoprenylation of cellular proteins, a crucial step for membrane attachment. The aim of this study was to evaluate the effect of HMG-CoA inhibition in combination with butyrate on cell proliferation, differentiation and apoptosis in the human colorectal adenocarcinoma cell line Caco-2. Methods. Caco-2 cells were incubated with butyrate (I mM), mevastatin,(a HRI) or mevalonate. Cell growth was determined by crystal violet staining, alkaline phosphatase (AP) was used as a differentiation marker. Cytotoxicity was excluded by a commercial kit measuring lactate dehydrogenase activity in the supernatant of damaged cells. Apoptosis was quantified by use of a photometric ELISA, measuring cytoplasmic histone-associated DNA fragments. Cell cycle analysis was performed by propidium iodine stanining and flow cytometry. Cyclin-dependent kinase (Cdk) 4 and Cdk 6 were detected by immunoblotting. Results. Inhibition of HMG-CoA-reductase by mevastatin potentiated the antiproliferative effect of butyrate in a doseand time-dependent manner (22861 :t2866 cells/em" vs. 4476S:t5470 in butyrate group, p
2917 EXPRESSION OF 5·LIPOXYGENASE DURING BUTYRATE·IN· DUCED CELL DIFFERENTIATION OF HUMAN CARCINOMA CELLS. Astrid Waechtershaeuser, Stefan M. Loitsch, Dieter Steinhilber, Wolfgang F. Caspary, Juergen Stein, J W Goethe Univ, Frankfurt/Main, Germany. Butyrate, a short-chain fatty acid produced by colonic fermentation of dietary fiber, modulates proliferation and differentiation of normal and neoplastic colonocytes. The underlying molecular mechanisms, however, are not clear. 5-Lipoxygenase (S-LO), besides its function as a key enzyme in leukotriene synthesis, has been shown to interact with cellular proteins, suggesting a possible role in the regulation of cellular growth. The aim of our studies was to investigate the differentiation-dependent expression of 5-LO and cyclooxygenase isoforms (COX-I, COX-2) in the human colorectal adenocarcinoma cell line Caco-2, which undergoes cell differentiation in the presence of sodium butyrate (NaBT). Methods. Human colon adenocarcinoma cells (Caco-2) were incubated with NaBT (2 mM). 5-LO, COX-I, COX-2, and FLAP (5-LO activating protein) on mRNA level were quantified by RT competitive multiplex PeR. The corresponding proteins were determined by immunoblotting. Cell differentiation was evaluated by analyzing alkaline phosphatase (AP) activity. TGF-13 secretion was measured by a commercial ELISA. Results. COX-2, COX-l and FLAP mRNA levels were not significantly influenced by NaBT treatment. In contrast, SoLOwas highly expressed on mRNA (78.3:t5.27-fold after 8d; p