Jörg M. Zeeh

Protective effect of epidermal growth factor in an experimental model of colitis in rats

BACKGROUND/AIMS The role of epidermal growth factor (EGF) in the maintenance of mucosal integrity in the lower gastrointestinal tract is unknown. The aim of this study was to determine the effect of EGF in experimental colitis. METHODS Colitis was induced with 2,4,6-trinitrobenzenesulfonic acid/ethanol enemas. Rats were pretreated with intraperitoneal administration of recombinant human EGF (600 micrograms/kg) or vehicle 1 hour before induction of colitis and daily thereafter until killed at 8 hours, 48 hours, and 1 week. A separate group received an identical dosage and administration of EGF or vehicle for 1 week with treatment initiated 24 hours after the induction of colitis. Colonic tissue was evaluated macroscopically, histologically, and for myeloperoxidase activity. RESULTS Pretreatment with EGF reduced microscopic erosions at 8 and 48 hours by 74% and 54%, respectively (P < 0.05). At 1 week, microscopic ulcerations and myeloperoxidase activity were reduced by 65% in the EGF-pretreated group (P < 0.05). No significant difference in macroscopic injury, histological damage, or myeloperoxidase activity was noted when EGF treatment was initiated after the induction of colitis. CONCLUSIONS Systemic EGF administration reduces mucosal damage and inflammation in a trinitrobenzenesulfonic acid/ethanol model of colitis in rats through a mechanism involving mucosal protection.

MYCOPHENOLATE MOFETIL IMPAIRS HEALING OF LEFT-SIDED COLON ANASTOMOSES 1

Introduction. Inadequate healing and consequent leakage from bowelanastomoses are a significant cause of postoperative morbidity and mortality.Immunosuppressive drugs are known to disturb healing processes and to impairthe mechanical stability of bowel anastomosis. Mycophenolate mofetil (MMF) isan immunosuppressive agent that selectively inhibits the proliferation of Tand B lymphocytes and has been shown to be effective in preventing allograftrejection after organ transplantation. Adverse effects are few; however,nothing is known in regard to possible adverse effects of MMF administrationon the healing of bowel anastomosis. The aim of the present study was toevaluate the effect of systemic MMF administration on the healing of colonanastomoses inrats. Methods. Rats underwent laparotomy, division of the left colon, andsigmoidostomy. MMF (25 mg/kg) or vehicle was administered intraperitoneally intwo groups (n=21 per group) 3 days before surgery and then once dailyuntil euthanization (7 animals per group; 2, 4, and 6 days after surgery).Bursting pressure measurements, histologic evaluation, morphometric analysis,mucin and collagen staining, and BrdU immunohistochemistry of the anastomoticsite were performed. Furthermore, matrix protein expression at the anastomoticsite was determined by collagen I and fibronectin Westernblots. Results. Administration of MMF significantly decreased anastomoticbursting pressure postoperatively. Accordingly, histology, mucin staining, andBrdU immunohistochemistry and measurements of the colonic crypt depth showedmore extended inflammation, a significantly lower proliferation rate, and asignificantly thinned mucosal layer in the MMF-treated groups when compared tocontrol animals, whereas matrix synthesis at the anastomotic site was notdifferent. Conclusions. The administration of the immunosuppressive agent MMFsignificantly impairs healing and mechanical stability of colon anastomosis inrats during the early postoperative period. MMF act to disturb host reparativeprocesses mainly by impairment of reparative colonic epithelium proliferationand less by a disturbance of matrixsynthesis.

Up-regulation of insulinlike growth factor I binding sites in experimental colitis in rats.

BACKGROUND/AIMS The gastrointestinal tract is a major target of insulinlike growth factor (IGF) I. IGF-I binds to two different receptors and to binding proteins (IGFBPs), which act as carriers and mediators. This study investigated the regulation of IGF-I binding sites in rat colitis. METHODS Colitis was induced by colonic instillation of 2,4,6-trinitrobenzenesulfonic acid in ethanol. IGF-I binding sites in colon sections were localized by incubation with 125I-IGF-I. The contribution of binding to the IGF-I receptor was estimated by competition with unlabeled IGF-I, IGF-II, and insulin. Colonic RNA was screened for IGFBPs by Northern hybridization. RESULTS IGF-I binding sites were increased more than two-fold in the muscularis propria of inflamed colon as soon as 12 hours and up to 1 week after injury. Insulin could not displace this elevated level of binding, even though it could displace IGF-I from the mucosa and muscularis mucosa. Northern hybridization showed a 2-3-fold increase in IGFBP-4 and IGFBP-5 messenger RNA from inflamed colon. CONCLUSIONS Experimental colitis in rats causes an increase in IGF-I binding to the muscularis propria, which represents increased levels of IGFBP-4 and IGFBP-5. These data suggest an important role for IGFBPs in modulating IGF effects during inflammation and tissue repair.

Differential expression and localization of IGF-I and IGF binding proteins in inflamed rat colon

Recent studies indicate increased insulin-like growth factor I (IGF-I) expression and altered expression of IGF binding proteins (IGFBP) in the bowel during experimental colitis. This study analyzes the cellular sites of altered IGF-I and IGFBP-expression in large bowel of rats with experimental colitis. Colitis was induced by colonic instillation of 2, 4, 6-trinitrobenzenesulfonic (TNB) acid in ethanol. Animals were sacrificed at 7 days after induction of colitis. Cryostat sections of colon from TNB-treated and control rats were hybridized with 35S-labeled antisense probes for IGF-I, IGFBP-3, IGFBP-4 and IGFBP-5. IGF-I mRNA was up-regulated in lamina propria cells, submucosa and smooth muscle of inflamed colon. IGFBP-3 mRNA was localized to lamina propria and was down-regulated in inflamed colon. IGFBP-4 and IGFBP-5 mRNAs were both up-regulated in inflamed colon. IGFBP-4 mRNA was increased in lamina propria, submucosa and smooth muscle, whereas IGFBP-5 mRNA was increased in smooth muscle. Increased IGF-I expression in mesenchymal layers of colon during experimental colitis supports the hypothesis that IGF-I contributes to hyperplasia and fibrosis in response to inflammation. Altered expression of IGFBP-3, IGFBP-4 and IGFBP-5 in specific bowel layers during colitis suggests that they play a role in modulating IGF-I action.

Lisofylline and Lysophospholipids Ameliorate Experimental Colitis in Rats

Background: Intestinal inflammatory processes initiate a chain reaction in which membrane-bound lipids generate eicosanoids and phospholipids. Bioactive lipid mediators play a pivotal role in the pathogenesis of intestinal inflammation and colonic mucosa from patients with inflammatory bowel disease contains high levels of phospholipids. Therefore, we investigated the effects of lysophosphatidic acid and lysophosphatidylethanolamine, two natural occurring phospholipids and lisofylline, which decreases lipid peroxidation, in an in-vivo model of intestinal inflammation. Methods: Colitis was induced by rectal administration of ethanol and trinitrobenzene sulfonic acid in rats. Rats were treated once daily with either lysophosphatidic acid or lysophosphatidylethanolamine rectally or twice daily intraperitoneally with lisofylline following induction of colitis. Rats were sacrificed after 7 days and the effect of lysophosphatidic acid, lysophosphatidylethanolamine, and lisofylline on colonic damage and inflammation were assessed using standardized macroscopical and histological injury scores. Results: Treatment with lysophosphatidic acid, lysophosphatidylethanolamine, and lisofylline significantly reduced the degree of inflammation and necrosis in the distal colon compared to control rats. In addition, the weight loss was significantly less in the treatment groups compared to controls. Histological studies revealed a significant reduction of epithelial damage and colonic inflammation. Conclusion: The administration of anti-inflammatory lysophospholipids and suppression of proinflammatory lipid metabolites by lisofylline may provide new approaches to ameliorate intestinal inflammation.

Expression of insulin-like growth factor binding proteins and collagen in experimental colitis in rats.

Objectives and design Crohns disease is complicated by smooth muscle hyperplasia and stricture formation. Insulin-like growth factor (IGF)-1 and insulin-like growth factor binding proteins (IGFBPs) may be involved in stimulating intestinal smooth muscle growth and collagen synthesis. Therefore, we investigated the expression of IGFBPs, collagen and collagenase activity in rat colitis and the effects of IGF-1 on IGFBP and collagen expression in rat colonic smooth muscle cells. Methods Animals were sacrificed during a 4-week time course of 2,4,6-trinitrobenzenesulphonic acid (TNBS)-induced colitis. RNA from the animals’ colons was blotted and hybridized with collagen-1 and IGFBP mRNA probes. Tissue proteins were screened for IGFBPs by Western ligand blotting. Collagenase activity was measured by zymography. Rat colonic smooth muscle cells in primary culture were incubated with IGF-1 then collagen-1, and IGFBP mRNAs and proteins were measured. Results In the rat tissue, IGFBP-3 mRNA and protein were increased 2 h after induction of colitis. IGFBP-4 mRNA was elevated after 2 h and IGFBP-4 protein after 4 h. IGFBP-5 mRNA was upregulated after 2 h with a peak at 12 h. IGFBP-5 protein was upregulated after 1 h and reached a peak at 3 days. Collagen-1 mRNA was increased after 5 days. Collagenase levels were decreased after 1 h and returned to normal by 28 days. In rat colonic smooth muscle cells, IGF-1 increased collagen-1 and IGFBP-5 expression. Conclusion We demonstrated an upregulation of IGFBP and collagen expression and a downregulation of collagenase in rat colitis. In colonic smooth muscle cells, we found an upregulation of collagen-1 and IGFBP-5 following IGF-1 incubation. These results suggest an important role of IGF-1 in the collagen synthesis in colitis, mediated by IGFBPs.

Epithelial cell-derived components induce transforming growth factor-α mRNA expression and epithelial cell proliferation in vitro

Background Epidermal growth factor (EGF) and transforming growth factor (TGF)-α protect the gastrointestinal mucosa against injury. In response to mucosal injury TGF-α, but not EGF, is locally increasingly expressed in the mucosa of the rat colon and stomach 4–8 h after injury. The aim of our present study was to characterize the possible signal for the induction of TGF-α expression. Methods Monolayers of the non-transformed intestinal epithelial cell (IEC)-6 and IEC-18 lines were harvested, homogenized and shock-frozen at − 80 °C for 1 h. Cell cultures of intact IEC-6 or IEC-18 cells were exposed to these cell homogenates and modulation of epithelial cell migration and proliferation was evaluated using standardized procedures as described previously. TGF-α mRNA expression in the exposed epithelial cell monolayers was assessed using reverse transcriptase–polymerase chain reaction (RT-PCR) and normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Results The proliferation of IEC-6 and IEC-18 epithelial cell monolayers was significantly inhibited if epithelial cell homogenates of 2000 cells/ml or more, and significantly induced if homogenates of less than 2000 cells/ml, were applied to the medium of the monolayers in vitro. In proliferating epithelial cells, a significant two-fold increase in TGF-α mRNA expression was obtained at 48 h and 72 h after application of the cell homogenates. The homogenate-induced epithelial cell proliferation was completely abolished after preincubation of the epithelial cell homogenates with neutralizing monoclonal anti-TGF-α antibodies. No effect on epithelial cell migration was noticed after application of epithelial-cell homogenates to the cell cultures. Conclusions Epithelial cell-derived components induce TGF-α mRNA expression and TGF-α-dependent cell proliferation of intact epithelial cells. We hypothesize that epithelial cell interaction bears one of the possible signals for the increased TGF-α mRNA expression after mucosal injury.

Determination of Various Molecular Forms of Cholecystokinin from Canine Mucosa by Radioimmunoassay and Bioassay

Bioassays using amylase release from isolated pancreatic acini measure only cholecystokinin (CCK) forms with an intact carboxyl terminus ending with phenylalanine amide, but it cannot be excluded that peptides not structurally related to CCK are also responsible for CCK-like bioactivity. CCK exists in several molecular forms in intestinal mucosa which are released into the circulating blood. We studied the molecular forms of CCK in canine intestinal extracts after separation by high performance liquid chromatography by bioassay and compared them with those detected by radioimmunoassay. For the radioimmunoassay, an antibody was used which needs the carboxyl terminal phenylalanine amide for recognition. Three immunoreactive peaks were reproducibly seen in HPLC eluates which eluted in the regions of synthetic CCK-8, purified porcine CCK-33-39 (which co-elute using this gradient) and purified canine CCK-58. All these peaks were bioactive for amylase release from isolated pancreatic acini. No further bioactive peaks were detected in the HPLC eluates. When an antibody was used which recognizes the midregion of CCK-58, an additional peak was detected which eluted between CCK-33-39 and CCK-58. This form presumably represents an amino terminal fragment of CCK lacking the carboxyl terminus. It can be concluded that bioassays of CCK measure only CCK bioactivity in intestinal mucosal extracts, whereas radioimmunoassays may detect biologically inactive forms depending on the antibody recognition site.

Transforming growth factor alpha-immunoreactivity in neural tissues of the rat stomach

We report TGF alpha immunoreactivity in neurons of the myenteric plexus and in nerve fibers in the muscle and submucosal layers of the rat stomach. Association of TGF alpha staining nerve fibers to vessels and smooth muscle cells gives morphological evidence that EGF/TGF alphas actions to increase mucosal blood flow and gastric motility may be mediated by TGF alpha derived from neural structures. These data suggest that TGF alpha plays a role in the neural control of the gastric function.

Epidermal growth factor increases basal mucosal blood flow in the rat colon, a prostaglandin dependent effect.

OBJECTIVE/BACKGROUND Non-mitogenic biological activity such as modulation of mucosal blood flow is suspected to convey the protective effects of epidermal growth factor (EGF) in vivo. The aims of our present study were to determine the effects of EGF on colonic mucosal blood flow and injury induced hyperaemia in rats. DESIGN/METHODS Rats were pretreated with i.p. injections of vehicle, EGF, or indomethacin and EGF prior to mucosal injury. Basal mucosal blood flow and injury induced hyperaemia at the border of the damaged mucosa was determined by using reflectance spectrophotometry. RESULTS EGF significantly increased basal mucosal blood flow but did not further enhance injury induced hyperaemia. The EGF induced increase in basal mucosal blood flow was completely abolished by indomethacin pretreatment. CONCLUSIONS EGF induces an increase of basal mucosal blood flow through induction of prostaglandin synthesis. We hypothesize that the increase in mucosal blood flow contributes to the ability of EGF to protect the colonic mucosa against injury.