Value of transesophageal echocardiography, esophago-gastroduodenoscopy and retinoscopy prior to intra-arterial fibrinolytic therapy in patients with peripheral arterial disease.

BACKGROUND: We prospectively investigated the need for esophagogastroduodenoscopy (EGD), transesophageal echocardiography (TEE) and retinoscopy for pre-interventional screening in patients with peripheral arterial occlusive disease (PAD) prior to intraarterial fibrinolytic therapy. PATIENTS AND METHODS: 212 consecutive patients suffering from PAD (164 male and 48 female, mean age: 64 +/- 11 years, 161 patients stage II of Fontaine's classification, 10 patients at stage III and 41 patients at stage IV) referred for interventional treatment were included. 173 EGDs, 169 TEEs and 188 retinoscopies were performed within one week prior to fibrinolysis. 114 patients had all three examinations. RESULTS: Pathologic findings were detected in 56 (49%) of the 114 patients: 23 erosions, 12 ulcers, 2 esophagites, 1 gastric carcinomata, 3 intracardiac thrombi, 5 aortic thrombi, 11 diabetic and 5 hypertensive retinopathies and 1 with retinal aneurysms. 30 patients (25%) received fibrinolytic therapy, despite a contraindication: The one patient with ventricular thrombus was treated as an ultimate therapy, and amputation was prevented. Two patients showing plaques covered by large thrombi in the descending thoracic aorta were treated because cranial embolism should not occur. Four diabetic patients with multilevel disease, severe claudication and prior retinal bleeding were treated. Fibrinolytic therapy was started on 23 patients after complete healing of the mucosal lesions. CONCLUSION: TEE revealed potential sources of embolization in 4% of the patients and is justified to reduce the individual risk. Retinoscopy should be done in patients with diabetes mellitus and hypertension, and if prior bleeding is present fibrinolysis should be done only if other treatment-regimes are not available.

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

BACKGROUND: Epidermal growth factor (EGF) and transforming growth factor (TGF)-alpha protect the gastrointestinal mucosa against injury. In response to mucosal injury TGF-alpha, 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-alpha expression. METHODS: Monolayers of the non-transformed intestinal epithelial cell (IEC)-6 and IEC-18 lines were harvested, homogenized and shock-frozen at -80 degrees 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-alpha 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-alpha 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-alpha 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-alpha mRNA expression and TGF-alpha-dependent cell proliferation of intact epithelial cells. We hypothesize that epithelial cell interaction bears one of the possible signals for the increased TGF-alpha mRNA expression after mucosal injury.

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

OBJECTIVES AND DESIGN: Crohn's 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.

Mycophenolate mofetil reduces tissue damage and inflammation in an experimental model of colitis in rats.

BACKGROUND: Lymphocytes are widely believed to be responsible for persistent intestinal inflammation in inflammatory bowel diseases. Mycophenolate mofetil (MMF) is a potent immunosuppressant that inhibits lymphocyte proliferation and has been shown to be effective in preventing allograft rejection after organ transplantation. The purpose of this study was to assess the modulating effects of MMF on intestinal inflammation in an experimental model of colitis in rats. METHODS: Colitis was induced by rectal instillation of trinitrobenzenesulfonic acid (TNBS) in ethanol in male Sprague-Dawley rats. One group of rats (n = 10) was treated with MMF i.p. (25 mg/kg b.w.) daily for 1 week starting 24 h after induction of colitis. A second group of rats (n = 10) was treated with MMF at the same dose 2 days, I day and 1 h prior to induction of colitis. Control animals (n = 10) received vehicle only. After being killed, colonic tissue was macroscopically evaluated for necrosis and microscopically for ulcerations. Sections were stained and examined for the presence of granulocytes. RESULTS: Administration of MMF after induction of TNBS colitis reduced macroscopic injury by 62% compared to control animals (P = 0.01). Microscopic ulcerations were reduced by 64% compared to controls (P = 0.009). In addition, posttreatment significantly reduced the number of granulocytes. MMF pretreatment did not significantly prevent macroscopic or microscopic tissue damage, or change the number of granulocytes. CONCLUSION: Systemic administration of MMF significantly ameliorates tissue damage in a model of experimental colitis in rats suggesting that this compound may play an important role as an immunosuppressant in the therapy of inflammatory bowel diseases.

Increased expression of epidermal growth factor-receptor in an experimental model of colitis in rats.

BACKGROUND: Epidermal growth factor and related proteins share some structural homology and bind to one common receptor. We have shown previously that exogenously applied EGF protects colonic mucosa against injury in an experimental model of colitis in rats and that the endogenously expressed ligands for the EGF-receptor are predominantly transforming growth factor alpha precursors. The aim of our present study was to evaluate the EGF-receptor expression in response to mucosal injury in the same model of colitis. METHODS: The trinitrobenzene sulphonic acid (TNBS)/ethanol-induced model of colitis in rats was used and EGF-receptor expression was evaluated using ribonuclease protection assay and Western blot analysis. The extent of mucosal injury and inflammation was characterized by using a microscopic and macroscopic damage score and by estimation of the myeloperoxidase activity in colonic specimens. RESULTS: Irritation of the colonic mucosa leads to severe colonic inflammation with tissue oedema, erosions and mucosal ulcers and to a significant increase in myeloperoxidase activity expressed by neutrophil granulocytes and macrophages. A significant increase in EGF-receptor mRNA expression was obtained at 8-24 h followed by an increased expression of the EGF-receptor protein at 1-5 days after the induction of colitis. On Western blot analysis only one immunoreactive band with a molecular weight of approximately 170 kDa was detected. CONCLUSIONS: Mucosal inflammation leads to a significant increase in the EGF-receptor expression in the early phases of colitis. These findings support our hypothesis that EGF and related proteins and their common receptor play a pivotal role in mucosal defence and repair.

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.

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.

Increased expression of transforming growth factor alpha precursors in acute experimental colitis in rats.

BACKGROUND AND AIM: Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha), members of the EGF family of growth factors, protect rat gastric and colonic mucosa against injury. Having shown previously that exogenously applied EGF protects rat colonic mucosa against injury, the aim of the present study was to evaluate the endogenously expressed ligand mediating the protective effect of EGF/TGF-alpha in vivo. METHODS: In an experimental model of trinitrobenzene sulphonic acid (TNBS)/ethanol induced colitis in rats EGF and TGF-alpha expression was evaluated using a ribonuclease protection assay, northern blot analysis, western blot analysis, and immunohistochemistry. RESULTS: TGF-alpha mRNA increased 3-4 times at 4-8 hours after induction of colitis and returned to control levels within 24 hours. TGF-alpha immunoreactive protein with a molecular size of about 28 kDa representing TGF-alpha precursors increased markedly after induction of colitis with a peak at 8-12 hours. No fully processed 5.6 kDa TGF-alpha protein was detected in normal or inflamed colon tissue. Only a weak signal for EGF mRNA expression was detected in the rat colon and no EGF protein was observed by immunohistochemistry or western blot analysis. CONCLUSIONS: TGF-alpha precursors are the main ligands for the EGF receptor in acute colitis. It is hypothesised that TGF-alpha precursors convey the biological activity of endogenous TGF-alpha peptides during mucosal defence and repair.

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 alpha's 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.

Expression of insulin-like growth factor I receptors and binding proteins by colonic smooth muscle cells.

We recently demonstrated upregulation of insulin-like growth factor I (IGF-I) binding sites in the smooth muscle layer of inflamed rat colon. The increase in binding sites was due to increased expression of IGF binding proteins (IGFBPs), which modulate the effects of IGF. To further study the role of IGF in the colon, we investigated whether cultured colonic smooth muscle cells (SMC) express IGF-I receptors and IGFBPs. SMC were isolated by collagenase digestion from rat colonic smooth muscle and grown in primary culture. Equilibrium binding experiments using (125)I-labeled IGF-I showed the presence of an IGF-I receptor with a dissociation constant of 1.96 nM and a maximal binding constant of 53,000 receptors/cell. Competition binding studies with IGF-II and insulin, together with chemical cross-linking experiments, corroborated this conclusion. Western ligand blotting of conditioned medium and Northern analysis of total RNA demonstrated that the cells expressed and secreted IGFBP-4, -5, and -3 with molecular masses of 25, 31, and 45 kDa, respectively. These results, together with our in vivo studies in the rat, support a role for IGF in tissue fibrosis and stricture formation during chronic intestinal inflammation.

Keratinocyte growth factor ameliorates mucosal injury in an experimental model of colitis in rats.

BACKGROUND & AIMS: Keratinocyte growth factor (KGF) is known to enhance tissue repair in the skin; however, its role in the gastrointestinal tract is largely unknown. The aim of this study was to evaluate the effects of exogenous KGF in an experimental model of colitis in rats. METHODS: KGF was administered before or after induction of colitis with 2,4,6-trinitrobenzenesulfonic acid/ethanol. In the first two study groups, KGF (5 mg/kg) was administered intraperitoneally 24 hours and 1 hour before induction of colitis; animals were killed 8 hours (n=10) and 1 week (n=10) after injury. In subsequent study groups, KGF or vehicle treatment was begun 24 hours after the induction of colitis at doses of 5 (n=20), 1 (n=10), and 0.1 (n=10) mg/kg intraperitoneally and continued once daily for 1 week. Colonic tissue samples were evaluated macroscopically and microscopically for mucosal injury and assayed for myeloperoxidase activity. RESULTS: Administration of KGF after but not before induction of colitis significantly ameliorated tissue damage. Macroscopic necrosis and microscopic ulcerations were reduced by 40%-50% at KGF doses of 1 and 5 mg/kg. CONCLUSIONS: Exogenous KGF has a key role in mucosal healing in an experimental model of colitis in rats.

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.

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.

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.

Influence of food on plasma cholecystokinin and gastrin in patients with partial gastric resections and Roux-en-Y anastomosis.

Cholecystokinin (CCK) is a polypeptid released postprandially by the upper intestinal mucosa. There are several biological active forms of CCK. Radioimmunological measurements of CCK may not detect all biological active forms or may have the disadvantage of crossreacting with gastrin. In the following we describe a modification of a bioassay for CCK which was first developed by Liddle et al. (J Clin Invest 1985). By means of this bioassay pre- and postprandial plasma CCK-levels of healthy male volunteers are compared with CCK-levels of patients with partial gastric resections and excluded duodenum. Both groups showed similar basal CCK-values (about 1 pM) and a food induced increase of this hormone by reaching maximal values after 15 to 30 min (control: 4.30 +/- 0.65 vs. operated: 13.37 +/- 2.83 pM). Patients with gastric resections, however, had about three times more CCK released over the 60 min time period studied as compared to controls. Thus exclusion of the duodenum, the supposed main place of CCK production, does not cause a lower but rather higher increase of postprandial CCK release.