Myenteric neuronal loss in rats with experimental colitis: role of tissue transglutaminase-induced apoptosis.

BACKGROUND AND AIMS: Transglutaminases are tissue enzymes involved in different neuronal processes including maintenance and signalling. However, their up-regulation elicited by a variety of noxae contributes to neurodegeneration. This study tested the hypothesis that experimental inflammation evoked transglutaminase up-regulation in myenteric neurons and that this event had an impact on neuronal survival. METHODS: Rats with or without trinitro-benzene-sulphonic acid-induced colitis were used. One week after colitis induction, longitudinal muscle-myenteric plexus preparations were obtained from left colon to assess tissue-transglutaminase activity, protein and mRNA expression. Double labelling immunofluorescence using antibodies to neuron-specific enolase and transglutaminase was performed to identify myenteric neurons expressing transglutaminase. Additional sets of experiments evaluated the involvement of transglutaminase in the apoptotic process of cultured myenteric neurons. RESULTS: Compared to controls, rats with colitis showed several tranglutaminase/neuron-specific enolase positive myenteric neurons. Western blot analysis and RT-PCR confirmed that in rats with colitis, the increased neuronal transglutaminase-immunoreactivity was associated with an increased enzyme expression. Similarly, transglutaminase activity was significantly higher than in controls (1100+/-280 m U/g vs. 725+/-119 m U/g, p<0.05). In cultured myenteric neurons incubation with the specific transglutaminase inducer, retinoic acid, significantly increased neuronal apoptosis, whereas the presence of cystamine significantly reduced the number of apoptotic neurons. CONCLUSIONS: Experimental colitis evoked transglutaminase up-regulation and increased activity in myenteric neurons. This mechanism enhances neuronal susceptibility to apoptosis and could contribute to neuropathic changes during gut inflammation.

Differential expression of multiple transglutaminases in human colon: impaired keratinocyte transglutaminase expression in ulcerative colitis.

BACKGROUND AND AIMS: Ulcerative colitis (UC) is characterised by refractory inflammatory ulceration and damage to the colon. The mechanisms underlying impaired healing have yet to be defined. As transglutaminase expression resulting in matrix protein cross linking is associated with increased wound healing in a rat model of colitis, we hypothesised that different types of transglutaminase might also play a role in UC. PATIENTS AND METHODS: Endoscopic and histological indices were studied in 26 patients with UC (10 active and 16 inactive) and in 20 normal controls undergoing colonoscopy. Transglutaminase activity was evaluated in plasma (factor XIIIa) by a radioenzymatic method. Factor XIIIa, tissue and keratinocyte transglutaminase protein content, and mRNA expression in the colon were evaluated by western blot analysis and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), respectively. Colonic location of transglutaminases and their reaction products, the epsilon-(gamma-glutamyl)lysine bonds, was evaluated by immunohistochemistry using specific monoclonal antibodies. RESULTS: Transglutaminase activity was significantly lower in the plasma of patients with active UC (4.2 (2.4) mU/ml; p<0.05 v controls) than in those with inactive UC and controls (10.6 (2.2) and 12.1 (1.7) mU/ml). As shown by western blot, protein levels of tissue transglutaminase and factor XIIIa were unchanged in active UC compared with inactive disease and controls, while the keratinocyte form was reduced in active UC. Tissue transglutaminase and factor XIIIa immunostaining was strongly present in damaged areas colocalising with isopeptide bonds. In contrast, the keratinocyte form was almost absent in active UC and localised in the upper part of the crypts in normal subjects. RT-PCR showed upregulation of tissue transglutaminase mRNA in active UC (320% compared with controls) while keratinocyte transglutaminase gene expression was downregulated in active UC. CONCLUSIONS: The results of the present study support the concept that, in the damaged colon, transglutaminases are needed in response to chronic injury and underline the key role of these enzymes in mucosal healing.

Glutathione supplementation improves oxidative damage in experimental colitis.

BACKGROUND: The pathogenesis of inflammatory bowel disease is due, in part, to enhanced free-radical production and reduced antioxidant potential in mucosa cells. AIM: We evaluated in a rat model of trinitrobenzensulphonic acid (TNBS) colitis to see whether parenteral administration of glutathione is able to improve mucosal oxidative damage at onset (study A) and during chronic phases of colitis (study B). METHODS: In study A, the rats were injected with a single dose of glutathione (200 mg/kg, i.p.) or saline (0,2 ml, i.p.) 1 h before colitis induction and killed 1 h later. In study B, rats with induced colitis were treated with daily injection of glutathione (50 mg/kg, i.p.) or saline (0,2 ml, i.p.), and killed at 1, 2, 4 and 8 weeks. We evaluated on mucosal samples the macroscopic and histological damage and the oxidative stress assessed by the mucosal levels of lipoperoxides, malonyldialdehyde, glutathione and cysteine. RESULTS: In study A, colitis induction caused a significant increase to the total histological score (p<0.05), lipoperoxide and malonyldialdehyde levels (p<0.001), but did not affect glutathione and cysteine content. Glutathione pre-treatment decreased both total histological score (p<0.05) and lipoperoxide and malonyldialdehyde values (p<0.001). In study B, the extensive macroscopic and histological colonic damage induced by TNBS was accompanied by a reduction of glutathione and cysteine mucosal levels (p<0.01) and increased lipid peroxidation. Glutathione supplementation significantly improved colonic damage (p<0.01), restored glutathione and cysteine levels, and decreased, and even, if not totally, abolished lipid peroxidation (p<0.001). CONCLUSION: This paper further supports the pathogenic role of the imbalance in oxidant/antioxidant content in inducing mucosal colonic damage.

Factor XIII improves gastric stress lesions in rats.

BACKGROUND/AIMS: Tissue transglutaminase has been reported to be involved in the healing of experimental gastric ulcer; nevertheless, other type(s) of transglutaminase could be involved. The present experiments aimed at examining whether plasma transglutaminase (factor XIIIa) contributes to such healing and at evaluating whether factor XIII supplementation improves gastric mucosal lesions. METHODS: The healing effect of 200 U/kg of factor XIII administered intravenously was examined using a water immersion restraint rat model of stress gastric damage. The rats were sacrified 0, 2, 4, and 12 h after stress. The gastric mucosa was examined macroscopically and microscopically, and the transglutaminase activities were assayed in serum and gastric mucosa. Factor XIIIa and tissue transglutaminase protein levels in the gastric mucosa were analyzed by immunoblot. Immunohistochemistry was used to identify the location of tissue transglutaminase, factor XIIIa, and fibronectin in the gastric mucosa. RESULTS: The transglutaminase activity, reduced by stress in the gastric mucosa, increased up to 12 h after stress, peaking at 4 h, when the ulcer index significantly decreased. The serum transglutaminase level was low at all time points. Exogenous administration of factor XIII allowed a faster reduction of the ulcer index that was coincident with an increased transglutaminase activity in the mucosa. Both tissue transglutaminase and factor XIIIa protein levels were reduced by 6 h of stress and increased after factor XIII administration. Immunohistochemistry showed a colocalization of both factor XIIIa and tissue transglutaminase with fibronectin in the extracellular matrix of the damaged area. CONCLUSIONS: Two forms of transglutaminase are involved in the healing of stress-induced gastric erosions, and factor XIII administration allows faster gastric mucosa healing.

Serum transglutaminase correlates with endoscopic and histopathologic grading in patients with ulcerative colitis.

Factor XIIIa, a circulating form of transglutaminase, plays a key role in intestinal mucosal repair. We found that transglutaminase levels are decreased in serum of patients with inflammatory bowel diseases and demonstrated in a rat model of chronic colitis that serum transglutaminase is closely related to the severity of intestinal damage. We aimed, therefore, to correlate serum transglutaminase levels with standard endoscopic and histopathologic grading systems in patients affected by ulcerative colitis (UC). In 249 patients with UC, we assayed serum transglutaminase activity by a radioenzymatic method and measured clinical activity index (CAI) according to modified Rachmilewitz's criteria. In a subset of 82 patients undergoing colonoscopy, endoscopic and histologic indices were studied. Biopsy specimens were also taken from 28 patients to measure myeloperoxidase (MPO) as a marker of mucosa inflammation. Serum transglutaminase levels significantly correlated with the CAI scoring (r = -0.63; P < 0.01); likewise serum transglutaminase showed the best correlation with endoscopic (r = -0.71; P < 0.001) and histologic (r = -0.79; P < 0.001) scores. Myeloperoxidase activity was significantly higher in patients with active UC than those in remission (P < 0.01), showing a significant correlation with serum transglutaminase levels (r = -0.68; P < 0.01). Immunohistochemistry showed factor XIIIa localization in the extracellular matrix of damaged mucosa. In conclusion, these results suggest that transglutaminase assay can be useful in managing UC as a serological, noninvasive indicator of intestinal mucosal status.

Recombinant factor XIII improves established experimental colitis in rats.

Factor XIII (FXIII) is the plasma-borne transglutaminase involved in fibrin clot stabilization and wound healing. FXIII levels in the plasma of patients with inflammatory bowel diseases are lower than normal and there is a significant inverse correlation of FXIII levels with clinical severity. Moreover, uncontrolled studies report beneficial effects of FXIII supplementation in patients resistant to conventional therapies. We investigated the effects of intravenous recombinant FXIII (rFXIII) treatment in experimentally induced rat colitis to verify that FXIII was the active agent in plasma FXIII concentrates and to better understand the potential therapeutic use of this protein. Colitis was induced by instillation of 12% 2.4,6-trinitrobenzenesulfonic acid (TNBS) in 50% ethanol into the colon of male Wistar rats. Rats were treated with 0.65 mg/kg rFXIII or vehicle (intravenously) daily for 10 days. Treatment was started either immediately after TNBS/EtOH instillation (to evaluate effects on developing lesions) or seven days later (to evaluate effects on established lesions). In both cases rats were killed either immediately after the end of treatment (to evaluate immediate effects) or 17 days later (to evaluate long-lasting effects). The effects of rFXIII were compared to positive (5-amino-2-hydroxybenzoic acid) control over a 35-day time course. The severity of lesions was determined by colon weight and macroscopic and histologic scores. Transglutaminase activity was measured in both colon tissue and serum. rFXIII treatment reduced lesion severity significantly not only in developing but also in established lesions. Improvements in healing persisted at least 18 days after treatment was discontinued. Serum and tissue transglutaminase levels were restored by rFXIII treatment. In conclusion, pure rFXIII is as effective as plasma FXIII concentrates in a rat model of experimental colitis. In addition, rFXIII significantly improves the healing of preexisting lesions, a characteristic useful in treatment of human inflammatory bowel diseases.

Direct evidence of oxidative damage in acute and chronic phases of experimental colitis in rats.

During inflammatory colitis in man and experimental animals, the production of free radicals increases. This study evaluated the histological pattern and biochemical parameters of oxidative damage during acute and chronic colitis induced by 2,4,-trinitrobenzenesulfonic acid + ethanol in rats. On the samples of scraped mucosa of six groups of rats, one not treated, one killed after 1 hr, and those killed one, two, four, and eight weeks after the induced-damage, we determined the histological and superoxide dismutase activity and the concentration of lipoperoxides, malonyldialdheyde, and reduced glutathione. After 1 hr, the mucosal damage and superoxide dismutase activity were slight; glutathione, lipoperoxides, and malonyldialdheyde were significantly increased. At one week, the histological damage was severe, decreasing progressively, and significantly correlated to superoxide dismutase activity. Lipoperoxides and malonyldialdheyde were high throughout the study. Glutathione was significantly increased at one and two weeks and dramatically decreased thereafter. Therefore, in experimental colitis the cascade of free-radical production induces a constant self-maintaining lipoperoxidation and consumes the cellular antioxidant capability.

Butyrate enemas in experimental colitis and protection against large bowel cancer in a rat model.

BACKGROUND & AIMS: Butyrate is effective in experimental colitis by increasing transglutaminase activity. Because ulcerative colitis increases the risk of colonic neoplasia, the aim of this study was to investigate whether butyrate treatment reduces mucosal sensitivity to colon cancer development in rats with experimental colitis. METHODS: Colon cancer was induced by azoxymethane injections in 10 rats with trinitrobenzensulfonic acid-induced colitis and 10 rats without colitis. Three additional groups of rats with colitis were treated with butyrate, mesalamine, and saline enemas, respectively, twice daily for 8 weeks; 1 week after colitis induction, tumors were induced. Biopsy specimens for assessment of proliferation pattern and transglutaminase activity were obtained during the latent period of cancer development. Characteristics of tumors were recorded 27 weeks after the first exposure to azoxymethane. RESULTS: Experimental colitis enhanced carcinogenesis; butyrate therapy reduced both incidence and size of tumors and also affected colonic proliferation pattern. Transglutaminase levels were restored by butyrate treatment in rats with colitis. CONCLUSIONS: The protective effect of butyrate against large bowel cancer in experimental colitis suggests its usefulness in long-term therapy to decrease disease relapses and to reduce colon cancer risk in ulcerative colitis.

Transglutaminases in Crohn's disease.

Transglutaminases are a family of Ca-dependent enzymes involved in various biological events. Circulating transglutaminase (factor XIIIa) is decreased in blood of patients with inflammatory bowel diseases. There is evidence that factor XIIIa and tissue type transglutaminase, present in cell cytosol, bind to various proteins of the extracellular matrix. This study examined the value of serum transglutaminase assay in the treatment and follow up of Crohn's disease and then investigated the intestinal location of both forms of transglutaminases by immunohistochemistry in normal and abnormal tissues. Serum transglutaminase activity was assayed in 36 patients with active Crohn's disease (CDAI > 150). Eighteen patients were studied prospectively from relapse into remission. A significant inverse correlation (p < 0.001) was found between circulating transglutaminase and Crohn's disease activity index; a correlation was also found between serum transglutaminase and serum orosomucoid (p < 0.01) and C reactive protein (p < 0.01). Patients were prospectively studied until clinical remission showed improvement in both their CDAI score mean (SD) (230 (46) to 72 (34), p < 0.01) and transglutaminase activity mean (SD) (0.61 (0.12) to 0.93 (0.13) mU/ml, p < 0.01). The immunohistochemistry assessment showed a colocalisation of factor XIIIa and tissue transglutaminase to the extracellular matrix of damaged tissues. In conclusion, these data confirm the value of serum transglutaminase assay as marker of Crohn's disease activity, extend the utility of serum transglutaminase assay to follow up of the disease, and emphasised the role of different types of transglutaminases in extracellular matrix assembly in the damaged tissues.

Transglutaminase in azoxymethane-induced colon cancer in the rat.

A widespread from of transglutaminase, tissue transglutaminase, has been identified in a number of mammalian cell types, both normal and transformed cells; its biological role is not well understood. We investigated the effect of experimentally induced colon cancer on transglutaminase activity in the rat. Azoxymethane (15 mg/kg for six weeks), given by a course of weekly intraperitoneal injections, produces tumors almost exclusively confined to the intestinal tract. Transglutaminase activity was assayed on tissue homogenates both during the period of treatment and, when the cancer had developed, on tumor tissue and on microscopically uninjured adjacent tissue. A transient proliferative phase was present in the intestine during azoxymethane treatment: in this phase we found a coincidentally increased transglutaminase levels. Transglutaminase activity in tumors of both small and large intestine was significantly higher than in adjacent tissue. Immunohistochemistry revealed higher levels of transglutaminase in tumors, mainly localized in the extracellular matrix, than in adjacent tissues, where it was widely distributed. The present study shows that transglutaminase, besides its potential role in intracellular process during early proliferative phase of carcinogenesis, may also play an important role in matrix processing during tumor growth and differentiation.

Butyrate, mesalamine, and factor XIII in experimental colitis in the rat: effects on transglutaminase activity.

BACKGROUND/AIMS: Butyrate and factor XIII may improve ulcerative colitis; they also affect tissue and serum transglutaminase levels. We investigated the therapeutic potential of sodium butyrate and factor XIII and the role of transglutaminase during mucosal repair in experimental colitis. METHODS: Rats with induced colitis were treated with sodium butyrate, mesalamine, sodium butyrate plus mesalamine, or saline enemas. Thromboxane B2 was monitored as index of inflammation. In a fifth group, the effectiveness of intravenous Factor XIII was assessed. RESULTS: Sodium butyrate, alone or plus mesalamine, reduced histological activity from 13.7 +/- 1.7 (saline) to 2.5 +/- 1.3 and 2.3 +/- 1.1 (P < 0.01), respectively. Transglutaminase, reduced in the colons of the saline group (783 +/- 157 vs. normal 1800 +/- 192 mU/g; P < 0.01), returned toward normal values in the sodium butyrate or sodium butyrate plus mesalamine groups (1390 +/- 228 and 1226 +/- 172 mU/g, respectively; P < 0.01 vs. saline). Furthermore, sodium butyrate plus mesalamine reduced thromboxane B2 levels by day 5 (0.92 +/- 0.16 vs. saline 1.85 +/- 0.34 ng/mL; P < 0.05). Factor XIII therapy improved the histological picture (2.7 +/- 2.1 vs. saline 13.8 +/- 1.7; P < 0.01) and increased transglutaminase levels both in serum (2.81 +/- 0.11 vs. saline 1.45 +/- 0.09 mU/mL; P < 0.01) and in colon (1503 +/- 127 vs. saline 747 +/- 103). CONCLUSIONS: Sodium butyrate and factor XIII improve colitis, sodium butyrate plus mesalamine reduce early thromboxane B2 synthesis, and transglutaminase(s) plays a role in ulcer healing.

Serum and tissue transglutaminase correlates with the severity of inflammation in induced colitis in the rat.

Simple rat models of acute and chronic colonic inflammation were used to study the behaviour in serum and mucosa of transglutaminase (TG), an enzyme recently found to be reduced in serum of patients with inflammatory bowel disease (IBD) and related to the activity index of the disease. In the first model the intraluminal administration of 400 mM lactic acid in the colon caused an acute inflammation resembling that of florid ulcerative colitis in humans. In the second, intraluminal administration of the hapten 2,4,6-trinitrobenzenesulphonic acid (TNB) (10 or 30 mg) in 0.25 ml of ethanol as a 'barrier breaker' produced a chronic inflammatory disease. The results showed a reduced TG activity in colon of rats in both acute and chronic induced colitis (447 +/- 75 versus 1344 +/- 59 mU/g protein (p less than 0.001) and 484 +/- 59 versus 1204 +/- 75 mU/g protein (p less than 0.001)). This decreased activity was related to the severity of mucosal damage, which was dose-dependent. Moreover, in severe colitis the immunohistochemistry showed a TG location in repairing tissue. Serum TG activity was decreased after TNB administration (1.36 +/- 0.05 versus 3.44 +/- 0.20 mU/ml (p less than 0.001)) but not after lactic acid treatment (3.97 +/- 0.11 versus 3.78 +/- 0.16 mU/ml). In summary, the reduction of TG activity in both tissue and serum when the damage is stabilized reflects the altered morphofunctional integrity of the colon and suggests that serum assay of this enzyme could be a simple marker of intestinal mucosal status in IBD.

Serum transglutaminase in inflammatory bowel diseases.

We evaluated the serum transglutaminase activity in patients with inflammatory bowel diseases (IBD) to correlate its level with clinical status. There were 49 patients with Crohn's disease (CD), 50 with ulcerative colitis (UC), 35 with diseases other than IBD as control group and 42 healthy subjects matched for sex and age. Enzyme activity was significantly lower in both IBD groups than in controls and in normal subjects (p less than 0.001); we found a significant negative correlation between serum transglutaminase (TG) activity and clinical severity of the disease in both IBD patient groups (r = -0.54 in CD, and r = -0.69 in UC). Moreover, in UC and CD patients, a serum TG value lower than 0.80 mU/ml retrospectively proved to predict the need for major surgery and/or total parenteral nutrition. These results suggest that serum TG may prove useful in the management of inflammatory intestinal diseases in predicting clinical outcome.

Behaviour of transglutaminase activity in intestine of starved and refed rats.

Starvation causes an intestinal mucosa atrophy which is greater in jejunum than in ileum. Hypoplasia is promptly reversed by refeeding. Transglutaminase (TG) has been controversially implicated in cell proliferation and its role in intestine is not defined. We investigate, by the above described model, the behaviour of TG in proximal and distal small bowel as well as in colon of rats after 4 days of starvation and at day 1, 2, 3, 4, 5, 7 and 10 of refeeding. Our results emphasize a significative reduction of TG in small bowel induced by starvation (day 0) and a prompt recovery of the enzyme activity after refeeding; furthermore, in the first intestinal tract TG activity reaches from day 2 to day 5 values which are significantly higher than basal. Four days of starvation do not affect TG in colon. In conclusion, our study demonstrates that in rats high values of TG activity are coincident with the intense proliferative phase in small intestine subsequent to starvation atrophy.