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Hepatocyte growth factor (HGF) has a protective effect on gastric epithelial integrity
A756
AGA ABSTRACTS
H E P A T O C Y T E G R O W T H FACTOR (HGF) HAS A PROTECTIVE EFFECT ON GASTRIC EPITHELIAL INTEGRITY M. Takahashi, S. eta, M. Kurita, K. Ogura, E. Hamada, T. Shimadal), A. Terano 2), M. Omata 1). 1) 2nd Department of Internal Medicine, University of Tokyo, Tokyo, Japan. 2) 2nd Department of Internal Medicine, University of Dokkyo, Tochigi, Japan. We have previously shown that endogenously produced HGF has a stimulative effect on proliferation and restitution of rabbit gastric epithelial cells in primary culture, which is in accordance with various studies in other organs (1994 DDW abstract #2176 and #2466). HGF is actually known to be a pleiotropic factor which has previously been shown to act as a mitogen, a motogen, a morphogen, and a tumor suppressor. In this study, we demonstrated the protective action of HGF On gastric epithelial integrity which is mediated by a pathway that has never been discussed before, using a primary culture system. METHODS: Primary culture of rabbit gastric epithelial cells was prepared from adult rabbit stomach, after enzyme digestion of gastric ceils. Cell proliferation was assessed by 3H-thymidine incorporation method. Restitution was assessed by in vitro round wound restituion model. Cellular viability was assessed by MTT assay. The integrity of monolayer culture was assessed by determining the denuded areas created in the monolayer sheet after incubating with noxious agents. Photomicrographs were taken and analysed by computered image analyzing system. The process was also observed by movie Pictures. RESULT: 1. Ethanol of low concentration, less than 4%, did not decrease cellular viability of gastric ePithelial cells. Sucralfate protected the decrease of cell viability caused by 15% ethanol, whil e HGF did not at all. 2. 2% ethanol caused disruption of gastric epithelial cell monolayer sheet, without damaging cellular viability. 3. HGF proteeted the monolayer significantly from disruption caused by 2% ethanol. Sucralfate had the same effect as was demonstrated elsewhere. 4. Sucralfate have little, if any, effect on the proliferation and migration of gastric epithelial ceils, while HGF had the strong effect, as was demonstrated previously. CONCLUSION: Ethanol of low concentration caused the disruption of gastric epithelial cells without damaging cellular viability which is an early stage of gastric damage. HGF has protective effect against early damage of gastric epithelial cells, which is mediated by neither proliferation nor restitution. As a result, HGF may contribute to maintain the integrity of the gastric mucosa.
• THE EFFECT OF DIETARY SULFATE ON COLONIC METHANE PRODUCTION IN MAN. A. Tanaerman. W. Wesseling*, M.B. Katan*, F.M. Nagengast. Dept. of Gastroenterology, University Hospital, Nijmegen, and *Agricultural University, Wageningen, The Netherlands. Hydrogen produced during colonic fermentation may be used by methanogens to produce methane or by sulfate-reducing bacteria (SRB), to form toxic hydrogen sulfide (H2S). Impairment of eolonocyte nutrition by H2S and involvement in the pathogenesis of ulcerative colitis have been postulated. It is controversial whether methanogens outnompete SRB for H2 consumption. Problems to quantitate sulfide in feces have hampered research in this field. We studied the role of SRB in the human colon in relation to that of the methanogens. Method~: In a cross-over study, twelve normal subjects with methanogenic activity (CH4-producers) and 8 subjects without (CH4-non-producers) received a one-week diet rich in dietary sulfate (S-rich period) or poor in dietary sulfate (S-poor period). Both periods were separated by a wash-out Of one week. At the end of each period, feces was sampled and fecal sulfide concentrations (free, bound and totall were measured by means of gaachmmatography. Results: No differences in total fecal sulfide levels were found between the S-rich ( 1 . 7 9 : 1 : 1 . 1 5 mmollkg dry weight, mean :t: SD) and the S-poor period (1.96 + 1.61 mmol/kg). The CH4-producers and non-producers had also similar sulfide levels. Nearly all sulfide Was bound to the insoluble particulate feces-fraction. During the S-rich period a significant reduction in breath methane was observed in the CH4-grou p. Of the 12 CH4-produeers, 4 became non-producer, 3 showed a strong reduction and 5 remained unchanged; This reduction was accompanied by a reduced CH 4production in the corresponding fecal samples during in vitro fecal incubation experiments, In vitro fecal incubation with 20raM Na2SO4 showed a huge increase in free sulfide concentrations, reaching levels of 30 mmollkg dry weight, both inthe S-rich and S-poor samples. This increase was even more pronounced in the CH4-producers, compared to the non-produears. In the CH4-producers, this increase in sulfide was accompanied with a decrease in CH4-production. Conclusions: Although a sulfate-rich diet did not result in elevated fecal sulfide levels, high sulfate levels might stimulate SRB-activity at the cost of methanogena, as also shown by in vitro incubation experiments. This study also showed that SRB-activity is not reserved to CH4-non-producers, but is at least equally active in CH4-producers.
GASTROENTEROLOGY, Vol. t O 8 , No. 4
K E R A T I N O C Y T E GROWTH FACTOR IS A N E N D O G E N O U S STIMULANT OF RABBIT GASTRIC EPITHELIAL C E L L PROLIFERATION A N D M I G R A T I O N IN PRIMARY CULTURE
M. Takahashi, S. eta, K. Ogura, E. Hamada, T. Shimadal), A. Terano 2), M. Omata 1). 1) 2nd Department of Internal Medicine, University of Tokyo, Tokyo, Japan. 2) 2nd Department of Internal Medicine, University of Dokkyo, Tochigi, Japan. Mesenchymal-epithelial interactions are important in the gastric mucosal repair. Although various growth factors have been suggested to be involved in the gastric mucosal repair, specific factors responsible for such interactions have not been established. In the present study, we have investigated the role of keratinocyte growth factor (KGF) in the gastric mueosal repair, using rabbit gastric epithelial cells in primary culture. METHODS: Primary culture of rabbit gastric epithelial ceUs and gastric fibroblasts were prepared from adult rabbit stomach, after enzyme digestion of gastric cells. Cell growth was assessed by 3H-thymidine uptake.. Restitution was assessed by a round wound restitution model of cultured gastric epithelial cells. The monolayer was wounded with a custom-made scraper which produced a round wound o f 1.5 mm in diameter, and cultured with agents. The denuded areas were measured in a time course. Cellular total RNA was extracted and mRNA was purified by oligo-DT ~01umn. Northern blot hybridization was performed, using 32p_ labelled KGF oligonucleotide. RESULTS: 1. KGF(1.56-50ng/ml) stimulated DNA synthesis of gastric epithelial cells significantly in a dose dependent manner. The effect of KGF(25ng/ml) was synergistic with HGF(10ng/ml), EGF(10ng/ml), and insulin(20mU/ml), indicating that the proliferative effect of KGF was mediated by distinct pathway. 2. KGF(2.5-20ng/ml) facilitated the restitution of gastric epithelial cells significantly in a dose dependent manner. 3. Northern blot analysis revealed that the expression of KGF mRNA was seen in the gastric fibroblasts. In contrast, it was not obvious in the gastric epithelial cells. CONCLUSION: These results suggest that KGF was produced at gastric fibroblasts and facilitated both proliferation and restitution of gastric epithelial cells, and as a result, KGF might be involved in the gastric mucosal repair, through mesenchymal-epithelial interaction.
• DEVELOPMENTAL EXPRESSION OF TREFOIL PEPTIDES IN THE RAT GASTROINTESTINAL TRACT. D.R. Taupin, A. Shnlkes , A.S. Giraud. University of Melbourne, Department of Medicine, Western Hospital, Australia. The trefoil peptides, intestinal trefoil factor (ITF) and spasmolytic peptide (SP) are h!ghly conserved and expressed, mucin-associated, secretory proteins of the gut. Since trefoil peptides are early pbenotypic markers in metaplastic gut mucosa, we have quantified the developmental expression of ITF, an intestinal peptide, and SP, an antral peptide, in rat stomach and intestine from fetal day 17 until after weaning. Methods: Timed pregnant Wistar rats were used to obtain fetal or posmatal (n=84) stomach, proximal intestine and pancreas from the last 6 days of gestation and postnatal days +16 and +27 (postweaning). Tissues were extracted for trefoil and gastrin RIA (values given as pmol/mg protein) and formalin fixed for immunohistocbemistry. Results: Gastric SP was present in levels comparable to the adult and weaned rat by day 17 (D17; 5.7_+2.1), with an appreciable peak before weaning (+16, 33.0-+3.3; +27, 18.2_+4,7, p=0.023). Levels of gastric ITF were static in late gestation and peaked after weaning, (D17, 4.8_+1.4 ; D19, 5.0_+2.0; D21, 6.7+-5.9; +16, 18.6_+7:8; +27, 141_+76). For both peptides, blebs of immunoreactivity overlying stratified epithelium and lining secondary lumena, and stippled material in some cohimnar ceUs were seen at DI9. By term positive ceils were present throughou t antral glands. After" weaning a mature pattern of l~redominantly basal gland and mucous neck cell positivity was seen; staining was more intense toward the duodenal pole, and by D20 corresponded to neutral mucin staining by PAS. In contrast, gastrin expression was <0.5 throughout the last 6 days of ontogeuy, with levels >2.5 at weaning. In the intestine, levels of SP were unchanged after I)17 (5.5_+3.0) with immunoreactivity demonstrable lining the epithelium and immature crypts, and within goblet ceils after weaning. Although appreciable intestinal ITF was present at days 17-18 (101.6_+25 and 79.2 _+43respectively)~ levels fell, then rose abruptly after day 21 (D19-21, median 20.2; D22, median 132; p=0.012, rank-sum test), and peaked before weaning. Dense ITF- lined clefts and primary and secondary lumena were observed by day 19 and strongly immunoreactlve immature goblet cells were readily identified at D22. Levels of rSP and rITF in the pancreas were<2 and <5 respectively at all gestational ages. Conclusions: 1. Appreciable levels of im/nunoreactive trefoils are present before morphological and cytochemical evidence of mucin and gash'in expression. 2. Trefoil expression in fetal gut shows the organ specificity and polarity of the mature gut. 3. Peak organ-specific trefoil expression occurs before weaning.
AGA ABSTRACTS
H E P A T O C Y T E G R O W T H FACTOR (HGF) HAS A PROTECTIVE EFFECT ON GASTRIC EPITHELIAL INTEGRITY M. Takahashi, S. eta, M. Kurita, K. Ogura, E. Hamada, T. Shimadal), A. Terano 2), M. Omata 1). 1) 2nd Department of Internal Medicine, University of Tokyo, Tokyo, Japan. 2) 2nd Department of Internal Medicine, University of Dokkyo, Tochigi, Japan. We have previously shown that endogenously produced HGF has a stimulative effect on proliferation and restitution of rabbit gastric epithelial cells in primary culture, which is in accordance with various studies in other organs (1994 DDW abstract #2176 and #2466). HGF is actually known to be a pleiotropic factor which has previously been shown to act as a mitogen, a motogen, a morphogen, and a tumor suppressor. In this study, we demonstrated the protective action of HGF On gastric epithelial integrity which is mediated by a pathway that has never been discussed before, using a primary culture system. METHODS: Primary culture of rabbit gastric epithelial cells was prepared from adult rabbit stomach, after enzyme digestion of gastric ceils. Cell proliferation was assessed by 3H-thymidine incorporation method. Restitution was assessed by in vitro round wound restituion model. Cellular viability was assessed by MTT assay. The integrity of monolayer culture was assessed by determining the denuded areas created in the monolayer sheet after incubating with noxious agents. Photomicrographs were taken and analysed by computered image analyzing system. The process was also observed by movie Pictures. RESULT: 1. Ethanol of low concentration, less than 4%, did not decrease cellular viability of gastric ePithelial cells. Sucralfate protected the decrease of cell viability caused by 15% ethanol, whil e HGF did not at all. 2. 2% ethanol caused disruption of gastric epithelial cell monolayer sheet, without damaging cellular viability. 3. HGF proteeted the monolayer significantly from disruption caused by 2% ethanol. Sucralfate had the same effect as was demonstrated elsewhere. 4. Sucralfate have little, if any, effect on the proliferation and migration of gastric epithelial ceils, while HGF had the strong effect, as was demonstrated previously. CONCLUSION: Ethanol of low concentration caused the disruption of gastric epithelial cells without damaging cellular viability which is an early stage of gastric damage. HGF has protective effect against early damage of gastric epithelial cells, which is mediated by neither proliferation nor restitution. As a result, HGF may contribute to maintain the integrity of the gastric mucosa.
• THE EFFECT OF DIETARY SULFATE ON COLONIC METHANE PRODUCTION IN MAN. A. Tanaerman. W. Wesseling*, M.B. Katan*, F.M. Nagengast. Dept. of Gastroenterology, University Hospital, Nijmegen, and *Agricultural University, Wageningen, The Netherlands. Hydrogen produced during colonic fermentation may be used by methanogens to produce methane or by sulfate-reducing bacteria (SRB), to form toxic hydrogen sulfide (H2S). Impairment of eolonocyte nutrition by H2S and involvement in the pathogenesis of ulcerative colitis have been postulated. It is controversial whether methanogens outnompete SRB for H2 consumption. Problems to quantitate sulfide in feces have hampered research in this field. We studied the role of SRB in the human colon in relation to that of the methanogens. Method~: In a cross-over study, twelve normal subjects with methanogenic activity (CH4-producers) and 8 subjects without (CH4-non-producers) received a one-week diet rich in dietary sulfate (S-rich period) or poor in dietary sulfate (S-poor period). Both periods were separated by a wash-out Of one week. At the end of each period, feces was sampled and fecal sulfide concentrations (free, bound and totall were measured by means of gaachmmatography. Results: No differences in total fecal sulfide levels were found between the S-rich ( 1 . 7 9 : 1 : 1 . 1 5 mmollkg dry weight, mean :t: SD) and the S-poor period (1.96 + 1.61 mmol/kg). The CH4-producers and non-producers had also similar sulfide levels. Nearly all sulfide Was bound to the insoluble particulate feces-fraction. During the S-rich period a significant reduction in breath methane was observed in the CH4-grou p. Of the 12 CH4-produeers, 4 became non-producer, 3 showed a strong reduction and 5 remained unchanged; This reduction was accompanied by a reduced CH 4production in the corresponding fecal samples during in vitro fecal incubation experiments, In vitro fecal incubation with 20raM Na2SO4 showed a huge increase in free sulfide concentrations, reaching levels of 30 mmollkg dry weight, both inthe S-rich and S-poor samples. This increase was even more pronounced in the CH4-producers, compared to the non-produears. In the CH4-producers, this increase in sulfide was accompanied with a decrease in CH4-production. Conclusions: Although a sulfate-rich diet did not result in elevated fecal sulfide levels, high sulfate levels might stimulate SRB-activity at the cost of methanogena, as also shown by in vitro incubation experiments. This study also showed that SRB-activity is not reserved to CH4-non-producers, but is at least equally active in CH4-producers.
GASTROENTEROLOGY, Vol. t O 8 , No. 4
K E R A T I N O C Y T E GROWTH FACTOR IS A N E N D O G E N O U S STIMULANT OF RABBIT GASTRIC EPITHELIAL C E L L PROLIFERATION A N D M I G R A T I O N IN PRIMARY CULTURE
M. Takahashi, S. eta, K. Ogura, E. Hamada, T. Shimadal), A. Terano 2), M. Omata 1). 1) 2nd Department of Internal Medicine, University of Tokyo, Tokyo, Japan. 2) 2nd Department of Internal Medicine, University of Dokkyo, Tochigi, Japan. Mesenchymal-epithelial interactions are important in the gastric mucosal repair. Although various growth factors have been suggested to be involved in the gastric mucosal repair, specific factors responsible for such interactions have not been established. In the present study, we have investigated the role of keratinocyte growth factor (KGF) in the gastric mueosal repair, using rabbit gastric epithelial cells in primary culture. METHODS: Primary culture of rabbit gastric epithelial ceUs and gastric fibroblasts were prepared from adult rabbit stomach, after enzyme digestion of gastric cells. Cell growth was assessed by 3H-thymidine uptake.. Restitution was assessed by a round wound restitution model of cultured gastric epithelial cells. The monolayer was wounded with a custom-made scraper which produced a round wound o f 1.5 mm in diameter, and cultured with agents. The denuded areas were measured in a time course. Cellular total RNA was extracted and mRNA was purified by oligo-DT ~01umn. Northern blot hybridization was performed, using 32p_ labelled KGF oligonucleotide. RESULTS: 1. KGF(1.56-50ng/ml) stimulated DNA synthesis of gastric epithelial cells significantly in a dose dependent manner. The effect of KGF(25ng/ml) was synergistic with HGF(10ng/ml), EGF(10ng/ml), and insulin(20mU/ml), indicating that the proliferative effect of KGF was mediated by distinct pathway. 2. KGF(2.5-20ng/ml) facilitated the restitution of gastric epithelial cells significantly in a dose dependent manner. 3. Northern blot analysis revealed that the expression of KGF mRNA was seen in the gastric fibroblasts. In contrast, it was not obvious in the gastric epithelial cells. CONCLUSION: These results suggest that KGF was produced at gastric fibroblasts and facilitated both proliferation and restitution of gastric epithelial cells, and as a result, KGF might be involved in the gastric mucosal repair, through mesenchymal-epithelial interaction.
• DEVELOPMENTAL EXPRESSION OF TREFOIL PEPTIDES IN THE RAT GASTROINTESTINAL TRACT. D.R. Taupin, A. Shnlkes , A.S. Giraud. University of Melbourne, Department of Medicine, Western Hospital, Australia. The trefoil peptides, intestinal trefoil factor (ITF) and spasmolytic peptide (SP) are h!ghly conserved and expressed, mucin-associated, secretory proteins of the gut. Since trefoil peptides are early pbenotypic markers in metaplastic gut mucosa, we have quantified the developmental expression of ITF, an intestinal peptide, and SP, an antral peptide, in rat stomach and intestine from fetal day 17 until after weaning. Methods: Timed pregnant Wistar rats were used to obtain fetal or posmatal (n=84) stomach, proximal intestine and pancreas from the last 6 days of gestation and postnatal days +16 and +27 (postweaning). Tissues were extracted for trefoil and gastrin RIA (values given as pmol/mg protein) and formalin fixed for immunohistocbemistry. Results: Gastric SP was present in levels comparable to the adult and weaned rat by day 17 (D17; 5.7_+2.1), with an appreciable peak before weaning (+16, 33.0-+3.3; +27, 18.2_+4,7, p=0.023). Levels of gastric ITF were static in late gestation and peaked after weaning, (D17, 4.8_+1.4 ; D19, 5.0_+2.0; D21, 6.7+-5.9; +16, 18.6_+7:8; +27, 141_+76). For both peptides, blebs of immunoreactivity overlying stratified epithelium and lining secondary lumena, and stippled material in some cohimnar ceUs were seen at DI9. By term positive ceils were present throughou t antral glands. After" weaning a mature pattern of l~redominantly basal gland and mucous neck cell positivity was seen; staining was more intense toward the duodenal pole, and by D20 corresponded to neutral mucin staining by PAS. In contrast, gastrin expression was <0.5 throughout the last 6 days of ontogeuy, with levels >2.5 at weaning. In the intestine, levels of SP were unchanged after I)17 (5.5_+3.0) with immunoreactivity demonstrable lining the epithelium and immature crypts, and within goblet ceils after weaning. Although appreciable intestinal ITF was present at days 17-18 (101.6_+25 and 79.2 _+43respectively)~ levels fell, then rose abruptly after day 21 (D19-21, median 20.2; D22, median 132; p=0.012, rank-sum test), and peaked before weaning. Dense ITF- lined clefts and primary and secondary lumena were observed by day 19 and strongly immunoreactlve immature goblet cells were readily identified at D22. Levels of rSP and rITF in the pancreas were<2 and <5 respectively at all gestational ages. Conclusions: 1. Appreciable levels of im/nunoreactive trefoils are present before morphological and cytochemical evidence of mucin and gash'in expression. 2. Trefoil expression in fetal gut shows the organ specificity and polarity of the mature gut. 3. Peak organ-specific trefoil expression occurs before weaning.