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Location of transforming growth factor-β1 and phenotypic modulation of fibroblasts during gastric ulcer healing in rats
April 1 9 9 5
Growth, Development, and Nutrition
• DEVELOPMENT OF INTRAEPITHELIAL LYMPHOCYTES (IEL) IN THE SMALL INTESTINE (SI) OF THE FETAL SHEEP- EFFECTS OF AGE AND ENTERAL DIET. F.M. Thompson, S.J. Wing, P. Sangild', J.F. Tmhair. Dep Anatomy & Histology, University of Adelaide, SA, AUSTRALIA; "Dep Clinical Studies, Reproduction, Royal Veterinary & Agricaltuml University of Copenhagen, DENMARK. Aims: To investigate the ontogeny of IELs in a well characterized model of SI development in a long gestation species (fetal sheep) 1. The effects of age, fetal swallowing and fetal growth retadation were studied. Methods: Normal fetal sheep were examined at 100(n=2), 125(7) and 140(3) days(d) gestation (term is 147d). Esophageal ligations and canuiations were carried out at 105d in 29 fetuses, preventing fetal ingestion. For 7d before collecting tissues at 125d, saline(n=4), milk(5) or colostral whey proteins(4), gaslrin releasing paptide (GRP, 3.6 nmol/day)(6), or amniotic fluid (5) was infused (4x20ml bolus par day). One group received no infusion(5). Non-pregnant ewes had endometrial caruncles removed prior to mating to produce fetuses(n=6) exhibiting chronic intraunterine growth retardation (IUGR) by 140d. Tissues from proximal SI were fixed in Bouin's fluid and wax embedded. IEL numbers per circumferential mm of mucosa were counted from H & E stained wax sections. Results: IEL count was significantly different between groups (ANOVA p<0.000). IELs increased as gestation proceeded (100, 125, 140days, significantly different from caeh other, p<0.05, Fishers pairwlse tes 0. All ligated fetsues had significantly lower IEL counts than normal (125d controls) @<0.05). Infusion with amniode fluid increased IELss (1><0.05)over ligation alone, or ligation plus eateral infusion. Growth retarded fetuses at 140 days had markedly depressed IEL counts (comparable to 100 day fetuses). IEL/mmmucosa(SEM)in fetalsheepSl 100d
125d
140d
liaated saline colts
28.2 (2.6)
49.2 (2.9)
65.0 11.8 17.6 (12.9) (1.18) (0.6)
12.2 (1.9)
milk
GRP
afluid IUGR
19.7 (1.9)
12.5 (3.0)
32.1 (1.8)
30.2 (2.7)
Conclusions: The fetal environment critically impacts on the development of IELs. Fetal ingestion of amniotie fluid is an important element in the development of the mueosal immune system. Growth retarded fetuses have impaired ontogeny of the IEL compartment. =J.F. Trahair & R. Harding (1987) Fetal gasttoiraestinalfunction and development. In: Animal models in fetal Medicine. Nathaliesz PW(ed) PerinatologyPress, Ithaca, 10p1-36.
• L O C A T I O N O F T R A N S F O R M I N G G R O W T H FACTOR-[~I AND P H E N O T Y P I C M O D U L A T I O N O F F I B R O B L A S T S D U R I N G G A S T R I C U L C E R H E A L I N G IN RATS. K. Tominaga, T. Arakawa, M. Tanaka, S. Kim,* T. Fukuda, K. Higuchi, H. Nakamura, H. Iwao,* K. Kobayashi. Third Dept. of Internal Medicine and *Dept. of Pharmacology, Osaka City University Medical School, Japan. Transforming growth factor-131 (TGF-B1) contributes to gastric ulcer healing (Gastroenterology 1994;104: A649). TGF-B1 modulates the phenotype of various cells in vitro; for example, it can transform fibroblasts into myofibmblasts. Myofibroblasts have smooth muscle actin (a contractile protein) and they contract granulation tissues, contributing to wound healing. The in vivo interaction between TGF-131 and fibroblasts during gastric ulcer healing is unknown. This study was done by immunohistochemical techniques to locate both TGF-B1 and fibroblasts in ulcerous areas of rat stomachs, and to identify phenotypie changes in such fibroblasts. Gastric ulcers were produced in male Wistar rats by the direct exposure of serosa during laparotomy to 100% acetic acid. Rats were killed 12 h later or on day 3, 5, 7, 18, or 32 (each, n = 6) after this treatment. Gastric ulcerous tissues and intact gastric tissues of sham-operated rats (controls) were stained by the immunoperoxidase method with chicken anti-human TGF-B1 antibody, mouse anti-swine vimentin monoclonal antibody, or mouse anti-human or-smooth muscle actin antibody. In intact gastric tissues, TGF-131 was found in some epithelial cells beneath the proliferative zone in the gastric glands. Fibroblasts in the intact submucosa were stained for vimentin. In ulcerous tissues on days 3 to 18, TGF-gl was found in most granulocytes on the ulcer bed, in most fibroblasts in the granulation tissues, and especially in marginal areas. Myofibroblasts (those stained for vimentin and a-smooth muscle actin) were not seen in intact submucosa, but appeared in marginal areas near the cells stained for TGF-131 by day 5 or 7, when the rate of decrease in the surface area of the ulcer ulcerous area was highest. By day 32, the number of myofibroblasts had decreased. That many myofibroblasts were found near cells stained for TGF-B1 in ulcerous areas suggested that TGF-gl contributes to gastric ulcer healing, possibly by phenotypic alteration of fibroblasts to myofibroblasts in a paracrinal interaction, leading to contraction of ulcerous tissues.
A757
• DIFFERENTIAL EFFECT OF GROWTH FACTORS ON INTESTINAL WALL COMPONENTS. J.S. Thomoson, Omaha VAMC, Depart of Surgery, University of Nebraska Medical Center, Omaha, Nebraska Both the mucosal and muscle layers respond to surgical manipulation of the small intestine. Various growth factors influence the mucosal response but less is known about changes in the muscle layers. Patching an intestinal defect with a serosal surface stimulates the adjacent intestine. Our aim was to evaluate the effect of epidermal growth factor (EGF) and octreotide (OCT) on changes in the intestinal wall induced by serosal patching. 24 NZ white rabbits (3-4 kg) were studied, GP I (n = 6) were unoparated controls. GP II (n = 6l underwent creation of a 2 x 5 cm serosal patch in the ileum. GP III (n=6) and GP IV (n=6) received EGF (1.5 pm/kg/hr SQ) or OCT (1000 h'g/d SQI after serosal patching. Wall composition, collagen content and mucosal proliferation and enzyme activity of normal adjacent ileum were studied after 7 days. GP I GP II GP III GP IV Mucosa (pm) 5 7 3 + 9 4 651_.+205 705._+_66* 542-+89@ Circ Muscle (pm) 86__+33 1 8 9 + 5 4 " 119-+37 161--+46" Long Muscle ~um) 55-+12 1051-17" 68-+17# 954-35 ~ Collagen Content 9 . 3 + 8 . 1 16.5-1- 8.4 5.6-+1,4"# 8.6-=-3.4 Crypt Cell Production Rate 6.1-t-.7 7.32~.2.1 11.5-+3.8 ~ 8.1-1-1.6 Ornithine decarb (sp act) 357-+84 1410-t-749" 1382__+434~ 652-+686#~ *p < . 0 5 v s G P I #p < . 0 5 v s G P I I ~p < . 0 5 v s G p l l l Serosal patching resulted in thickening of the mucosa and muscle layers with increased collagen content. EGF augmented the mucosal thickening and stimulated mucosal proliferation but inhibited the changes in muscle and collagen content. OCT inhibited the mucosal thickening and proliferation but did not affect the muscle. Disaccharidase activity was similar to unoperated animals in all gps. Conclusion: (1) Growth factors have differential effects on the intestinal wall components. (2) EGF stimulates the mucosa but has inhibitory effects on induced muscle changes. (3) Octreotide inhibits mucosal proliferation without apparent effect on muscle, (4) These observations have important implications for the therapeutic use of these agents, particularly after intestinal surgery.
O E V I D E N C E FOR A pH-DEPENDENT, DIDS-SENSITIVE C A R R I E R M E D I A T E D F O L A T E U P T A K E M E C H A N I S M IN THE HUMAN C O L O N I C LUMINAL M E M B R A N E VESICLES. S.A. Torania, H.F. Naom, H.M. Said* and P.K. Dudeja. Department of Medicine, University of Illinois at Chicago, Chicago, IL and *University of California-Irvine, Long Beach, CA Colonic bacteria have been shown to synthesize significant amounts of folate in the lumen. Furthermore, recent Northern blot studies in our laboratory using mouse colonic poly (A) + RNA have shown clear hybridization with labeled eDNA of the recently cloned small intestinal folate transporter. The current studies were, therefore, undertaken: i) to test the hypothesis that a folate transporter is present in apical membranes of the human colon, and if present, ii) to characterize this transporter. Apical membrane vesicles were isolated and purified from mucosal scrapings of organ donor proximal colons using a differential centrifugation and divalent cation (Mg+2) precipitation technique. 3H folate uptake was measured by a rapid filtration technique. Our results are summarized as follows: i) 3H folate uptake significantly increased with decreasing pH of the incubation buffer; ii) DIDS and SITS (0.5 raM) inhibited 3H relate uptake (in the presence of a pH gradient, 7.5in/5.5out) by -80%, whereas other transport inhibitors e.g. acetazolamide, amiloride, bumetanide, furosemide and DPC had no significant effect; iii) folate uptake was sensitive to temperature and osmolarity of the incubation medium; iv) 3H folate uptake was markedly inhibited by structural analogs amethopterin and 5methyltetrahydrofolate; v) 3H relate uptake into these vesicles exhibited trans-stimulation phenomenon; vi) 3H folate uptake was potential insensitive as voltage clamping of the vesicles or making them inside positive with K+/ valinomycin failed to influence the uptake; vii) 3H folate uptake demonstrated saturation kinetics with an apparent Km for relate of 8.2 _+ 1.7 ~tM and a V m a x of t9.8 -+ 2.9 pmol/mg prot./10 sec. Studies with distal colonic apical membrane vesicles also demonstrated the presence of a folate transporter in this region of the colon with similar transport characteristics (Kin : 5.0 ± 0.4 aM; Vmax : 12.5 ± 1.2 pmol/mg prot./10 see). Conclusion: These results demonstrate, for the first time, the existence of a pH-dependent, DIDS-sensitive, electroneutral carrier-mediated mechanism for folate absorption in the human colon. Our studies also indicate that the folate synthesized by colonic luminal bacteria might be nutritionally available for the host. (Supported by BRSG, NIDDK and the Dept. of Veterans Affairs)
Growth, Development, and Nutrition
• DEVELOPMENT OF INTRAEPITHELIAL LYMPHOCYTES (IEL) IN THE SMALL INTESTINE (SI) OF THE FETAL SHEEP- EFFECTS OF AGE AND ENTERAL DIET. F.M. Thompson, S.J. Wing, P. Sangild', J.F. Tmhair. Dep Anatomy & Histology, University of Adelaide, SA, AUSTRALIA; "Dep Clinical Studies, Reproduction, Royal Veterinary & Agricaltuml University of Copenhagen, DENMARK. Aims: To investigate the ontogeny of IELs in a well characterized model of SI development in a long gestation species (fetal sheep) 1. The effects of age, fetal swallowing and fetal growth retadation were studied. Methods: Normal fetal sheep were examined at 100(n=2), 125(7) and 140(3) days(d) gestation (term is 147d). Esophageal ligations and canuiations were carried out at 105d in 29 fetuses, preventing fetal ingestion. For 7d before collecting tissues at 125d, saline(n=4), milk(5) or colostral whey proteins(4), gaslrin releasing paptide (GRP, 3.6 nmol/day)(6), or amniotic fluid (5) was infused (4x20ml bolus par day). One group received no infusion(5). Non-pregnant ewes had endometrial caruncles removed prior to mating to produce fetuses(n=6) exhibiting chronic intraunterine growth retardation (IUGR) by 140d. Tissues from proximal SI were fixed in Bouin's fluid and wax embedded. IEL numbers per circumferential mm of mucosa were counted from H & E stained wax sections. Results: IEL count was significantly different between groups (ANOVA p<0.000). IELs increased as gestation proceeded (100, 125, 140days, significantly different from caeh other, p<0.05, Fishers pairwlse tes 0. All ligated fetsues had significantly lower IEL counts than normal (125d controls) @<0.05). Infusion with amniode fluid increased IELss (1><0.05)over ligation alone, or ligation plus eateral infusion. Growth retarded fetuses at 140 days had markedly depressed IEL counts (comparable to 100 day fetuses). IEL/mmmucosa(SEM)in fetalsheepSl 100d
125d
140d
liaated saline colts
28.2 (2.6)
49.2 (2.9)
65.0 11.8 17.6 (12.9) (1.18) (0.6)
12.2 (1.9)
milk
GRP
afluid IUGR
19.7 (1.9)
12.5 (3.0)
32.1 (1.8)
30.2 (2.7)
Conclusions: The fetal environment critically impacts on the development of IELs. Fetal ingestion of amniotie fluid is an important element in the development of the mueosal immune system. Growth retarded fetuses have impaired ontogeny of the IEL compartment. =J.F. Trahair & R. Harding (1987) Fetal gasttoiraestinalfunction and development. In: Animal models in fetal Medicine. Nathaliesz PW(ed) PerinatologyPress, Ithaca, 10p1-36.
• L O C A T I O N O F T R A N S F O R M I N G G R O W T H FACTOR-[~I AND P H E N O T Y P I C M O D U L A T I O N O F F I B R O B L A S T S D U R I N G G A S T R I C U L C E R H E A L I N G IN RATS. K. Tominaga, T. Arakawa, M. Tanaka, S. Kim,* T. Fukuda, K. Higuchi, H. Nakamura, H. Iwao,* K. Kobayashi. Third Dept. of Internal Medicine and *Dept. of Pharmacology, Osaka City University Medical School, Japan. Transforming growth factor-131 (TGF-B1) contributes to gastric ulcer healing (Gastroenterology 1994;104: A649). TGF-B1 modulates the phenotype of various cells in vitro; for example, it can transform fibroblasts into myofibmblasts. Myofibroblasts have smooth muscle actin (a contractile protein) and they contract granulation tissues, contributing to wound healing. The in vivo interaction between TGF-131 and fibroblasts during gastric ulcer healing is unknown. This study was done by immunohistochemical techniques to locate both TGF-B1 and fibroblasts in ulcerous areas of rat stomachs, and to identify phenotypie changes in such fibroblasts. Gastric ulcers were produced in male Wistar rats by the direct exposure of serosa during laparotomy to 100% acetic acid. Rats were killed 12 h later or on day 3, 5, 7, 18, or 32 (each, n = 6) after this treatment. Gastric ulcerous tissues and intact gastric tissues of sham-operated rats (controls) were stained by the immunoperoxidase method with chicken anti-human TGF-B1 antibody, mouse anti-swine vimentin monoclonal antibody, or mouse anti-human or-smooth muscle actin antibody. In intact gastric tissues, TGF-131 was found in some epithelial cells beneath the proliferative zone in the gastric glands. Fibroblasts in the intact submucosa were stained for vimentin. In ulcerous tissues on days 3 to 18, TGF-gl was found in most granulocytes on the ulcer bed, in most fibroblasts in the granulation tissues, and especially in marginal areas. Myofibroblasts (those stained for vimentin and a-smooth muscle actin) were not seen in intact submucosa, but appeared in marginal areas near the cells stained for TGF-131 by day 5 or 7, when the rate of decrease in the surface area of the ulcer ulcerous area was highest. By day 32, the number of myofibroblasts had decreased. That many myofibroblasts were found near cells stained for TGF-B1 in ulcerous areas suggested that TGF-gl contributes to gastric ulcer healing, possibly by phenotypic alteration of fibroblasts to myofibroblasts in a paracrinal interaction, leading to contraction of ulcerous tissues.
A757
• DIFFERENTIAL EFFECT OF GROWTH FACTORS ON INTESTINAL WALL COMPONENTS. J.S. Thomoson, Omaha VAMC, Depart of Surgery, University of Nebraska Medical Center, Omaha, Nebraska Both the mucosal and muscle layers respond to surgical manipulation of the small intestine. Various growth factors influence the mucosal response but less is known about changes in the muscle layers. Patching an intestinal defect with a serosal surface stimulates the adjacent intestine. Our aim was to evaluate the effect of epidermal growth factor (EGF) and octreotide (OCT) on changes in the intestinal wall induced by serosal patching. 24 NZ white rabbits (3-4 kg) were studied, GP I (n = 6) were unoparated controls. GP II (n = 6l underwent creation of a 2 x 5 cm serosal patch in the ileum. GP III (n=6) and GP IV (n=6) received EGF (1.5 pm/kg/hr SQ) or OCT (1000 h'g/d SQI after serosal patching. Wall composition, collagen content and mucosal proliferation and enzyme activity of normal adjacent ileum were studied after 7 days. GP I GP II GP III GP IV Mucosa (pm) 5 7 3 + 9 4 651_.+205 705._+_66* 542-+89@ Circ Muscle (pm) 86__+33 1 8 9 + 5 4 " 119-+37 161--+46" Long Muscle ~um) 55-+12 1051-17" 68-+17# 954-35 ~ Collagen Content 9 . 3 + 8 . 1 16.5-1- 8.4 5.6-+1,4"# 8.6-=-3.4 Crypt Cell Production Rate 6.1-t-.7 7.32~.2.1 11.5-+3.8 ~ 8.1-1-1.6 Ornithine decarb (sp act) 357-+84 1410-t-749" 1382__+434~ 652-+686#~ *p < . 0 5 v s G P I #p < . 0 5 v s G P I I ~p < . 0 5 v s G p l l l Serosal patching resulted in thickening of the mucosa and muscle layers with increased collagen content. EGF augmented the mucosal thickening and stimulated mucosal proliferation but inhibited the changes in muscle and collagen content. OCT inhibited the mucosal thickening and proliferation but did not affect the muscle. Disaccharidase activity was similar to unoperated animals in all gps. Conclusion: (1) Growth factors have differential effects on the intestinal wall components. (2) EGF stimulates the mucosa but has inhibitory effects on induced muscle changes. (3) Octreotide inhibits mucosal proliferation without apparent effect on muscle, (4) These observations have important implications for the therapeutic use of these agents, particularly after intestinal surgery.
O E V I D E N C E FOR A pH-DEPENDENT, DIDS-SENSITIVE C A R R I E R M E D I A T E D F O L A T E U P T A K E M E C H A N I S M IN THE HUMAN C O L O N I C LUMINAL M E M B R A N E VESICLES. S.A. Torania, H.F. Naom, H.M. Said* and P.K. Dudeja. Department of Medicine, University of Illinois at Chicago, Chicago, IL and *University of California-Irvine, Long Beach, CA Colonic bacteria have been shown to synthesize significant amounts of folate in the lumen. Furthermore, recent Northern blot studies in our laboratory using mouse colonic poly (A) + RNA have shown clear hybridization with labeled eDNA of the recently cloned small intestinal folate transporter. The current studies were, therefore, undertaken: i) to test the hypothesis that a folate transporter is present in apical membranes of the human colon, and if present, ii) to characterize this transporter. Apical membrane vesicles were isolated and purified from mucosal scrapings of organ donor proximal colons using a differential centrifugation and divalent cation (Mg+2) precipitation technique. 3H folate uptake was measured by a rapid filtration technique. Our results are summarized as follows: i) 3H folate uptake significantly increased with decreasing pH of the incubation buffer; ii) DIDS and SITS (0.5 raM) inhibited 3H relate uptake (in the presence of a pH gradient, 7.5in/5.5out) by -80%, whereas other transport inhibitors e.g. acetazolamide, amiloride, bumetanide, furosemide and DPC had no significant effect; iii) folate uptake was sensitive to temperature and osmolarity of the incubation medium; iv) 3H folate uptake was markedly inhibited by structural analogs amethopterin and 5methyltetrahydrofolate; v) 3H relate uptake into these vesicles exhibited trans-stimulation phenomenon; vi) 3H folate uptake was potential insensitive as voltage clamping of the vesicles or making them inside positive with K+/ valinomycin failed to influence the uptake; vii) 3H folate uptake demonstrated saturation kinetics with an apparent Km for relate of 8.2 _+ 1.7 ~tM and a V m a x of t9.8 -+ 2.9 pmol/mg prot./10 sec. Studies with distal colonic apical membrane vesicles also demonstrated the presence of a folate transporter in this region of the colon with similar transport characteristics (Kin : 5.0 ± 0.4 aM; Vmax : 12.5 ± 1.2 pmol/mg prot./10 see). Conclusion: These results demonstrate, for the first time, the existence of a pH-dependent, DIDS-sensitive, electroneutral carrier-mediated mechanism for folate absorption in the human colon. Our studies also indicate that the folate synthesized by colonic luminal bacteria might be nutritionally available for the host. (Supported by BRSG, NIDDK and the Dept. of Veterans Affairs)