Morio Takahashi

Hepatocyte growth factor is the most potent endogenous stimulant of rabbit gastric epithelial cell proliferation and migration in primary culture.

Various growth factors are suggested to be involved in gastric mucosal repair. Our previous studies have shown that exogenous hepatocyte growth factor (HGF) has a proliferative effect on gastric epithelial cells. In the present study, comparison of the maximum proliferative effects and the optimum concentrations of several growth factors revealed that HGF was the most potent mitogen for gastric epithelial cells, as is the case for hepatocytes. Restitution of gastric epithelial cell monolayers was assessed using a round wound restitution model. HGF was the most effective agent for facilitating gastric epithelial restitution among those tested. A binding assay revealed specific binding of HGF to its receptor on gastric epithelial cells. Northern blot analysis confirmed the expression of specific HGF receptor mRNA (c-met) by gastric epithelial cells but not by gastric fibroblasts. To investigate endogenous HGF production, we determined the effect of gastric fibroblast-conditioned medium on epithelial proliferation and restitution. The conditioned medium produced similar effects to HGF and its activity was neutralized by an anti-HGF antibody. In addition, expression of HGF mRNA was detected in gastric fibroblasts but not in gastric epithelial cells. Our immunohistochemical study confirmed these in vitro data by means of demonstrating the existence and localization of HGF at human native gastric mucosa. HGF was localized at fibroblasts under the epithelial cell layer around gastric ulcers. These results suggest that HGF may be a potent endogenous promotor of gastric epithelial cell proliferation and migration, and may contribute to gastric mucosal repair through a paracrine mechanism.

HEPATOCYTE GROWTH FACTOR AS A KEY TO MODULATE ANTI-ULCER ACTION OF PROSTAGLANDINS IN STOMACH

Although the clinical efficacy of prostaglandins (PGs), especially on gastric mucosal injuries induced by nonsteroidal antiinflammatory drugs, is widely appreciated, their mechanism of action, apart from acid suppression, is quite unclear. In this study, we have established a primary culture system of human gastric fibroblasts and clearly demonstrated that PGs strongly induce the expression of hepatocyte growth factor (HGF) in the fibroblasts, which is mediated by PGE specific receptor, EP2 or EP4. Since HGF facilitates repair and protection of gastric epithelial cells in a paracrine manner, it is assumed that some of the beneficial effects of PGs may be mediated by HGF. To confirm this assumption, we established a simplified in vitro culture gastric mucosal model which consists of gastric epithelial cells and gastric fibroblasts. Using the model, we performed a round wound restitution assay. PGE1 remarkably accelerated restitution which was completely inhibited by anti-HGF antibody, indicating that the action was mediated by HGF. To confirm these in vitro data, we further demonstrated that HGF mRNA expression is downregulated at the edges of nonsteroidal antiinflammatory drug-induced gastric ulcers where PGs should be depleted. In summary, we proposed that gastric fibroblasts are newly recognized targets of PGs, and HGF produced by human gastric fibroblasts may be a key factor for anti-ulcer action of PGs in the stomach.

High Seropositivity of Anti-CagA Antibody in Helicobacter pylori-Infected Patients Irrelevant to Peptic Ulcers and Normal Mucosa in Japan

CagA-positive H. pylori is reported to beassociated with gastroduodenal disease in Westerncountries. To evaluate the relationship between CagA anddisease, cloning of the entire cagA gene (3771 bp),insertion of a partial fragment (1272 bp) into anexpression vector, purification of the recombinantprotein, production of an antibody against therecombinant CagA protein through rabbits, and use of therecombinant CagA protein as an antigen, detection of theanti-CagA antibody by western blotting were allperformed. Sera of 132 H. pylori-infected patientsundergoing endoscopy were studied. Anti-CagA antibodieswere detected in 90%, 87%, 90%, 94%, and 93% ofpatients with gastric ulcer (N = 34), duodenal ulcer (N= 27), chronic gastritis (N = 31), gastric cancer (N =17), and normal mucosa (N = 15), respectively. High seropositivity of anti-CagA antibody even inindividuals with normal mucosa indicated that CagA maynot be a unique marker for disease by H. pyloriinfection in Japan.

Interleukin-8 Production in Primary Cultures of Human Gastric Epithelial Cells Induced by Helicobacter pylori

Interleukin-8 (IL-8) production by the gastricmucosa is increased in Helicobacter pylori infection.Previous studies indicated that H. pylori induces IL-8synthesis in cancer cell lines, and the ability of H. pylori to stimulate IL-8 production issupposed to be associated with cag A and other cagpathogenicity island genes, including pic B gene. In thepresent study, we investigated the induction of IL-8 in primary cultures of normal human gastricepithelial cells to elucidate the IL-8 induction by wildtype strains and by the pic B knockout strain. Humangastric epithelial cells were obtained from surgically resected specimens from four patients. Three H.pylori strains (TN2F4; type 1 clinical isolate, TN2F4m1;isogenic pic B mutant of TN2F4, Tx30a; type 2 strain)were cocultured with the normal gastric epithelial cells or the transformed MKN-28. IL-8 levels inculture medium were determined by enzyme immunoassay.Human gastric epithelial cells produced IL-8 at a 10 -50times higher level than MKN-28 did when cocultured with TN2F4. The mutant TN2F4m1 induced IL-8 atsignificantly lower levels than the parent strain. Cellsfrom four patients behaved similarly on IL-8 production.The results of the present study demonstrated the induction of IL-8 in normal gastricepithelial cells, suggesting that pic B gene product mayplay an essential role in vivo.

A case of primary gastric choriocarcinoma and a review of the Japanese literature

A 63-year old woman who had experienced melena for 2 weeks was admitted to Tokyo University Hospital. Gastric adenocarcinoma was diagnosed endoscopically and histologically, and a total gastrectomy was performed soon thereafter. Pathological examination of the resected stomach revealed choriocarcinoma of the stomach. Although chemotherapy was administered after surgery, she died 3 months after admission. Autopsy confirmed the diagnosis of primary gastric choriocarcinoma, a rare, but highly malignant tumor. It is characteristic; macroscopically it forms a necrotic mass with bleeding, and microscopically it often consists of adenocarcinoma and choriocarcinoma. Since its prognosis is extremely poor, we must take into account the possibility of primary gastric choriocarcinoma when a hemorrhagic gastric tumor with necrosis is found.

Promoters of epithelialization induce expression of vascular endothelial growth factor in human gastric epithelial cells in primary culture

Both epithelialization and angiogenesis are indispensable processes in gastric ulcer healing. Coordination between these processes has not been well studied. In the present study, we have established a new primary culture system of human gastric epithelial cells and investigated the effect of epithelialization stimulants on a specific angiogenic factor, vascular endothelial growth characterized as epithelial cells. Both epithelialization stimulants, hepatocyte growth factor (HGF) and epidermal growth factor (EGF), significantly stimulated vascular EGF expression in gastric epithelial cells. HGF and EGF receptors were expressed by the cells, suggesting that regulation may be mediated through specific receptors.

Growth regulation of rabbit gastric epithelial cells and protooncogene expression

We recently developed a primary culture system for gastric epithelial cells from adult rabbits that allows the investigation of growth regulation at the cellular level. In this study, we demonstrated that epidermal growth factor (EGF), insulin, and dibutyryl adenosine 3′,5′-cyclic monophosphate (dBcAMP) all stimulated cell proliferation. Insulin and dB-cAMP potentiated the stimulation of cell proliferation by EGF, while transforming growth factor-β1 (TGF-β1) inhibited it. Expression of c-fos and c-myc was induced in response to the stimulation by these growth regulators, but the degree of expression did not necessarily correlate with the effects of these agents on cell proliferation. In conclusion, EGF, insulin, and dBcAMP were positive growth regulators, while TGF-β1 was a negative regulator in gastric epithelial cells. These growth modulators may exert their effects by distinct pathways from a standpoint of the expression of c-fos and c-myc.

Antiulcer drugs and gastric prostaglandin E2: an in vitro study.

Prostaglandin (PG) has been reported to be an important protective and acid-suppressive factor in the gastric mucosa. Although the mechanisms of some antiulcer drugs are attributed to their stimulatory effects on endogenous prostaglandins, an understanding of these actions has not been established. In the present study we investigated the effects of antiulcer drugs on PGE2 using cultured gastric mucosal cells. Rabbit gastric mucosal cells were cultured after isolation with collagenase and ethylenediaminetetraacetic acid. PGE2 was measured by enzyme-linked immunoassay. Histamine H2-blockers (cimetidine, ranitidine, famotidine), omeprazole, and sucralfate did not modulate the media content of PGE2, whereas sofalcone dose- and time-dependently increased it. Sofalcone-induced increase of PGE2 was dose-dependently prevented by indomethacin. Sofalcone did not affect intracellular Ca2+ as assessed by the calcium-sensitive probe indo-1. Deprivation of Ca2+ in the media did not modulate the action of sofalcone. Sofalcone significantly suppressed 15-OH-PG dehydrogenase. These results suggest that among the various antiulcer drugs only sofalcone increases PGE2, which may be a factor in its therapeutic effect against peptic ulcer diseases.

The possible role of vascular endothelial growth factor (VEGF) in gastric ulcer healing: effect of sofalcone on VEGF release in vitro.

Angiogenesis plays an important role in gastric ulcer repair. Several growth factors are involved in angiogenesis and, of these, vascular endothelial growth factor (VEGF) has received considerable attention because it is the only factor that acts specifically on endothelial cells and, unlike other angiogenic growth factors, has the hydrophobic signal peptide required for extracellular transport according to classical secretory pathways. We have lately demonstrated the role of VEGF in gastric ulcer repair. Previous reports confirmed that sofalcone has remarkable effects on gastric ulcer healing, which may be mediated by its stimulatory effect on prostaglandin (PG) release in gastric cells. Our data indicate that PGs stimulate VEGF expression in gastric fibroblasts. In this report we hypothesize that the clinical effect of sofalcone is mediated by VEGF expression and we demonstrate that sofalcone stimulates VEGF release by gastric fibroblasts in primary culture.

Effect of caffeine on mucus secretion and agonist-dependent Ca2+ mobilization in human gastric mucus secreting cells.

Caffeine is known to stimulate gastric acid secretion, but, the effects of caffeine on gastric mucus secretion have not been clarified. To elucidate the action of caffeine on gastric mucin-producing cells and its underlying mechanism, the effects of caffeine on mucus glycoprotein secretion and agonist-induced [Ca2+]i mobilization were examined in human gastric mucin secreting cells (JR-I cells). The measurement of [Ca2+]i using Indo-1 and the whole cell voltage clamp technique were applied. Mucus glycoprotein secretion was assessed by release of [3H]glucosamine. Caffeine by itself failed to increase [Ca2+]i and affect membrane currents, while it dose-dependently inhibited agonist (acetylcholine (ACh) or histamine)-induced [Ca2+]i rise, resulting in inhibiting activation of Ca2+-dependent K+ current (I(K.Ca)) evoked by agonists. The effect of caffeine was reversible, and the half maximal inhibitory concentration was about 0.5 mM. But, caffeine did not suppress [Ca2+]i rise and activation of I(K.Ca) induced by A23187 or inositol trisphosphate (IP3). Theophylline or 3-isobutyl-1-methyl-xanthine (IBMX) did not mimic the effect of caffeine. Caffeine failed to stimulate mucus secretion, while it significantly decreased ACh-induced mucus secretion. These results indicate that caffeine selectively inhibits agonist-mediated [Ca2+]i rise in human gastric epithelial cells, probably through the blockade of receptor-IP3 signaling pathway, which may affect the mucin secretion.